| Objective and Significance:Hepatocellular carcinoma(HCC)is the most common type of liver cancer,accounting for 75-85%of primary liver cancer cases,and there are approximately 850,000 new cases worldwide each year.Due to insidious disease,high degree of malignancy,rapid progress,easy metastasis and low drug sensitivity,the prognosis of patients is poor,and the 5-year survival rate is less than 20%.Although surgical resection is currently the most effective method,most HCC is already advanced or metastatic at the time of diagnosis,and only 10-20%of patients have the chance of surgical resection.Xiaoai Jiedu recipe(XJR)is an effective anti-tumor prescription created by the master of Chinese medicine Zhou Zhongying through years of clinical practice experience.It can effectively inhibit liver cancer,but its molecular mechanism is still unclear.This study is based on genomics analysis of its regulated miRNA and downstream targets,and explores the possible mechanism of XJR’s anti-hepatocellular carcinoma.Methods:(1)Construct H22 liver cancer tumor-bearing mouse models,which are divided into normal saline group(negative control group),XJR low(XJRL),medium(XJRM),high dose group(XJRH),and cisplatin group(positive control group).Observe the curative effect of each group,and use gene chip technology to analyze the differentially expressed miRNA in the blood after XJR treatment,and use RT-PCR to verify.(2)Transfect miR-200b-3p inhibitor(anti-miR-shRNA)and miR-200b-3p inhibitor NC(NC-shRNA)into HepG2 cells,and use RT-PCR to verify the intracellular miR-200b-3p after transfection expression.The cells are divided into four groups:control group(blank serum),XJR group(XJR medicated serum),anti-miR-shRNA group(anti-miR-200b-3p shRNA plasmid-treated cells),XJR+anti-miR-shRNA Group(XJR medicated serum+anti-miR-200b-3p shRNA plasmid-treated cells),the proliferation ability,migration ability and invasion ability of each group were determined by MTT experiment,scratch experiment and Transwell experiment.(3)Predict the possible targets of miR-200b-3p through bioinformatics analysis,and detect its activity through the luciferase reporter gene.It was further verified by Western Blot(WB)and RT-PCR.Results:(1)In vitro experiments showed that XJRH can significantly inhibit tumor growth.Using gene chip technology,it was found that there were 233 differentially expressed miRNAs(75 up-regulated and 158 down-regulated)in the blood after XJRH treatment(Fold change>2 or<0.5,P-value<0.05).Through RT-PCR verification,miR-453,miR-200b-3p,miR-135a-1-3p,miR-1960(up-regulated)and miR-466f(down-regulated)are consistent with the chip results.Among them,we chose miR-200b-3p for follow-up research.(2)In vivo experiments showed that the cells were successfully transfected with anti-miR-shRNA and NC-shRNA.XJR significantly inhibited the proliferation,migration and metastasis of HepG2 cells,and transfection with anti-miR-shRNA eliminated the inhibitory effect of XJR on cell proliferation,migration and metastasis.(3)Using bioinformatics to predict that Notch1 may be the target of miR-200b-3p.The luciferase report shows that Notchl is the direct target of miR-200b-3p.It was further verified by WB and RT-PCR that XJR can significantly reduce the expression of Notch1 in HepG2 cells,and transfection of anti-miR-shRNA reversed the decrease of Notch1 expression induced by XJR.Conclusions:(1)XJRH can effectively inhibit tumor growth and increase the level of miR-200b-3p in the blood;(2)XJR can effectively inhibit the proliferation,migration,and invasion of HepG2 cells,and reducing the content of miR-200b-3p in the cells can eliminate the anti-tumor effect of XJR;(3)Notchl is the target of miR-200b-3p.XJR may exert its anti-tumor effect through miR-200b-3p/Notch1. |
PDF Full Text Request