Regulatory Mechanism Of CKAP4 In Proliferation Of Rat Hepatocytes BRL-3A

Cytoskeleton Associated Protein 4(CKAP4)is a type II transmembrane protein,which mainly exists in the endoplasmic reticulum to maintain the stability of the endoplasmic reticulum.At the same time,it was found that CKAP4 is also located on the plasma membrane,as a protein-related antiproliferative factor(APF),surfactant protein A(SP-A),tissue plasminsogen activator(t PA),secreted protein Dickkopf 1(DKK 1)and other protein receptors are involved in the occurrence of disease.CKAP4 may promote the proliferation of normal cells and carcinoma cells by activating the PI3K/ AKT pathway in pancreatic cancer,lung cancer,renal cancer and esophageal squamous cancer.However,it has been reported that CKAP4 may act as a tumor suppressor to inhibit the occurrence of Wnt pathway in hepatocellular carcinoma,and CKAP4 may inhibit the metastasis of hepatocellular carcinoma cells and is associated with favorable prognosis.CKAP4 has been extensively studied as a diagnostic marker or therapeutic target for a variety of cancers.In the Rat Genome 230 2.0 database in our laboratory,CKAP4 expression was significantly up-regulated during 12 h to 72 h after partial hepatectomy(PH),that is,during the proliferative stage of liver regeneration after PH,which indicating that CKAP4 is closely related to the proliferation of hepatocytes in the process of liver regeneration.However,there are few reports on the role of CKAP4 in liver regeneration.Therefore,this study took rat hepatocyte BRL-3A cells as the research material to explore the effect of CKAP4 on the proliferation of hepatocytes and its mechanism.First,CKAP4 overexpressed plasmid was constructed in this study,and CKAP4 overexpressed BRL-3A cell lines were obtained by lentivirus-mediated method.The overexpression efficiency of CKAP4 was detected by RT-q PCR and Western Blot,and the fluorescence expression intensity of CKAP4 was detected by immunofluorescence technique.MTT,CCK8,flow cytometry and Western Blot were used to detect the effect of CKAP4up-regulation on cell viability,proliferation and cell cycle.The results showed that CKAP4 overexpression enhanced BRL-3A cell viability and significantly accelerated cell proliferation and cell cycle.Secondly,CKAP4 expression was knocked down by transfection with siRNA,Knockdown of CKAP4 expression by siRNA transfection was also detected by the above method.The results showed that down-regulation of CKAP4 inhibited the viability and proliferation of BRL-3A cells,which was consistent with the experimental results of up-regulation of CKAP4.In order to further illustrate the mechanism of CKAP4 regulating cell proliferation in BRL-3A cells,PI3 K pathway inhibitor LY294002 was used to treat CKAP4 overexpression and knockdown BRL-3A cells,respectively.Cell viability was detected by MTT,CCK8 and flow cytometry,subsequently.The results show that cell proliferation and cycle were inhibited,and the inhibition effect after CKAP4 knockdown was even more significant.Western Blot results showed that both p-PI3K(p58?)and p-Akt(Ser 473)were down-regulated in both CKAP4 overexpression group and knockdown group,and P-PI3K(p58?)and P-AKT in the CKAP4 knockdown group(Ser 473)expression was significantly reduced.In conclusion,we found that the expression changes of CKAP4 are related to the proliferation of BRL-3A cells.The high expression of CKAP4 can promote the vitality and proliferation of BRL-3A cells,while the down-regulated expression of CKAP4 can inhibit the proliferation of BRL-3A cells.CKAP4 may regulate the proliferation of hepatocytes through the PI3K/ AKT pathway.