Roles Of MiR-214-3p In Hepatocellular Carcinoma And Its Underlying Mechanisms

Objectives: Hepatocellular caicinoma?HCC?is one of the most common malignant tumors in the world and more than half of the new and dead cases happened in China.More and more studies have found that micro RNAs expressed in primary hepatocellular carcinoma are associated with multiple processes in development of hepatocellular carcinoma such as proliferation,metastasis,angiogenesis,epithelial-mesenchymal transition,and so on.Therefore,the purpose of this study is to find out the micro RNAs that are differentially expressed in primary hepatocellular carcinoma in a large number of micro RNAs and to study their effects on the important biological functions such as proliferation,migration and invasion in the development of primary hepatocellular carcinoma and then to research the regulation mechanism in the development and progression of primary hepatocellular carcinoma by predicting and comfirming the specific target genes of the micro RNAs.Method: 1.The micro RNA chip data published in the National Center for Biotechnology Information?NCBI?GEO database?https://www.ncbi.nlm.nih.gov/geo/?was analyzed using a variety of methods.Micro RNAs were screened out and the target micro RNAs further studied were identified by reviewing the literature and analyzing the micro RNA chip data.2.Collected samples of primary hepatocellular carcinoma and adjacent normal tissues,six hepatocellular carcinoma cell lines ——BEL-7402,SMMC-7721,SK-HEP-1,Hu H-7,Hep G2,Li7,and one normal liver cell line L02 were used in real-time quantitative PCR experiments to verify whether mi R-214-3p is indeed down-regulated in hepatocellular carcinoma tissues and cell lines 3.The relationship between mi R-214-3p down-foldchange and the parameters of primary hepatocellular carcinoma patients was analyzed.4.Mi R-214-3p mimics ware transfected into BEL-7402,SMMC-7721 and SK-HEP-1,and the levels of mi R-214-3p were upregulated.Then MTS proliferation assay,transwell migration assay and transwell invasion assay were used to detect the effect of mi R-214-3p on proliferation,migration,invasion and other important biological functions in the development and progression of primary hepatocellular carcinoma.5.SK-HEP-1 cells co-incubated with mi R-214-3p agomir were injected into nude mice by tail vein,and IVIS Spectrum in vivo imaging system was used to observe the nude mice.Then the nude mice were executed and the lungs of the nude mice were removed and IVIS Spectrum in vivo imaging system was used to observe the lungs of the nude mice.HE pathological staining on the lungs of the nude mice was used to observe and analyzed the metastasis of SK-HEP-1 cells.6.Two target gene online prediction program Target Scan?http://www.targetscan.org /vert₇1/?and mi Randa?http://www.microrna.org/microrna/home.do?were used to predict the direct target gene of mi R-214-3p.7.Fluorescence in situ hybridization and immunofluorescence experiments were used to initially verifiy whether HMGA1 is the direct target gene of mi R-214-3p,and then the dual luciferase reporter system was used to determine whether HMGA1 is the direct target gene of mi R-214-3p.8.The HMGA1 overexpression vector was constructed and co-transfected into BEL-7402,SMMC-7721 and SK-HEP-1 with mi R-214-3p mimics,and the MTS proliferation assay,transwell migration assay and transwell invasion assay were used to detect the specific mechanism of mi R-214-3p in the development and progression of primary.Result: 1.By analyzing the chip data GSE36915 we found that compared with non-tumor liver tissue,the expression of 49 micro RNAs in primary hepatocellular carcinoma tissue was significantly reduced,of which,the expression of mi R-214-3p?199a-5p?199a-3p cluster was very similar.The expression of mi R-214-3p was the most downregulated micro RNA in the three micro RNAs in mi R-214-3p / 199a-5p / 199a-3p cluster.And compared with non-tumor liver tissue,the expression of mi R-214-3p was down-regulated in 85%?58/68?hepatocellular carcinoma tissues.2.The expression of mi R-214-3p in hepatocellular carcinoma was significantly lower than that in adjacent normal tissues.The expression of mi R-214-3p in 6 human hepatocellular carcinoma cells was significantly lower than that in L02.3.Mi R-214-3p expression down-regulation was correlated with TNM stage?P = 0.0002?and distant metastasis?P = 0.0046?,but not significantly correlated with age,sex,AFP,tumor size and cirrhosis.4.Compared with mi R-control group,the proliferation of BEL-7402,SMMC-7721 and SK-HEP-1 cells was significantly inhibited after 48 hours from transfection with mi R-214-3p mimics.Compared with mi R-control group,the number of BEL-7402,SMMC-7721 and SK-HEP-1 cells migrated was significantly decreased after transfection with mi R-214-3p mimics.Compared with mi R-control group,the number of BEL-7402,SMMC-7721 and SK-HEP-1 cells ied was significantly decreased after transfection with mi R-214-3p mimics.5.The nude mice imaging fluorescence signal of mi R-214-3p agomir group was significantly lower than that of mi R-mi R-214-3p agomir group?P<0.01?.The nude mice lung imaging fluorescence signal of mi Rcontrol agomir group was significantly lower than that of mi Rcontrol agomir group?P<0.05?.The number of lung metastases of mi R-214-3p agomir group was significantly lower than that of mi Rcontrol agomir group?P<0.05?.6.HMGA1 is the predicted as the target gene of mi R-214-3p in two online prediction program.7.The result of fluorescence in situ hybridization and immunofluorescence assay showed that the expression level of mi R-214-3p in adjacent normal tissue was higher than that in HCC tissues,while the expression level of HMGA1 in adjacent normal tissues was lower than that in HCC.Mi R-214-3p mimics could significantly inhibited the activity of the enzyme of the HMGA1 3'-UTR wild type reporter vector group?P<0.01?,the activity of the enzyme in HMGA1 3'-UTR mutant reporter vector group was not significantly inhibited.8.HMGA1 expression could restore the proliferation,migration and invasion of BEL-7402,SMMC-7721 and SK-HEP-1 cells overexpressing mi R-214-3p.Conclusion: The expression level of mi R-214-3p?199a-5p?199a-3p cluster in hepatocellular carcinoma was down-regulated,and the expression level of mi R-214-3p was down-regulated.The proliferation,migration and invasion of BEL-7402,SMMC-7721 and SK-HEP-1 cells in vitro could be inhibited by upregalation of the mi R-214-3p expression level.The metastasis and growth of SK-HEP-1 cells in vivo could also be inhibited by mi R-214-3p.And the inhibition of the proliferation,migration and invasion of BEL-7402,SMMC-7721 and SK-HEP-1 cells by mi R-214-3p was through downregulating the expression of HMGA1.