Mir-153Inhibits Epithelial-mensenchymal Transition In Hepatocellular Carcinoma By Targeting SNAI1

Part1Low Expression of MiR-153in HCC Tissue Cells and Plasmid’s Transfected and Cell model’s IdentificationHepatocellular carcinoma (HCC) is one of the most common and the worst prognosis of cancer in China. According to the statistics, HCC is the fifth most common malignant carcinoma and the third most common cause of cancer-related deaths in the world. The incidence of HCC is increasing, about600,000new cases each year. Unfortunately, cause the symptom of HCC in the early stage is not obvious, lots of HCC patients diagnosed in advanced stages. although the treatment strategies develop rapidly, including surgery, radiotherapy, chemotherapy and photodynamic therapy, the5-years survival rate for HCC patients is still very low, with only15%to30%of advanced HCC patients survived after surgical operation. What’s more, the HCC seen extremely vulnerable to invasion the capsule and blood vessels, leading to local spread and distant metastases. Autopsy report shows that the extrahepatic cancer metastasis of HCC as high as40%to71.6%. Thus, it is necessary to study the mechanism in HCC to understand its development and find new prognostic marker for HCC diagnosis and theraptic target for clinical treatments.MicroRNA-153(miR-153) was found to play important roles in the occurrence and development of various cancers, and was considered as an oncogene or tumor suppressor gene. Growing number of experimental studies have shown miR-153regulates tumor occurrence and development, however, the mechanisms between miR-153and its regulated gene is unclear. Purposes:To investigate miR-153expression in HCC, we used real-time PCR assay to analyze miR-153expression in human normal liver cells and8HCC cell lines and14pairs of HCC patients and adjacent non-cancerous tissues(ANT).Construct miR-153cell models of overexpression and low expression.Results:Real-time PCR analyses observed that miR-153was down-regulated in14HCC samples paired with adjacent non-cancerous tissues from the same patient. Furthermore, comparative analysis revealed that miR-153was down-regulated in all8examined HCC cancer cell lines, as compared with that in normal liver cell line. Down-regulation of miR-153was identified in165patients from the microarray data with accession number GSE31384. These results indicated that miR-153is down-regulated in HCC. After transfection of miR-153mimic to hepatoma Huh7and HepG2cell lines, miR-153in lab group is150times expression of negative control group. After transfection of miR-153inhibitor to HepG2and Huh, compared with the negative control group. the expression of the miR-153in the lab group is only20%.Conclusion:Compared with normal human liver tissues and normal liver cells (NLC), miR-153was significantly downregulated in HCC tissues and cell lines. By transfection, miR-153cell models of overexpression and low expression.are established. Part2The Relationship between MiR-153and Metastasis and Invasion Ability of Hepatoma CellsEpithelial-to-mesenchymal transition (EMT) is a process by which epithelial cells lose their cell polarity and adhesion, and gain migratory and invasive properties to become mesenchymal cells, it has been implicated in dynamic cellular process in embryonic development and invasion of human cancers.Purposes:To investigate the biological role of miR-153expression in the EMT phenotype and progression of HCC, we next verified the protein expression of E-cadherin, N-cadherin, vimentin and a-catenin in HCC cells Huh-7and HepG2with hsa-miR-153mimic oligonucleotides and miR-153inhibitor. Further, wound healing, matrigel-coated Boyden chamber invasion assays and3D culture assays were used to inspect the correlation between invasion and miR-153in HCC.Results:Western blot showed that the expression of epithelial makers E-cadherin and-Catenin was significantly upregulated in miR-153overexpressed cell, while they were downregulated after miR-153was inhibited. Conversely, ectopic expression of miR-153evidently decreased the expression of mesenchymal markers including N-cadherin, Vimentin and Fibronectin, while inhibition of miR-153obviously upregulated these markers. Upregulation of miR-153decreased invasion of liver cancer cells, while inhibiting miR-153enhanced the ability of cell invasion.Conclusion:These results suggested that upregulation of miR-153could inhibit the migration, invasion and EMT phenotys of HCC cells in vitro, but inhibition of miR-153show the opposite. Part3MiR-153Directly Targeted to SNAI13’UTR to Decrease it’s ExpressionSnail1is a critical convergence hub in EMT regulation which transcriptionally represses E-cadherin expression. Currently, upregulation of Snail is mainly due to dually regulation by protein stability and cellular location, and partially by transcriptionally activation. However, whether alternative regulatory mechanism exists remains unclear.Purposes:To study the mechanism of miR-153on regulating cell invasion. SNAI1was a theoretical target according to targetscan software; we further validated that miR-153inhibited the expression of SNAI1by western blotting and luciferase assay; we explored the effect of inhibiting miR-153on SNAI1by western blotting and luciferase assay; we analyze the relevance of miR-153and SNAI1with clinical samples.Results:Analysis using three publicly available microRNA target predictors (TargetScan, Pictar, miRANDA) revealed a highly conserved region in the3’-UTR of SNAI1mRNA that matches to the seed region of miR-153. Western blotting analysis confirmed that ectopic expression of miR-153in HepG2and Huh7cells decreased the expression of SNAI1protein level, while inhibition of miR-153in these cells increased the Snail protein expression. Ectopic expression of miR-153in HepG2and Huh7cells dramatically inhibited the GFP protein expression, but not the expression of GFP--tubulin that was used as a control for transfection efficiency. Further more, a consistent and dose-dependent reduction in luciferase activity upon miR-153transfection was observed in HepG2and Huh7cells, which could be reversed by transfection with the miR-153inhibitor. Point mutations in the tentative miR-153-binding seed region abrogated the aforementioned repressive effect of miR-153. We examined the expression levels of miR-153and Snail in ten cases of freshly prepared human HCC biopsies. The result showed that SNAI1protein level was relatively lower in tissues displayed higher miR-153expression.Conclusion:In liver cancer cells, miR-153directly targeted to SNAI13’UTR to decrease its expression,while inhibiting miR-153increased. SNAI1played an important role in miR-153suppressed invasion.