Potassium Channel KCa3.1 Promotes Cell Cycle Progression By Targeting SKP2 And Invasiveness Through EMT Pathway In Hepatocellular Carcinoma

Background:Primary hepatocellular carcinoma(HCC)has a high incidence in China,which accounting for about 55%new cases and more than 40%death toll of liver cancer in the world each year.And the incidence of HCC in the world shows an increasing trend recent years.Although HCC comprehensive treatment system which based on surgical resection has formed,but the high rate of recurrence after surgery is still a major threat to long-term survival of HCC patients.In addition,patients who suffered late stage of HCC could not be treated by surgery.In recent years,with the deep understanding of the mechanism of tumorigenesis,the molecular-targeted drugsare gradually applied in clinical practice,which brings hope to patients with advanced HCC.However,poor efficacy and side effects still occurred when using molecular-targeted drugs.Therefore,exploring new pathogenesis and new therapeutic targets of HCC have extremely important clinical and scientific significance.Objectives:The aberrant expressed potassium channels in HCC tissues and normal liver tissues were screened by gene chip technique and verified by RT-PCR and Western Blot.We systematically studied the expression pattern and function of potassium channel KCa3.1 in human hepatocellular carcinoma.The relationship between the expression of KCa3.1 and the clinicopathological features of HCC was been analyzed.Further in vivoand in vitro studies were performedto elucidate the role and potential molecular mechanism of potassium channel in the pathogenesis and development of HCC,which would provide a new theoretical strategy for the treatment of HCC.Result:1.Compared with adjacent tissues,the expression of KCa3.1 mRNA in HCC tissues was not significantly altered.Western Blot results showed that KCa3.1 was highly expressed in poorly differentiated HCC tissues.Immunohistochemistry results showed that KCa3.1 protein was mainly expressed in the cytoplasm and membrane of cell,and the expression of KCa3.1 was correlated with the histological grade of HCC.2.After silencing the expression of KCa3.1 in Huh7 and LM3 cell lines,theabilities of proliferation,migration and invasion were weakened,thenumber of apoptotic cells was increased,and the cell cycle wasarrested in those two cell lines.When overexpressing KCa3.1 in Huh7 and LM3 cell lines,the abilities of proliferation,migration and invasion were enhanced,the number of apoptotic cells wasdecreased,and the cell cycle was progressed in those two cell lines.3.Animals studies showed that knockdown of KCa3.1 in Huh7 and LM3 cells could reduce the ability of subcutaneous tumor formation in nude mice.However,the ability of subcutaneous tumor formation in nude mice was enhanced after overexpression of KCa3.1.4.The results from gene chip suggest that KCa3.1 could activate cell cycle signaling pathway.In addition,the expressions of SKP2 and RELN gene change significantly after KCa3.1 silence or overexpression.5.Western Blot results showed that potassium channel KCa3.1 can activate the cell cycle-related pathway and EMT-related pathway.6.The rescue experiment of SKP2 confirmed that KCa3.1 could control the cell cycle progression of HCC cells by regulating SKP2/p21 and p27 signaling pathways,and the rescue experiments of RELN confirmed that KCa3.1 could regulate the migration and invasion of HCC cells by targeting RELN.Conclusion:The expression of KCa3.1 was elevated in low-differentiated HCC tissues compared to tumor-adjacent tissues.And the abilities of proliferation,migration,and invasion was reduced,the number of apoptosis cells was increased,cell cycle was retardant in HCC cells lines when silencing the expression of KCa3.1,while these cellular function effects were reversed when overexpressing KCa3.1 in HCC cells lines.In accordance with results of cellular experiments,in vivo animal experiments showed HCC tumor growth was reduced when silencing the expression of KCa3.1,while HCC tumor growth was promoted when overexpressing KCa3.1 in HCC cell lines.Mechanism study suggested that KCa3.1 could influence cell cycle progression of HCC cell by targeting SKP2,and KCa3.1 could regulate invasiveness of HCC cell through EMT pathway and RELN.Our study demonstrated potassium channel KCa3.1 which play important roles in the formation and development of HCC might be promising therapeutic targets in HCC.