| Background and AimsEpithelial-Mesenchymal Transition (EMT) is phenomenon that epithelial cells transform into mesenchymal cell in both morphology and phenotype under physiological and (or) pathological conditions. This process involves profound phenotypic changes that include the loss of cell-cell adhesion, the loss of cell polarity, the changes in cytoskeleton, the changes in extracellular matrix and the acquisition of migratory and invasive properties. EMT widely presents in various stages of development in the organism, and is a crucial part in embryonic differentiation and maturation, tissue and organ repair and tumor metastasis.In mature liver, EMT plays variety of roles. Under variety of adverse stimuli or injury, hepatocyte appears with damage even death at the time. EMT is involved in the progress of tissue repair, by promoting regeneration of the damaged hepatocye or cholangiocyte and shifting epithelial cells into mesenchymal cells that migrate to fix the damage part. But when the stimulus persists or the extent of damage exceeds repair capacity, EMT will exacerbate liver fibrosis, and eventually lead to cirrhosis. In hepatocellular carcinoma (HCC), malignant cells that undergo EMT can change the constitution and get rid of intercellular connections, then invade into the neighboring tissue. Carcinoma shows an emergence of aggressive, more metastasis and proliferation. In patients this process ultimately lead to liver metastasis, tumor recurrence and a poor prognosis.HCC is one of common malignant tumors in China, rank sixth in the incidence of tumors, third mortality. Although surgical techniques and research of medical improved in the last years, the problem of the poor long-term prognosis has not been fundamentally changed. The intrahepatic metastasis and the high recurrence rate after treatment are still the factors that deteriorate the long-term prognosis of HCC patients. Even in the patients with tumor size< 5cm, liver metastases is still nearly 60% and the 1 year survival rate after surgery is less than 40%. Researches for effective targets and specific markers have become one of the key points that improve treetment efficacy.EMT is a complex biological process that tumor microenvironment, growth factors, transcription factors, microRNA (miRNA) are involved in. Transforming growth factor-β (TGF-β), epidermal growth factor (EGF), fibroblast growth factors (FGF), vascular endothelial growth factor (VEGF) and other growth factors, signaling pathways interact to induce or activate EMT by associating conversion factors, such as Snail 1, Snail2, Twist, ZEB1, ZEB2 and so on. At last, attributed to the downregulation of E-cadherin and upregulation of N-calcium, epithelial cells lose phenotype and show mesenchymal characteristics. Zinc transporter LIV-1 which located in the cell membrane regulate intracellular Zn2+concentration, also function as a signal transporter participating in a variety of signal pathway. In the EMT process, LIV-1 increase the nuclear translocation of Snaill, which can further activate ZEB2 (Zinc finger E-box binding protein 2) and inhibit E-cadherin expression and finally promote EMT.miRNAs are small endogenous non-coding RNA molecules that function as gene regulatory by specifically identifying and combining the 3’-uncoding region of the target mRNA. miRNA ultimately degrades the target mRNA or repress the translation of target mRNA, reduce protein levels. In recent years, studies on the regulation of miRNA confirm its participation in various liver diseases especially in HCC. In the process of EMT, miRNA plays an important role.miRNA-192, a class of miRNA that abundant in liver, was found to be associated with the activation of p53 and involved in cell cycle regulation. Research has shown that in the kidney, miRNA-192 and EMT related fibrotic process. From biological database, we found that miRNA-192-5p may combine to mRNA 3’UTR region of LIV-land ZEB2. Therefore miRNA-192-5p possibly regulate through these two factors in the process of EMT in HCC. This study was carried out by the hypothesis that miRNA-192 may play a regulatory role in HCC, and reveal the molecular mechanisms of EMT.Methods1. Detect the target gene transcription level in normal cell line and liver cancer cell lines by RT-PCR.2. Analysis with Target Scan, microrna database, forecast the possible miRNA that may combine 3’UTR of the target gene mRNA sequence.3. Transfect hepatocellular carcinoma cell lines with miRNA-192-5p mimics and inhibitor.4. Detect mRNA of target genes in cell lines by RT-PCR after transfection.5. Detect the expression levels of the target proteinby western blot.6. We detect the level of downstream effective proteins in EMT that associated with the target genes by western blot.7. Test the function of migration and invasion of transfected cells by transwell assay.8. Hepatocellular carcinoma tumor xenografts were established by subcutaneous injection of tumor cells into the right axilla of BALB/C mice. After the model successfully constructed, local intratumoral injection was carried out once every three days with miRNA agomir for two weeks. Observe survival and tumor growth in mice regularly.9. By the endpoint, collect tumor mass samples, HE staining, immunohistochemical observateof tumor cell morphology and expression of target proteins.10. We Co-transfected 293T cell with miRNA-192-5p and luciferase plasmid vector. Then we detect the combination of miRNA-192-5p with 3’UTR of target mRNA by dual luciferase reporter assay.results1. Bioinformatic analysis shows that, miRNA-192-5p have possible binding sites with sequences of 3’UTR of LIV-land ZEB2 mRNA, suggesting that miRNA-192-5p may be involved in the regulation of LIV-1, ZEB2 transcription.2. LIV-land ZEB2 mRNA level is higher in the carcinoma cell lines than in normal liver cell line. That is, these two genes are highly express in HCC.3. miRNA-192-5p is an important molecules that can regulate EMT by repressing LIV-1 and ZEB2. 3.1 Transfected with exogenous miRNA-192-5p mimics, LIV-1 and ZEB2 mRNA levels showed to be downregulated. 3.2 Transfected with exogenous miRNA-192-5p inhibitor, LIV-1 and ZEB2 mRNA levels increase. 3.3 After transfecting of miRNA-192-5p mimics and inhibitor, LIV-1 and ZEB2 proteins changed in the same trend compared with mRNA levels.4. Transfected with miRNA-192-5p mimics, E-cadherin,the key molecule in EMT, was increased.5. With transfection of miRNA-192-5p mimics, capacity of migration and invasion diminished in carcinoma cell lines Hep3B and MHCC97H.6. Tumor xenografts of HCC were established successfully. Injected with miRNA agomir, the growth of carcinoma xenografts in nude mice slowed down in both diameters and volume but showed no significant different between two groups (P>0.05).7. After 2 weeks’miRNA agomir locally injection, HE staining showed necrotic area in the tumor.8. Immunohistochemical staining showed that, compared with the control group, LIV-1, ZEB2 were inhibited, E-cad, Ki67 were increased.9. miRNA-192-5p can specifically bind to the 3’UTR region of ZEB2 mRNA, but not LIV-1.conclusionIn this study, based on the results of bioinformatics analysis, we explored the possible regulate mechanisms of EMT in HCC. and the role of miRNA-192-5p, an important molecule in EMT, and intended to validate by in vivo and in vitro experiments.The following conclusions:1. In the hepatocellular carcinoma cell lines, both LIV-1 and ZEB2, the important molecular in EMT increases. That is, in carcinoma cell lines EMT tends to occour.2. In vivo and in vitro, miRNA-192-5p can repress LIV-1 and ZEB2, leads to the upregulation of E-cadherin.3. Targeting the 3’UTR of ZEB2 mRNA, miRNA-192-5p inhibits ZEB2 level, while reducing the LIV-1 level indirectly.4. miRNA-192-5p regulate EMT in HCC through LIV-1-ZEB2 pathway.5. miRNA-192-5p may also affect cell cycle in hepatocellular carcinoma xenografts.In hepatocellular carcinoma, EMT leads to tumor metastasis, affecting the prognosis of HCC patients. MiRNA-192-5p, as a regulatory factor of LIV-1 pathway, a key signaling in EMT process, can suppresses the expression of both LIV-1 and ZEB2 and protect intercellular adhesion proteins, reduce tumor cell invasion and migration. |
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