| Objective Hepatocellular carcinoma is one of the malignant tumors with the highest morbidity and mortality in China,which is resistant to both present chemotherapeutic and targeted drugs,and is prone to relapse and metastasis.Therefore,it is difficult to achieve satisfactory treatment effect.The research of drug therapy in liver cancer is focus on seeking for drugs with specific targets and low toxicity from the natural products,and finding the new drug targets.The Matrine inj ection or Kangai injection,whose main component is matrine,have been used to combined drug therapy of liver cancer with other chemotherapy drugs,and it have achieved a certain effect.But the specific role and mechanism of matrine in hepatocellular carcinoma is still not clear.This study was to investigate the effects of matrine on the proliferation,migration and invasion of hepatoma cells in vitro,and to explore its mechanisms in the regulation of miR-199a-5p/HIF-1?/EMT and Wnt/?-catenin pathwaysMethods 1.The effects of matrine on the proliferation,migration and invasion of human hepatocellular carcinoma HepG2 and Huh7 cells were detected by MTT assay,colony formation and Transwell assay.2.Real-time quantitative PCR was used to measure the expression of miR-199a-5p in human normal hepatocellular cell line L02,HepG2 and Huh7 cells and to detect the effect of matrine on the expression of miR-199a-5p in human hepatocellular carcinoma cells.MiR-199a-5p mimic and miR-199a-5p inhibitor were transfected into hepatocarcinoma cells to investigate whether matrine inhibit the migration and invasion of HCC cells through regulation of miR-199a-5p.4.The target of miR-199a-5p was predicted by three softwares(TargetScan,PicTar and miRanda).The effects of matrine and miR-199a-5p on the migration and invasion of HepG2 and Huh7 cells under hypoxia were investigated by Transwell assay.Western blottings were applied to investigate the effects of matrine and miR-199a-5p on the expression of hypoxia-inducible factor-1?,Snail1,twist,E-cadherin and N-cadherin proteins involved in EMT.5.The effects of matrine and miR-199a-5p on expression of p-AKT,AKT,p-GSK3?,GSK3?,p-?-catenin,?-catenin proteins involved in Wnt/?-cantenin pathway were detected by Western blotting.Result 1.Matrine decreased the cell viability of HepG2 and Huh7 cells in a concentration-dependent manner.The IC50 of matrine is 2369,1565 and 909.1?g/mL when the HepG2 cells was incubated with matrine for 24 h,48 h and 72 h.The IC50 of matrine is 1355,781.8 and 612.8 ?g/mL when the Huh7 cells was incubated with matrine for 24 h,48 h and 72 h.Matrine inhibited the colony formation of HepG2 and Huh7 cells.The inhibitory rate of 400 ?g/mL matrine on the colony formation of HepG2 cells was 49.49±4.68%.The inhibitory rates of 200 ?g/mL matrine on the colony formation of Huh7 cells was 35.80±4.62%.Matrine significantly inhibited the migration and invasion of hepatocellular carcinoma cells.The inhibitory rates of 800 ?g/mL matrine on the migration and invasion of HepG2 cells were 52.12±4.58%and 56.58±3.43%respectively.The inhibitory rates of 800?g/mL matrine on the migration and invasion of Huh7 cells were 55.051±6.50%and 60.29±5.84%respectively.2.MiR-199a-5p was lowly and differentially expressed in L02,HepG2 and Huh7 cells.The expression of miR-199a-5p in hepatocellular carcinoma cells HepG2 and Huh7 were significantly up-regulated by matrine.After transfection of miR-199a-5p mimic,the proliferation of HepG2 and Huh7 cells was not affected significantly,but the migration and invasion of HepG2 was inhibited.The inhibion rate is 30.55±1.67%and 30.01±3.29%.MiR-199a-5p mimic had a mild promoting effect on the migration and invasion of Huh7 cells,but the effect was not significant.MiR-199a-5p inhibitor could promote the migration and invasion of HepG2 cells slightly compared with the control group,though the effect was not statistically different.MiR-199a-5p have no effect on the migration and invasion of Huh7 cells.Down-regulated expression of miR-199a-5p in HepG2 cells could weaken the inhibition of matrine on migration and invasion.3.All of the three database software(Targets can,PicTar and miRanda)predicted that there was a conserved site in the 3'UTR region of HIF-1? mRNA could bind to the miR-199a-5p seed sequence.4.Matrine inhibited the migration and invasion of HepG2 and Huh7 cells under hypoxia.The inhibitory rates of 800?g/mL matrine on the migration and invasion of HepG2 cells were 63.28±5.04%and 57.82±4.75%respectively.The inhibitory rates of 800 ?g/mL matrine on the migration and invasion of Huh7 cells were 79.65±1.29%and 86.43±0.51%respectively.MiR-199a-5p inhibited the migration and invasion of HepG2 cells under hypoxia,and the inhibition rates were 42.07±3.22%and 37.36±5.625%respectively.MiR-199a-5p promoted the migration of Huh7 cells,but the promotion showed no statistical significance.5.In HepG2 cells,matrine and miR-199a-5p inhibited the expression of HIF-1?,Twist,Snaill and N-cadherin,but promoted the expression of E-cadherin.In Huh7 cells,matrine inhibited the expression of HIF-1?,N-cadherin,Snail1,?-catenin.It could also inhibit the phosphorylation of AKT,GSK3? and ?-catenin,and promoted the expression of E-cadherin and p-?-catenin.MiR-199a-5p had no significant effect on the expression of HIF-1?,N-cadherin and Snail1 in Huh7 cells,but significantly inhibited the expression of E-cadherin and increased the expression of twist.Conclusion 1.Matrine can inhibit the proliferation,colony formation,migration and invasion of hepatocellular carcinoma cells HepG2 and Huh7 cells.The cytotoxicity of matrine occurs at higher concentrations,while inhibitioin of migration and invasion are significant at lower concentrations.2.Matrine can up-regulate the expression of miR-199a-5p in HepG2 and Huh7 cells.The overexpression of miR-199a-5p inhibits the migration and invasion of HepG2 cells,but causes no effect on Huh7 cells.The inhibitory effect of matrine on migration and invasion was impaired after down-regulating of miR-199a-5p expression in HepG2 cells.MiR-199a-5p has no significant effect on the proliferation of hepatoma cells.In conclusion,matrine supresses the migration and invasion of HepG2 cells by increasing the expression of miR-199a-5p.3.The inhibitory effect of matrine on the migration and invasion of HepG2 and Huh7 cells,and supressing effect of miR-199a-5p on the migration and invasion of HepG2 cells under hypoxia was stronger than that under normoxic Matrine may inhibit EMT through miR-199a-5p/HIF-1?/Twist/Snail1 pathway,and thus inhibit migration and invasion of HepG2 cells.In Huh7 cells,matrine inhibits EMT through AKT/HIF-la and AKT/GSK3?/?-catenin pathway,thereby inhibiting cell migration and invasion. |
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