Investigation On Potential Pharmacological Mechanisms Of Fufang Biejia Ruangan Pills Acting On The Malignant Transformation From Liver Fibrosis To Hepatocellular Carcinoma Based On The Regulatory Network Of Competing Endogenous RNAs

Background: Fufang-Biejia-Ruangan Pill(FBRP)is the first clinically approved anti-fibrosis herbal formula in China.However,whether FBRP could be used to treat hepatocellular carcinoma(HCC)remain unclear.Competing endogenouseRNA(ceRNA)regulation has been received an increasing attention due to their involvement in the carcinogenesis and cancer progression of the liver.The aim of the present study was to identify a novel ceRNA regulatory axis involved into HCC,and to explore the pharmacological mechanisms of FBRP acting on the malignant transformation from liver fibrosis to HCC through this axis.Methods: Expression profiles of lncRNAs,miRNAs and m RNAs in clinically derived HCC tissues and self-paired adjacent non-cancerous tissues were detected using microarray analysis.The candidate lncRNA-miRNA-m RNA axis involved into the-malignant transformation from liver fibrosis to HCC was screened according to bioinformatics prediction based on miRanda and Target Scan databases,real-time PCR and dual-luciferase reporter gene assay.Subsequently,the clinical significance of theRNAs in the candidate ceRNA regulatory axis were statistically evaluated based on The Cancer Genome Atlas(TCGA)data set and our clinical cohort.Both gain-of-function and loss-of-function analyses were performed to determine the the biological functions(cell proliferation,cell migration,cell apoptosis and cell cycle)of the candidate ceRNA regulatory axis using two human HCC cell lines(HUH7,MHCC97H)in vitro.In addition,the in situ nude mouse model and the diethylnitrosamine-induced rat HCC model were established and were used to explore whether FBRP could play a role in intervening the malignant transformation from liver fibrosis to HCC by regulating the candidate ceRNA regulatory axis.Results: Compared to the adjacent non-cancerous tissues,miR-328-3p expression was significantly down-regulated,but SRSF9 expression was on the contrary(both P<0.05).In addition,both SRSF9 and lncRNA·TUG1 were predicted to be one of the candidate putative target m RNAs and lncRNAs of miR-328-3p by binding with the same 3′‐UTR site of this miRNA.Clinically,the overall and disease-free survivals of HCC patients with elevated expression of SRSF9 and lncRNA·TUG1 were both shorter than those with low expression.Functionally,the knockdown of SRSF9 and the enforced expression of miR-328-3p obviously inhibited HCC cell proliferation,migration,cell cycle and induced HCC cell apoptosis,which were all reversed by SRSF9 restoration.Moreover,lncRNA·TUG1 was demonstrated to be an endogenous “sponge” by binding with miR-328-3p,subsequently affecting its modulation on SRSF9 in HCC cells.Then,the dysregulation of lncRNA·TUG1-micro RNA-328-3p-SRSF9 axis in HCC tissues was effectively reversed by the administration of FBRP in vivo..Conclusions: Our data revealed that lncRNA·TUG1-miRNA-328-3p-SRSF9 m RNA axis may be the novel ceRNA regulatory axis involved into the malignant transformation from liver fibrosis to HCC,and FBRP may effectively reverse this malignant process by regulating this axis,suggesting that FBRP may be a promising candidate drug for reduction of fibrogenesis and prevention of HCC.