MiR-590-5p Promotes Proliferation In Human HCCs By Directly Targeting PDCD4

BackgroundHepatocellular carcinoma (HCC) is one of the most common cancer and the second leading cause of death from cancer in China, causing serious problems to the public health. Just like any other cancer, mutation to the cellular machinery causes a higher rate of cell replication and/or a capability of avoiding cell apoptosis, which leading to Hepatocellular carcinoma ultimately. MicroRNAs (miRNAs) are a class of small non-coding RNAs with21-25nucleotides that are highly conserved throughout metazoan species. MicroRNAs suppress gene expression by inhibition of translation or facilitation of mRNA degradation by binding to the3’-Untranslated Region (3’-UTR) of a target mRNAs with perfect or near perfect complementarity. Recent evidence has shown that miRNA mutations or mis-expression correlate with various human cancers and indicates that miRNA can function as tumor suppressors and oncogenes. Therefore, an understanding of the molecular mechanisms by which they regulate the behavior of cancer cells is needed. It has been reported that miR-590-5p regulates proliferation in HCC by targeting TGF-βRII. Yet, paradoxically, the TGF-Psignaling pathway has been considered as both a tumor suppressor pathway and a promoter of tumor progression. Therefore, it was speculated that miR-590regulates proliferation in HCC by targeting multiple genes. The programmed cell death4(PDCD4) gene was originally identified as a tumor-related gene in humans and a possible tumor suppressor in HCC. However, the connection between miR-590and PDCD4in HCC is still unclear.Objectives1. To investigate the relationship of miR-590expression and clinicopathological features of HCC and evaluate its prognostic role in HCC.2. To observe the influence of miR-590expression on proliferation, colony formation, cell cycle of HCC cell line.3. To investigate the role of miR-590and its relationship with PDCD4in HCCtumorigenesis.4. To analyze the correlations between miR-590, PDCD4and clinicopathological features of HCC to evaluate its prognostic role in HCC.Methods1. Real-time PCR were performed to compare the expression of miR-590between HCC tissues and their paired adjacent non-cancerous liver tissues, with each pair surgically taken from a same patient, and between normal liver cell line LO-2and6HCC cell lines.2.64frozen, archived HCC tissue (Nan Fang Hospital) were detected by Real-time PCR. SPSS15.0statistical software were used to analysis the correlation between miR-590expression and clinicopathological parameters of HCC.3. MTT, colony formation assays and flow cytometry analysis were used to observe the influence of miR-590expression on proliferation, colony formation, cell cycle of HCC cell line.4. Western blot and luciferase reporter assay were used to detected whether miR-590directly targets PDCD4in HCC cells or not. 5. Transfection and colony formation assays were used to certificated whether PDCD4is necessary for miR-590regulating proliferation in HCC.Results1. miR-590is up-regulated in HCC tissues and cell lines Real-time PCR analyses showed that the level of miR-590was higher in HCC tissues than their paired adjacent non-cancerous liver tissues, and the expression levels of miR-590strongly correlated with the clinical stage of HCC. The level of miR-590expression was higher in HCC cell lines than normal liver cell line LO-2(p<0.05). Furthermore, miR-590high expression correlates with HCC patients overall survival(p<0.01).2. Ectopically expressed miR-590promotes HCC cell proliferation Ectopically expressed miR-590promotes HCC cell proliferation.To determine whether miR-590plays a role in the development and progression of HCC, we examined the effect of miR-590overexpression on HCC cell proliferation. miR-590up-regulation significantly increased the growth rate of MHCC97L HCC cell, as analyzed by MTT and colony formation assays. Furthermore, flow cytometric analysis showed that ectopic expression of miR-590increased the percentage of S-phase cells, thus suggesting that miR-590up-regulation promotes the tumorigenicity of HCC cells in vitro. On the other hand, miR-590inhibition reduced HCC cell proliferation. We examined the effect of miR-590inhibition on HCC cell proliferation. We observed that inhibition of miR-590decreases the growth rate of MHCC97H HCC cells, as determined by MTT and colony formation assays. These data indicate that miR-590inhibition decreases HCC cell proliferation(p<0.05). Furthermore, flow cytometry assays showed that miR-590inhibition significantly decreases the percentage of S-phase cells and increases the percentage of G1/G0-phase cells, thus indicating that miR-590suppression inhibits G1/S transition. Taken together, our results indicate that miR-590down-regulation plays an important role in repressing the development of HCC.3. miR-590directly targets PDCD4in HCC cell Analysis using TargetScan indicates that PDCD4is a potential target of miR-590. As predicted, Western blotting analysis revealed that PDCD4expression decreases in MHCC97L cells transfected with a miR-590mimic and increases in MHCC97H cell transfected with the miR-590inhibitor. To examine whether miR-590-mediated PDCD4down-regulation occurs through a miR-590-binding site located in the PDCD43’-untranslated region (3’UTR), we cloned the PDCD43’UTR into the pGL3dual luciferase reporter vectors. We observed that miR-590overexpression reduced, and miR-590inhibition increased, PDCD43’UTR luciferase reporter activity in a consistent and dose-dependent manner (p<0.05). Collectively, these results demonstrate that PDCD4is a bona fide target of miR-590.4. PDCD4plays an important role in miR-590-induced proliferation Colony formation assay showed that co-transfection of miR-590and PDCD4significantly slowed the colony formation rate of HCC cell line. However, the combination of PDCD4-3’UTR and miR-590in the HCC cell had no obvious effect on the colony formation rate over that of HCC cell co-transfected with miR-590and vector. Furthermore, statistical analysis demonstrated that miR-590expression inversely correlated with PDCD4(r=-0.617, p<0.05)ConclusionOur study provides an important link between miR-590-mediated proliferation of HCC cells and down-regulation of PDCD4, and the overexpression of miR-590correlates with HCC patients overall survival. We report that expression of miR-590was markedly up-regulated in HCC cells and HCC cancer tissues compared with normal liver cells and normal liver tissues. Ectopic expression of miR-590induced the proliferation of HCC cancer cells, while inhibition of miR-590reduced this effect. Furthermore, we demonstrated that miR-590down-regulated PDCD4expression by directly targeting the PDCD43’-untranslated region. Moreover, overexpressed PDCD4can reduce the effect of miR-590and miR-590expression inversely correlated with PDCD4. Taken together, our results suggest that miR-590may play an important role in promoting proliferation of HCC cancer cells and present a novel mechanism of miRNA-mediated direct suppression of PDCD4expression in cancer cells. Our findings suggest an essential role of miR-590in the regulation of HCC cells proliferation. Understanding the precise role played by miR-590in HCC progression will not only increase our knowledge of the biology of the tumor but its inhibition may also allow development of a novel therapeutic strategy.