Regulation Of IL-6R Expression By JAK2/STAT3 Pathway And Mechanism Research In Hepatocellular Carcinoma

Background and Ami:Hepatocellular carcinoma（HCC）is a prevalent subtype of primary liver cancer,ranking the 6th among all cancers in terms of fatality rate globally.Although systemic treatment has made great progress,the progress of HCC requires the interaction of multiple factors and steps,and its pathogenesis is relatively difficult to detect.Especially for patients with advanced HCC,the therapeutic effect of traditional therapy is very limited,while the efficacy of targeted therapeutic drugs is limited by drug resistance.Numerous research studies have indicated that the interleukin-6 receptor（IL-6R）plays a crucial role in the initiation and progression of various types of cancer,but insufficient studies have been conducted on its function and the mechanism of its regulation of HCC expression.Therefore,this study aims to explore the biological effects of IL-6R in HCC cells and its regulated expression mechanism.In this context,this study can provide new treatment strategies and more effective methods for the early diagnosis and treatment of HCC.Methods:In this study,the protein levels of IL-6R in normal hepatocytes（THLE-2,THLE-5）and hepatocellular carcinoma cells（Hep G2,Huh7,SK-Hep1）were examined using Western blot analysis.Immunocytochemical methods were employed to qualitatively and quantitatively characterize and localize IL-6R in each cell group.We also successfully constructed SK-Hep1^IL-6R-and SK-Hep1^STAT3-cell lines by transfection with lentivirus.The morphological differences of cells were observed using hematoxylin and eosin staining,while JC-1 method was used to evaluate the differences in apoptosis of different cell groups under the action of sorafenib,and the proliferative capacity differences between treatment groups were evaluated using cloning formation assay and Edu.Scratch healing assay and Transwell assay were utilized to determine the invasive and migratory abilities of each cell group.Furthermore,a nude mouse model of transplanted human hepatocellular carcinoma was established and immunohistochemical staining and Western blotting were conducted to validate the variations in IL-6R protein levels between groups.The tumor growth was calculated through regular monitoring of tumor volume and the effect of IL-6R on tumor growth of xenotransplantation model of liver cancer in vivo was evaluated to verify the regulated expression mechanism of IL-6R.Results:Research studies have shown that IL-6R is expressed at low levels in normal liver cells,while abnormally over-expressed in HCC cells.The expression level of IL-6R in SK-Hep1 is high,while that in Hep G2 is low.Conversely,the expression level of IL-6R was found to be reduced in SK-Hep1STAT3-cell line.The study also found that in Hep G2^IL-6 cell line,IL-6 promoted cell proliferation and migration by up-regulating proliferation molecules p-P70S6K and migration molecules MMP2 and MMP9,and enhanced the anti-apoptosis ability of cells by down-regulating apoptosis molecules C-Caspase3 and C-Caspase7.In contrast,SK-Hep1^IL-6R-cell lines that interfere with the expression of IL-6R and SK-Hep1^TCZ cell lines that inhibit the expression of IL-6R by using the inhibitor of IL-6R Tocilizumab attenuated the proliferation,migration and invasion of cells by down-regulating the proliferation molecules p-P70S6K,migration molecules MMP2 and MMP9,and up-regulating the apoptosis molecules C-Caspase3 and C-Caspase7 was found to decrease the anti-apoptotic capacity of cells.By further studying the regulatory mechanism of IL-6R,we found that by activating the transcription factor STAT3,JAK2/STAT3 signaling pathway facilitates the binding of IL-6R to the promoter region,thereby enhancing the transcriptional expression levels of IL-6R.In animal experiments,the fastest growing tumor was in the Hep G2^IL-6 treatment group,with the largest terminal volume;The slowest growth of tumor was in the SK-Hep1 group which interfered with STAT3 expression after tumor loading,and the terminal volume was the smallest.Western blot showed that the activation level of JAK2/STAT3 signaling pathway and the expression level of IL-6R protein analyzed using Western blotting were up-regulated in Hep G2^IL-6 treatment group and SK-Hep1 tumor bearing group.Conclusion:The findings from this study demonstrate that the upregulation of IL-6R is mediated through the JAK2/STAT3 signaling cascade,following exposure to IL-6stimulation,thereby enhancing the proliferation,migration and anti-sorafenib capabilities of HCC cells,and thus promoting the progression of HCC.Figure[28]Table[0]Reference[45]...