CFIm25 Inhibits Hepatocellular Carcinoma Proliferation And Metastasis By Suppressing The JNK/c-Jun And P38 Signaling Pathways

Objective: Hepatocellular carcinoma(HCC)is one of the common and dangerous malignant tumors.Its incidence rate ranks fifth in malignant tumors and ranks third in cancer-related deaths.The occurrence and development of HCC is a complex process that involves multiple factors,and its mechanism is not fully understood.Therefore,in-depth research on the molecular mechanism of HCC might discover a potential intervention therapy.Researches has showed that CFIm25 is the most important "switch" of Alternative polyadenylation(APA)in pre-transcriptional modification.Inhibition of CFIm25 can regulate m RNA formation with a short 3' untranslated region(3'UTR),which can escape negative regulations and promote cell proliferation/transformation.At present,there are no reports about the function of CFlm25 in hepatocellular carcinoma.This study will explore the role and molecular mechanism of CFIm25 in the proliferation and metastasis of HCC.Methods: The expression of CFIm25 in human hepatocellular carcinoma and its adjacent tissues was detected by real-time quantitative PCR(RT-q PCR),Western blot and immunohistochemical(IHC).Then we analyzed the relationship between the expression of CFIm25 with the pathological features and prognosis of patients with liver cancer surgery.CFIm25 silenced by small interference RNA(si RNA)and plasmids carrying CFIm25 gene were transfected in Hepatoma cell line.The effects of high or low expression of CFIm25 on the proliferation,invasion and metastasis of hepatocellular carcinoma cells were detected by plate cloning,CCK8 and Transwell invasion migration assay and cell scratch assay respectively.The effect of CFIm25 on proliferation,invasion and metastasis of HCC in vivo were analyzed by the mouse subcutaneous tumor and tail vein injection metastasis model.The cell cycle and cytoskeletal changes after high/low expression of CFIm25 were detected by flow cytometry and F-actin staining.The molecular mechanism of the impact of CFIm25 on the proliferation,invasion and metastasis of HCC were analyzed by RT-q PCR,Western blot,and luciferase reporter gene assays.Results: The results of RT-PCR and Western blot showed that the expression level of CFIm25 in HCC tissues was significantly lower than that in the adjacent tissues.In immunohistochemical,there was no significant difference in the expression of CFIm25 in cirrhosis,chronic hepatitis,and fatty liver compared with normal liver tissue.The expression of CFIm25 in these paracancerous tissues was significantly higher than that in liver cancer tissues,and its expression was localized in nucleus.Combined with the clinicopathologic features of patients undergoing liver cancer surgery,the expression of CFIm25 was closely related to TNM stage and lymph node metastasis of liver cancer,and the overall survival of CFIm25 high expression group was significantly higher than that of CFIm25 low expression group.According to the result of RT-PCR and Western bot,CFIm25 was lowly expressed in the high-metastatic potential cell lines Sk-hep-1 and HCC-LM3 but was highly expressed in SMMC-7721 and Hep G2 which with low metastatic potential.Inhibiting the expression of CFIm25 of SMMC-7721 and Hep G2,the proliferation activity and the ability of invasion and migration of SMMC-7721 and Hep G2 were significantly increased.And over-expression of CFIm25 of Sk-hep-1 and HCC-LM3,it went an apparent decline in the proliferation activity and the ability of invasion and migration of these cells.In the subcutaneous xenograft model of nude mice,the volume of subcutaneous tumors of the group which was overexpressing CFIm25 was significantly smaller than that of the control group,moreover in the tail vein injection metastasis model,the number of metastatic tumors in liver and lung of the overexpressing CFIm25 group was significantly less than that of the control group.Further studies showed that over-expression of CFIm25 can inhibit the expression of cyclin D1(CCND1)and cell cycle-dependent kinase 4(CDK4)and cause G1 arrest in hepatocellular carcinoma cells,whereas inhibiting the expression of CFIm25,the opposite result occurred.The relationship between CFIm25 and epithelial mesenchymal transition(EMT)in HCC showed that overexpression of CFIm25 could induce the expression of E-cadherin,a key factor in EMT,and decrease the expression of N-cadherin.And inhibiting the expression of CFIm25,the expression of E-cadherin decreases,while the expression of N-cadherin increases.At the same time,CFIm25 caused rearrangement of F-actin and changed the cytoskeleton.The study of the molecular mechanism involved in the regulation of CFIm25 showed that: overexpression of CFIm25 induced the expression of phosphorylated JNK/c-Jun and P38 decreased;and inhibiting expression of CFIm25 caused the expression of phosphorylated JNK/c-Jun and P38 increased.The results of the luciferase reporter gene showed that the transcriptional activity of the transcription factor AP-1 was increased by inhibiting CFIm25,and decreased by overexpression of CFIm25.Conclusions: We conclude that CFIm25 is lowly expressed in human hepatocellular carcinoma.And the expression of CFIm25 is closely related to TNM stage,lymph node metastasis,and prognosis of patients with liver cancer.At the cellular and animal levels,CFIm25 can inhibit the proliferation,invasion and metastasis of liver cancer.CFIm25 can inhibit the expression of CCND1 and CDK4,leading to cell G1 arrest.At the same time,CFIm25 can increase the expression of E-cadherin,a key factor of EMT,and reduce N-cadherin,causing rearrangement of F-actin and altering the cytoskeleton,thus inhibiting EMT in liver cancer.CFIm25 inhibits the proliferation,invasion and metastasis of hepatoma cells by inhibiting the JNK/c-Jun and P38 MAPK signaling pathways and inhibiting the activity of the transcription factor AP-1.