MicroRNA200a Inhibits TGF-β Induced Liver Fibrosis Induced By Schistosomiasis

[Objective]To investigate the effect of microRNA200a(miR-200a)on the activation and proliferation of hepatic stellate cells(LX-2)induced by TGF-β,and to explore the mechanism of miR-200a inhibiting TGF-βto improve liver fibrosis of schistosomiasis.[Methods]The cell experiment was performed with LX-2,and the cells were cultured to logarithmic growth phase.TGF-β1 with different concentrations(0,5,10,15ng/mL)was used at different time points(0,24,48,72hours).LX-2 was stimulated,total RNA and protein were extracted by kit,and mRNA of miR-200a,α-SMA,Colla I and TGF-β2 in LX-2 were detected by fluorescent quantitative PCR;LX-2 was detected by western-blot method.Protein expression of TGF-β2 in the medium;cell proliferation rate was measured by MTT assay.Subsequent experiments were carried out using the TGF-β1 optimal stimulation concentration(5ng/mL)and the optimal stimulation time(48hours),which was obtained in the above experiment.The miR-200a mimics-related plasmids were constructed and transfected into LX-2 cells by transient transfection technique.The cell experiments were divided into:PBS group,TGF-β1 group,miR-200a mimics group,miR-200a mimicsNC group,miR-200a inhibitor group and miR-200a inhibitor NC group Six of group,were cultured for 6 hours.Except PBS group,other groups were added with TGF-β1 for 48 hours,and the total RNA and protein were extracted by kit.The mRNA of miR-200a,α-SMA,Colla I and TGF-β2 in LX-2was detected by real-time PCR.The protein expression of TGF-β2 in LX-2 was detected by western-blot method.The cell proliferation rate was detected by MTT assay.In the animal experiment,64 Balb/c female mice with 6-8 weeks old were randomly divided into four groups,there were 16 mice in each group:the normal control group was uninfected with schistosomiasis,and the other three groups were all passed through the tail sac.The infection was infected with schistosomiasis(16±2scorpions per mouse)and the infected group.They were:PBS group(schistosomiasis model group);Lenti-NC group(lentiviral negative control group);Lenti-miR-200a group(lentiviral experimental group).Each group of mice was injected with 60μL of normal saline,PBS,Lenti-NC(1×10~9TU/mL)and Lenti-miR-200a(1×10~9TU/mL)through the tail vein,and after infection the mice were routinely fed.At four time points(4w,6w,8w and 10w),4 mice in each group were randomly anesthetized and sacrificed by cervical vertebrae.Blood was taken from the eyeball and liver tissue was removed.The mRNA expression levels of miR-200a,α-SMA,Colla I and TGF-β2 in liver tissues of different groups were detected by real-time PCR.The serum ALT level was detected by biochemical quantitative analysis.The pathological changes of liver tissue in mice were observed by HE and Masson staining.[Results]The results of cell experiments showed that:(1)Different concentrations of TGF-β1(0,5,10,15ng/mL)stimulated LX-2,and the expression of miR-200a decreased in a dose-dependent manner,and at 5ng/mL The most obvious decline was the proliferation rate of LX-2,the mRNA and protein expression levels of TGF-β2 in LX-2,and the mRNA expression levels of fibrosis factorsα-SMA and Colla I,which gradually increased with the increase of TGF-β1 concentration.(2)LX-2 was stimulated at the optimal concentration of 5ng/mL TGF-β1.The expression of miR-200a decreased in a time-dependent manner,and decreased significantly at 0∽48 hours.There was no significant difference at 48∽72 hours,48hours.For the best stimulation time.(3)After miR-200a mimics was transfected into LX-2 cells,the expression of miR-200a in LX-2 was significantly increased,while the mRNA expression levels ofα-SMA,colla I and TGF-β2 were significantly decreased.The proliferation rate of LX-2 was significantly decreased,which was statistically significant compared with the other groups(P<0.05).The results of animal experiments showed that:(1)liver tissue miR-200a levels:expression levels of miR-200a in liver tissue with PBS group(schistosomiasis model group)and Lenti-NC group(lentiviral negative control group)with prolonged infection time.The gradual down-regulation was significantly different from the normal control group(uninfected schistosomiasis group)(P<0.05),while the expression of miR-200a in the liver tissue of the Lenti-miR-200a group(lentiviral experimental group)was significantly increased.(2)Changes of liver fibrosis index:The mRNA expression levels ofα-SMA and TGF-β2 in PBS group and Lenti-NC group gradually increased with the prolongation of infection time,and the expression level of CollaI was at the 6th week of infection.The peak expression ofα-SMA,collaI and TGF-β2 in Lenti-miR-200a group was significantly lower than that in PBS group and Lenti-NC group(P<0.05).(3)Serum ALT levels:PBS group and Lenti-NC group began to increase at the 4th day of infection,peaked at6w;while Lenti-miR-200a group was significantly lower than PBS at 4w and 6w after infection.Lenti-NC group(P<0.05).(4)HE and Masson staining of liver tissue:from the 6th week after infection,different degrees of inflammatory cells and blue collagen fibers appeared in the liver tissues of PBS group and Lenti-NC group,and it was more obvious at the 8th week,and the infection time was prolonged and the condition was aggravated,while the lesions in the Lenti-miR-200a group were significantly reduced.[Conclusion]miR-200a can inhibit TGF-βinduced schistosomiasis liver fibrosis by down regulating TGF-β2.