The Expression And Function Of MicroRNA-340 In Hepatocellular Carcinoma

Background:MicroRNAs are short RNAs that play a crucial role in origination and progression of hepatocellular carcinoma(HCC).MiR-340 is identified as a novel tumor suppressor recent years,such as colorectal cancer,ovarian cancer and breast cancer.However,the association between miR-340 and hepatocellular carcinoma is not clear.Methods:In the present study,the expression of miR-340 in four HCC cell lines(HepG2,Hep3 B,Bel-7402,SMMC-7721),one normal hepatocyte(HL-7702)and clinically resected human HCC tissues ensured by pathology was evaluated by qRT-PCR,according to Chi-square test,we analysed the association of the expression of miR-340 with HCC patients' age,gender,HBs Ag,HBV DNA,AFP,ALT,AST,tumor size,cirrhosis and TNM staging.Using the transfection to increase or decrease the expression of miR-340 in SMMC-7721,and than exploring the proliferation and apoptosis of miR-340 in SMMC-7721 by CCK-8 and Flow Cytometry.Further,the roles of miR-340 in SMMC-7721 migration and invasion was detected by Transwell invasion assay.Finally,the underlying mechanism of miR-340 for HCC development was investigated by qRT-PCR and Western Blot.Result:qRT-PCR revealed that the expression of mi R-340 is lower in four HCC cell lines(Hep3B,Bel-7402,HepG2,SMMC-7721)than one normal hepatocyte(HL-7702),the decreased expression level of miR-340 compared with normal hepatocyte has the statistical significance(p value apartlly : p<0.05,p<0.001,p<0.001,p<0.001).The expression of miR-340 in SMMC-7721 cell lines is the lowest so that we select SMMC-7721 as the target for the next experiment.The miR-340 level in tumor tissues is lower than adjacent non-tumor tissues in all 45 pairs samples(p<0.05).Relying on statistical analysis,the miR-340 level in tumor tissues is lower than adjacent non-tumor tissues in all 45 pairs samples,and the difference was statistically significant(p < 0.001).The expression of miR-340 correlates with the HBs Ag(p=0.01),HBV DNA(p=0.002),tumor size(p=0.001)and the TNM staging of HCC(p=0.002),while unrelated with age(p=0.751),gender(p=0.179),AFP(p=0.079),ALT(p=0.6),AST(p=1.0),cirrhosis(p=0.053).CCK-8 test showes that after increased the expression of miR-340,the proliferation ratio of cells in miR-340 mimic group is lower than miR-340 mimic control group at 24 hours,48 hours,72 hours,while after decreased the expression of miR-340,the proliferation ratio of cells in miR-340 inhibitor group is higher than miR-340 inhibitor control group at 24 hours,48 hours,72 hours.Flow Cytometry indicated that after increased the expression of miR-340,the apoptosis ratio of cells in miR-340 mimic group is higher than miR-340 mimic control group(p<0.01),while after decreased the expression of miR-340,the apoptosis ratio of cells in miR-340 inhibitor group is lower than miR-340 inhibitor control group(p<0.001).Transwell invasion assay manifested that after increased the expression of miR-340,the migration and invision cells in miR-340 mimic group is lower than miR-340 mimic control group(p<0.001),while after decreased the expression of miR-340,the migration and invision cells in miR-340 inhibitor group is higher than miR-340 inhibitor control group(p<0.001).Finally,qRT-PCR and Western Blot make clear that after increased the expression of miR-340,the expression of SKP2 mRNA and protein in miR-340 mimic group is lower than miR-340 mimic control group(p<0.05),while after decreased the expression of mi R-340,the expression of SKP2 mRNA and protein in miR-340 inhibitor group is higher than miR-340 inhibitor control group(p<0.05).Conclusion:In the present study,we detected that compared with normal hepatocyte(HL-7702)and HCC patients' adjacent non-tumor tissues,miR-340 was significantly decreased in four human HCC cell lines(Hep3B,Bel-7402,HepG2,SMMC-7721)and HCC cancer tissues.The expression of miR-340 correlated with HCC patients' HBs Ag,HBV DNA,tumor size(?5 cm)and TNM staging(?).We elaborated that miR-340 could suppress the proliferation,migration,invasion and induce apoptosis in SMMC-7721 cell lines.In other words,miR-340 may play the significant antitumor functions in HCC development.The potential mechanism may be putted down to the suppression effect of miR-340 for SKP2 inducing the aforesaid cell biological behaviour changed.These results suggest that miR-340 takes part in the HCC develepement as a potential tumor suppressor gene.Further,the miR-340 level may relate with the HBV infection,we may predict tumor progression or prognosis by the expression of miR-340 after further experiment.