Hypoxia-related MiR-210-5p And MiR-210-3p Regulate Hypoxia-induced Migration And Epithelial-mesenchymal Transition In Hepatoma Cells

Objective:To investigate the expression of miR-210-5p,miR-210-3p in Hepatocellular carcinoma cells（hepatoma cells,HCC）under normoxia and hypoxia and its effects on migration and epithelial mesenchymal transformation（EMT）.Methods:Hepatoma cells cultured at 37℃,20%O₂,5%CO₂,75%N₂ and suitable humidity in normal cell incubator were set up as normoxia group.Hypoxia was established by two methods:using hypoxic AnaeroPack and CoCl₂treated respectively and these were set up as AnaeroPack group and CoCl₂treated group.The expression of miR-210-5p,miR-210-3p and hypoxia inducible factor（HIF-1α）in HCC cells of normoxic group and hypoxic group was detected by real-time quantitative PCR（qPCR）.The migration in vitro of normoxic group and hypoxic group was detected by Transwell method.The expression of HIF-1αand EMT related molecules such as E-cadherin,N-cadherin and Twist1 in normoxic and hypoxic group were detected by Western Blot.MiR-210-5p inhibitor,miR-210-3p inhibitor and inhibitor NC were transfected into hepatoma cell lines SMMC-7721,Huh-7 and set the final concentration of 100 nM,and they were divided into three groups:miR-210-5p inhibitor group,miR-210-3p inhibitor group and inhibitor NC group（control group）.The effect of miR-210-5p inhibitor,miR-210-3p inhibitor and inhibitor NC on migiration of hepatoma cells was also detected by Transwell assay.Western Blot assay was used to detect the expression of EMT related molecules in miR-210-5p inhibitor group,miR-210-3p inhibitor group and inhibitor NC group,and the effect of miR-210-5p inhibitor and miR-210-3p inhibitor on proliferation of hepatoma cells was detected by CCK-8 assay.Results:（1）The results of qPCR showed that miR-210-5p,miR-210-3p were highly expressed in hepatoma cells in normoxia.MiR-210-5p was highly expressed in SMMC-7721,Huh-7,HepG2,MHCC97H than that in normal hepatocytes（L02）,and miR-210-3p was highly expressed in SMMC-7721,Huh-7 than that in normal hepatocytes（L02）.The relative expression of miR-210-5p in SMMC-7721,Huh-7 was significantly higher than that of miR-210-3p（p<0.05）.（2）Theand CoCl₂ treated group showed better experimental stability than that of the AnaeroPack group,and the levels of HIF-1αin CoCl₂ treated group were significantly increased（p<0.05）.（3）In hypoxia,the relative expression of miR-210-5p and miR-210-3p in SMMC-7721,Huh-7 were up-regulated,and the relative expression of miR-210-5p was still much higher than that of miR-210-3p.（4）The result of Transwell showed that the number of perforating cells of SMMC-7721,Huh-7 in hypoxia group increased significantly,and the migration ability in vitro was significantly enhanced;Western Blot showed that the expression of EMT-related markers N-Cadherin,Slug,Twis1 and MMP2 in hypoxia group was up-regulated,while E-Cadherin was down-regulated（p<0.05）.（5）Inhibition of miR-210-5p and miR-210-3p significantly inhibited the migration in vitro and EMT of SMMC-7721 and Huh-7（p<0.05）.（6）The result of CCK-8 assay showed that hypoxia could promote cell proliferation of SMMC-7721 and Huh-7,but inhibition of miR-210-5p and miR-210-3p had no significant effect on the proliferation of SMMC-7721 and Huh-7（p>0.05）.Conclusion:（1）MiR-210-5p and miR-210-3p were highly expressed in SMMC-7721 and Huh-7 under normoxia,and the relative expression of miR-210-5p was much higher than that of miR-210-3p.（2）Hypoxia could up-regulate the expression of miR-210-5p,miR-210-3p,HIF-1αin SMMC-7721 and Huh-7,and enhance the migration ability in vitro and EMT of SMMC-7721 and Huh-7.（3）MiR-210-5p and miR-210-3p could regulate hypoxia-induced migration in vitro and EMT in SMMC-7721 and Huh-7,but the proliferation was not regulated by miR-210-5p and miR-210-3p.