MicroRNA-630 Regulates Tumor Metastasis Through The TGF-?-miR-630-slug Signaling Pathway In Hepatocellular Carcinoma

Objective:To investigate the clinical significance of miR-630 expression in HCC, we measure 97 tumor tissues and analyze the relationship between the miR-630 and clinicopathological features and evaluate the contribution to the progression of HCC.Material and Methods:Ninety-seven human liver tumor tissues were obtained between 2010 and 2012 from patients who underwent liver resection surgery at the Hepatic Surgery Center of Tongji Hospital affiliated with the Huazhong University of Science and Technology. Expression of miR-630 was analyzed by quantitative real-time PCR.Result:Compared with non-metastatic tumor tissues, the relative expression of miR-630 was significantly reduced in metastatic tumor tissues. Furthermore, the expression levels of miR-630 in patients with incomplete encapsulation of their tumors had a significantly lower miR-630 expression compared with patients with completely encapsulated tumors. Moreover, the miR-630 expression level was inversely associated with the tumor Edmondson-Steiner stage (?-?/?-?), tumor-node-metastasis (TNM) stage (?-?/?-?) and Barcelona-Clinic Liver Cancer (BCLC) stage (0+A/B+C). To determine the relationship between miR-630 expression levels and clinicopathological features, the 97 patients in the study were divided into two groups according to the median level of miR-630 expression among them. High miR-630 levels were negatively associated with AFP, tumor number, vascular invasion, Edmondson-Steiner stage and BCLC stage but not with tumor size. Kaplan-Meier curves showed that patients with low miR-630 expression had a higher recurrence rate and shorter overall survival (OS) compared with patients with high miR-630 expression. However, clinicopathological features were not correlated with disease-free survival as determined by multivariate analysis.Conclusion:Decreased miR-630 expression levels correlate with poor HCC prognosis, suggesting that inhibition of miR-630 expression may contribute to the progression of HCC.Objectives:To explore the function roles of miR-630 in hepatocellular carcinoma progression.Material and Methods:The Be17402 and HLF cells were transiently transfected with the mimics, inhibitors or vector. Then, CCK-8, Transwell assay, wound healing Immunofluorescence assay were performed to compare the difference of proliferation, migration, invasion, and the epithelial-mesenchymal transition between control group and mimics/inhibitors group in vitro. Furthermore, we established stable Be17402-shmiR-630 cell lines using lentivirus. We assessed the tumorigenicity and metastasis between the control group and Be17402-shmiR-630 group in vivo.Result:MiR-630 suppresses HCC cell migration and invasion but not proliferation in vitro. Moreover, miR-630 can reverse EMT in HCC cells. MiR-630 functions as a tumor suppressor in HCC by suppressing metastatic colonization but not tumorigenesis in vivo.Conclusion:MiR-630 inhibits HCC cells migration, invasion, and EMT in vitro. Besides, Reduction of miR-630 expression increases HCC metastasis in vivo.Objective:To elucidate the underlying molecular mechanism by which miR-630 suppresses metastasis in HCC.Methods:We used publicly available databases, including TargetScan (http://www.targetscan.orp/) and PicTar (http://pictar.mdc-berlin.de/) to search for the genes targeted by miR-630. We performed immunohistochemistry, Western blot, and RT-PCR assays to investigate whether Slug was the target of miR-630 in HCC. Furthermore, to examine whether miR-630 abrogated migration, invasion, and EMT by targeting Slug, we carried out Transwell, wound healing, immunofluorescent staining, Western blot, and RT-PCR assays in HCC.Result:Publicly available databases, including TargetScan (http://www.targetscan.org/) and PicTar (http://pictar.mdc-berlin.de/) predicted Slug (SNAI2) as one of the target genes. Luciferase Reporter assays showed that the activity of luciferase linked with the 3'UTR of Slug was suppressed in Be17402 cells transfected with miR-630 mimics compared with control cells. Conversely, miR-630 inhibition caused a significant increase in luciferase activity. Furthermore, Slug expression was inversely correlated with miR-630 expression in 80 HCC tumor tissues as determined by Spearman's correlation analysis. The up-regulation of Slug in HCC is associated with poor clinical outcomes. Western blot and RT-PCR analysis consistently indicated that the expression levels of Slug, N-Cadherin and vimentin were reduced in miR-630-overexpressing cells, whereas their levels were elevated in cells transfected with miR-630 inhibitors. Conversely, up-regulation of miR-630 caused a significant increase in E-Cadherin, whereas inhibition of miR-630 resulted in a significant decrease in E-Cadherin. Transwell assays showed the knockdown of Slug decreased the cell migration and invasion induced by miR-630 inhibitors. Similarly, the wound healing assay also revealed that the reduction of Slug rescued the effect of miR-630 inhibitors on the migration. Moreover, immunofluorescent staining consistently showed the down-regulation of Slug partly suppressed vimentin and increased E-Cadherin initiated by miR-630 inhibitors. Western blot and RT-PCR analysis consistently showed that the levels of Slug, N-Cadherin, and vimentin were increased after transfected with miR-630 inhibitors, whereas co-transfection with siRNA against Slug reversed these effects.Conclusion:These results imply that miR-630 suppresses Slug and in turn attenuates the expression of EMT-related genes. Furthermore, miR-630 might suppress migration and invasion by inhibiting EMT. Slug serves as a target of miR-630, contributing to the repression effect of miR-630 on metastasis.Objectives:To investigate the upstream signaling pathway that miR-630 was regulated in HCC.Methods:To investigate whether TGF-? represses miR-630 expression, we first examined the levels of miR-630 in TGF-?-treated Be17402 and HLF cells by RT-PCR. To determine which signaling pathway activated by TGF-P exerted this effect, we treated Be17402 and HLF cell with the following kinase inhibitors:SB431542, (a receptor type I TGF-? (T0RI, ALK5) serine/threonine kinase inhibitor), SB203580 (a p38 MAPK inhibitor), SP600125 (a JNK inhibitor), U0126 (a MEK1/2 inhibitor that suppresses Erk signaling) and SIS3 (a SMAD3 inhibitor). Western blot analysis to confirm TGF-P could activate the non-SMAD dependent signaling pathways in HCC cell lines. To further determine how miR-630 is transcriptionally regulated by TGF-?, representative regions, covering 1.0-kb upstream of the transcriptional initiation site of the miR-630, were investigated using NCBI (http://www.ncbi.nlm.nih.gov/pubmed/) and Jaspar (http://jaspar.genereg.net/) databases. To identify the response element essential for the down-regulation of miR-630 promoter activity in response to TGF-P, we introduced a series of miR-630 promoter reporter constructs into Be17402 cells. Additionally, we carried out chromatin immunoprecipitation assays to identify SP1 and c-Jun could bind the upstream promoter region (-79-0) of the miR-630 gene. Furthermore, we using SP1, c-Jun and SP1+c-Jun mutant constructs, we identified these response elements participated in the TGF-?-repression of miR-630 promoter activity. Next, we knocked down SP1 and c-Jun using siRNA in HCC cells to determine whether the activation of the ERK/SP1 and JNK/c-Jun signaling pathways by TGF-? is responsible for the repression of miR-630 transcription. Furthermore, we performed Western blot assay to identify the reduction of SP1 and c-Jun in Be17402 cells: repressed the ability of TGF-? to induce Slug expression. Moreover, to investigate whether miR-630 reversed the effects induced by TGF-? in HCC, we performed gain-of-function analyses such as Transwell assays, wound healing assay, immunofluorescence staining assay, Western blot and RT-PCR assays in TGF-? treated cells by up-regulating miR-630( expression. We used Transwell assays, wound healing assay, immunofluorescence staining assay, Western blot and RT-PCR assays to confirm that miR-630 attenuated the TGF-?-induced EMT of HCC cells.Result:TGF-P treatment resulted in a significant decrease in miR-630 expression in a time-dependent manner. The repression of miR-630 by TGF-? was reversed by treatment with U0126, SB203580, SP600125and SB431542 kinase inhibitors but not SIS3 kinas inhibitor. Western blot analysis confirmed that TGF-? activated the non-SMAD dependent signaling pathways in HCC cell lines. NCBI (http://www.ncbi.nlm.nih.gov/pubmed/) and Jaspar (http://jaspar.genereg.net/) databases showed that the 1.0-kb region upstream of the transcriptional initiation site of miR-630 contained binding sites for the following TGF-?/non-SMAD dependent pathway transcription factors:ELK-1, CREB1, SP1 and c-Jun. Luciferase Reporter assays showed that the region of the miR-630 promoter from-79 to 0 bp was critical for TGF-?-mediated transcriptional repression of miR-630( expression. Chromatin immunoprecipitation assays revealed that, in response to TGF-? SP1 and c-Jun bind the upstream promoter region (-79-0) of the miR-630 gene. Using SP1 c-Jun and SP1+c-Jun mutant constructs, we identified these response elements participated in the TGF-?-repression of miR-630 promoter activity. Consistently, RT-PCR analysis confirmed the functional role of SP1 and c-Jun in down-regulating miR-630 expression in Be17402 cells. Knockdown of SP1 or c-Jun could up-regulate miR-630, whereas co-treatment with TGF-p reversed this effect. Furthermore, Western blot showed that the reduction of SP1 and c-Jun in Bel7402 cells repressed the ability of TGF-? to induce Slug expression. Transwell assays showed that the ectopic expression of miR-630 significantly antagonized the TGF-?-induced migration and invasion of Bel7402 and HLF cells. Similarly, the wound healing assay also confirmed that the overexpression of miR-630 suppressed the TGF-?-induced migration. Furthermore, immunofluorescence staining showed that miR-630 overexpression reversed the TGF-?-mediated up-regulation of vimentin and down-regulation of E-Cadherin. In addition, E-Cadherin was increased, while Slug, N-Cadherin, and vimentin were decreased after transfection with miR-630 in TGF-?-treated HCC cells.Conclusion:The Erk/SP1 and JNK/c-Jun signaling pathways exert a critical role for TGF-? in down-regulating miR-630 transcription. MiR-630 could attenuate the TGF-?-induced EMT of HCC cells.