| Purpose:The incidence and mortality of liver cancer are high,and the existing treatment methods are not so effective,which seriously harm people’s health.Previous studies have found that diosgenin,the main active component of traditional Chinese medicine fenugreek,has a certain anti-liver cancer effect,but the specific mechanism of action is not clear.In addition,studies have suggested that the Hippo signaling pathway plays an important role in the pathogenesis of liver cancer and other tumors.Through network pharmacology and molecular docking technology,we found that the core gene of diosgenin’s anti-hepatocellular carcinoma effect was SRC,so it was speculated that diosgenin could exert anti-hepatocellular carcinoma effect through c-Src/Hippo signaling pathway.Therefore,this study aims to explore the anti-hepatocellular carcinoma activity and action mechanism of diosgenin.The completion of this project is expected to provide new ideas for the clinical diagnosis and treatment of liver cancer.Method:1.To investigate the main active ingredients and potential targets of trigonellae semen in the treatment of hepatocellular carcinoma based on network pharmacology and molecular docking methods.2.To explore the expression level of c-Src/Hippo signaling pathway related molecules in hepatocellular carcinoma and its relationship with the occurrence and development of the disease.In this study,RT-q PCR and Western Blot were used to detect the expression differences of related molecules in fresh liver cancer tissues and paired paracancer tissues to verify the bioinformatics data.The protein expression levels and cell localization of cSrc and YAP in paraffin sections of liver cancer tissue were detected by IHC technology,and their correlation with clinical indicators(serum AFP level,clinical stage of tumor,survival time,etc.)was retrospectively analyzed to evaluate their clinical significance.3.To explore the anti-hepatocellular carcinoma activity of diosgenin and its regulatory effect on c-Src/Hippo signaling pathway.Two human hepatoma cell lines,MHCC-97 H and Hep G2,were used in this study.The cells were divided into control group,diosgenin low and high concentration treatment group,Hippo signaling pathway inhibitor group,and diosgenin and Hippo signaling pathway inhibitor co-culture group.The inhibitory effects of diosgenin on hepatocellular carcinoma cells were investigated from multiple directions of proliferation(CCK-8 method,Ed U staining method),apoptosis(TUNEL method),invasion and metastasis(Cell scratch test,Transwell test).The regulatory effects of diosgenin on apoptosis-related molecular markers and c-Src/Hippo signaling pathway were detected by Western Blot.Finally,Hippo signaling pathway inhibitor XMU-MP-1 was used to perform response experiments to further verify that diosgenin can play an antihepatocellular carcinoma role by regulating c-Src/Hippo signaling pathway.Results:1.With the help of network pharmacology and molecular docking methods,we finally learned that the main active component of trigonellae semen in the treatment of hepatocellular carcinoma is diosgenin and the docking core gene is SRC.2.Clinical studies showed that compared with normal liver tissues,c-Src/Hippo signaling pathway related molecules c-Src,Lats1 and YAP were highly expressed in cancer tissues,and the difference was statistically significant(P<0.001);Correlation analysis showed that the expression level of c-Src was closely related to small volume tumors and cirrhosis(P=0.043,P=0.016),while YAP expression was only related to large volume tumors(P=0.03),and no correlation was found between the two and other clinical and pathological features;Prognostic analysis indicated that high expression of YAP was an independent risk factor affecting the prognosis of liver cancer.3.In vitro cell experiments showed that compared with the control group,the proliferation,invasion and metastasis of human hepatoma cell lines MHCC-97 H and Hep G2 were significantly inhibited by diosgenin treatment,and cell apoptosis was increased(P<0.05).However,after pretreatment with Hippo signaling pathway inhibitor XMU-MP-1,compared with diosgenin group,the proliferation,invasion and metastasis of cells in the co-culture group were increased,and the apoptosis process was slowed down(P<0.05),which effectively reversed the anti-hepatocellular carcinoma effect of diosgenin.In addition,XMU-MP-1 pretreatment can increase the expression of p-Src and YAP protein down-regulated by diosgenin,and inhibit the expression of p-Lats1 and p-YAP protein upregulated by diosgenin,and the rescue effect is obvious.Conclusion:The main active component of trigonellae semen in the treatment of hepatocellular carcinoma is diosgenin and c-Src is the target protein;The expression level of c-Src/Hippo signaling pathway related molecules is closely related to disease severity and prognosis survival of patients with hepatocellular carcinoma;Diosgenin can play an antihepatocellular carcinoma role by regulating c-Src/Hippo signaling pathway. |
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