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MALAT1 promotes the proliferation and metastasis of gallbladder cancer cells by activating the ERK/MAPK pathway Xiang-Song Wu Ding
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, Xu-An Wang abc
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, Zhu-Jun Tan
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, Tian-Yu Liu d
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, Yun-Ping Hu
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, Feng Tao & Ying-Bin Liu
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, Mao-Lan Li
, Yang Cao
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Department of General Surgery; Xinhua Hospital; Shanghai Jiao Tong University School of Medicine; Shanghai, PR China b
Laboratory of General Surgery, Xinhua Hospital; Shanghai Jiao Tong University School of Medicine; Shanghai, PR China
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Institute of Biliary Tract Disease; Shanghai Jiao Tong University School of Medicine; Shanghai, PR China d
Department of Gastrointestinal Surgery; Shaoxing People’s Hospital; Shaoxing, PR China Published online: 21 Mar 2014.
To cite this article: Xiang-Song Wu, Xu-An Wang, Wen-Guang Wu, Yun-Ping Hu, Mao-Lan Li, Qian Ding, Hao Weng, Yi-Jun Shu, Tian-Yu Liu, Lin Jiang, Yang Cao, Run-Fa Bao, Jia-Sheng Mu, Zhu-Jun Tan, Feng Tao & Ying-Bin Liu (2014) MALAT1 promotes the proliferation and metastasis of gallbladder cancer cells by activating the ERK/MAPK pathway, Cancer Biology & Therapy, 15:6, 806-814, DOI: 10.4161/cbt.28584 To link to this article: http://dx.doi.org/10.4161/cbt.28584
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Research Paper Research Paper
Cancer Biology & Therapy 15:6, 806–814; June 2014; © 2014 Landes Bioscience
MALAT1 promotes the proliferation and metastasis of gallbladder cancer cells by activating the ERK/MAPK pathway Xiang-Song Wu1,2,3,†, Xu-An Wang1,2,3,†, Wen-Guang Wu1,2,3,†, Yun-Ping Hu1,2,3,†, Mao-Lan Li1,2,3, Qian Ding1,2,3, Hao Weng1,2,3, Yi-Jun Shu1,2,3, Tian-Yu Liu1,2,3, Lin Jiang1,2,3, Yang Cao1,2,3, Run-Fa Bao1,2,3, Jia-Sheng Mu1,2,3, Zhu-Jun Tan1,2,3, Feng Tao4,*, and Ying-Bin Liu1,2,3,* Department of General Surgery; Xinhua Hospital; Shanghai Jiao Tong University School of Medicine; Shanghai, PR China; 2Laboratory of General Surgery, Xinhua Hospital; Shanghai Jiao Tong University School of Medicine; Shanghai, PR China; 3Institute of Biliary Tract Disease; Shanghai Jiao Tong University School of Medicine; Shanghai, PR China; 4Department of Gastrointestinal Surgery; Shaoxing People’s Hospital; Shaoxing, PR China These authors contributed equally to this work.
†
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Keywords: gallbladder carcinoma, MALAT1, lncRNA, ERK, MAPK, proliferation, metastasis Abbreviations: MALAT1, metastasis-associated lung adenocarcinoma transcript 1; GBC, gallbladder carcinoma; lncRNA, long non-coding RNA; EMT, epithelial–mesenchymal transition; ERK, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; HOTAIR, HOX antisense intergenic RNA; PCGEM1, prostate-specific transcript 1; CUDR, cancer upregulated drug resistant; MVIH, lncRNA associated with micro-vascular invasion in HCC; NSCLC, non-small cell lung cancer; qRT-PCR, quantitative real-time PCR analysis; FBS, fetal calf serum; shRNA, short hairpin RNA; CMV, cytomegalovirus; EGFP, enhanced green fluorescent protein; CCK-8, cell counting kit-8; GPC6, glypican 6; CXCL5, C-X-C motif chemokine 5
Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), a long non-coding RNA (lncRNA), is associated with metastasis and is an independent prognostic factor for lung cancer. Recent studies have demonstrated that MALAT1 plays an important role in other malignancies. However, little is known about the role of MALAT1 in gallbladder carcinoma (GBC), which is the most common cancer of the biliary tract and has an extremely poor prognosis. In this study, we focused on the expression, biological functions and mechanism of MALAT1 in GBC and found that MALAT1 was significantly upregulated in GBC tissues compared with corresponding non-cancerous tissues. Knockdown of MALAT1 in GBC cell lines using lentivirus-mediated RNA interference significantly inhibited the proliferation and metastasis of the GBC cells both in vitro and in vivo. Furthermore, ERK/MAPK pathway was found to be inactivated in the GBC cell lines after MALAT1 knockdown. These results indicated that MALAT1 might serve as an oncogenic lncRNA that promotes proliferation and metastasis of GBC and activates the ERK/MAPK pathway.
Introduction Gallbladder carcinoma (GBC) is the most common cancer of the biliary tract and the sixth most common gastrointestinal cancer.1-9 Curative resection is the only cure for this highly lethal malignancy. However, owing to its non-specific symptoms and highly invasive nature, most patients are at an advanced stage when they are diagnosed.10 Therefore, only a minority of patients are candidates for curative resection. As a result, GBC has an extremely poor prognosis. The mean survival for this malignancy ranges from 5.2 to 24.4 mo, according to different reports.9,11-13 Most of these patients die from tumor metastasis and recurrence. Therefore, an understanding of the molecular mechanisms of GBC metastasis and recurrence is indispensable for the development of effective adjuvant therapy.
The human genome sequencing project has found that 70% of the genome is transcribed, but only up to 2% of the human genome serves as blueprints for proteins.14,15 Long non-coding RNAs (lncRNAs) are defined as endogenous cellular RNAs more than 200 nucleotides in length that lack an open reading frame of significant length.16 In recent years, several lncRNAs have been shown to be involved in carcinogenesis and cancer progression.17 Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) was initially found to be highly expressed in lung cancer and is a prognostic factor for the survival of patients with stage I non-small cell lung cancer.18 Moreover, studies have revealed that MALAT1 can promote cancer cell migration and the epithelial–mesenchymal transition (EMT) by regulating the expression of relevant genes.19,20 However, little is known about the role of MALAT1 in GBC progression.
*Correspondence to: Feng Tao; Email: [email protected]; Yingbin Liu; Email: [email protected] Submitted: 12/23/2013; Revised: 03/17/2014; Accepted: 03/18/2014; Published Online: 03/21/2014 http://dx.doi.org/10.4161/cbt.28584 806
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1
In the present study, MALAT1 was found to be overexpressed in GBC lesions compared with paired non-tumor samples. In addition, GBC cell lines (NOZ and SGC-996) infected with MALAT1-siRNA expressing lentivirus (Lv-siMALAT1) exhibited a reduced growth rate, lowered invasion ability, and inactivation of the ERK/MAPK pathway.
Results
Table 1. Clinical and pathological information of 20 gallbladder cancer patients Variable
Number or mean
Total number
20
Sex Male
6
Female
14
Age (mean)
60.1
Well
4
Moderately
10
Poorly
6
Tumor invasion T1
2
T2
5
T3
7
T4
6
Lymphnode metastasis Absence
7
Presence
12
Not available
1
Distant metastasis Absence
18
Presence
2
TNM stage I
2
II
4
III
8
IV
6
Figure 1. MALAT1 was upregulated in GBC tissues. (A) MALAT1 expression in 20 pairs of GBC tissues (Tumor) and corresponding non-tumorous tissues (NT). MALAT1 expression was calculated and is expressed as the MALAT1/GAPDH expression ratio (i.e., 2–ΔCt). The mean MALAT1 expression levels in the tumor and NT samples were 11.32 and 3.72, respectively. MALAT1 expression was significantly increased in the tumor tissues (**P < 0.01). (B) Comparison of the MALAT1 expression level between the CRC tissues and their corresponding non-tumorous tissues.
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Tumor differentiation
MALAT1 was upregulated in GBC tissues First, we performed qRT-PCR to evaluate the expression of MALAT1 in 20 GBC tissues and corresponding non-tumor tissues. All patients were diagnosed as gallbladder adenocarcinoma. Relevant clinical and pathological information of these 20 patients are shown in Table 1. MALAT1 was upregulated approximately 3-fold in GBC tissue samples compared with the corresponding non-tumor tissues (Fig. 1). Lentiviral siRNA effectively and specifically inhibited MALAT1 expression in SGC-996 and NOZ cells Because MALAT1 was overexpressed in GBC, we investigated the function of MALAT1 using lentivirus siRNA-mediated disruption of MALAT1 expression in two GBC cell lines (SGC-996 and NOZ). The lentiviral transfection efficiencies of the SGC-996 and NOZ cells were determined by examining GFP expression under a microscope 72 h after transfection (Fig. 2A). The efficiency of lentiviral transfection in both the SGC-996 and NOZ cells was higher than 90%. MALAT1 expression in SGC-996 and NOZ cells was examined using real-time PCR analysis after transfection with lentivirus. The levels of MALAT1 expression in the SGC-996 and NOZ cells transfected with MALAT1-siRNA lentivirus (siMALAT1) were decreased by 61.5% (P < 0.01) and 75.7% (P < 0.001), respectively, compared with the control cells (Fig. 2B). These results demonstrated that the lentivirus-mediated
siRNA effectively and specifically reduced MALAT1 expression in the SGC-996 and NOZ cells. Effects of MALAT1 knockdown on SGC-996 and NOZ cell proliferation To investigate whether MALAT1 knockdown could influence the proliferation of gallbladder cancer cells in vitro, CCK-8 and colony formation assays were performed. Figure 3A and B show that the proliferation abilities of the SGC-996 and NOZ cells decreased significantly after incubation with si-MALAT1 for 4 d (P < 0.001 for SGC-996, P < 0.01 for NOZ). Additionally, the colony formation assay also showed that silencing of MALAT1 significantly decreased the number of colonies formed by the SGC-996 and NOZ (Fig. 3C and D) cells compared with the control and si-Control group (P < 0.01, respectively). To understand the effects of MALAT1 knockdown on cell cycle distribution, gallbladder cancer cells were analyzed by flow cytometry. The results indicated that MALAT1 knockdown increased the percentage of G2 /M cells (P < 0.001, respectively, Fig. 3E–G). Furthermore, to probe the effects of MALAT1 on gallbladder cancer cell growth in vivo, MALAT1-depleted or control SGC996 cells were injected into the left axilla of nude mice. Our results showed that the growth of tumors from the MALAT1-depleted
xenografts was significantly inhibited compared with that of tumors formed from the mock-infected or control cells (P < 0.05, respectively, Fig. 3H–J). Effects of MALAT1 knockdown on SGC-996 and NOZ cell metastasis Transwell migration and invasion assays were performed to investigate the role of MALAT1 in the metastasis of GBC cells. Figure 4A and C shows that MALAT1 disruption significantly reduced the migration of SGC-996 and NOZ cells (P < 0.01 and P < 0.001, respectively). Invasiveness was also inhibited by MALAT1 knockdown (P < 0.01 and P < 0.001, respectively, for SGC-996 and NOZ; Fig. 4B and D). Accordingly, the expression of matrix metalloproteinase 9 (MMP-9), an enzyme that is involved in the breakdown of the extracellular matrix during cancer cell invasion, was also found to be significantly reduced after MALAT1 downregulation (Fig. 4E). To confirm these findings in vivo, we used a peritoneal metastasis model in nude mice. Mice injected with MALAT1-depleted NOZ cells exhibited few ascites (Fig. 4F) at 8 wk after inoculation. After dissecting the peritoneal metastatic tumor, we found that the total tumor weight was significantly lower in the si-MALAT1 group than in the si-Control group (P < 0.01; Fig. 4G).
Figure 3 (See opposite page). MALAT1 increased proliferation in the GBC cell lines. (A and B) Cellular proliferation of untransfected or transfected SGC-996 and NOZ cells were measured using a CCK-8 assay daily for 4 d. (C and D) SGC-996 and NOZ cells were seeded at 400 cells/well, and the cells were allowed to form colonies. The colony numbers were counted and recorded. (E) Untransfected or transfected SGC-996 and NOZ cells were stained by propidium iodide and analyzed by flow cytometry. (F and G) The percentage of cells in the G0/G1, S, and G2/M phases of the cell cycle were calculated. Results are expressed as mean ± SD from three independent experiments. (H) A subcutaneous implanted model of human GBC was established using SGC-996 cells. (I) Photograph of a tumor developed in the subcutaneous implanted model. (J) A statistical plot of average tumor weights in the subcutaneous implanted model. The graph shows the mean ± SD; ***P < 0.001, **P < 0.01, *P < 0.05. The data represent one of three separate experiments. Control, blank control; si-control, cells transduced with lentivirus-mediated scr-siRNA; si-MALAT1, cells transduced with lentivirus-mediated MALAT1-siRNA. 808
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Figure 2. Lentivirus-mediated siRNA decreased the expression of MALAT1 in the SGC-996 and NOZ cells. (A) The transfection efficiency was determined 3 d after incubation with lentivirus at an MOI of 50. The transfected cells labeled with GFP were observed under a light microscope and a fluorescence microscope. Light micrograph (upper); Fluorescent micrograph (lower) (200×). (B and C) Total RNA was extracted 5 d after infection, and the relative MALAT1 expression was determined using quantitative real-time PCR. GAPDH was used as an internal control. The data represent the mean ± SD of three independent experiments. **P < 0.001, compared with si-Control. Control, blank control; si-control, cells transduced with lentivirus-mediated scr-siRNA; si-MALAT1, cells transduced with lentivirus-mediated MALAT1-siRNA.
Figure 3. For figure legend, see page 808.
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Suppression of MAPK kinase pathways by MALAT1 knockdown To determine the possible mechanism by which MALAT1 regulated proliferation and metastasis of GBC cells, we performed western blot analysis to investigate the effects of MALAT1 knockdown on the extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) pathway, which is often aberrantly activated in human cancers and contributes to enhanced cell proliferation and metastasis.21,22 Western blot analysis showed that MALAT1 downregulation significantly reduced the levels of phosphorylated MEK1/2, ERK 1/2, MAPK, and JNLK 1/2/3, while no detectable changes were observed in the total levels of MEK1/2, ERK 1/2, MAPK, and JNLK 1/2/3 (Fig. 5). These results indicated that the ERK/MAPK pathway
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might participate in the MALAT1-induced proliferation and metastasis of GBC cells.
Discussion GBC is a highly invasive and rapidly proliferative cancer with a poor prognosis.9 Therefore, the identification of novel methods that can effectively inhibit GBC growth and metastasis is needed. Although lncRNAs were initially argued to be spurious transcriptional noise, recent evidence suggests that they may play a major biological role in cellular development and human diseases.23,24 During recent years, an increasing number of studies have focused on the role of lncRNAs in tumorigenesis. LncRNAs
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Figure 4. MALAT1 promoted metastasis in the GBC cell lines. (A) Representative photographs of crystal violet-stained SGC-996 and NOZ cells that migrated through polycarbonate membranes. (B) Representative photographs of crystal violet-stained SGC-996 and NOZ cells that invaded through matrigel. (C) A statistical plot of the average number of migrated SGC-996 and NOZ cells in each group. (D) A statistical plot of the average number of invasive SGC-996 and NOZ cells in each group. (E) Western blot analysis indicated that MMP-9 was downregulated in the SGC-996 and NOZ cells after MALAT1 knockdown. (F) A peritoneal metastasis model of human GBC was established using NOZ cells. Mice receiving the MALAT1-depleted NOZ cells exhibited little ascites at 8 wk after inoculation. (G) A statistical plot of the average tumor weights in the peritoneal metastasis model. The graph shows the mean ± SD; ***P < 0.001, **P < 0.01, *P < 0.05. The data represent one of three separate experiments. Control, blank control; si-control, cells transduced with lentivirus-mediated scr-siRNA; si-MALAT1, cells transduced with lentivirus-mediated MALAT1-siRNA.
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Figure 5. MALAT1 activated the ERK/MAPK pathway in the GBC cell lines. Western blot analysis of the ERK/MAPK pathway-related proteins in the SGC-996 and NOZ cells. Representative blots are shown. Control, blank control; si-control, cells transduced with lentivirus-mediated scr-siRNA; si-MALAT1, cells transduced with lentivirus-mediated MALAT1-siRNA.
MALAT1 knockdown significantly reduced the expression of phosphorylated MEK1/2, ERK 1/2, MAPK, and JNK 1/2/3. However, no detectable changes in total MEK1/2, ERK 1/2, MAPK, or JNK 1/2/3 protein expression were observed. Therefore, impaired GBC cell growth and metastasis due to MALAT1 knockdown could be explained, at least in part, by the inactivation of the ERK/MAPK pathway. Unfortunately, a direct link between MALAT1 and the ERK/MAPK pathway remain unelucidated. According to Gutschner et al.’s study,34 knockout of MALAT1 in lung cancer cells could significantly reduce the expression level of several metastasis related genes, which included Glypican 6 (GPC6) and C-X-C motif chemokine 5 (CXCL5). Furthermore, previous studies35,36 have indicated that depletion of GPC6 or CXCL5 could lead to the inactivation of MAPK pathway. Therefore, we can hypothesize that MALAT1 activates ERK/MAPK pathway via regulation the expression of GPC6 or CXCL5 genes. In conclusion, we found that MALAT1 was significantly upregulated in GBC tissues. Knockdown of MALAT1 could inhibit GBC cell proliferation and metastasis both in vitro and in vivo. Moreover, knockdown of MALAT1 led to the inactivation of the ERK/MAPK pathway. Therefore, MALAT1 might serve as an oncogenic lncRNA that promotes proliferation and metastasis of GBC and activates the ERK/MAPK pathway.
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such as HOTAIR (HOX antisense intergenic RNA), PCGEM1 (prostate-specific transcript 1), CUDR (cancer upregulated drug resistant), and MVIH (lncRNA associated with microvascular invasion in HCC) have been shown to be involved in the biological hallmarks of cancer, including sustaining proliferative signaling, evading growth suppressors, enabling replicative immortality, activating invasion and metastasis, inducing angiogenesis, and resisting cell death.17,25 MALAT1, which is also known as HCN, NEAT2, PRO2853, and NCRNA00 047, is located at chromosome 11q13.1 and encodes a polyadenylated non-coding RNA (ncRNA) of ~8 kb.18,26 Ji’s work demonstrated that MALAT1 influenced metastasis and patient survival in non-small cell lung cancer (NSCLC).18 This ncRNA is extremely abundant in many human cell types and is highly conserved across several species, which underscores its functional importance.27 Additionally, Ying et al. showed that MALAT1 was upregulated in the metastatic tissue of bladder cancer and contributed to bladder cancer cell migration.20 Lai et al. found that MALAT1 was an independent prognostic factor for HCC recurrence after liver transplantation.28 However, MALAT1 has not so far been linked to GBC or any other malignancy in the biliary tract. In this study, we found that MALAT1 was highly expressed in GBC tissue samples. To understand its biological role in GBC, we used lentivirus-mediated siRNA to knock down MALAT1. We observed that, in SGC-996 and NOZ cells, MALAT1 downregulation significantly inhibited cell proliferation in vitro according to the CCK8 and colony formation assays. Additionally, cell migration and invasion were also inhibited in vitro by MALAT1 knockdown. Furthermore, the subcutaneous and peritoneal implanted models were used to confirm these findings in vivo, which showed that both proliferation and metastasis were suppressed by MALAT1 knockdown. In general, the exact mechanism of MALAT1 function is still unknown. MALAT1 specifically localizes to nuclear speckles and regulates the alternative splicing of pre-mRNAs by modulating the levels of active serine/arginine splicing factors.26,29 Depletion of MALAT1 alters the processing of a subset of pre-mRNAs that play important roles in cancer biology.30 This evidence supports the hypothesis that MALAT1 could be a regulator of posttranscriptional RNA processing or modification.17 Moreover, MALAT1 was found to interact with the unmethylated form of CBX4, which controls the relocation of growth-control genes between the polycomb bodies and interchromatin granules, sites of silent or active gene expression, respectively.31 In addition, MALAT1 was shown to promote cancer cell migration by inducing the EMT, and the activation of the Wnt pathway participated in this process. To elucidate the possible mechanism by which MALAT1 regulates GBC cell proliferation and metastasis, western blot analysis of the key molecular factors of cancerrelated pathways, such as NFκB, Smad2, Akt, and others (data not shown), was performed. The ERK/MAPK pathway is one of the most important signal transduction pathways, and MALAT1 promotes tumor growth and metastasis by activating this signaling cascade.32,33 We observed that in SGC-996 and NOZ cells,
Patients and pathological data This study was approved by the ethics committee of Xinhua Hospital, and all patients provided informed consent. The data do not contain any information that could identify the patients. Fresh GBC tissue samples and paired non-cancerous tissue samples were obtained from 20 GBC patients who underwent radical cholecystectomy without prior radiotherapy or chemotherapy between 2012 and 2013 at the Department of General Surgery, Xinhua Hospital, School of Medicine, Shanghai Jiao Tong University, PR China. These samples were used for quantitative realtime PCR analysis (qRT-PCR). Fresh tissues were collected in the operating room and were processed within 15 min after they were removed from the body. Each sample was frozen and stored at –80 °C. The paired non-cancerous tissues were dissected at least 2 cm away from the tumor border and were confirmed to lack tumor cells by microscopy. The diagnoses of GBC were confirmed by histopathological examination. Quantitative real-time PCR (qRT-PCR) Total RNA was extracted from the tissue samples or cultured cells with Trizol reagent (Takara), and first-strand cDNA was synthesized from 2 μg of total RNA using random primers and the M-MLV Reverse Transcriptase (Invitrogen). The following primers were used to detect the expression of MALAT1 and GAPDH (internal control): MALAT1 (sense): 5′-AAAGCAAGGT CTCCCCACAA G-3′; MALAT1 (antisense): 5′-GGTCTGTGCT AGATCAAAAG GCA-3′; GAPDH (sense): 5′-AGAAGGCTGG GGCTCATTTG-3′; GAPDH (antisense): 5′-AGGGGCCATC CACAGTCTTC-3′. RNA expression was measured by qRT-PCR using the SYBRGreen method (Takara) according to the manufacturer’s instructions. The results were normalized to the expression of GAPDH. Cell culture Two GBC cell lines (SGC-996 and NOZ) were used in the present study. The SGC-996 cell line was provided by the Academy of Life Science, Tongji University and was cultured in RPMI-1640 medium (GIBCO) supplemented with 10% fetal calf serum (FBS; GIBCO) and 100 U/mL penicillin/streptomycin. The NOZ cell line was purchased from the Health Science Research Resources Bank and was cultured in Williams’ Medium E (GIBCO) supplemented with 10% FBS and 100 U/mL penicillin/streptomycin. All cells were cultivated in a humidified incubator at 37 °C and 5% CO2. Lentivirus-mediated RNA interference The following short hairpin RNA (shRNA)19 was used to target human MALAT1: sense: 5′-CACAGGGAAA GCGAGTGGTT GGTAA-3′; antisense: 5′-TTACCAACCA CTCGCTTTCC CTGTG-3′. The sequence of the negative control shRNA was 5′-TTCTCCGAAC GTGTCACGT-3′. These shRNAs were synthesized and inserted into the pFH1UGW lentivirus core vector containing a cytomegalovirus (CMV)driven enhanced green fluorescent protein (EGFP) reporter gene; expression of the shRNA was driven by the H1 promoter. Recombinant lentivirus expressing MALAT1-siRNA or control siRNA (si-MALAT1 or si-Control) was produced by the Genechem
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Company. SGC-996 and NOZ cells were infected with concentrated virus in medium without FBS. The supernatant was replaced with complete culture medium after 24 h. The expression of MALAT1 in the infected cells was validated by qRT-PCR analysis after 120 h. Cell proliferation assay Cell proliferation was quantified using the Cell Counting Kit-8 (CCK-8; Dojindo). Briefly, 100 μL of cells from the three groups (control, si-Control, and si-MALAT1) were seeded into a 96-well plate at a concentration of 1000 cells per well and were incubated at 37 °C. At daily intervals (days 1, 2, 3, and 4), the optical density was measured at 450 nm using a microtiter plate reader (Quant BioTek Instruments), and the cell survival rate was expressed as the absorbance relative to that of the control group. The results represent the average of three replicates under the same conditions. Colony formation assay Both non-transfected and transfected SGC-996 and NOZ cells (400 cells/well) were seeded in 6-well plates. The cells were cultured for approximately 12 d, fixed with 4% paraformaldehyde, and stained with 0.1% crystal violet (Sigma-Aldrich). After washing, the plates were air-dried, and the stained colonies were photographed using a microscope (Leica). The total number of colonies (>50 cells/colony) was counted. The experiments were performed in triplicate. Cell cycle analysis Both non-transfected and transfected SGC-996 and NOZ cells were then harvested by trypsinization, washed twice in cold PBS, and fixed in 70% ethanol at 4 °C overnight. After fixation, the cells were washed and resuspended in cold PBS and incubated in a solution of 10 mg/mL RNase and 1 mg/mL propidium iodide (Sigma-Aldrich) at 37 °C for 30 min in the dark. Finally, the DNA content was determined by flow cytometry (BD Biosciences). The percentage of cells in the G0 /G1, S, and G2 /M phases was determined using Cell Quest acquisition software (BD Biosciences). Transwell migration and invasion assay Cell migration ability was assessed using 6.5-mm transwell chambers with a pore size of 8 μm. Cell invasion was assessed using the Chamber matrigel invasion 24-well DI kit (BD). The assays were performed according to the manufacturer’s instructions. Briefly, 2.5 × 104 cells (SGC-996 and NOZ) from each group were suspended in serum-free medium and were seeded into the upper chamber. The lower chamber was filled with medium containing 10% FBS. After incubation for 24 h, the migrated/ invaded cells in the lower chamber (below the filter surface) were fixed in 4% paraformaldehyde, stained with 0.1 mg/mL crystal violet solution, and counted under a microscope (20× objective lens). Five random visual fields were counted for each well, and the average was determined. The experiments were performed in triplicate. Subcutaneous and peritoneal implanted models To explore the effects of MALAT1 on tumor growth in vivo, 2 × 106 SGC-996 cells from the three groups (control, si-Control, and si-MALAT1) were subcutaneously injected into the left axilla of 5 BALB/C nude mice. After 3 wk, all mice were sacrificed and
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Materials and Methods
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Statistical analysis Statistical analyses were conducted using SPSS software, version 18.0 (SPSS Inc.). The data are expressed as the mean ± SD. An independent Student t test was used to determine statistical significance, and a P value of less than 0.05 was considered statistically significant. Disclosure of Potential Conflict of Interests
There was no conflict of interests in the present study. Acknowledgments
This study was supported by National Natural Science Foundation of China (81172026, 81272402, 81301816, and 81172029), National High Technology Research and Development Program (863 Program) (2012AA022606), Foundation for Interdisciplinary research of Shanghai Jiao Tong University (YG2011ZD07), Shanghai science and technology commission inter-governmental international cooperation project (12410705900), Shanghai science and technology commission medical-guiding project (12401905800), Program for Changjiang Scholars, Natural Science Research Fundation of Shanghai Jiao Tong University School of Medicine (13XJ10037), Leading Talent program of Shanghai and Specialized Research Fundation for PhD Program of Higher Education-Priority Development Field (20130073130014).
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the tumors were dissected and weighed. In addition, to investigate the effects of MALAT1 on tumor metastasis in vivo, 1 × 105 NOZ cells (si-Control and si-MALAT1) were suspended in 1 mL of FBS-free medium and were peritoneally injected into 5 BALB/C nude mice. After 8 wk, all mice were sacrificed and the peritoneal tumors were dissected and collected. The animal procedures were approved by the Institutional Animal Care and Use Committee of Xinhua Hospital, School of Medicine, Shanghai Jiao Tong University. Western blot analysis Cellular proteins were extracted in lysis buffer (Beyotime) from SGC-996 and NOZ cells (control, si-Control, and siMALAT1). The proteins were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes (Millipore). The membranes were blocked and then probed with primary antibodies overnight at 4 °C. The following primary antibodies were used in this study: anti-p-ERK1/2, anti-ERK1/2, antip-MEK1/2, anti-MEK1/2, anti-p-MAPK, anti-MAPK, anti-pJNK1/2/3, anti-JNK1/2/3, and anti-MMP9 (1:500, all from Bioworld Technology). β-actin (Beyotime) was used as a loading control. After washing, the membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse IgG (Beyotime) and were visualized using an enhanced chemiluminescence detection reagent from Pierce.
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22. Xander P, Brito RR, Pérez EC, Pozzibon JM, de Souza CF, Pellegrino R, Bernardo V, Jasiulionis MG, Mariano M, Lopes JD. Crosstalk between B16 melanoma cells and B-1 lymphocytes induces global changes in tumor cell gene expression. Immunobiology 2013; 218:1293-303; PMID:23731774; http:// dx.doi.org/10.1016/j.imbio.2013.04.017 23. Mercer TR, Dinger ME, Mattick JS. Long noncoding RNAs: insights into functions. Nat Rev Genet 2009; 10:155-9; PMID:19188922; http://dx.doi. org/10.1038/nrg2521 24. Wilusz JE, Sunwoo H, Spector DL. Long noncoding RNAs: functional surprises from the RNA world. Genes Dev 2009; 23:1494-504; PMID:19571179; http://dx.doi.org/10.1101/gad.1800909 25. Yuan SX, Yang F, Yang Y, Tao QF, Zhang J, Huang G, Yang Y, Wang RY, Yang S, Huo XS, et al. Long noncoding RNA associated with microvascular invasion in hepatocellular carcinoma promotes angiogenesis and serves as a predictor for hepatocellular carcinoma patients’ poor recurrencefree survival after hepatectomy. Hepatology 2012; 56:2231-41; PMID:22706893; http://dx.doi. org/10.1002/hep.25895 26. Hutchinson JN, Ensminger AW, Clemson CM, Lynch CR, Lawrence JB, Chess A. A screen for nuclear transcripts identifies two linked noncoding RNAs associated with SC35 splicing domains. BMC Genomics 2007; 8:39-54; PMID:17270048; http:// dx.doi.org/10.1186/1471-2164-8-39 27. Gutschner T, Baas M, Diederichs S. Noncoding RNA gene silencing through genomic integration of RNA destabilizing elements using zinc finger nucleases. Genome Res 2011; 21:1944-54; PMID:21844124; http://dx.doi.org/10.1101/gr.122358.111
Cancer Biology & Therapy Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/kcbt20
MALAT1 promotes the proliferation and metastasis of gallbladder cancer cells by activating the ERK/MAPK pathway Xiang-Song Wu Ding
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, Xu-An Wang abc
, Hao Weng
Jia-Sheng Mu
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, Zhu-Jun Tan
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, Tian-Yu Liu d
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, Yun-Ping Hu
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, Lin Jiang
, Feng Tao & Ying-Bin Liu
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, Mao-Lan Li
, Yang Cao
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Department of General Surgery; Xinhua Hospital; Shanghai Jiao Tong University School of Medicine; Shanghai, PR China b
Laboratory of General Surgery, Xinhua Hospital; Shanghai Jiao Tong University School of Medicine; Shanghai, PR China
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Institute of Biliary Tract Disease; Shanghai Jiao Tong University School of Medicine; Shanghai, PR China d
Department of Gastrointestinal Surgery; Shaoxing People’s Hospital; Shaoxing, PR China Published online: 21 Mar 2014.
To cite this article: Xiang-Song Wu, Xu-An Wang, Wen-Guang Wu, Yun-Ping Hu, Mao-Lan Li, Qian Ding, Hao Weng, Yi-Jun Shu, Tian-Yu Liu, Lin Jiang, Yang Cao, Run-Fa Bao, Jia-Sheng Mu, Zhu-Jun Tan, Feng Tao & Ying-Bin Liu (2014) MALAT1 promotes the proliferation and metastasis of gallbladder cancer cells by activating the ERK/MAPK pathway, Cancer Biology & Therapy, 15:6, 806-814, DOI: 10.4161/cbt.28584 To link to this article: http://dx.doi.org/10.4161/cbt.28584
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Research Paper Research Paper
Cancer Biology & Therapy 15:6, 806–814; June 2014; © 2014 Landes Bioscience
MALAT1 promotes the proliferation and metastasis of gallbladder cancer cells by activating the ERK/MAPK pathway Xiang-Song Wu1,2,3,†, Xu-An Wang1,2,3,†, Wen-Guang Wu1,2,3,†, Yun-Ping Hu1,2,3,†, Mao-Lan Li1,2,3, Qian Ding1,2,3, Hao Weng1,2,3, Yi-Jun Shu1,2,3, Tian-Yu Liu1,2,3, Lin Jiang1,2,3, Yang Cao1,2,3, Run-Fa Bao1,2,3, Jia-Sheng Mu1,2,3, Zhu-Jun Tan1,2,3, Feng Tao4,*, and Ying-Bin Liu1,2,3,* Department of General Surgery; Xinhua Hospital; Shanghai Jiao Tong University School of Medicine; Shanghai, PR China; 2Laboratory of General Surgery, Xinhua Hospital; Shanghai Jiao Tong University School of Medicine; Shanghai, PR China; 3Institute of Biliary Tract Disease; Shanghai Jiao Tong University School of Medicine; Shanghai, PR China; 4Department of Gastrointestinal Surgery; Shaoxing People’s Hospital; Shaoxing, PR China These authors contributed equally to this work.
†
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Keywords: gallbladder carcinoma, MALAT1, lncRNA, ERK, MAPK, proliferation, metastasis Abbreviations: MALAT1, metastasis-associated lung adenocarcinoma transcript 1; GBC, gallbladder carcinoma; lncRNA, long non-coding RNA; EMT, epithelial–mesenchymal transition; ERK, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; HOTAIR, HOX antisense intergenic RNA; PCGEM1, prostate-specific transcript 1; CUDR, cancer upregulated drug resistant; MVIH, lncRNA associated with micro-vascular invasion in HCC; NSCLC, non-small cell lung cancer; qRT-PCR, quantitative real-time PCR analysis; FBS, fetal calf serum; shRNA, short hairpin RNA; CMV, cytomegalovirus; EGFP, enhanced green fluorescent protein; CCK-8, cell counting kit-8; GPC6, glypican 6; CXCL5, C-X-C motif chemokine 5
Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), a long non-coding RNA (lncRNA), is associated with metastasis and is an independent prognostic factor for lung cancer. Recent studies have demonstrated that MALAT1 plays an important role in other malignancies. However, little is known about the role of MALAT1 in gallbladder carcinoma (GBC), which is the most common cancer of the biliary tract and has an extremely poor prognosis. In this study, we focused on the expression, biological functions and mechanism of MALAT1 in GBC and found that MALAT1 was significantly upregulated in GBC tissues compared with corresponding non-cancerous tissues. Knockdown of MALAT1 in GBC cell lines using lentivirus-mediated RNA interference significantly inhibited the proliferation and metastasis of the GBC cells both in vitro and in vivo. Furthermore, ERK/MAPK pathway was found to be inactivated in the GBC cell lines after MALAT1 knockdown. These results indicated that MALAT1 might serve as an oncogenic lncRNA that promotes proliferation and metastasis of GBC and activates the ERK/MAPK pathway.
Introduction Gallbladder carcinoma (GBC) is the most common cancer of the biliary tract and the sixth most common gastrointestinal cancer.1-9 Curative resection is the only cure for this highly lethal malignancy. However, owing to its non-specific symptoms and highly invasive nature, most patients are at an advanced stage when they are diagnosed.10 Therefore, only a minority of patients are candidates for curative resection. As a result, GBC has an extremely poor prognosis. The mean survival for this malignancy ranges from 5.2 to 24.4 mo, according to different reports.9,11-13 Most of these patients die from tumor metastasis and recurrence. Therefore, an understanding of the molecular mechanisms of GBC metastasis and recurrence is indispensable for the development of effective adjuvant therapy.
The human genome sequencing project has found that 70% of the genome is transcribed, but only up to 2% of the human genome serves as blueprints for proteins.14,15 Long non-coding RNAs (lncRNAs) are defined as endogenous cellular RNAs more than 200 nucleotides in length that lack an open reading frame of significant length.16 In recent years, several lncRNAs have been shown to be involved in carcinogenesis and cancer progression.17 Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) was initially found to be highly expressed in lung cancer and is a prognostic factor for the survival of patients with stage I non-small cell lung cancer.18 Moreover, studies have revealed that MALAT1 can promote cancer cell migration and the epithelial–mesenchymal transition (EMT) by regulating the expression of relevant genes.19,20 However, little is known about the role of MALAT1 in GBC progression.
*Correspondence to: Feng Tao; Email: [email protected]; Yingbin Liu; Email: [email protected] Submitted: 12/23/2013; Revised: 03/17/2014; Accepted: 03/18/2014; Published Online: 03/21/2014 http://dx.doi.org/10.4161/cbt.28584 806
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1
In the present study, MALAT1 was found to be overexpressed in GBC lesions compared with paired non-tumor samples. In addition, GBC cell lines (NOZ and SGC-996) infected with MALAT1-siRNA expressing lentivirus (Lv-siMALAT1) exhibited a reduced growth rate, lowered invasion ability, and inactivation of the ERK/MAPK pathway.
Results
Table 1. Clinical and pathological information of 20 gallbladder cancer patients Variable
Number or mean
Total number
20
Sex Male
6
Female
14
Age (mean)
60.1
Well
4
Moderately
10
Poorly
6
Tumor invasion T1
2
T2
5
T3
7
T4
6
Lymphnode metastasis Absence
7
Presence
12
Not available
1
Distant metastasis Absence
18
Presence
2
TNM stage I
2
II
4
III
8
IV
6
Figure 1. MALAT1 was upregulated in GBC tissues. (A) MALAT1 expression in 20 pairs of GBC tissues (Tumor) and corresponding non-tumorous tissues (NT). MALAT1 expression was calculated and is expressed as the MALAT1/GAPDH expression ratio (i.e., 2–ΔCt). The mean MALAT1 expression levels in the tumor and NT samples were 11.32 and 3.72, respectively. MALAT1 expression was significantly increased in the tumor tissues (**P < 0.01). (B) Comparison of the MALAT1 expression level between the CRC tissues and their corresponding non-tumorous tissues.
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Tumor differentiation
MALAT1 was upregulated in GBC tissues First, we performed qRT-PCR to evaluate the expression of MALAT1 in 20 GBC tissues and corresponding non-tumor tissues. All patients were diagnosed as gallbladder adenocarcinoma. Relevant clinical and pathological information of these 20 patients are shown in Table 1. MALAT1 was upregulated approximately 3-fold in GBC tissue samples compared with the corresponding non-tumor tissues (Fig. 1). Lentiviral siRNA effectively and specifically inhibited MALAT1 expression in SGC-996 and NOZ cells Because MALAT1 was overexpressed in GBC, we investigated the function of MALAT1 using lentivirus siRNA-mediated disruption of MALAT1 expression in two GBC cell lines (SGC-996 and NOZ). The lentiviral transfection efficiencies of the SGC-996 and NOZ cells were determined by examining GFP expression under a microscope 72 h after transfection (Fig. 2A). The efficiency of lentiviral transfection in both the SGC-996 and NOZ cells was higher than 90%. MALAT1 expression in SGC-996 and NOZ cells was examined using real-time PCR analysis after transfection with lentivirus. The levels of MALAT1 expression in the SGC-996 and NOZ cells transfected with MALAT1-siRNA lentivirus (siMALAT1) were decreased by 61.5% (P < 0.01) and 75.7% (P < 0.001), respectively, compared with the control cells (Fig. 2B). These results demonstrated that the lentivirus-mediated
siRNA effectively and specifically reduced MALAT1 expression in the SGC-996 and NOZ cells. Effects of MALAT1 knockdown on SGC-996 and NOZ cell proliferation To investigate whether MALAT1 knockdown could influence the proliferation of gallbladder cancer cells in vitro, CCK-8 and colony formation assays were performed. Figure 3A and B show that the proliferation abilities of the SGC-996 and NOZ cells decreased significantly after incubation with si-MALAT1 for 4 d (P < 0.001 for SGC-996, P < 0.01 for NOZ). Additionally, the colony formation assay also showed that silencing of MALAT1 significantly decreased the number of colonies formed by the SGC-996 and NOZ (Fig. 3C and D) cells compared with the control and si-Control group (P < 0.01, respectively). To understand the effects of MALAT1 knockdown on cell cycle distribution, gallbladder cancer cells were analyzed by flow cytometry. The results indicated that MALAT1 knockdown increased the percentage of G2 /M cells (P < 0.001, respectively, Fig. 3E–G). Furthermore, to probe the effects of MALAT1 on gallbladder cancer cell growth in vivo, MALAT1-depleted or control SGC996 cells were injected into the left axilla of nude mice. Our results showed that the growth of tumors from the MALAT1-depleted
xenografts was significantly inhibited compared with that of tumors formed from the mock-infected or control cells (P < 0.05, respectively, Fig. 3H–J). Effects of MALAT1 knockdown on SGC-996 and NOZ cell metastasis Transwell migration and invasion assays were performed to investigate the role of MALAT1 in the metastasis of GBC cells. Figure 4A and C shows that MALAT1 disruption significantly reduced the migration of SGC-996 and NOZ cells (P < 0.01 and P < 0.001, respectively). Invasiveness was also inhibited by MALAT1 knockdown (P < 0.01 and P < 0.001, respectively, for SGC-996 and NOZ; Fig. 4B and D). Accordingly, the expression of matrix metalloproteinase 9 (MMP-9), an enzyme that is involved in the breakdown of the extracellular matrix during cancer cell invasion, was also found to be significantly reduced after MALAT1 downregulation (Fig. 4E). To confirm these findings in vivo, we used a peritoneal metastasis model in nude mice. Mice injected with MALAT1-depleted NOZ cells exhibited few ascites (Fig. 4F) at 8 wk after inoculation. After dissecting the peritoneal metastatic tumor, we found that the total tumor weight was significantly lower in the si-MALAT1 group than in the si-Control group (P < 0.01; Fig. 4G).
Figure 3 (See opposite page). MALAT1 increased proliferation in the GBC cell lines. (A and B) Cellular proliferation of untransfected or transfected SGC-996 and NOZ cells were measured using a CCK-8 assay daily for 4 d. (C and D) SGC-996 and NOZ cells were seeded at 400 cells/well, and the cells were allowed to form colonies. The colony numbers were counted and recorded. (E) Untransfected or transfected SGC-996 and NOZ cells were stained by propidium iodide and analyzed by flow cytometry. (F and G) The percentage of cells in the G0/G1, S, and G2/M phases of the cell cycle were calculated. Results are expressed as mean ± SD from three independent experiments. (H) A subcutaneous implanted model of human GBC was established using SGC-996 cells. (I) Photograph of a tumor developed in the subcutaneous implanted model. (J) A statistical plot of average tumor weights in the subcutaneous implanted model. The graph shows the mean ± SD; ***P < 0.001, **P < 0.01, *P < 0.05. The data represent one of three separate experiments. Control, blank control; si-control, cells transduced with lentivirus-mediated scr-siRNA; si-MALAT1, cells transduced with lentivirus-mediated MALAT1-siRNA. 808
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Figure 2. Lentivirus-mediated siRNA decreased the expression of MALAT1 in the SGC-996 and NOZ cells. (A) The transfection efficiency was determined 3 d after incubation with lentivirus at an MOI of 50. The transfected cells labeled with GFP were observed under a light microscope and a fluorescence microscope. Light micrograph (upper); Fluorescent micrograph (lower) (200×). (B and C) Total RNA was extracted 5 d after infection, and the relative MALAT1 expression was determined using quantitative real-time PCR. GAPDH was used as an internal control. The data represent the mean ± SD of three independent experiments. **P < 0.001, compared with si-Control. Control, blank control; si-control, cells transduced with lentivirus-mediated scr-siRNA; si-MALAT1, cells transduced with lentivirus-mediated MALAT1-siRNA.
Figure 3. For figure legend, see page 808.
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Suppression of MAPK kinase pathways by MALAT1 knockdown To determine the possible mechanism by which MALAT1 regulated proliferation and metastasis of GBC cells, we performed western blot analysis to investigate the effects of MALAT1 knockdown on the extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) pathway, which is often aberrantly activated in human cancers and contributes to enhanced cell proliferation and metastasis.21,22 Western blot analysis showed that MALAT1 downregulation significantly reduced the levels of phosphorylated MEK1/2, ERK 1/2, MAPK, and JNLK 1/2/3, while no detectable changes were observed in the total levels of MEK1/2, ERK 1/2, MAPK, and JNLK 1/2/3 (Fig. 5). These results indicated that the ERK/MAPK pathway
810
might participate in the MALAT1-induced proliferation and metastasis of GBC cells.
Discussion GBC is a highly invasive and rapidly proliferative cancer with a poor prognosis.9 Therefore, the identification of novel methods that can effectively inhibit GBC growth and metastasis is needed. Although lncRNAs were initially argued to be spurious transcriptional noise, recent evidence suggests that they may play a major biological role in cellular development and human diseases.23,24 During recent years, an increasing number of studies have focused on the role of lncRNAs in tumorigenesis. LncRNAs
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Figure 4. MALAT1 promoted metastasis in the GBC cell lines. (A) Representative photographs of crystal violet-stained SGC-996 and NOZ cells that migrated through polycarbonate membranes. (B) Representative photographs of crystal violet-stained SGC-996 and NOZ cells that invaded through matrigel. (C) A statistical plot of the average number of migrated SGC-996 and NOZ cells in each group. (D) A statistical plot of the average number of invasive SGC-996 and NOZ cells in each group. (E) Western blot analysis indicated that MMP-9 was downregulated in the SGC-996 and NOZ cells after MALAT1 knockdown. (F) A peritoneal metastasis model of human GBC was established using NOZ cells. Mice receiving the MALAT1-depleted NOZ cells exhibited little ascites at 8 wk after inoculation. (G) A statistical plot of the average tumor weights in the peritoneal metastasis model. The graph shows the mean ± SD; ***P < 0.001, **P < 0.01, *P < 0.05. The data represent one of three separate experiments. Control, blank control; si-control, cells transduced with lentivirus-mediated scr-siRNA; si-MALAT1, cells transduced with lentivirus-mediated MALAT1-siRNA.
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Figure 5. MALAT1 activated the ERK/MAPK pathway in the GBC cell lines. Western blot analysis of the ERK/MAPK pathway-related proteins in the SGC-996 and NOZ cells. Representative blots are shown. Control, blank control; si-control, cells transduced with lentivirus-mediated scr-siRNA; si-MALAT1, cells transduced with lentivirus-mediated MALAT1-siRNA.
MALAT1 knockdown significantly reduced the expression of phosphorylated MEK1/2, ERK 1/2, MAPK, and JNK 1/2/3. However, no detectable changes in total MEK1/2, ERK 1/2, MAPK, or JNK 1/2/3 protein expression were observed. Therefore, impaired GBC cell growth and metastasis due to MALAT1 knockdown could be explained, at least in part, by the inactivation of the ERK/MAPK pathway. Unfortunately, a direct link between MALAT1 and the ERK/MAPK pathway remain unelucidated. According to Gutschner et al.’s study,34 knockout of MALAT1 in lung cancer cells could significantly reduce the expression level of several metastasis related genes, which included Glypican 6 (GPC6) and C-X-C motif chemokine 5 (CXCL5). Furthermore, previous studies35,36 have indicated that depletion of GPC6 or CXCL5 could lead to the inactivation of MAPK pathway. Therefore, we can hypothesize that MALAT1 activates ERK/MAPK pathway via regulation the expression of GPC6 or CXCL5 genes. In conclusion, we found that MALAT1 was significantly upregulated in GBC tissues. Knockdown of MALAT1 could inhibit GBC cell proliferation and metastasis both in vitro and in vivo. Moreover, knockdown of MALAT1 led to the inactivation of the ERK/MAPK pathway. Therefore, MALAT1 might serve as an oncogenic lncRNA that promotes proliferation and metastasis of GBC and activates the ERK/MAPK pathway.
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such as HOTAIR (HOX antisense intergenic RNA), PCGEM1 (prostate-specific transcript 1), CUDR (cancer upregulated drug resistant), and MVIH (lncRNA associated with microvascular invasion in HCC) have been shown to be involved in the biological hallmarks of cancer, including sustaining proliferative signaling, evading growth suppressors, enabling replicative immortality, activating invasion and metastasis, inducing angiogenesis, and resisting cell death.17,25 MALAT1, which is also known as HCN, NEAT2, PRO2853, and NCRNA00 047, is located at chromosome 11q13.1 and encodes a polyadenylated non-coding RNA (ncRNA) of ~8 kb.18,26 Ji’s work demonstrated that MALAT1 influenced metastasis and patient survival in non-small cell lung cancer (NSCLC).18 This ncRNA is extremely abundant in many human cell types and is highly conserved across several species, which underscores its functional importance.27 Additionally, Ying et al. showed that MALAT1 was upregulated in the metastatic tissue of bladder cancer and contributed to bladder cancer cell migration.20 Lai et al. found that MALAT1 was an independent prognostic factor for HCC recurrence after liver transplantation.28 However, MALAT1 has not so far been linked to GBC or any other malignancy in the biliary tract. In this study, we found that MALAT1 was highly expressed in GBC tissue samples. To understand its biological role in GBC, we used lentivirus-mediated siRNA to knock down MALAT1. We observed that, in SGC-996 and NOZ cells, MALAT1 downregulation significantly inhibited cell proliferation in vitro according to the CCK8 and colony formation assays. Additionally, cell migration and invasion were also inhibited in vitro by MALAT1 knockdown. Furthermore, the subcutaneous and peritoneal implanted models were used to confirm these findings in vivo, which showed that both proliferation and metastasis were suppressed by MALAT1 knockdown. In general, the exact mechanism of MALAT1 function is still unknown. MALAT1 specifically localizes to nuclear speckles and regulates the alternative splicing of pre-mRNAs by modulating the levels of active serine/arginine splicing factors.26,29 Depletion of MALAT1 alters the processing of a subset of pre-mRNAs that play important roles in cancer biology.30 This evidence supports the hypothesis that MALAT1 could be a regulator of posttranscriptional RNA processing or modification.17 Moreover, MALAT1 was found to interact with the unmethylated form of CBX4, which controls the relocation of growth-control genes between the polycomb bodies and interchromatin granules, sites of silent or active gene expression, respectively.31 In addition, MALAT1 was shown to promote cancer cell migration by inducing the EMT, and the activation of the Wnt pathway participated in this process. To elucidate the possible mechanism by which MALAT1 regulates GBC cell proliferation and metastasis, western blot analysis of the key molecular factors of cancerrelated pathways, such as NFκB, Smad2, Akt, and others (data not shown), was performed. The ERK/MAPK pathway is one of the most important signal transduction pathways, and MALAT1 promotes tumor growth and metastasis by activating this signaling cascade.32,33 We observed that in SGC-996 and NOZ cells,
Patients and pathological data This study was approved by the ethics committee of Xinhua Hospital, and all patients provided informed consent. The data do not contain any information that could identify the patients. Fresh GBC tissue samples and paired non-cancerous tissue samples were obtained from 20 GBC patients who underwent radical cholecystectomy without prior radiotherapy or chemotherapy between 2012 and 2013 at the Department of General Surgery, Xinhua Hospital, School of Medicine, Shanghai Jiao Tong University, PR China. These samples were used for quantitative realtime PCR analysis (qRT-PCR). Fresh tissues were collected in the operating room and were processed within 15 min after they were removed from the body. Each sample was frozen and stored at –80 °C. The paired non-cancerous tissues were dissected at least 2 cm away from the tumor border and were confirmed to lack tumor cells by microscopy. The diagnoses of GBC were confirmed by histopathological examination. Quantitative real-time PCR (qRT-PCR) Total RNA was extracted from the tissue samples or cultured cells with Trizol reagent (Takara), and first-strand cDNA was synthesized from 2 μg of total RNA using random primers and the M-MLV Reverse Transcriptase (Invitrogen). The following primers were used to detect the expression of MALAT1 and GAPDH (internal control): MALAT1 (sense): 5′-AAAGCAAGGT CTCCCCACAA G-3′; MALAT1 (antisense): 5′-GGTCTGTGCT AGATCAAAAG GCA-3′; GAPDH (sense): 5′-AGAAGGCTGG GGCTCATTTG-3′; GAPDH (antisense): 5′-AGGGGCCATC CACAGTCTTC-3′. RNA expression was measured by qRT-PCR using the SYBRGreen method (Takara) according to the manufacturer’s instructions. The results were normalized to the expression of GAPDH. Cell culture Two GBC cell lines (SGC-996 and NOZ) were used in the present study. The SGC-996 cell line was provided by the Academy of Life Science, Tongji University and was cultured in RPMI-1640 medium (GIBCO) supplemented with 10% fetal calf serum (FBS; GIBCO) and 100 U/mL penicillin/streptomycin. The NOZ cell line was purchased from the Health Science Research Resources Bank and was cultured in Williams’ Medium E (GIBCO) supplemented with 10% FBS and 100 U/mL penicillin/streptomycin. All cells were cultivated in a humidified incubator at 37 °C and 5% CO2. Lentivirus-mediated RNA interference The following short hairpin RNA (shRNA)19 was used to target human MALAT1: sense: 5′-CACAGGGAAA GCGAGTGGTT GGTAA-3′; antisense: 5′-TTACCAACCA CTCGCTTTCC CTGTG-3′. The sequence of the negative control shRNA was 5′-TTCTCCGAAC GTGTCACGT-3′. These shRNAs were synthesized and inserted into the pFH1UGW lentivirus core vector containing a cytomegalovirus (CMV)driven enhanced green fluorescent protein (EGFP) reporter gene; expression of the shRNA was driven by the H1 promoter. Recombinant lentivirus expressing MALAT1-siRNA or control siRNA (si-MALAT1 or si-Control) was produced by the Genechem
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Company. SGC-996 and NOZ cells were infected with concentrated virus in medium without FBS. The supernatant was replaced with complete culture medium after 24 h. The expression of MALAT1 in the infected cells was validated by qRT-PCR analysis after 120 h. Cell proliferation assay Cell proliferation was quantified using the Cell Counting Kit-8 (CCK-8; Dojindo). Briefly, 100 μL of cells from the three groups (control, si-Control, and si-MALAT1) were seeded into a 96-well plate at a concentration of 1000 cells per well and were incubated at 37 °C. At daily intervals (days 1, 2, 3, and 4), the optical density was measured at 450 nm using a microtiter plate reader (Quant BioTek Instruments), and the cell survival rate was expressed as the absorbance relative to that of the control group. The results represent the average of three replicates under the same conditions. Colony formation assay Both non-transfected and transfected SGC-996 and NOZ cells (400 cells/well) were seeded in 6-well plates. The cells were cultured for approximately 12 d, fixed with 4% paraformaldehyde, and stained with 0.1% crystal violet (Sigma-Aldrich). After washing, the plates were air-dried, and the stained colonies were photographed using a microscope (Leica). The total number of colonies (>50 cells/colony) was counted. The experiments were performed in triplicate. Cell cycle analysis Both non-transfected and transfected SGC-996 and NOZ cells were then harvested by trypsinization, washed twice in cold PBS, and fixed in 70% ethanol at 4 °C overnight. After fixation, the cells were washed and resuspended in cold PBS and incubated in a solution of 10 mg/mL RNase and 1 mg/mL propidium iodide (Sigma-Aldrich) at 37 °C for 30 min in the dark. Finally, the DNA content was determined by flow cytometry (BD Biosciences). The percentage of cells in the G0 /G1, S, and G2 /M phases was determined using Cell Quest acquisition software (BD Biosciences). Transwell migration and invasion assay Cell migration ability was assessed using 6.5-mm transwell chambers with a pore size of 8 μm. Cell invasion was assessed using the Chamber matrigel invasion 24-well DI kit (BD). The assays were performed according to the manufacturer’s instructions. Briefly, 2.5 × 104 cells (SGC-996 and NOZ) from each group were suspended in serum-free medium and were seeded into the upper chamber. The lower chamber was filled with medium containing 10% FBS. After incubation for 24 h, the migrated/ invaded cells in the lower chamber (below the filter surface) were fixed in 4% paraformaldehyde, stained with 0.1 mg/mL crystal violet solution, and counted under a microscope (20× objective lens). Five random visual fields were counted for each well, and the average was determined. The experiments were performed in triplicate. Subcutaneous and peritoneal implanted models To explore the effects of MALAT1 on tumor growth in vivo, 2 × 106 SGC-996 cells from the three groups (control, si-Control, and si-MALAT1) were subcutaneously injected into the left axilla of 5 BALB/C nude mice. After 3 wk, all mice were sacrificed and
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Materials and Methods
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Statistical analysis Statistical analyses were conducted using SPSS software, version 18.0 (SPSS Inc.). The data are expressed as the mean ± SD. An independent Student t test was used to determine statistical significance, and a P value of less than 0.05 was considered statistically significant. Disclosure of Potential Conflict of Interests
There was no conflict of interests in the present study. Acknowledgments
This study was supported by National Natural Science Foundation of China (81172026, 81272402, 81301816, and 81172029), National High Technology Research and Development Program (863 Program) (2012AA022606), Foundation for Interdisciplinary research of Shanghai Jiao Tong University (YG2011ZD07), Shanghai science and technology commission inter-governmental international cooperation project (12410705900), Shanghai science and technology commission medical-guiding project (12401905800), Program for Changjiang Scholars, Natural Science Research Fundation of Shanghai Jiao Tong University School of Medicine (13XJ10037), Leading Talent program of Shanghai and Specialized Research Fundation for PhD Program of Higher Education-Priority Development Field (20130073130014).
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the tumors were dissected and weighed. In addition, to investigate the effects of MALAT1 on tumor metastasis in vivo, 1 × 105 NOZ cells (si-Control and si-MALAT1) were suspended in 1 mL of FBS-free medium and were peritoneally injected into 5 BALB/C nude mice. After 8 wk, all mice were sacrificed and the peritoneal tumors were dissected and collected. The animal procedures were approved by the Institutional Animal Care and Use Committee of Xinhua Hospital, School of Medicine, Shanghai Jiao Tong University. Western blot analysis Cellular proteins were extracted in lysis buffer (Beyotime) from SGC-996 and NOZ cells (control, si-Control, and siMALAT1). The proteins were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes (Millipore). The membranes were blocked and then probed with primary antibodies overnight at 4 °C. The following primary antibodies were used in this study: anti-p-ERK1/2, anti-ERK1/2, antip-MEK1/2, anti-MEK1/2, anti-p-MAPK, anti-MAPK, anti-pJNK1/2/3, anti-JNK1/2/3, and anti-MMP9 (1:500, all from Bioworld Technology). β-actin (Beyotime) was used as a loading control. After washing, the membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse IgG (Beyotime) and were visualized using an enhanced chemiluminescence detection reagent from Pierce.
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