| Objective: The HPV16 E6/E7 protein is the major carcinogenic gene in the development of tumors,and the long-term continuous infection of E6/E7 can cause lung cancer.Studies have found that E6 inhibits apoptosis mainly by degrading p53 gene,and E7 promotes cell proliferation mainly by inhibiting retinoblastoma protein(p Rb).Therefore,the signaling pathways regulated by E6 and E7 proteins may not be identical in tumorigenesis,but the differences between the two in the regulation of tumor mechanisms have rarely been reported.Liver kinase b1(liver kinase b1 LKB1)is a cancer gene that is a key barrier to multiple tumors in the body and controls the occurrence,differentiation and metastasis of tumors.Our previous study found that the E6 and E7 proteins,which can significantly reduce the expression of LKB1 in lung cancer cells,but the difference of molecular mechanism that E6/E7 protein regulates LKB1 is not clear.Wong et al.found that KIF7 the seven family members of the protein family is a cancer suppressor in prostate cancer,and could promote the anti-tumor activity of LKB1 by inducing the phosphorylation of LKB1 at ser428 site to raise the expression of the p-LKB1(ser428).However,it is unclear whether KIF7 regulates LKB1 and the subcellular localization of activated P-LKB1(SER428)in HPV-associated lung cancer.Cui et al.found that miR-106 a is a carcinogenic gene in cervical cancer,which can be raised by HPV16 E7,and inhibits the expression of LKB1 and promotes the proliferation of cancer cells,but E6 has no regulation on miR-106 a.However,it is not clear whether there is a regulatory relationship between E7 and miR-106 a in HPV-associated lung cancer.Therefore,the research on the regulation relationship between E6/E7?KIF7 and LKB1,and the regulation relationship between miR-106 a and LKB1 ? the subcell localization of p-LKB1(ser428)in HPV-associated lung cancer are the main target of this study,and aim for providing new treatment targets and research directions for the targeted treatment of HPV related lung cancer.Methods: 1.Using transfection and sequence interference techniques to regulate the expression of HPV16 E6 and E7 in lung adenocarcinoma cells,Western blot were used to detect the expression levels of KIF7,LKB1 and p-LKB1(ser428),the PCR(q RT-PCR)method was used to detect the level of m RNA expression of the miR-106a?KIF7 and LKB1.2.Regulating the expression of KIF7 and miR-106 a in the lung adenocarcinoma cells,Western blot were used to detect the expression levels of LKB1 and p-LKB1(ser428),q RT-PCR method was used to detect the level of m RNA expression of LKB1.3.Regulating the expression of KIF7 and miR-106 a in the lung adenocarcinoma cells,nuclear protein and cytoplasmic protein were separated by nuclear cytoplasmic separation technique,western blot were used to detect the protein expression level of LKB1 and p-LKB1(ser428)in the nucleus and cytoplasm,explore the subcellular location of the activated p-LKB1(ser428),and detect the determining factors of the LKB1 anti-tumor activity.4.CCK8 experiment and clone formation assay were used to evaluate the cell proliferation of the cancer cells,and cell apoptosis was detected by flow cytometry.5.The spss 22.0 software is used to handle this data,and make P<0.05 that the results were statistically significant.Results: 1.Screening of lung cancer cell lines.According to our previous results,the expression of HPV16 E6 and E7 proteins was lower in H1299 cell line and higher in A549 cell line.On the basis of these results,we selected H1299 cell line to transfect p EGFP-N1-HPV16 E6 and p EGFP-N1-HPV16 E7 plasmids,while in A549 cell line,si E6 and si E7 interfered with the expression of endogenous E6 and E7.Western Blot was used to detect the expression of KIF7 protein in normal human bronchial epithelial cells(HBE)and three lung cancer cell lines(H460,A549,H1299).The expression of miR-106 a in normal human bronchial epithelial cells(HBE)and three lung cancer cell lines(H460,A549 and H1299)were detected by RT-PCR.The results showed that the protein expression levels of KIF7 in all three lung cancer cell lines were lower than those of HBE cells.The expression level was higher in H1299 cell line but lower in A549 and H460 cell line.The expression level of miR-106 a in both lung adenocarcinoma cell lines(H1299 and A549)was higher than that in HBE cells,and the expression level of miR-106 a was higher in H1299 cell line,lower in A549 cell line,and lower in H460 cell line than that in HBE cells.Based on the above results,we selected A549 cell line to transfect p EF6/ V5-HIS-KIF7 plasmid,and H1299 cell line to transfect si RNA to interfere with endogenous KIF7 expression.A549 cell line was selected to transfect miR-106 a mimics and H1299 cell line was selected to transfect miR-106 a inhibitors.2.HPV16 E6/E7 up-regulated the expression of miR-106 a and down-regulated the expression of LKB1 and P-LKB1(Ser428);E6 down-regulated the expression of KIF7 protein,and E7 did not regulate the expression of KIF7.p EGFP-N1-E6 and p EGFP-N1-E7 plasmids were transfected into H1299 cell lines which low expression of E6/E7,respectively.The control group was simulated transfection and empty vector of E6/E7.The results showed that overexpression of E6/E7 significantly up-regulated the expression level of miR-106 a,down-regulated the expression level of p-LKB1(ser428),and down-regulated the protein and m RNA levels of LKB1.Overexpression of E6,protein levels of KIF7 were significantly down-regulated while m RNA levels remained unchanged.Overexpression of E7,protein and m RNA expression levels of KIF7 remained unchanged.3.Knocked down E6/E7 down-regulate the expression of miR-106 a and the expression of LKB1 and p-LKB1(SER428)was up-regulated;Knocked down E6up-regulated expression of KIF7 protein,but knockdown E7 did not regulate KIF7 expression.To further verify the regulatory effect of E6 / E7 on miR-106 a,KIF7,LKB1 and p-LKB1(SER428),E6 / E7-specific si RNA was transferred into A549 cell line which high expression of E6 / E7.The control group was E6 /E7 non-specific si RNA and simulated specific si RNA.The results showed that inhibition of E6/E7 significantly down-regulated the expression level of miR-106 a,significantly up-regulated the expression level of p-LKB1(ser428),and significantly up-regulated the protein and m RNA levels of LKB1.The inhibition of E6 significantly up-regulated KIF7 protein levels,while m RNA levels remained unchanged.Inhibition of E7,the expression levels of KIF7 protein and m RNA level remained unchanged.4.KIF7 inhibited the proliferation of lung cancer cells,promote apoptosis of lung cancer cells.The p EF6/ V5-HIS-KIF7 plasmid was transfected into A549 cell lines with low expression of KIF7,and p EF6/ V5-HIS empty vector and simulated transfection were used as control group.KIF7 specific si RNA was transfected into H1299 cells with high expression of KIF7,and KIF7 non-specific si RNA and simulated specific si RNA were used as control group.CCK-8 assay?cell clonogenesis assay and flow cytometry were used for detection.The results showed that the proliferation ability of A549 cells was decreased significantly after KIF7 overexpression.However,the proliferation ability of H1299 cells was significantly increased after KIF7 interference,the ability of cell cloning was significantly increased,and the proportion of apoptosis was significantly decreased.5.KIF7 up-regulated the expression of LKB1 and p-LKB1(Ser428).To verify whether E6 regulates LKB1 by affecting KIF7 protein expression and whether KIF7 induce LKB1 phosphorylation,we transitively transferred p EF6/V5-HIS-KIF7 plasmid into A549 cell line with low KIF7 expression,and the control group was empty vector p EF6/V5-HIS and simulated transfection.The results showed that overexpression of KIF7,LKB1 protein and m RNA levels were significantly up-regulated.The expression level of p-LKB1(ser428)was significantly up-regulated.6.Knockdown KIF7 could down-regulate the expression of LKB1 and p-LKB1(ser428).The KIF7-specific si RNA was transferred into H1299 cell line with high expression of KIF7.The control group was composed of KIF7 non-specific si RNA and simulated specific si RNA.The results showed that the protein and m RNA expression levels of KIF7 and LKB1 were significantly down-regulated by inhibition of KIF7.The expression of p-LKB1(ser428)was significantly down-regulated.7.KIF7 up-regulated the expression of p-LKB1(ser428)in cytoplasm.To verify KIF7 have impacting on the subcellular localization of p-LKB1(ser428),we transitively transfected p EF6/V5-HIS-KIF7 plasmid into A549 cell lines with low expression of KIF7.The control group was empty vector p EF6/ V5-HIS and simulated transfection.Western Blot results showed that the expression of p-LKB1(ser428)in cytoplasm was significantly up-regulated by overexpression of KIF7.The expression of p-LKB1(ser428)in the nucleus was basically unchanged.8.Knockdown KIF7 could down-regulate the expression of p-LKB1(ser428)in cytoplasm.The KIF7-specific si RNA was transferred into H1299 cell line with high expression of KIF7.The control group was composed of KIF7 non-specific si RNA and simulated specific si RNA.Western Blot results showed that the inhibition of KIF7 significantly down-regulated the expression of p-LKB1(ser428)in cytoplasm.The expression level of p-LKB1(ser428)in the nucleus was basically unchanged.9.miR-106 a promotes proliferation of lung adenocarcinoma cells,and inhibits the apoptosis of lung cancer cells.miR-106 a mimics were transfected into A549 cell lines with low expression of miR-106 a,and miRNA-mimics and mimics were used as control group.miR-106 a inhibitors were transfected into H1299 cells with high expression of miR-106 a,and miRNA-Inhibit and mimic inhibit were used as control group.CCK-8 assay ? cell clonogenesis assay and flow cytometry were used for detection.The results showed that the proliferation ability of A549 cells was significantly increased after transfection with mir-106 a mimics.After transfection with mir-106 a inhibitor,the proliferation ability of H1299 cells was significantly decreased,the cell clonogenesis ability was significantly decreased,and the apoptosis ratio was significantly increased.10.miR-106 a down-regulates the expression of LKB1 and p-LKB1(ser428).miR-106 a mimics were transfected into A549 cells with low expression of miR-106 a,and the control group was miRNA-mimics and mimics.The results showed that overexpression of miR-106 a,LKB1 protein and m RNA expression levels were significantly down-regulated.The expression level of p-LKB1(ser428)was significantly down-regulated.11.miR-106 a up-regulated the expression of LKB1 and p-LKB1(ser428).miR-106 a inhibitors were transfected into H1299 cells with high expression of miR-106 a,and the control group was made of miRNA-Inhibit and mimics inhibit.The results showed that ihibition of miR-106 a,LKB1 protein and m RNA expression levels were significantly up-regulated.The expression level of p-LKB1(ser428)was upregulated.12.miR-106 a down-regulates the expression of LKB1 and p-LKB1(ser428)both in cytoplasm and nucleus.To verify the effect of miR-106 a on p-LKB1(ser428)subcellular localization,we transfected miR-106 a mimics into A549 cells with low miR-106 a expression,and the control group was miRNA-mimics and mimics.After plasma and nuclear proteins isolation,Western Blot results showed that overexpression of miR-106 a significantly down-regulated the expression levels of LKB1 and p-LKB1(ser428)in plasma and nucleus.13.Knockdown miR-106 a up-regulated the expression of LKB1 and p-LKB1(ser428) both in cytoplasm and nucleus.miR-106 a inhibitors were transfected into H1299 cells with high expression of miR-106 a,and the control group was made of miRNA-Inhibit and mimics inhibit.Western Blot results showed that inhibition of miR-106 a significantly up-regulated the expression levels of LKB1 and p-LKB1(ser428)in cytoplasm and nucleus after separation of cytoplasm and nuclear proteins.Conclusion: 1.The E6 protein in HPV16 inhibited the anti-tumor activity of LKB1 in lung cancer cells by down-regulating the expression of KIF7 protein,while E6 protein did not regulate the expression of KIF7 m RNA.2.Although E7 protein in HPV16 can also inhibit the anti-tumor activity of LKB1,but not by regulating KIF7.3.Both E6 and E7 proteins in HPV16 can inhibit the anti-tumor activity of LKB1 in lung cancer cells by up-regulating the expression of miR-106 A,which is inconsistent with the conclusion that E6 protein in cervical cancer has no regulatory effect on miR-106 a reported in the literature.4.Over-expression or interference of KIF7 could regulate the expression level of p-LKB1(ser428)in cytoplasm,there is no change in the expression level of p-LKB1(ser428)in the nucleus.5.The anti-tumor activity of LKB1 is determined by two factors,one is the phosphorylation of LKB1 and the other is subcellular localization,that is,p-LKB1(ser428)must be located in the cytoplasm to exert antitumor activity.One is indispensable. |
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