Tumor Biol. DOI 10.1007/s13277-014-2631-4
RESEARCH ARTICLE
MALAT1 promotes the proliferation and metastasis of osteosarcoma cells by activating the PI3K/Akt pathway Yongqiang Dong & Guojun Liang & Bo Yuan & Chaoqun Yang & Rui Gao & Xuhui Zhou
Received: 8 August 2014 / Accepted: 10 September 2014 # International Society of Oncology and BioMarkers (ISOBM) 2014
Abstract Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), one of the first found cancer-associated long noncoding RNAs (lncRNAs), involves in the development and progression of many types of tumors. An aberrant expression of MALAT1 was observed in hepatocellular carcinoma, cervical cancer, breast cancer, ovarian cancer, and colorectal cancer. However, the exact effects and molecular mechanisms of MALAT1 in osteosarcoma progression are still unknown up to now. Here, we investigated the role of MALAT1 in human osteosarcoma cell lines and clinical tumor samples in order to determine the function of this molecule. In our research, the MALAT1 messenger RNA (mRNA) was highly expressed in human osteosarcoma tissues, and its expression level was closely correlated with pulmonary metastasis. Then, we employed lentivirus-mediated knockdown of MALAT1 in U-2 OS and SaO2 to determine the role of
MALAT1 in osteosarcoma cell lines. Lentivirus-mediated MALAT1 small interfering RNA (siRNA) could efficiently downregulated the expression level of MALAT1 in osteosarcoma cell lines. Knockdown of MALAT1 inhibited the proliferation and invasion of human osteosarcoma cell and suppressed its metastasis in vitro and vivo. At the same time, the proliferating cell nuclear antigen (PCNA), matrix metallopeptidase 9 (MMP-9), phosphorylated PI3Kp85α, and Akt expressions were significantly inhibited in MALAT1-deleted cells. These findings indicated that MALAT1 might suppress the tumor growth and metastasis via PI3K/AKT signaling pathway. Taken together, our data indicated that MALAT1 might be an oncogenic lncRNA that promoted proliferation and metastasis of osteosarcoma and could be regarded as a therapeutic target in human osteosarcoma. Keywords MALAT1 . Osteosarcoma . Growth . Migration
Yongqiang Dong and Guojun Liang contributed equally to this work. Y. Dong : G. Liang : B. Yuan Department of Spine Surgery, Xinchang County People’s Hospital, 117 Gushanzhong Road, Xinchang County, Zhejiang Province 312500, China Y. Dong e-mail: [email protected] G. Liang e-mail: [email protected] B. Yuan e-mail: [email protected] C. Yang : R. Gao (*) : X. Zhou (*) Department of Orthopedic Surgery, Changzheng Hospital, Second Military Medical University, 415 Fengyang Road, Shanghai 200003, People’s Republic of China e-mail: [email protected] e-mail: [email protected] C. Yang e-mail: [email protected]
Introduction Osteosarcoma accounts for approximately 20 % of all primary bone cancers and is the second highest cause of cancer-related death in the pediatric age group [1, 2]. Despite several improvements in the curative protocols, the total survival trends of osteosarcoma have not been substantially improved and approximately 35 % of patients will die within 5 years [3]. To overcome the malignant osteosarcoma, research has been performed to detect and validate a variety of molecules associated with osteosarcoma cell proliferation, differentiation, invasion, and metastasis. However, only a few molecular mechanisms have been revealed and translated into clinical application so far. Thus, it is indispensable to understand the molecular mechanisms of osteosarcoma metastasis and recurrence for the development of an effective adjuvant therapy.
Tumor Biol.
Long noncoding RNAs (lncRNAs) are kinds of transcriptional products of the eukaryotic genome that are composed of more than 200 nucleotides in length [4, 5]. Metastasisassociated lung adenocarcinoma transcript 1 (MALAT1), one of the first found cancer-associated lncRNAs [6], was highly expressed in metastasizing non-small cell lung cancer [7, 8]. Subsequently, research found that MALAT1 was widely expressed in human tissues including the heart, kidney, spleen, and brain [9]. Progressing findings suggested that MALAT1 played a pivotal role in the progression of human cancers, including hepatocellular carcinoma, cervical cancer, breast cancer, ovarian cancer, and colorectal cancer [10–14]. It was demonstrated that MALAT1 influenced metastasis and patient survival in colorectal cancer [15] and hepatocellular carcinoma [16]. MALAT1 was also upregulated in the metastatic tissue of bladder cancer and contributed to bladder cancer cell migration [17]. Furthermore, Myc-6, a tumorsuppressive regulator, could repress tumor growth via downregulation of MALAT1 [18]. Recent evidence supported the hypothesis that MALAT1 could be a regulator of posttranscriptional RNA processing or modification in cancer [19]. Collectively, MALAT1 is a newly identified gene associated with cancer growth and metastasis, representing a new therapeutic target for the treatment of cancer. However, the effects and molecular mechanisms of MALAT1 on osteosarcoma progression have not yet been comprehensively explored, although a previous study found that MALAT1 level was correlated with chemotherapy sensitivity in high-grade osteosarcoma [20]. In the present study, we investigated the expression and clinical significance of MALAT1 in osteosarcoma tissues and adjacent non-tumor corresponding tissues (ANCTs). By knockdown, MALAT1 using RNA interference in osteosarcoma cells with relatively high MALAT1 expression, cell proliferation, apoptosis, and cycle distribution was determined. We further explore the underlying signaling pathway involved in the progression of osteosarcoma relative to MALAT1.
Materials and methods Patient samples Osteosarcoma tissues and paired adjacent non-tumor corresponding tissues (ANCTs) were obtained from 19 patients diagnosed as osteosarcoma between 2009 and 2011 in the Department of Orthopedic Surgery, Changzheng Hospital (Shanghai Province) and Xinchang People’s Hospital (Zhejiang Province). Surgically resected specimens were formalin fixed, paraffin embedded, and cut into 5-μM sections. The sections were stained with hematoxylin and eosin and reviewed by Guojun Liang to determine tumor grade and
stage proposed by Edmonson and Steiner [21]. The margins of surgical specimens were inked and checked under a microscope. This study was approved by the ethics committee of Second Military Medical University, and all patients provided informed consent. Cells and reagents MG63, HOS, SaO2, and U-2 OS osteosarcoma cell lines were purchased from American Type Culture Collection (ATCC, USA). TRIzol reagent and Lipofectamine 2000 were from Invitrogen (NY, USA), SYBR Green Master Mixture was from Takara (Otsu, Japan), Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were from Gibco Invitrogen Corporation (NY, USA), and 2-(2methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium (WST-8, Beyotime, Haimen, China) was from Beyotime (Haimen, China). Lentivirus-mediated RNA interference The following short hairpin RNA (shRNA)19 was used to target human MALAT1: sense: 5-CACAGGGAAAGCGA GTGGTTGGTAA-3′ and antisense: 5′-TTACCAACCA CTCGCTTTCCCTGTG-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 small interfering RNA (siRNA) or control siRNA (siMALAT1 or siCON) was designed and produced by GeneChem (Shanghai, China). Cell proliferation assay Cell proliferation was analyzed with the cell count assay using WST-8 kits. Briefly, cells infected with siMALAT1 were incubated in 96-well plates at a density of 1×105 cells per well with DMEM medium supplemented with 10 % FBS. Cells were treated with 10 μl WST at 12, 24, 48, and 72 h. The color reaction was measured at 570 nm with enzyme immunoassay analyzer (Bio-Rad, American). The proliferative activities were calculated for each clone. Real-time PCR To quantitatively determine the messenger RNA (mRNA) expression level of MALAT1 in osteosarcoma cell lines, real-time PCR was used. Total RNA of each clone was extracted with TRIzol according to the manufacturer’s protocol. The genes were amplified using specific oligonucleotide
Tumor Biol.
primer, and human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used as an endogenous control. 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′, and GAPDH (antisense): 5′-AGGGGCCATC CACAGTCT TC-3′. Data were analyzed using the comparative Ct method (2−ΔΔCt). Three separate experiments were performed for each clone. Colony formation assay Both non-transfected and transfected U2-OS and SaO2 cells (500 cells/well) were seeded in six-well plates. The cells were cultured for approximately 10 days and fixed with 4 % Fig. 1 MALAT1 was upregulated in osteosarcoma tissues. a MALAT1 expression in 19 pairs of osteosarcoma tissues (Tumor) and ANCT. MALAT1 expression was calculated, and the data were normalized against GAPDH expression. The mean MALAT1 expression levels in the tumor and ANCT samples were 11.5 and 5.6, respectively. MALAT1 expression was significantly increased in the tumor tissues (**P
RESEARCH ARTICLE
MALAT1 promotes the proliferation and metastasis of osteosarcoma cells by activating the PI3K/Akt pathway Yongqiang Dong & Guojun Liang & Bo Yuan & Chaoqun Yang & Rui Gao & Xuhui Zhou
Received: 8 August 2014 / Accepted: 10 September 2014 # International Society of Oncology and BioMarkers (ISOBM) 2014
Abstract Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), one of the first found cancer-associated long noncoding RNAs (lncRNAs), involves in the development and progression of many types of tumors. An aberrant expression of MALAT1 was observed in hepatocellular carcinoma, cervical cancer, breast cancer, ovarian cancer, and colorectal cancer. However, the exact effects and molecular mechanisms of MALAT1 in osteosarcoma progression are still unknown up to now. Here, we investigated the role of MALAT1 in human osteosarcoma cell lines and clinical tumor samples in order to determine the function of this molecule. In our research, the MALAT1 messenger RNA (mRNA) was highly expressed in human osteosarcoma tissues, and its expression level was closely correlated with pulmonary metastasis. Then, we employed lentivirus-mediated knockdown of MALAT1 in U-2 OS and SaO2 to determine the role of
MALAT1 in osteosarcoma cell lines. Lentivirus-mediated MALAT1 small interfering RNA (siRNA) could efficiently downregulated the expression level of MALAT1 in osteosarcoma cell lines. Knockdown of MALAT1 inhibited the proliferation and invasion of human osteosarcoma cell and suppressed its metastasis in vitro and vivo. At the same time, the proliferating cell nuclear antigen (PCNA), matrix metallopeptidase 9 (MMP-9), phosphorylated PI3Kp85α, and Akt expressions were significantly inhibited in MALAT1-deleted cells. These findings indicated that MALAT1 might suppress the tumor growth and metastasis via PI3K/AKT signaling pathway. Taken together, our data indicated that MALAT1 might be an oncogenic lncRNA that promoted proliferation and metastasis of osteosarcoma and could be regarded as a therapeutic target in human osteosarcoma. Keywords MALAT1 . Osteosarcoma . Growth . Migration
Yongqiang Dong and Guojun Liang contributed equally to this work. Y. Dong : G. Liang : B. Yuan Department of Spine Surgery, Xinchang County People’s Hospital, 117 Gushanzhong Road, Xinchang County, Zhejiang Province 312500, China Y. Dong e-mail: [email protected] G. Liang e-mail: [email protected] B. Yuan e-mail: [email protected] C. Yang : R. Gao (*) : X. Zhou (*) Department of Orthopedic Surgery, Changzheng Hospital, Second Military Medical University, 415 Fengyang Road, Shanghai 200003, People’s Republic of China e-mail: [email protected] e-mail: [email protected] C. Yang e-mail: [email protected]
Introduction Osteosarcoma accounts for approximately 20 % of all primary bone cancers and is the second highest cause of cancer-related death in the pediatric age group [1, 2]. Despite several improvements in the curative protocols, the total survival trends of osteosarcoma have not been substantially improved and approximately 35 % of patients will die within 5 years [3]. To overcome the malignant osteosarcoma, research has been performed to detect and validate a variety of molecules associated with osteosarcoma cell proliferation, differentiation, invasion, and metastasis. However, only a few molecular mechanisms have been revealed and translated into clinical application so far. Thus, it is indispensable to understand the molecular mechanisms of osteosarcoma metastasis and recurrence for the development of an effective adjuvant therapy.
Tumor Biol.
Long noncoding RNAs (lncRNAs) are kinds of transcriptional products of the eukaryotic genome that are composed of more than 200 nucleotides in length [4, 5]. Metastasisassociated lung adenocarcinoma transcript 1 (MALAT1), one of the first found cancer-associated lncRNAs [6], was highly expressed in metastasizing non-small cell lung cancer [7, 8]. Subsequently, research found that MALAT1 was widely expressed in human tissues including the heart, kidney, spleen, and brain [9]. Progressing findings suggested that MALAT1 played a pivotal role in the progression of human cancers, including hepatocellular carcinoma, cervical cancer, breast cancer, ovarian cancer, and colorectal cancer [10–14]. It was demonstrated that MALAT1 influenced metastasis and patient survival in colorectal cancer [15] and hepatocellular carcinoma [16]. MALAT1 was also upregulated in the metastatic tissue of bladder cancer and contributed to bladder cancer cell migration [17]. Furthermore, Myc-6, a tumorsuppressive regulator, could repress tumor growth via downregulation of MALAT1 [18]. Recent evidence supported the hypothesis that MALAT1 could be a regulator of posttranscriptional RNA processing or modification in cancer [19]. Collectively, MALAT1 is a newly identified gene associated with cancer growth and metastasis, representing a new therapeutic target for the treatment of cancer. However, the effects and molecular mechanisms of MALAT1 on osteosarcoma progression have not yet been comprehensively explored, although a previous study found that MALAT1 level was correlated with chemotherapy sensitivity in high-grade osteosarcoma [20]. In the present study, we investigated the expression and clinical significance of MALAT1 in osteosarcoma tissues and adjacent non-tumor corresponding tissues (ANCTs). By knockdown, MALAT1 using RNA interference in osteosarcoma cells with relatively high MALAT1 expression, cell proliferation, apoptosis, and cycle distribution was determined. We further explore the underlying signaling pathway involved in the progression of osteosarcoma relative to MALAT1.
Materials and methods Patient samples Osteosarcoma tissues and paired adjacent non-tumor corresponding tissues (ANCTs) were obtained from 19 patients diagnosed as osteosarcoma between 2009 and 2011 in the Department of Orthopedic Surgery, Changzheng Hospital (Shanghai Province) and Xinchang People’s Hospital (Zhejiang Province). Surgically resected specimens were formalin fixed, paraffin embedded, and cut into 5-μM sections. The sections were stained with hematoxylin and eosin and reviewed by Guojun Liang to determine tumor grade and
stage proposed by Edmonson and Steiner [21]. The margins of surgical specimens were inked and checked under a microscope. This study was approved by the ethics committee of Second Military Medical University, and all patients provided informed consent. Cells and reagents MG63, HOS, SaO2, and U-2 OS osteosarcoma cell lines were purchased from American Type Culture Collection (ATCC, USA). TRIzol reagent and Lipofectamine 2000 were from Invitrogen (NY, USA), SYBR Green Master Mixture was from Takara (Otsu, Japan), Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were from Gibco Invitrogen Corporation (NY, USA), and 2-(2methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium (WST-8, Beyotime, Haimen, China) was from Beyotime (Haimen, China). Lentivirus-mediated RNA interference The following short hairpin RNA (shRNA)19 was used to target human MALAT1: sense: 5-CACAGGGAAAGCGA GTGGTTGGTAA-3′ and antisense: 5′-TTACCAACCA CTCGCTTTCCCTGTG-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 small interfering RNA (siRNA) or control siRNA (siMALAT1 or siCON) was designed and produced by GeneChem (Shanghai, China). Cell proliferation assay Cell proliferation was analyzed with the cell count assay using WST-8 kits. Briefly, cells infected with siMALAT1 were incubated in 96-well plates at a density of 1×105 cells per well with DMEM medium supplemented with 10 % FBS. Cells were treated with 10 μl WST at 12, 24, 48, and 72 h. The color reaction was measured at 570 nm with enzyme immunoassay analyzer (Bio-Rad, American). The proliferative activities were calculated for each clone. Real-time PCR To quantitatively determine the messenger RNA (mRNA) expression level of MALAT1 in osteosarcoma cell lines, real-time PCR was used. Total RNA of each clone was extracted with TRIzol according to the manufacturer’s protocol. The genes were amplified using specific oligonucleotide
Tumor Biol.
primer, and human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used as an endogenous control. 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′, and GAPDH (antisense): 5′-AGGGGCCATC CACAGTCT TC-3′. Data were analyzed using the comparative Ct method (2−ΔΔCt). Three separate experiments were performed for each clone. Colony formation assay Both non-transfected and transfected U2-OS and SaO2 cells (500 cells/well) were seeded in six-well plates. The cells were cultured for approximately 10 days and fixed with 4 % Fig. 1 MALAT1 was upregulated in osteosarcoma tissues. a MALAT1 expression in 19 pairs of osteosarcoma tissues (Tumor) and ANCT. MALAT1 expression was calculated, and the data were normalized against GAPDH expression. The mean MALAT1 expression levels in the tumor and ANCT samples were 11.5 and 5.6, respectively. MALAT1 expression was significantly increased in the tumor tissues (**P
