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MicroRNA-590-5p regulates cell viability, apoptosis, migration and invasion of renal cell carcinoma cell lines through targeting ARHGAP24 Lei Wang,†a Wan-qing Wei,†b Zi-yu Wua and Gong-cheng Wang
*c
Renal cell carcinoma (RCC) is the leading cause of death in renal malignancies. MicroRNA-590-5p (miR-590-5p) is of great importance in the processes of many cancers regarding regulation of cancer cell invasion and proliferation. In our study, alternation of miR-590-5p expression in RCC cell lines through transfection with pre-miR-590-5p (up-regulation) or anti-miR-590-5p (down-regulation) was performed. Apoptosis and viability of RCC cell lines were measured by flow cytometry and CCK-8 analysis, respectively. Cell invasion and migration were estimated by Transwell assay. The association of miR-590-5p with ARHGAP24 expression was evaluated using luciferase assays, real-time PCR and western blot assay. The expressions of apoptosis and migration-related protein were also measured by western blotting. We found that pre-miR-590-5p transfection in Caki-2 and 786-O cells showed significant increases in cell viability, invasion and migration, which were accompanied by decreased cell apoptosis, while anti-miR-590-5p transfection obviously inhibited the cell viability, migration and Received 7th July 2017, Accepted 13th September 2017 DOI: 10.1039/c7mb00406k
invasion of Caki-2 and 786-O cells as well as induced apoptosis, compared with the negative control group. Furthermore, bioinformatics combined with luciferase reporter assays indicated that ARHGAP24 is directly targeted by miR-590-5p. ARHGAP24 overexpression in 786-O and Caki-2 cells phenocopied the effects of anti-miR-590-5p transfection along with enhanced expression of active Caspase-3 and Bax/Bcl-2 ratio as well as decreased expression of MMP-2 and MMP-9. These findings suggested that
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miR-590-5p/ARHGAP24 seems to function as a potentially beneficial target for RCC treatment.
Introduction Renal cell carcinoma (RCC) is one of the three most common tumors in the urinary system, with the highest mortality in the case of parenchymal urinary tumors in adults,1,2 and accounts for about 3% of adult malignancies, with 2% of incidence each year.3 About 90% of kidney cancer is RCC, more than 80% of which is clear cell renal cell carcinoma originating from the renal proximal tubule. Different pathological types of RCC have different clinical features, treatment options and prognosis. The 3-year survival rate of RCC is calculated to be about 47%,4 and the incidence of patients with RCC is about 10%, with metastasis.5 Previous studies have shown that metastasis is universal in patients with RCC and functions as a major prognostic factor.6,7 a
Department of Urology, The Affiliated Huai’an Hospital of Xuzhou Medical University and The Second People’s Hospital of Huai’an, Huai’an 223200, China b Department of Pediatric Surgery, Maternal and Children Health Hospital of Huai’an, Huai’an 223002, China c Department of Urology, Huai’an First People’s Hospital, Nanjing Medical University, 1 Huanghe West Road, Huaiyin District, Huai’an 223300, China. E-mail: [email protected]; Fax: +86-0517-80872660; Tel: +86-0517-80872660 † Co-first authors.
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Clinically, 30–50% of patients are asymptomatic patients who have been found incidentally, while most of the patients with clinical symptoms have metastatic disease at the time of diagnosis. At present, the main treatment of RCC is surgical resection because late or metastatic RCC is relatively resistant to conventional chemotherapy or radiotherapy.8 However, some of the RCC patients have metastasis, including to the brain, lungs, liver and bone, even if they are treated at the early stage.9 After tumor resection, tumor recurrence occurs in 20–40% of patients.10 Therefore, effective control of the growth and metastasis of RCC cells, especially at the late stages, is a major challenge. The early diagnosis and treatment of RCC, especially in advanced RCC patients with recurrence tendency, is still the preface to the study of kidney cancer. Because of the complexity of the cause, development and migration of RCC, a lot of studies are related to a variety of mechanisms and genes, molecules and their respective roles involved. MicroRNAs (miRNAs, miR) compromise a specialized subset of noncoding RNAs with a length from 19 to 25 nucleotides, mainly combined with the 30 -UTR domain of a specific gene to form the complementary sequence, and then exert their regulatory effects at the stability of transcription and the post-transcriptional
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level.11 miRNAs are associated with various biological processes such as cell apoptosis, differentiation, proliferation, immune response and metabolism.12,13 Changes in miRNA expression can mediate the critical biological processes of cells regarding tumor development,14 and an assessment of alternation in miRNA expression may supply insights into the underlying mechanisms by which miRNA regulates the occurrence and process of cancer. In addition, the therapeutic regulation of miRNA can simultaneously affect multiple target genes and achieve excellent clinical outcomes,15 and miRNA expression profiling is therefore becoming a potentially powerful tool for disease diagnosis and prognosis. miR-590-3p was found to promote tumorigenesis of hepatocellular carcinoma (HCC) by down-regulating PTEN to activate the PI3K-AKT signaling pathway16 and regulate proliferation and invasion in HCC cells by targeting TGF-b RII.17 miR-590-5p promotes cervical cancer cell growth and invasion by targeting CHL1.18 miR-590-3p promotes cell growth of colon cancer by targeting TFAM.19 miR-590 promotes RCC cell invasion and proliferation through down-regulating PBRM1.20 The members of the Rho GTPase family are key regulators of actin dynamics and are known to be involved in many biological processes of cells including cell differentiation, adhesion, and migration.21 Several Rho GTPases have been shown to contribute to the tumor initiation and malignant progression, which may serve as a tumor suppressor or oncogene.22,23 Rho GTPase-activating protein 24 (ARHGAP24), also known as FilGAP, is highly conserved and enriched during podocyte differentiation and in kidney tissues.24 ARHGAP24 has been shown to down-regulate in RCC tissues and indicate a better prognosis, along with inhibition of cell cycle processes and invasion and induction of apoptosis of RCC cells.25 It is a potential target of miR-590-5p based on the analysis of Targetscan (http://www.targetscan.org/ vert_71/). But whether miR-590-5p along with ARHGAP24 also plays a crucial role in RCC progression remains largely unknown. In our work, the functions and mechanisms by which miR-590-5p regulated the cell viability, apoptosis, invasion and migration of RCC cells were assessed. We observed that ARHGAP24 was targeted by miR-590-5p. ARHGAP24 up-regulation mimicked the regulatory action of anti-miR-590-5p transfection in RCC cells, along with inhibited cell viability, invasion and migration, but promoted apoptosis. miR-590-5p/ARHGAP24 may serve as a target for therapy in treatment of RCC.
Materials and methods Cell culture Human clear cell renal cell carcinoma cell lines (Caki-2 and 786-O cells) were obtained from the ATCC (Rockville, USA) and cultured in DMEM (Life technology) complemented with 10% FBS (Life Technologies) along with 1% Penicillin–Streptomycin Solution (100; 100 U ml1 penicillin and 0.1 mg ml1 streptomycin for working; Solarbio) separately incubated at 37 1C with 5% CO2 and 95% air.
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Cell transfection pre-miR-590-5p, anti-miR-590-5p, and negative control (NC) were purchased from Beyotime (Shanghai, China) and transfected into Caki-2 and 786-O cells using a HiPerFect transfection reagent (Qiagen, CA) following the manufacturer’s recommendations. pLVX-AcGFP-C1 lentiviral vector carrying the ARHGAP24 coding sequence was obtained from GenePharma (Suzhou, China) and transfected into Caki-2 and 786-O cells for overexpression of ARHGAP24 using Lipofectamine 2000 (Invitrogen, Shanghai, China) following the instructions. At 6 h posttransfection, the medium was replaced with fresh medium. The transfected Caki-2 and 786-O cells were harvested for subsequent experiments 48 h post-transfection. CCK-8 assay Cell viability was analyzed by Cell Counting Kit CCK-8/WST-8 assay,25 which was carried out following the standard procedure in 96 well plates. Briefly, cells were added into a 96-well plate and grown to 80% confluence. After 24 h of incubation, the medium was removed. Then pre-miR-590-5p in the absence or presence of pLVX-AcGFP-C1-ARHGAP24 or anti-miR-590-5p or negative control (NC) was transfected into cells. The CCK-8 (10 ml per well) reagent was added at 0, 12, 24, 48 and 72 h post-transfection. The reaction system was incubated for 1 h under the same incubator conditions after which the absorbance readings were obtained at 450 nm. Cell apoptosis assay The cell apoptosis was assayed by flow cytometric assay.25 In brief, cells were added into a 6-well plate until grown to 60–80% confluence, and washed with 1 ml Binding Buffer (3 ml 10 Binding Buffer in 27 ml ddH2O). Subsequently, cells were mixed with 195 ml Annexin V-FITC for 10 min as well as 5 ml propidium iodide (PI) in darkness for 5 min. The fluorescence intensities were assessed by FACS using a FL-2A filter (BD FACS Calibur, USA). Data were studied using the Cyflogic software (CyFlo Ltd, Turku, Finland). Transwell assay At 48 h post-transfection, cells (5 104 cells per ml) were moved into DMEM supplemented with 1% FBS and plated into the upper chambers of Transwell plates (Millipore, Beijing, China), and the medium supplemented with 10% FBS was plated into the lower chambers. The membrane was fixed in methanol, and then stained using hematoxylin after 24 h of incubation. Stained cells were visualized and counted under a microscope (200 magnification). The analysis of cell invasion was consistent with that of cell migration expect that the cell invasion was detected by Transwell assay using Matrigel Invasion Chambers (BD Biosciences). Dual-luciferase reporter assay 24-Well plates were used for Caki-2 and 786-O cultivation for 24 h before transfection to ensure a density of 2 105 cells per well. pmiRGLO-ARHGAP24-3 0 UTR-wt or pmiRGLO-ARHGAP243 0 UTR-mut containing both firefly and renilla luciferases and
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pre-miR-590-5p or NC was transfected into Caki-2 and 786-O cells using Lipofectaime 2000t (Invitrogen). Post-transfection for 24 h, cells were lysed and activities of renilla luciferase were detected by a dual-luciferase reporter system (Promega, USA) following the provided protocols.
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Real-time PCR Total RNA extraction was performed using a miRNeasy kit (QIAGEN) and stored at 80 1C in a RNA secure RNase Inactivation Reagent (Thermo Fisher Scientific) following the manufacturer’s instructions. Total RNA (500 ng) was converted to complementary DNA. Real-time PCR was performed via SYBR Green LightCycler PCR (LC-PCR) assay on a 7900HT system. The relative quantification of miR-590-5p is given as ratios to 5S by using a 2DDCt cycle threshold method, respectively. Western blot assay Total protein extraction was performed with RIPA lysis buffer added with protease inhibitors 48 h after transfection and then centrifugation was carried out at 10 000g for 30 min at 4 1C. 50 mg of the protein extracted were loaded into 12% SDS-PAGE and were shifted onto a PVDF membrane (Millipore, USA). Then blots were incubated at 4 1C overnight with ARHGAP24, Bax, Bcl-2, active Caspase-3, MMP-2, MMP-9, or GAPDH antibodies (1 : 1000; Abcam, Cambridge, MA, USA). The horseradish peroxidase-conjugated IgG antibody (Jackson ImmunoResearch) was further used for 60 min at 25 1C. Then, membranes were developed using an enhanced chemiluminescence (ECL) substrate (Pierce, USA) and spotted using an Image Quant LAS 4010 Imaging System (GE Healthcare, Piscataway, NJ). Statistical analysis All quantitative values were expressed as mean SD of at least three repeated individual experiments for each group and they were statistically analyzed with One-way ANOVA, followed by Tukey’s post hoc test. All of the statistical analyses were made using GraphPad Prism v5.0 (GraphPad Software, Inc.). P o 0.05 was considered statistically significant.
Results miR-590-5p strengthens cell viability and inhibits apoptosis of RCC cells To estimate the function of miR-590-5p in RCC tumorigenesis, pre-miR-590-5p, anti-miR-590-5p, or negative control (NC) was transfected into the Caki-2 and 786-O cells, respectively. The expression of miR-590-5p in Caki-2 and 786-O cells is shown in Fig. 1A and B. The cell viability of Caki-2 and 786-O at 0, 12, 24, 48 and 72 h post-transfection was measured by CCK-8 assay and is displayed in Fig. 1C and D. pre-miR-590-5p transfection enhanced the viability of Caki-2 and 786-O cells in a timedependent manner. However, anti-miR-590-5p transfection timedependently decreased the viability of Caki-2 and 786-O cells, with the lowest viability of Caki-2 and 786-O cells detected at 72 h (51.3 2.3% of Caki-2-NC and 42.1 2.1% of 786-O-NC).
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Meanwhile, the cell viability of Caki-2 and 786-O at 12 h posttransfection was not changed by pre-miR-590-5p transfection compared with NC. Based on the influence of miR-590-5p on the cell viability of RCC, the cell apoptosis of Caki-2 and 786-O induced by miR-590-5p was also assessed. Our results revealed that pre-miR-590-5p transfection markedly attenuated Caki-2 and 786-O cell apoptosis by 48.5 3.1% and 54.4 3.8% compared with NC, respectively (Fig. 1E–H). However, anti-miR-590-5p transfection significantly promoted apoptosis of Caki-2 and 786-O cells by 13.8 1.3-fold and 8.5 0.8-fold compared with NC, respectively (Fig. 1E–H). Our findings speculate that miR-590-5p promotes the viability of RCC cell lines, in part, through inhibiting cell apoptosis. miR-590-5p enhances migration and invasion in RCC cells Next, the cell invasion and migration ability of RCC cell lines was measured by Transwell assay. Fig. 2A and B display that pre-miR-590-5p transfection significantly promoted Caki-2 cell migration by 50.9 4.4% compared with NC. However, antimiR-590-5p transfection significantly inhibited Caki-2 cell migration by 64.9 5.3% compared with NC. Meanwhile, it is figured out that pre-miR-590-5p transfection significantly strengthened invasion of 786-O cells by 27.2 1.5% compared with NC (Fig. 2C and D). However, anti-miR-590-5p transfection significantly inhibited 786-O cell invasion by 39.5 2.5% compared with NC. ARHGAP24 is a target of miR-590-5p in RCC cells To further elucidate the underlying mechanisms through which miR-590-5p regulates the cell viability, invasion and migration of RCC cells, Targetscan was introduced for predicting miR-590-5p targets (Fig. 3A). The luciferase activity assay showed that overexpression of miR-590-5p inhibited the protein of ARHGAP24 through binding the 3 0 UTR site of the latter in Caki-2 and 786-O cells, but did not decrease the activity of ARHGAP24 with 3 0 UTR-mut (Fig. 3B). Next we confirmed that ARHGAP24 is a target gene of miR-590-5p in Caki-2 and 786-O cells. As expected, our findings demonstrated that pre-miR-590-5p transfection obviously down-regulated ARHGAP24 protein expression compared with NC (Fig. 3C). These data suggested that ARHGAP24 is a target of miR-590-5p in RCC cell lines. ARHGAP24 overexpression inhibits cell viability and induces apoptosis of RCC To further validate the correlation between miR-590-5p and ARHGAP24 described above, pre-miR-590-5p in the absence or presence of pLVX-AcGFP-C1-ARHGAP24 was transfected into Caki-2 and 786-O cells. It was observed that ARHGAP24 overexpression up-regulated the ARHGAP24 protein level by 1.3 0.08-fold and 0.72 0.04-fold in Caki-2 and 786-O cells in comparison with blank vector transfection, respectively (Fig. 4A and B). Cell viability assay demonstrated that overexpression of ARHGAP24 markedly inhibited pre-miR-590-5p induced increase in cell viability of Caki-2 and 786-O cells at 24, 48 and 72 h, respectively (Fig. 4C and D). Flow cytometry assay
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Fig. 1 miR-590-5p promoted cell viability and inhibited apoptosis of RCC cell lines. Caki-2 and 786-O cells were transfected with pre-miR-590-5p, anti-miR-590-5p, or negative control (NC) for 1, 12, 24, 48 and 72 h. (A and B) The expression of miR-590-5p in Caki-2 and 786-O cells was measured by real-time PCR. (C and D) The cell viability of Caki-2 and 786-O cells was measured by CCK-8 assay. (E–H) The cell apoptosis of Caki-2 and 786-O cells was measured by flow cytometry assay. *P o 0.01 compared with NC.
demonstrated that overexpression of ARHGAP24 markedly inhibited pre-miR-590-5p induced decrease in cell apoptosis
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of Caki-2 and 786-O cells by 14.5 0.8-fold and 12.8 0.7-fold compared with blank vector transfection, respectively
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Fig. 2 miR-590-5p promoted cell migration and invasion of RCC cell lines. Caki-2 and 786-O cells were transfected with pre-miR-590-5p, anti-miR590-5p, or negative control (NC) for 48 h. The cell migration of Caki-2 (A and B) and cell invasion of 786-O cells (C and D) were measured by Transwell assay. *P o 0.01 compared with NC.
(Fig. 4E and F). These findings suggested the important role of ARHGAP24 in cell viability and apoptosis of RCC cell lines induced by miR-590-5p. ARHGAP24 overexpression inhibits cell migration and invasion of RCC Transwell assay found that overexpression of ARHGAP24 markedly inhibited pre-miR-590-5p induced increase in Caki-2 cell migration and 786-O cell invasion by 86.4 5.8% and 33.8 2.6% compared with blank vector transfection, respectively (Fig. 5A–C). These results suggested the important role of ARHGAP24 in cell invasion and migration of RCC cell lines induced by miR-590-5p. Effects of miR-590-5p and ARHGAP24 up-regulation on regulation of protein expression in RCC cells Then, the protein expression of active Caspase-3, Bcl-2, Bax, MMP-2 and MMP-9 was measured by western blot assay. As shown in Fig. 6A and B, pre-miR-590-5p transfection in Caki-2 cells obviously decreased the Bax/Bcl-2 ratio and active Caspase-3, and increased protein expression of MMP-2 and MMP-9. Meanwhile, ARHGAP24 overexpression significantly increased the
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Bax/Bcl-2 ratio and active Caspase-3 expression, and decreased the levels of MMP-2 and MMP-9 protein in Caki-2 cells with pre-miR-590-5p transfection, which is in contrast to the regulatory function of pre-miR-590-5p. Moreover, similar findings were also demonstrated in 786-O cells (Fig. 6C and D). This suggests that the effects of miR-590-5p up-regulation in RCC cells depend specifically on ARHGAP24 decrease.
Discussion Targeted therapies designed against molecules participating in the pathogenesis of cancer were reported as effective ways in cancer recently. Growing evidence has shown that miRNAs are often deregulated in many cancers.26,27 Specific cellular characterization of miRNAs in cancer cells has been intensively studied for clinical biomarkers and novel therapeutic targets. The pattern of miRNA expression appears with high tissue specificity, and is more precise in cancer diagnosis in comparison with the analysis based on mRNA.28,29 At present, although the causes of RCC, its diagnosis, and treatment are clear, the effective treatment of advanced RCC is limited. Although surgical
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Fig. 3 ARHGAP24 is a validated target of miR-590-5p. (A) ARHGAP24 as a potential target of miR-590-5p using Target Scan. (B) Luciferase reporter assays showed that up-regulation of miR-590-5p obviously reduced the relative luciferase activity of ARHGAP24 3 0 UTR. (C) In both Caki-2 and 786-O cells, pre-miR-590-5p transfection decreased the protein expression of ARHGAP24. *P o 0.01 compared with NC.
Fig. 4 ARHGAP24 overexpression inhibited cell viability and induced apoptosis of RCC cell lines. (A and B) Caki-2 and 786-O cells were transfected with pLVX-AcGFP-C1-ARHGAP24 for 48 h, and the protein expression of ARHGAP24 was measured by western blot assay. After 0, 12, 24, 48 and 72 h of transfection, the cell viability of Caki-2 and 786-O cells was measured by CCK-8 assay (C and D), and apoptosis of Caki-2 and 786-O cells was measured by flow cytometry assay (E and F). *P o 0.01 compared with pre-miR-590-5p+vector.
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Fig. 5 ARHGAP24 overexpression inhibited cell migration and invasion of RCC cell lines. (A and B) Caki-2 and 786-O cells were transfected with pLVXAcGFP-C1-ARHGAP24 for 48 h; the cell migration of Caki-2 cells (A and C) and cell invasion of 786-O cells (B and C) were measured by Transwell assay. *P o 0.01 compared with pre-miR-590-5p+vector.
resection, along with radiotherapy as well as chemotherapy, is of great importance in the treatment of RCC, the effect is not yet satisfactory. With the development of tumor molecular biology, finding a specific target gene has become a major research direction. In the present study, we observed that ARHGAP24 was targeted by miR-590-5p. ARHGAP24 up-regulation phenocopied the effects of anti-miR-590-5p transfection in RCC cells, along with inhibited cell viability, invasion and migration, but promoted apoptosis. New therapies are urgently needed to be developed because the treatment strategy of RCC has not been effective enough recently. The occurrence of a tumor is a combination of many oncogenes and tumor suppressor genes associated with oncogene activation, tumor suppressor gene inactivation, epigenetic changes and other complex changes in pathogenesis. miR-590-5p dysregulation was observed in many types of cancer via the regulation of diverse critical cancer-associated pathways,30–32 which indicated that miR-590-5p is an oncogene implicated in these malignancies. However, in contrast to these findings, several other studies have reported the tumor-suppressive role of miR-5905p in colorectal cancer,33 breast cancer34 and hepatocellular carcinoma.35 Recent reports showed miR-590-5p up-regulation in tumor tissues from patients with RCC36 and in the examined
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RCC cell lines,20 including SN12C, ACHN, TK10, A704 and 786-O, compared with normal kidney tissues and normal HEK293 cells, which indicated that miR-590-5p was possibly related to RCC progression. Therefore, pre-miR-590-5p and anti-miR-590-5p were introduced into the Caki-2 and 786-O cells for elucidating the regulatory action of miR-590-5p in RCC. One of the most prominent features of tumor progression is abnormal cell proliferation. The close relationship between cell proliferation and tumorigenesis has been realized; since tumorigenesis is a long-term, multifactor and multistep process, the regulatory factor of cell proliferation in the effect and mechanism of tumor evolution has rarely been fully clarified. Thus, in this study the functions of miR-590-5p in cell proliferation of RCC were investigated. We figured out that pre-miR-590-5p significantly boosted cell viability and inhibited apoptosis of Caki-2 and 786-O cells, while anti-miR-590-5p significantly inhibited cell viability of Caki-2 and 786-O and induced apoptosis. In line with our findings, previous studies highlighted that miR-590-5p enhanced cell proliferation and survival in RCC,20 hepatocellular carcinoma16 and cervical cancer.18 Moreover, the expression of apoptosis-related proteins showed that pre-miR-590-5p significantly decreased Bax/Bcl-2 and active Caspase-3 in Caki-2 and 786-O cells, while anti-miR-590-5p
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Fig. 6 miR-590-5p and ARHGAP24 regulated protein expression in RCC cell lines. Caki-2 and 786-O cells were transfected with pre-miR-590-5p in the absence or presence of ARHGAP24 overexpression; the protein expression of Bax, Bcl-2, Caspase-3, MMP-2 and MMP-9 in Caki-2 (A and B) and 786-O cells (C and D) was measured by western blot assay. *P o 0.01 compared with NC. #P o 0.01 compared with pre-miR-590-5p+vector.
significantly increased those. Once activated, Caspase-3 is responsible for the proteolytic cleavage of a broad spectrum of cellular targets, which ultimately leads to cell death, and Bcl-2 overexpression allows for continued cellular proliferation through antagonism of apoptotic signals imparted by the pro-apoptotic Bax.37 In addition to abnormal cell proliferation, invasion and migration are major characteristics of human cancer. In this study, it was revealed that pre-miR590-5p obviously increased Caki-2 cell migration and 786-O cell invasion, while anti-miR-590-5p significantly inhibited Caki-2 cell migration and 786-O cell invasion. The enhancement of migration and invasion of RCC cells by miR-590-5p is related to its up-regulation of MMP-2 and MMP-9 expression, which are of great importance in cancer cell invasion and migration through enzymatic degradation of the extracellular matrix.38,39 MMP-2 and MMP-9 have increased expression in
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RCC and this is associated with a poor prognosis in patients with RCC.40 miR-590-5p increased cell invasion and proliferation as well as decreased the sensitivity of gastric cancer cells to cisplatin and paclitaxel through inversely regulating RECK expression.32 miR-590-5p promoted proliferation, G1/S transition and invasion of RCC cells through targeting PBRM1.20 In our study, we explored the functional role and the target of miR-590-5p in Caki-2 and 786-O cells and found that it can be an efficient regulator for ARHGAP24. Studies have shown that ARHGAP24 was involved in regulating the cell cycle, cell proliferation, apoptosis and invasion in distinguished types of cancers,25,41 suggesting a potential correlation with miR-590-5p. In this research, it was observed that miR-590-5p significantly inhibited ARHGAP24 expression in Caki-2 and 786-O cells at protein levels, and ARHGAP24 overexpression significantly reduced cell
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viability, migration and invasion and induced apoptosis, suggesting a tumor suppressor role in the RCC process. In agreement with our results, Xu et al.25 reported that ARHGAP24 induces apoptosis, along with inhibition of the cell cycle and invasion through regulating expression of PCNA, CDK1/2, Bax, Caspase-3, and Bcl-2 in RCC cells. However, ARHGAP24 was significantly higher in malignant lymphocytes, positively correlated with Rac1 expression, and associated with a poor overall survival rate.42 The interaction between ARHGAP24 and other molecules is possible, depending on the different molecular pathways of different types of cancers. Consequently, for the first time, we demonstrated that ARHGAP24 was down-regulated in RCC cells by miR-590-5p, which promotes the cell viability, invasion and migration of RCC cells through targeting ARHGAP24. Therefore, our demonstration suggested that targeting the miR-590-5p–ARHGAP24 interaction provides us with a new treatment strategy for RCC.
Conflicts of interest The authors declare that they have no competing interests.
Acknowledgements This study was supported by the Huai’an 2016 Annual Promotion Project for Science and Technology International Cooperation (HAC201620).
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Cite this: DOI: 10.1039/c7mb00406k
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MicroRNA-590-5p regulates cell viability, apoptosis, migration and invasion of renal cell carcinoma cell lines through targeting ARHGAP24 Lei Wang,†a Wan-qing Wei,†b Zi-yu Wua and Gong-cheng Wang
*c
Renal cell carcinoma (RCC) is the leading cause of death in renal malignancies. MicroRNA-590-5p (miR-590-5p) is of great importance in the processes of many cancers regarding regulation of cancer cell invasion and proliferation. In our study, alternation of miR-590-5p expression in RCC cell lines through transfection with pre-miR-590-5p (up-regulation) or anti-miR-590-5p (down-regulation) was performed. Apoptosis and viability of RCC cell lines were measured by flow cytometry and CCK-8 analysis, respectively. Cell invasion and migration were estimated by Transwell assay. The association of miR-590-5p with ARHGAP24 expression was evaluated using luciferase assays, real-time PCR and western blot assay. The expressions of apoptosis and migration-related protein were also measured by western blotting. We found that pre-miR-590-5p transfection in Caki-2 and 786-O cells showed significant increases in cell viability, invasion and migration, which were accompanied by decreased cell apoptosis, while anti-miR-590-5p transfection obviously inhibited the cell viability, migration and Received 7th July 2017, Accepted 13th September 2017 DOI: 10.1039/c7mb00406k
invasion of Caki-2 and 786-O cells as well as induced apoptosis, compared with the negative control group. Furthermore, bioinformatics combined with luciferase reporter assays indicated that ARHGAP24 is directly targeted by miR-590-5p. ARHGAP24 overexpression in 786-O and Caki-2 cells phenocopied the effects of anti-miR-590-5p transfection along with enhanced expression of active Caspase-3 and Bax/Bcl-2 ratio as well as decreased expression of MMP-2 and MMP-9. These findings suggested that
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miR-590-5p/ARHGAP24 seems to function as a potentially beneficial target for RCC treatment.
Introduction Renal cell carcinoma (RCC) is one of the three most common tumors in the urinary system, with the highest mortality in the case of parenchymal urinary tumors in adults,1,2 and accounts for about 3% of adult malignancies, with 2% of incidence each year.3 About 90% of kidney cancer is RCC, more than 80% of which is clear cell renal cell carcinoma originating from the renal proximal tubule. Different pathological types of RCC have different clinical features, treatment options and prognosis. The 3-year survival rate of RCC is calculated to be about 47%,4 and the incidence of patients with RCC is about 10%, with metastasis.5 Previous studies have shown that metastasis is universal in patients with RCC and functions as a major prognostic factor.6,7 a
Department of Urology, The Affiliated Huai’an Hospital of Xuzhou Medical University and The Second People’s Hospital of Huai’an, Huai’an 223200, China b Department of Pediatric Surgery, Maternal and Children Health Hospital of Huai’an, Huai’an 223002, China c Department of Urology, Huai’an First People’s Hospital, Nanjing Medical University, 1 Huanghe West Road, Huaiyin District, Huai’an 223300, China. E-mail: [email protected]; Fax: +86-0517-80872660; Tel: +86-0517-80872660 † Co-first authors.
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Clinically, 30–50% of patients are asymptomatic patients who have been found incidentally, while most of the patients with clinical symptoms have metastatic disease at the time of diagnosis. At present, the main treatment of RCC is surgical resection because late or metastatic RCC is relatively resistant to conventional chemotherapy or radiotherapy.8 However, some of the RCC patients have metastasis, including to the brain, lungs, liver and bone, even if they are treated at the early stage.9 After tumor resection, tumor recurrence occurs in 20–40% of patients.10 Therefore, effective control of the growth and metastasis of RCC cells, especially at the late stages, is a major challenge. The early diagnosis and treatment of RCC, especially in advanced RCC patients with recurrence tendency, is still the preface to the study of kidney cancer. Because of the complexity of the cause, development and migration of RCC, a lot of studies are related to a variety of mechanisms and genes, molecules and their respective roles involved. MicroRNAs (miRNAs, miR) compromise a specialized subset of noncoding RNAs with a length from 19 to 25 nucleotides, mainly combined with the 30 -UTR domain of a specific gene to form the complementary sequence, and then exert their regulatory effects at the stability of transcription and the post-transcriptional
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level.11 miRNAs are associated with various biological processes such as cell apoptosis, differentiation, proliferation, immune response and metabolism.12,13 Changes in miRNA expression can mediate the critical biological processes of cells regarding tumor development,14 and an assessment of alternation in miRNA expression may supply insights into the underlying mechanisms by which miRNA regulates the occurrence and process of cancer. In addition, the therapeutic regulation of miRNA can simultaneously affect multiple target genes and achieve excellent clinical outcomes,15 and miRNA expression profiling is therefore becoming a potentially powerful tool for disease diagnosis and prognosis. miR-590-3p was found to promote tumorigenesis of hepatocellular carcinoma (HCC) by down-regulating PTEN to activate the PI3K-AKT signaling pathway16 and regulate proliferation and invasion in HCC cells by targeting TGF-b RII.17 miR-590-5p promotes cervical cancer cell growth and invasion by targeting CHL1.18 miR-590-3p promotes cell growth of colon cancer by targeting TFAM.19 miR-590 promotes RCC cell invasion and proliferation through down-regulating PBRM1.20 The members of the Rho GTPase family are key regulators of actin dynamics and are known to be involved in many biological processes of cells including cell differentiation, adhesion, and migration.21 Several Rho GTPases have been shown to contribute to the tumor initiation and malignant progression, which may serve as a tumor suppressor or oncogene.22,23 Rho GTPase-activating protein 24 (ARHGAP24), also known as FilGAP, is highly conserved and enriched during podocyte differentiation and in kidney tissues.24 ARHGAP24 has been shown to down-regulate in RCC tissues and indicate a better prognosis, along with inhibition of cell cycle processes and invasion and induction of apoptosis of RCC cells.25 It is a potential target of miR-590-5p based on the analysis of Targetscan (http://www.targetscan.org/ vert_71/). But whether miR-590-5p along with ARHGAP24 also plays a crucial role in RCC progression remains largely unknown. In our work, the functions and mechanisms by which miR-590-5p regulated the cell viability, apoptosis, invasion and migration of RCC cells were assessed. We observed that ARHGAP24 was targeted by miR-590-5p. ARHGAP24 up-regulation mimicked the regulatory action of anti-miR-590-5p transfection in RCC cells, along with inhibited cell viability, invasion and migration, but promoted apoptosis. miR-590-5p/ARHGAP24 may serve as a target for therapy in treatment of RCC.
Materials and methods Cell culture Human clear cell renal cell carcinoma cell lines (Caki-2 and 786-O cells) were obtained from the ATCC (Rockville, USA) and cultured in DMEM (Life technology) complemented with 10% FBS (Life Technologies) along with 1% Penicillin–Streptomycin Solution (100; 100 U ml1 penicillin and 0.1 mg ml1 streptomycin for working; Solarbio) separately incubated at 37 1C with 5% CO2 and 95% air.
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Cell transfection pre-miR-590-5p, anti-miR-590-5p, and negative control (NC) were purchased from Beyotime (Shanghai, China) and transfected into Caki-2 and 786-O cells using a HiPerFect transfection reagent (Qiagen, CA) following the manufacturer’s recommendations. pLVX-AcGFP-C1 lentiviral vector carrying the ARHGAP24 coding sequence was obtained from GenePharma (Suzhou, China) and transfected into Caki-2 and 786-O cells for overexpression of ARHGAP24 using Lipofectamine 2000 (Invitrogen, Shanghai, China) following the instructions. At 6 h posttransfection, the medium was replaced with fresh medium. The transfected Caki-2 and 786-O cells were harvested for subsequent experiments 48 h post-transfection. CCK-8 assay Cell viability was analyzed by Cell Counting Kit CCK-8/WST-8 assay,25 which was carried out following the standard procedure in 96 well plates. Briefly, cells were added into a 96-well plate and grown to 80% confluence. After 24 h of incubation, the medium was removed. Then pre-miR-590-5p in the absence or presence of pLVX-AcGFP-C1-ARHGAP24 or anti-miR-590-5p or negative control (NC) was transfected into cells. The CCK-8 (10 ml per well) reagent was added at 0, 12, 24, 48 and 72 h post-transfection. The reaction system was incubated for 1 h under the same incubator conditions after which the absorbance readings were obtained at 450 nm. Cell apoptosis assay The cell apoptosis was assayed by flow cytometric assay.25 In brief, cells were added into a 6-well plate until grown to 60–80% confluence, and washed with 1 ml Binding Buffer (3 ml 10 Binding Buffer in 27 ml ddH2O). Subsequently, cells were mixed with 195 ml Annexin V-FITC for 10 min as well as 5 ml propidium iodide (PI) in darkness for 5 min. The fluorescence intensities were assessed by FACS using a FL-2A filter (BD FACS Calibur, USA). Data were studied using the Cyflogic software (CyFlo Ltd, Turku, Finland). Transwell assay At 48 h post-transfection, cells (5 104 cells per ml) were moved into DMEM supplemented with 1% FBS and plated into the upper chambers of Transwell plates (Millipore, Beijing, China), and the medium supplemented with 10% FBS was plated into the lower chambers. The membrane was fixed in methanol, and then stained using hematoxylin after 24 h of incubation. Stained cells were visualized and counted under a microscope (200 magnification). The analysis of cell invasion was consistent with that of cell migration expect that the cell invasion was detected by Transwell assay using Matrigel Invasion Chambers (BD Biosciences). Dual-luciferase reporter assay 24-Well plates were used for Caki-2 and 786-O cultivation for 24 h before transfection to ensure a density of 2 105 cells per well. pmiRGLO-ARHGAP24-3 0 UTR-wt or pmiRGLO-ARHGAP243 0 UTR-mut containing both firefly and renilla luciferases and
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pre-miR-590-5p or NC was transfected into Caki-2 and 786-O cells using Lipofectaime 2000t (Invitrogen). Post-transfection for 24 h, cells were lysed and activities of renilla luciferase were detected by a dual-luciferase reporter system (Promega, USA) following the provided protocols.
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Real-time PCR Total RNA extraction was performed using a miRNeasy kit (QIAGEN) and stored at 80 1C in a RNA secure RNase Inactivation Reagent (Thermo Fisher Scientific) following the manufacturer’s instructions. Total RNA (500 ng) was converted to complementary DNA. Real-time PCR was performed via SYBR Green LightCycler PCR (LC-PCR) assay on a 7900HT system. The relative quantification of miR-590-5p is given as ratios to 5S by using a 2DDCt cycle threshold method, respectively. Western blot assay Total protein extraction was performed with RIPA lysis buffer added with protease inhibitors 48 h after transfection and then centrifugation was carried out at 10 000g for 30 min at 4 1C. 50 mg of the protein extracted were loaded into 12% SDS-PAGE and were shifted onto a PVDF membrane (Millipore, USA). Then blots were incubated at 4 1C overnight with ARHGAP24, Bax, Bcl-2, active Caspase-3, MMP-2, MMP-9, or GAPDH antibodies (1 : 1000; Abcam, Cambridge, MA, USA). The horseradish peroxidase-conjugated IgG antibody (Jackson ImmunoResearch) was further used for 60 min at 25 1C. Then, membranes were developed using an enhanced chemiluminescence (ECL) substrate (Pierce, USA) and spotted using an Image Quant LAS 4010 Imaging System (GE Healthcare, Piscataway, NJ). Statistical analysis All quantitative values were expressed as mean SD of at least three repeated individual experiments for each group and they were statistically analyzed with One-way ANOVA, followed by Tukey’s post hoc test. All of the statistical analyses were made using GraphPad Prism v5.0 (GraphPad Software, Inc.). P o 0.05 was considered statistically significant.
Results miR-590-5p strengthens cell viability and inhibits apoptosis of RCC cells To estimate the function of miR-590-5p in RCC tumorigenesis, pre-miR-590-5p, anti-miR-590-5p, or negative control (NC) was transfected into the Caki-2 and 786-O cells, respectively. The expression of miR-590-5p in Caki-2 and 786-O cells is shown in Fig. 1A and B. The cell viability of Caki-2 and 786-O at 0, 12, 24, 48 and 72 h post-transfection was measured by CCK-8 assay and is displayed in Fig. 1C and D. pre-miR-590-5p transfection enhanced the viability of Caki-2 and 786-O cells in a timedependent manner. However, anti-miR-590-5p transfection timedependently decreased the viability of Caki-2 and 786-O cells, with the lowest viability of Caki-2 and 786-O cells detected at 72 h (51.3 2.3% of Caki-2-NC and 42.1 2.1% of 786-O-NC).
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Meanwhile, the cell viability of Caki-2 and 786-O at 12 h posttransfection was not changed by pre-miR-590-5p transfection compared with NC. Based on the influence of miR-590-5p on the cell viability of RCC, the cell apoptosis of Caki-2 and 786-O induced by miR-590-5p was also assessed. Our results revealed that pre-miR-590-5p transfection markedly attenuated Caki-2 and 786-O cell apoptosis by 48.5 3.1% and 54.4 3.8% compared with NC, respectively (Fig. 1E–H). However, anti-miR-590-5p transfection significantly promoted apoptosis of Caki-2 and 786-O cells by 13.8 1.3-fold and 8.5 0.8-fold compared with NC, respectively (Fig. 1E–H). Our findings speculate that miR-590-5p promotes the viability of RCC cell lines, in part, through inhibiting cell apoptosis. miR-590-5p enhances migration and invasion in RCC cells Next, the cell invasion and migration ability of RCC cell lines was measured by Transwell assay. Fig. 2A and B display that pre-miR-590-5p transfection significantly promoted Caki-2 cell migration by 50.9 4.4% compared with NC. However, antimiR-590-5p transfection significantly inhibited Caki-2 cell migration by 64.9 5.3% compared with NC. Meanwhile, it is figured out that pre-miR-590-5p transfection significantly strengthened invasion of 786-O cells by 27.2 1.5% compared with NC (Fig. 2C and D). However, anti-miR-590-5p transfection significantly inhibited 786-O cell invasion by 39.5 2.5% compared with NC. ARHGAP24 is a target of miR-590-5p in RCC cells To further elucidate the underlying mechanisms through which miR-590-5p regulates the cell viability, invasion and migration of RCC cells, Targetscan was introduced for predicting miR-590-5p targets (Fig. 3A). The luciferase activity assay showed that overexpression of miR-590-5p inhibited the protein of ARHGAP24 through binding the 3 0 UTR site of the latter in Caki-2 and 786-O cells, but did not decrease the activity of ARHGAP24 with 3 0 UTR-mut (Fig. 3B). Next we confirmed that ARHGAP24 is a target gene of miR-590-5p in Caki-2 and 786-O cells. As expected, our findings demonstrated that pre-miR-590-5p transfection obviously down-regulated ARHGAP24 protein expression compared with NC (Fig. 3C). These data suggested that ARHGAP24 is a target of miR-590-5p in RCC cell lines. ARHGAP24 overexpression inhibits cell viability and induces apoptosis of RCC To further validate the correlation between miR-590-5p and ARHGAP24 described above, pre-miR-590-5p in the absence or presence of pLVX-AcGFP-C1-ARHGAP24 was transfected into Caki-2 and 786-O cells. It was observed that ARHGAP24 overexpression up-regulated the ARHGAP24 protein level by 1.3 0.08-fold and 0.72 0.04-fold in Caki-2 and 786-O cells in comparison with blank vector transfection, respectively (Fig. 4A and B). Cell viability assay demonstrated that overexpression of ARHGAP24 markedly inhibited pre-miR-590-5p induced increase in cell viability of Caki-2 and 786-O cells at 24, 48 and 72 h, respectively (Fig. 4C and D). Flow cytometry assay
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Fig. 1 miR-590-5p promoted cell viability and inhibited apoptosis of RCC cell lines. Caki-2 and 786-O cells were transfected with pre-miR-590-5p, anti-miR-590-5p, or negative control (NC) for 1, 12, 24, 48 and 72 h. (A and B) The expression of miR-590-5p in Caki-2 and 786-O cells was measured by real-time PCR. (C and D) The cell viability of Caki-2 and 786-O cells was measured by CCK-8 assay. (E–H) The cell apoptosis of Caki-2 and 786-O cells was measured by flow cytometry assay. *P o 0.01 compared with NC.
demonstrated that overexpression of ARHGAP24 markedly inhibited pre-miR-590-5p induced decrease in cell apoptosis
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of Caki-2 and 786-O cells by 14.5 0.8-fold and 12.8 0.7-fold compared with blank vector transfection, respectively
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Fig. 2 miR-590-5p promoted cell migration and invasion of RCC cell lines. Caki-2 and 786-O cells were transfected with pre-miR-590-5p, anti-miR590-5p, or negative control (NC) for 48 h. The cell migration of Caki-2 (A and B) and cell invasion of 786-O cells (C and D) were measured by Transwell assay. *P o 0.01 compared with NC.
(Fig. 4E and F). These findings suggested the important role of ARHGAP24 in cell viability and apoptosis of RCC cell lines induced by miR-590-5p. ARHGAP24 overexpression inhibits cell migration and invasion of RCC Transwell assay found that overexpression of ARHGAP24 markedly inhibited pre-miR-590-5p induced increase in Caki-2 cell migration and 786-O cell invasion by 86.4 5.8% and 33.8 2.6% compared with blank vector transfection, respectively (Fig. 5A–C). These results suggested the important role of ARHGAP24 in cell invasion and migration of RCC cell lines induced by miR-590-5p. Effects of miR-590-5p and ARHGAP24 up-regulation on regulation of protein expression in RCC cells Then, the protein expression of active Caspase-3, Bcl-2, Bax, MMP-2 and MMP-9 was measured by western blot assay. As shown in Fig. 6A and B, pre-miR-590-5p transfection in Caki-2 cells obviously decreased the Bax/Bcl-2 ratio and active Caspase-3, and increased protein expression of MMP-2 and MMP-9. Meanwhile, ARHGAP24 overexpression significantly increased the
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Bax/Bcl-2 ratio and active Caspase-3 expression, and decreased the levels of MMP-2 and MMP-9 protein in Caki-2 cells with pre-miR-590-5p transfection, which is in contrast to the regulatory function of pre-miR-590-5p. Moreover, similar findings were also demonstrated in 786-O cells (Fig. 6C and D). This suggests that the effects of miR-590-5p up-regulation in RCC cells depend specifically on ARHGAP24 decrease.
Discussion Targeted therapies designed against molecules participating in the pathogenesis of cancer were reported as effective ways in cancer recently. Growing evidence has shown that miRNAs are often deregulated in many cancers.26,27 Specific cellular characterization of miRNAs in cancer cells has been intensively studied for clinical biomarkers and novel therapeutic targets. The pattern of miRNA expression appears with high tissue specificity, and is more precise in cancer diagnosis in comparison with the analysis based on mRNA.28,29 At present, although the causes of RCC, its diagnosis, and treatment are clear, the effective treatment of advanced RCC is limited. Although surgical
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Fig. 3 ARHGAP24 is a validated target of miR-590-5p. (A) ARHGAP24 as a potential target of miR-590-5p using Target Scan. (B) Luciferase reporter assays showed that up-regulation of miR-590-5p obviously reduced the relative luciferase activity of ARHGAP24 3 0 UTR. (C) In both Caki-2 and 786-O cells, pre-miR-590-5p transfection decreased the protein expression of ARHGAP24. *P o 0.01 compared with NC.
Fig. 4 ARHGAP24 overexpression inhibited cell viability and induced apoptosis of RCC cell lines. (A and B) Caki-2 and 786-O cells were transfected with pLVX-AcGFP-C1-ARHGAP24 for 48 h, and the protein expression of ARHGAP24 was measured by western blot assay. After 0, 12, 24, 48 and 72 h of transfection, the cell viability of Caki-2 and 786-O cells was measured by CCK-8 assay (C and D), and apoptosis of Caki-2 and 786-O cells was measured by flow cytometry assay (E and F). *P o 0.01 compared with pre-miR-590-5p+vector.
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Fig. 5 ARHGAP24 overexpression inhibited cell migration and invasion of RCC cell lines. (A and B) Caki-2 and 786-O cells were transfected with pLVXAcGFP-C1-ARHGAP24 for 48 h; the cell migration of Caki-2 cells (A and C) and cell invasion of 786-O cells (B and C) were measured by Transwell assay. *P o 0.01 compared with pre-miR-590-5p+vector.
resection, along with radiotherapy as well as chemotherapy, is of great importance in the treatment of RCC, the effect is not yet satisfactory. With the development of tumor molecular biology, finding a specific target gene has become a major research direction. In the present study, we observed that ARHGAP24 was targeted by miR-590-5p. ARHGAP24 up-regulation phenocopied the effects of anti-miR-590-5p transfection in RCC cells, along with inhibited cell viability, invasion and migration, but promoted apoptosis. New therapies are urgently needed to be developed because the treatment strategy of RCC has not been effective enough recently. The occurrence of a tumor is a combination of many oncogenes and tumor suppressor genes associated with oncogene activation, tumor suppressor gene inactivation, epigenetic changes and other complex changes in pathogenesis. miR-590-5p dysregulation was observed in many types of cancer via the regulation of diverse critical cancer-associated pathways,30–32 which indicated that miR-590-5p is an oncogene implicated in these malignancies. However, in contrast to these findings, several other studies have reported the tumor-suppressive role of miR-5905p in colorectal cancer,33 breast cancer34 and hepatocellular carcinoma.35 Recent reports showed miR-590-5p up-regulation in tumor tissues from patients with RCC36 and in the examined
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RCC cell lines,20 including SN12C, ACHN, TK10, A704 and 786-O, compared with normal kidney tissues and normal HEK293 cells, which indicated that miR-590-5p was possibly related to RCC progression. Therefore, pre-miR-590-5p and anti-miR-590-5p were introduced into the Caki-2 and 786-O cells for elucidating the regulatory action of miR-590-5p in RCC. One of the most prominent features of tumor progression is abnormal cell proliferation. The close relationship between cell proliferation and tumorigenesis has been realized; since tumorigenesis is a long-term, multifactor and multistep process, the regulatory factor of cell proliferation in the effect and mechanism of tumor evolution has rarely been fully clarified. Thus, in this study the functions of miR-590-5p in cell proliferation of RCC were investigated. We figured out that pre-miR-590-5p significantly boosted cell viability and inhibited apoptosis of Caki-2 and 786-O cells, while anti-miR-590-5p significantly inhibited cell viability of Caki-2 and 786-O and induced apoptosis. In line with our findings, previous studies highlighted that miR-590-5p enhanced cell proliferation and survival in RCC,20 hepatocellular carcinoma16 and cervical cancer.18 Moreover, the expression of apoptosis-related proteins showed that pre-miR-590-5p significantly decreased Bax/Bcl-2 and active Caspase-3 in Caki-2 and 786-O cells, while anti-miR-590-5p
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Fig. 6 miR-590-5p and ARHGAP24 regulated protein expression in RCC cell lines. Caki-2 and 786-O cells were transfected with pre-miR-590-5p in the absence or presence of ARHGAP24 overexpression; the protein expression of Bax, Bcl-2, Caspase-3, MMP-2 and MMP-9 in Caki-2 (A and B) and 786-O cells (C and D) was measured by western blot assay. *P o 0.01 compared with NC. #P o 0.01 compared with pre-miR-590-5p+vector.
significantly increased those. Once activated, Caspase-3 is responsible for the proteolytic cleavage of a broad spectrum of cellular targets, which ultimately leads to cell death, and Bcl-2 overexpression allows for continued cellular proliferation through antagonism of apoptotic signals imparted by the pro-apoptotic Bax.37 In addition to abnormal cell proliferation, invasion and migration are major characteristics of human cancer. In this study, it was revealed that pre-miR590-5p obviously increased Caki-2 cell migration and 786-O cell invasion, while anti-miR-590-5p significantly inhibited Caki-2 cell migration and 786-O cell invasion. The enhancement of migration and invasion of RCC cells by miR-590-5p is related to its up-regulation of MMP-2 and MMP-9 expression, which are of great importance in cancer cell invasion and migration through enzymatic degradation of the extracellular matrix.38,39 MMP-2 and MMP-9 have increased expression in
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RCC and this is associated with a poor prognosis in patients with RCC.40 miR-590-5p increased cell invasion and proliferation as well as decreased the sensitivity of gastric cancer cells to cisplatin and paclitaxel through inversely regulating RECK expression.32 miR-590-5p promoted proliferation, G1/S transition and invasion of RCC cells through targeting PBRM1.20 In our study, we explored the functional role and the target of miR-590-5p in Caki-2 and 786-O cells and found that it can be an efficient regulator for ARHGAP24. Studies have shown that ARHGAP24 was involved in regulating the cell cycle, cell proliferation, apoptosis and invasion in distinguished types of cancers,25,41 suggesting a potential correlation with miR-590-5p. In this research, it was observed that miR-590-5p significantly inhibited ARHGAP24 expression in Caki-2 and 786-O cells at protein levels, and ARHGAP24 overexpression significantly reduced cell
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viability, migration and invasion and induced apoptosis, suggesting a tumor suppressor role in the RCC process. In agreement with our results, Xu et al.25 reported that ARHGAP24 induces apoptosis, along with inhibition of the cell cycle and invasion through regulating expression of PCNA, CDK1/2, Bax, Caspase-3, and Bcl-2 in RCC cells. However, ARHGAP24 was significantly higher in malignant lymphocytes, positively correlated with Rac1 expression, and associated with a poor overall survival rate.42 The interaction between ARHGAP24 and other molecules is possible, depending on the different molecular pathways of different types of cancers. Consequently, for the first time, we demonstrated that ARHGAP24 was down-regulated in RCC cells by miR-590-5p, which promotes the cell viability, invasion and migration of RCC cells through targeting ARHGAP24. Therefore, our demonstration suggested that targeting the miR-590-5p–ARHGAP24 interaction provides us with a new treatment strategy for RCC.
Conflicts of interest The authors declare that they have no competing interests.
Acknowledgements This study was supported by the Huai’an 2016 Annual Promotion Project for Science and Technology International Cooperation (HAC201620).
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