The Role And Mechanism Of PDIA6 In Control Of Cell Proliferation And Cell Cycle Of Lung Squamous Cell Carcinoma

Objective:Through the construction of over expression of PDIA6 gene lentiviral recombinant plasmid,The cell model stably expressing PDIA6 was established.To investigate the effect of PDIA6 gene on proliferation and cell cycle of lung squamous cell carcinoma NCI-H520 cells and study on the molecular mechanisms of PDIA6 on the cell proliferation and cell cycle.Methods:1.Use Western blotting to detect the expressions of PDIA6 in 7 kinds of lung cancer cell lines:A549,H157,NCI-H460,NCI-H520,973,SPCA-1 cell lines and HBE cell line and futher screen the cell line with lower expression of PDIA6.2.According to the specific sequence of PDIA6,PCR primers were designed to amplifial the target fragment,NCI-H520 cell RNA was extracted and reverse transcribed into CDNA.Through cloning connected to subcloned PDIA6 into the lentiviral vector,pCDH-CMV-MCS-EF1-copGFP-T2A-Puro,to generate the lentiviral expression vector.After gel electrophoresis to identify connected products,finally,the positive clones were obtained by enzyme digestion,and the sequence were determined.3.The recombinant plasmid combined withpsPAX2和 pMD2G were transfected into 293T cells for virus packaging,divided into lentiviral group,which containing PDAI6 gene and Green fluorescent protein,and control group.The harvest of lentivirus were used to infect NCI-H520 cells,PDIA6 expression in NCI-H520 cell were detected by fluorescence microscopy and western blotting.4.The proliferation ability of lung squamous cell carcinoma NCI-H520 cells was detected by CCK-8 and plate cloning experiment,some proteins were detected by western blotting.5.The changes of cell cycle of lung squamous cell carcinoma NCI-H520 cells were detected by flow cytometry western blotting was used to detect the proteins associated with cell cycle.Results:1.The result of Western blotting screening the expression in cell lines showed that PDIA6 protein was lower expressed in lung squmous cancer NCI-H520 cell line,which can be used to subsequent test.2.Control group and experimental group were all visible green fluorescence under fluorescence microscope,and the infection rate was close to 90%.The positive clones were identified by Western blotting method.The high expression of NCI-H520/pCDH-PDIA6 stable cell lines and NCI-H520/pCDH vector were established.3.Cell growth curve and colony forming experiment showed that,after transfection,cell proliferation ability increased(p<0.05),the difference is statistically significant.Experimental group NCI-H520/pCDH-PDIA6 of cell clone cluster volume greater than that of the control group NCI-H520/pCDH and the negative control group NCI-H520.The detection results of proliferation related protein PCNA and ERK and phosphorylated ERK showed that the expression levels of PCNA and phosphorylated ERK in the experimental group were all increased compared with the control group.4.The results of flow cytometry showed that compared with the control group NCI-H520/pCDH and the negative control group NCI-H520,the G0/G1 phase of the experimental group NCI-H520/pCDH-PDIA6 was decreased,while the S phase was significantly increased.At the same time,the expression of cell cycle related proteins was found by Blot Western.The expression of CyclinD1,CDK4/CDK6,pRb were up-regulated,and the expression of p27 was down regulated.Conclusion:1 The human lung squamous cell carcinoma NCI-H520 cell line was successfully constructed with the overexpression of PDIA6.2.Overexpression of PDIA6 promotes the proliferation of human lung squamous cell carcinoma NCI-H520 cell line in vitro,that is,PDIA6 has a role in promoting tumor cell growth.3.The results of cell cycle showed that the G0/G1 phase of the experimental group NCI-H520/pCDH-PDIA6 was decreased,while the S phase was increased,and the G2/M phase had no significant change.This shows that PDIA6 is likely to be involved in the regulation of the G1/S phase of the cell cycle,and at the same time,it may increase the synthesis of DNA by some way,so as to play a role in promoting the growth of tumor cells.4.The expression of genes associated with the development of tumor is accompanied by changes in PDIA6,such as PCNA,p-ERK,CyclinD1,CDK4/CDK6,p27,pRb and so on.The expression of CyclinD1,CDK4/CDK6,pRb were up-regulated,and the expression of p27 was down regulated.5.PDIA6 gene may promote the growth of lung squamous cell carcinoma cells by influencing the ERK signaling pathway,and may further activating Ras-Raf-MEK-ERK,thus promoting lung cancer proliferation.6.PDIA6 gene may be involved in cell cycle regulation by affecting the CDK4/CDK6 CyclinD1-signaling pathway,thus promoting the regulation of G 1/S in tumor cells.