HPV16 E6/E7 Improve The Glucose Uptake Of GLUT1 By Repressing NDRG2 Expression And Further Nuclear Translocation Of β-catenin In Lung Cancer Cells

Objective: HPV16 is the most common infection subtype,among which E6 and E7 proteins are the most common carcinogenic proteins.Our previous studies found that both E6 and E7 proteins regulated the expression and the glucose uptake of GLUT1 through multiple molecular signaling pathways in lung cancer.However,the potential molecular mechanism has not been identified.According to reports,N-Myc downstream regulated gene 2(NDRG2)had an regulatory effect on GLUT1 expression and its glucose uptake.Nevertheless,the association between HPV16 and NDRG2 has not been proved.Therefore,the purpose of this study was to verify the regulatory relationship among E6 or E7,NDRG2,GLUT1 in lung cancer and their potential mechanisms.Methods: 1.Using transfection and interference techniques to regulate the expression of HPV16 E6 and E7 proteins respectively,detected the expression levels of downstream genes NDRG2,β-catenin and GLUT1 at protein and m RNA by Western blotting and q RT-PCR.2.NDRG2 si RNA was used to inhibit the expression of NDRG2.The changes of downstream genes β-catenin and GLUT1 in protein and m RNA expression were detected by Western blotting and q RT-PCR,respectively.3.β-catenin-specific si RNA was used to knocked down the expression of β-catenin,and the changes of GLUT1 in protein and m RNA expression were detected by Western blotting and q RT-PCR.4.After suppressing the expression of NDRG2 by interference technique,the nuclear translocation of β-catenin was observed by immunofluorescence.5.Glucose uptake assay was used to detect the changes of glucose uptake by GLUT1 after overexpression E6 or E7 and knockdown of E6 or E7,NDRG2,β-catenin respectively.Results: 1.Screening of lung cancer cell lines: NDRG2 protein was highly expressed in H460 cell line,and β-catenin protein was highly expressed in A549 cell line.2.Both E6 and E7 inhibited the expression of NDRG2 but promoted the expression of β-catenin and GLUT1.3.The knockdown of E6 or E7 increased the expression of NDRG2,while decreased the expression of β-catenin and GLUT1.4.The repression of NDRG2 led to upregulate the expression of β-catenin and GLUT1.5.Knocked NDRG2 promotedβ-catenin nuclear translocation.6.The knockdown of β-catenin inhibited the expression of GLUT1.7.The overexpression of both E6 and E7,as well as the knockdown of NDRG2 significantly promoted the glucose uptake of GLUT1,whereas the knockdown of E6,E7 and β-catenin obviously inhibited the glucose uptake of GLUT1.Conclusion: 1.Both E6 and E7 in HPV16 had inhibitory effects of NDRG2 which further resulted in enhanced β-catenin expression and nuclear translocation,furthermore improved the expression and glucose uptake of GLUT1.2.Our findings confirmed the regulatory role of NDRG2 in the occurrence and development of lung cancer,and we further demonstrate the detail relationships among E6/E7,NDRG2,β-catenin,and GLUT1.This discovery is expected to provided a new basis for future lung cancer treatment.