Study On The Function Of CKLF1 Upregulated By HBV In The Development Of Primary Hepatocellular Carcinoma

Objective Hepatocellular carcinoma(HCC),one of the most common and deadliest malignancies worldwide,has a poor prognosis,owing to its high potential for vascular invasion and metastasis and the lack of biomarkers for early diagnosis.The chronic inflammation caused by HBV is a significant risk factor for HCC.The secretion,accumulation and interaction of inflammatory cytokines induced by chemokines lead to the proliferation and transformation of cells and angiogenesis,and finally result in the development and progression of HCC.Transcript variant 2 of the chemokine-like factor,named CKLF1,is a newly discovered CC chemokine.CKLF1 plays essential roles in inflammatory and autoimmune diseases.However,the relationship between CKLF1 and cancer is uncertain.In this study,we investigated the function of CKLF1 in HBV-induced HCC and explored the mechanism of CKLF1 in the development and the worse clinical outcomes of HCC.We revealed a new mechanism of the development of HBV-related HCC and reported an attractive target for cancer therapy.Methods 1.The level of CKLF1 was investigated in Hep G2 cell line and Hep G2.2.15 cell line.The expression of CKLF1 was detected after the transfection of HBV by q PCR and western blotting.2.Bioinformatics analysis,q PCR,western blotting and IHC were performed to detect the expression of CKLF1 in HCC and the relationship between CKLF1 and HCC.The expression level of CKLF1 was also detected in different cell lines.3.The functions of CKLF1 in cell proliferation,migration,invasion,tumorigenicity were demonstrated by a series of in vitro and in vivo experiments,such as RTCA,CCK-8,wound healing assay,Transwell,foci formation assay and subcutaneous xenograft experiment.4.Bioinformatics analysis predicted the correlation between CKLF1 and IL-6/STAT3 signaling pathway.The expression levels of CKLF1,IL-6,STAT3,p-STAT3(Y705)and the downstream genes of STAT3 were detected by q PCR,ELASA and western blotting after the transfection of CKLF1 plasmid or sh RNA for CKLF1.The pyruvate kinase activity and lactate production were also detected.Additionally,the mechanism of CKLF1-enhanced HCC was investigated through a series of experiment with the treatment of IL-6 si RNA or Stattic,the inhibitor of p-STAT3(Y705).5.The function and mechanism of CKLF1 in DOX-induced apoptosis were revealed by flow cytometry,q PCR and western blotting after the different treatments.Results 1.CKLF1 was higher in HepG2.2.15 cell line compared with Hep G2 cell line.The level of CKLF1 was up-regulated by HBV.2.CKLF1 was highly expressed in HCC tissues and related to vascular invasion,tumor size,tumor stage and poor overall survival.There was a significant positive correlation between the expression of CKLF1 and STAT3.3.CKLF1 promoted cell proliferation,migration and invasion,tumorigenicity aerobic glycolysis through the IL-6/STAT3 pathway.4.CKLF1 prevented DOX-induced apoptosis through IL-6/STAT3 signaling.Conclusion CKLF1,up-regulated by HBV,promoted malignant transformation through the IL-6/STAT3 pathway,and ultimately enhanced the development and metastasis of HCC.Additionally,the overexpression of CKLF1 protected HCC cells from the apoptosis and induced chemotherapy resistance of HCC through IL-6/STAT3 signaling.Thus,our work revealed that CKLF1 was a significant prognostic factor of HCC and might be a potential molecular therapeutic target for HCC.