MSI2 Overexpressed In Cancer Associated Fibroblast And Promote Hepatocellular Carcinoma Progression

Background and objective:Tumor microenvironment,as the external environment of tumor cells,is a key factor influencing the biological behavior of tumor cells.As the main mesenchymal cell in the tumor microenvironment,myofibroblast in HCC,also called cancer associated fibroblasts(CAF)plays an important role in tumor progression.In our study,we found that Musashi2(MSI2)is highly expressed in tumor-associated fibroblasts and associated with the proliferation and invasion of hepatocellular carcinoma(HCC)cells.Therefore,this study was intended to explore the role of myofibroblast derived MSI2 in the progression of HCC.Methods:Sirius red,immunohistochemical and immunofluorescence staining were used to evaluate collagen deposition and detect α-SMA and MSI2 expression in tumor tissues of HCC patients and mice orthotopic transplanted tumors.The conditional knockout Msi2 of myofibroblast mouse model were constructed by the Cre-LoxP system.Density gradient centrifugation was performed to isolate primary hepatic stellate cells(HSCs).The myofibroblast was induced from HSCs via in vitro culturing and activating.Conditioned medium were used to determine that if myofibroblast affected the biological behavior of HCC cells.CCl4 induced liver fibrosis orthotopic implantation HCC mouse model was used to investigate the affection of myofibroblast Msi2 knockout on HCC progression.RNA-seq on Msi2 knockout mouse myofibroblast was performed to detect the gene expression change in myofibroblast after Msi2 knockout.Kaplan-Meier survival curve was used to analyze the effect of tumor collagen deposition on the prognosis of HCC patients and the survival of mice after orthotopic transplanted tumor cells.Results:In our database of 117 liver cancer cases,60.7%of HCC patients with tumor collagen deposition.Survival analysis and multivariate analysis of variance indicated that tumor collagen deposition was an independent risk factor for patients’ prognosis.Through the public database of GEO,HBV related fibrosis(GSE84044)and NAFLD liver fibrosis(GSE49541)gene expression data analysis.In HBV associated and NAFLD fibrosis,as the fibrosis is aggravating,accompanied with MSI2 expression increased.Suggest that MSI2 expression in the myofibroblasts.In addition,there was a positive relationship between the expression of a-SMA and collagen deposition in HCC patients,indicating that the collagen fibers mainly came from myofibroblasts in HCC.Immunofluorescence co-staining of MSI2 with α-SMA in HCC patients showed co-localization of α-SMA and MSI2.In addition,MSI2 expression was also detected in the isolated human primary cancer associated fibroblast by immunofluorescence staining.We construct the conditional knokout Msi2 of myofibroblast(Msi2 ΔCollal)mouse.And established the CCl4 induced liver fibrosis orthotopic implantation HCC mouse model.compared with the control group Msi2F/F mice,conditional knockout myofibroblast Msi2,the mice survival significantly prolonged(P=0.0021).And conditioned knockout Msi2 in myofibroblast,tumor growth,intrahepatic metastasis and pulmonary metastasis of liver tumors in situ could be significantly inhibited.In vitro experiments also showed that knockout Msi2 in myofibroblast significantly inhibited the proliferation and invasion ability of HCC cells after conditioned medium treatment RNA sequencing was performed on Msi2-knockout mouse myofibroblast to analyze the gene expression profile of myofibroblast related to Msi2 gene.Bioinformatics was used to analyze the sequencing results,and suggested that MSI2 promote myofibroblast secrete cytokines 116,Il11,etc.through the ERK1/2 signaling pathway.Further in vitro experiments also showed that Msi2 could promote myofibroblasts to secrete cytokines 116 and Il11 through the ERK1/2 signaling pathway.Conclusion:Deposition of collagen and myofibroblasts in tumor tissues suggests a poor prognosis of HCC patients.MSI2 facilitates the myofibroblast secretion of cytokines IL6 and IL11 by the ERK1/2 signaling pathway,and further promotes the invasion and metastasis of HCC cells.In addition,CAF-derived MSI2,not HCC-derived MSI2,played the major role in promoting HCC progression in fibrotic liver.