| Primary liver cancer is one of the most common malignant tumors in China.It mainly includes cholangiocarcinoma and hepatocellular carcinoma(HCC),and the incidence of HCC in primary liver cancer is about 90%.HCC is the most common invasive human malignant tumor in the world.It is characterized by high malignancy,low early diagnosis rate,poor prognosis and high recurrence rate.Although significant progress has been made in the study of the occurrence and development of HCC,but the precise mechanism of HCC is still unclear.In the past few years,the 5 year survival rate of HCC has changed little.Therefore,early diagnosis and treatment of hepatocellular carcinoma is particularly important.MicroRNA is a tiny RNA,consisting of 20 to 22 nucleotides,which is combined with the mRNA of target gene through base complementary pairing.It inhibits the translation of target genes into corresponding proteins or leads to their degradation,thus regulating the expression of genes after transcription.MicroRNA-320a(miR-320a)is a member of the miR-320 family,it has been found in a variety of tumors.In addition to playing the basic suppressor in the tumor,miR-320 a is also involved in the basic process of tumor chemoradiotherapy and drug therapy,but the effect of miR-320 a on hepatocellular carcinoma and the main mechanism needs further study.Ribonucleic acid II is a drug that can enhance the immune function of human body,and the drug itself is a ribonucleic acid and has an antitumor effect.In previous studies,a gene chip has been found that the drug contained a variety of tiny RNA including miR-320 a.Therefore,we envisage whether the antitumor effect of Ribonucleic Acid II is associated with miR-320 a,and whether the changes of mi R-320 a expression level in the hepatoma cells may affect the antitumor effect of Ribonucleic Acid II.Objective: The first part of this study is aims to investigate the expression level of miR-320 a in human HCC cell lines and surgically resected human hepatocellular carcinoma tissues,and to study the effect of miR-320 a in HCC cell lines;The second part of the study is to explore the possible mechanism of miR-320 a on hepatocellular carcinoma;The third part of the study is to investigate the effect of mi R-320 a on the apoptosis and migration of Bel-7402 cells induced by ribonucleic acid II.It will provide a theoretical basis for the clinical treatment of hepatocellular carcinoma and the study of drug targets.Methods: The first part: normal human liver cell line HL-7702 and hepatocellular carcinoma cell lines(Bel-7402,SMMC-7721,Hep3 B and HepG2)were cultured in vitro,and collected human hepatocellular carcinoma tissues and corresponding noncancerous tissues which were used Reverse Transcription-Polymerase Chain Reaction(RT-PCR)to detect the expression level of mi R-320 a.The miR-320 a mimics were transfected into SMMC-7721 and Bel-7402 cells,then the miR-320 a expression levels were measured by RT-PCR after 48 h.Flow cytometry(FCM)was used to detect the effect of miR-320 a on the cell apoptosis and cell cycle;Scratch Test and transwell were used to detect the effect of miR-320 a on cell migration and invasion;The second part: target genes of mi R-320 a were analyzed by Targetscan,microRNA.org and mi RBase;then the luciferase reporter assay was performed by chemical synthesis and plasmid construction;RT-PCR and Western-blot were used to detected the expression level of ?-catenin in HCC cells;the expression of cell cycle related protein CDK4 and Cyclin D1,apoptotic related proteins MDM2,Bax,Bcl-2,p53 and caspase3,migration related protein matrix metalloproteinase MMP3,MMP9 were detected by Western-blot.The third part: the expression level of miR-320 a in Bel-7402 and Bel-7402+Ribonucleic cells were detected by RT-PCR;the effect of ribonucleic acid II on proliferation of Bel-7402 and Bel-7402-miR-320 a cells was measured by CCK-8 assay;FCM was used to detect cell cycle and apoptosis;The migration and invasion ability of ribonucleic acid II induced Bel-7402 cells was tested by Transwell method.The expression of p53,Cyclin D1,Bax,Bcl-2,MMP-3,?-catenin proteins were examined by Western-blot.Results: The first part1.miR-320 a expression levels in HCC cell lines and HCC tissue were significantly lower than that in normal cell line and adjacent non-tumor tissue;2.Flow cytometric analysis indicated that cell cycle arrest in G0/G1 phase and cell apoptosis rate increased significantly in overexpression miR-320 a group compared with the control group;3.Scratch test results showed that: in the negative control(NC)group,compared with 0 h,the scratch distance narrows after 24 h and 48 h,suggesting that cells had strong migration ability;and in the overexpression miR-320 a group,the scratch distance changed little after 24 h and 48 h compared with 0h.These results suggested that overexpression of miR-320 a could inhibit cell migration;4.Transwell results further showed that over expression of mi R-320 a could significantly reduce the migration and invasion ability compared with the control group.The second part1.Bioinformatics analysis and luciferase report assay were determine that CTNNB1 is the target genes of miR-320a;2.RT-PCR results showed that the expression of ?-catenin was up regulated in four hepatocellular carcinoma cell lines compared with normal hepatocyte line HL-7702,while overexpression miR-320 a in SMMC-7721 and Bel-7402 cells could reduce the expression of ?-catenin;3.Western-blot results showed that overexpression of miR-320 a could significantly inhibit the expression of ?-catenin protein(CTNNB1);4.The results of Western-blot suggested that over expression of miR-320 a could make the expression of CDK4,CyclinD1,MDM2,Bcl-2,MMP3 and MMP9 protein significantly decreased,but the expression of p53,Bax and Caspase3 obviously increased.The third part1.RT-PCR showed that the expression of miR-320 a in HCC cell could be increased by ribonucleic acid II;2.CCK8 results showed that ribonucleic acid II could inhibite the growth of Bel-7402 and Bel-7402-miR-320 a cells in a dose and time dependent manner;especially miR-320 a overexpression,the inhibition effect is more obvious;3.Flow cytometric analysis indicated that ribonucleic acid II could cause cell cycle arrest in G0/G1 and promoted HCC cell apoptosis,while miR-320 a overexpression could inhance the effects;4.Transwell results showed that compared with the Control group,the migration and invasion ability of cells induced by Ribonucleic Acid II were weakened,especially in the overexpressed miR-320 a group,the migration and invasion ability of the cells weakened more significantly;5.Western blot analysis further suggested that the expression of ?-catenin protein in ribonucleic acid II group was changed,and the expression level of apoptotic,cell cycle-associated and migration related proteins were also changed,especially in the group of overexpressed miR-320 a.Conclusion1.MiR-320 a is down-regulated in HCC cell lines and tissues;overexpression of miR-320 a can arrest cell cycle in G0/G1 phase,promote apoptosis and inhibit cell migration and invasion in HCC;2.The luciferase assay showed that ?-catenin was the target gene of miR-320 a,and the expression of ?-catenin in hepatocellular carcinoma cells was upregulation,mi R-320 a overexpression could significantly downregulate the expression of ?-catenin in HCC cells.The biological effect of miR-320 a on hepatocellular carcinoma maybe achieved by inhibiting the expression of ?-catenin;3.MiR-320 a overexpression could increase the sensitivity of Bel-7402 to ribonucleic acid II and enhance the pharmacological effects of ribonucleic acid II on HCC cells. |
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