HPV16 E6/E7 Promotes GLUT1 Plasma Membrane Translocation And Glucose Uptake By Inhibiting NBR2 Phosphorylation Of AMPK In Lung Cancer Cells

Objective:According to the global statistical analysis of cancer in 2018,both the incidence and mortality of lung cancer ranked first.Although smoking is considered to be the most important and confirmed risk factor for lung cancer,the number of non-smoking patients is increasing year by year,so it is necessary to explore the causes of cancer other than smoking.In recent years,the correlation between HPV and lung cancer has gradually attracted researchers’ attention.HPV16 E6/E7 protein is the main oncogene.Cancer cells use the Warburg effect to consume more glucose and gain energy through aerobic glycolysis,in which activated GLUT1 is the main glucose transporter.Our previous study showed that HPV16 E6/E7 protein up-regulated GLUT1 expression in lung cancer cells.However,whether E6 and E7 proteins promote GLUT1 glucose uptake during this process and the molecular mechanisms are still unclear.Mechanisms suggested that intracellular GLUT1 overexpression was not the key to GLUT1 activation.Activate GLUT1 by stimulating increased GLUT1 transport from the cytoplasm to the cell membrane,or by stimulating the activity of GLUT1 transporters already present on the cell membrane.Therefore,GLUT1 translocates to the plasma membrane,and the potential molecular mechanism involved in this process is the main objective of the current study.Barnes et al.showed that the mechanism regulating GLUT1 activation is related to the stimulation of AMP-activated protein kinase(AMPK)activity.It has been reported that NBR2 is an lnc RNA induced by glucose deficiency.Under the condition of glucose starvation,the interaction between NBR2 and AMPK can promote the activity of AMPK kinase.At the same time,some researchers proposed that lnc RNA NBR2 regulates the sensitivity of cancer cells to biguanides through GLUT1.Based on the above data,we proposed that HPV16E6/E7,NBR2,AMPK and GLUT1 play a regulatory role in HPVassociated lung cancer cell lines,especially to clarify whether HPV16E6/E7 can upregulate GLUT1 expression by inhibiting NBR2 phosphorylation of AMPK.It also promoted the plasma membrane translocation and glucose uptake of GLUT1 in lung cancer cells.By exploring the role of lung cancer development and the possible molecular mechanism,we can provide a new way for the diagnosis and treatment of HPV-related lung cancer.Research Methods:1.All bronchoscopy examinations were performed by two experienced bronchoscopy physicians using standard video bronchoscopy,flexible long biopsy forceps,and a direct brush.Bronchoscopy biopsy and brush examination were taken from all subjects.Histological and cytological specimens were processed in separate laboratories and reviewed and reported by two different pathologists at different times of the day.Suspected malignant areas were brushed 2-3 times,smear fixed with 95% alcohol,and then pap staining was performed.Next,biopsy specimens were clamps from the same brush site for histological examination,followed by hematoxylin-eosin(H&E)staining and/or immunohistochemical staining.TBNA specimens are used for histological wax blocks and cytological smears.According to the histological diagnosis of clamp biopsy,TBNA and excised specimens,and the cytological diagnosis of brush examination and TBNA,the diagnostic results were recorded into the histological and cytological groups,respectively.2.We biologically regulated the expression of HPV16 E6 and E7 in lung cancer cells by transfection and interference techniques,and detected the protein expression changes of GLUT1,AMPK and p-AMPK(Thr-172)by Western blotting.The m RNA expression levels of GLUT1,AMPK and NBR2 were detected by real-time quantitative polymerase chain reaction(Real-time PCR).3.Transfection and interference techniques were used to bibitively regulate the expression of NBR2 in lung cancer cells.Western blotting and real-time PCR were used to detect the expression levels of AMPK,p-AMPK(Thr-172)and GLUT1,respectively.4.Interfering technology was used to regulate the expression of AMPK in lung cancer cells,and the expression levels of GLUT1 protein and m RNA were detected by Western blotting and Real-time PCR.5.Immunofluorescence assay and glucose uptake assay were used to observe the plasma membrane distribution of GLUT1 and glucose uptake by confocal microscopy.6.The SPSS22.0 statistical analysis software was applied to process and analyze the experimental data,using p<0.05 indicates a significant difference.Results:1.We found that the presence of ciliated cells in the normal cell population,which is easily confused with adenocarcinoma cells,is an important clue.We identified four cell types of fibroblasts,including mucinous columnar cells,ciliated cubic cells,ciliated columnar cells,and reactive ciliated cells.The results of cytological diagnosis are closely related to nuclear enlargement and cell arrangement.Enlargement of single nucleus,arrangement of multistage papilla and general enlargement of nucleus are the main basis for the explanation of adenocarcinoma cells,while escape cells are the main clues for the explanation of suspected cancer cells and dysplastic cells.2.Screenings of lung cancer cell lines Our previous research results indicated that human lung adenocarcinoma cell lines(A549)and human lung squamous cell cell lines(SK)were cell lines with high expression of E6 and E7,respectively,while human lung adenocarcinoma cell lines(H1299)were cell lines with low expression of E6 and E7.Meanwhile,GLUT1 expression on the plasma membrane was observed in A549,SK and H1299 cell lines,and low glucose uptake occurred in all of them.Therefore,E6 and E7 small si RNA interferers were transfected in A549 and SK cell lines,while p EGFP-N1-HPV16 E6 and p EGFP-N1-HPV16 E7 plasmids were transfected in H1299 cell lines.Secondly,the expressions of NBR2 and p-AMPK(Thr-172)in human lung adenocarcinoma(A549),human lung squamous cell carcinoma(SK)and human lung adenocarcinoma(H1299)were detected by Real-time PCR and Western blotting.Human bronchial epithelial cells(HBE)were selected as the control for NBR2 and p-AMPK(Thr-172)expression levels,higher than HBE was considered high expression,and lower than HBE was considered low expression.We found that the expression level of NBR2 was higher in SK,but lower in A549 and H1299.The expression level of p-AMPK(Thr-172)was higher in A549,H1299 and SK lung cancer cell lines.Based on these results,NBR2 mimics were transfected in A549 and H1299 cell lines,and NBR2 inhibitors were transfected in SK cell lines.The expression of endogenous p-AMPK(Thr-172)was interfered with by transfection of small si RNA in A549,H1299 and SK cell lines.3.HPV16E6 /E7 down-regulated the expression of NBR2 and p-AMPK(Thr-172),but up-regulated the expression of GLUT1.We transiently transferred p EGFP-N1-E6/E7 into H1299 with low expression of E6/E7,using empty vector of E6/E7 and simulated transfection as control.Western blotting and real-time PCR were used to detect the transfection efficiency of E6 and the expression of AMPK,p-AMPK(Thr-172),NBR2 and GLUT1,respectively.The results showed that overexpression of E6/E7 significantly downregulated the expression of NBR2 m RNA,AMPK m RNA and p-AMPK(Thr-172)protein,while up-regulated the expression of GLUT1 protein and m RNA,and the protein expression of AMPK showed little or no change.4.E6/E7 knockout up-regulated the expression of NBR2 and p-AMPK(Thr-172),but down-regulated the expression of GLUT1.In order to further verify the regulatory effect of E6/E7 on NBR2,p-AMPK(Thr-172),AMPK and GLUT1,E6/E7 specific si RNA was transfected into A549 and SK cells with high expression of E6/E7,and E6 non-specific si RNA and simulated specific si RNA were used as controls.Western blotting and real-time PCR were used to detect the transfection efficiency of E6/E7 and the expression of AMPK,p-AMPK(Thr-172),NBR2 and GLUT1,respectively.The results showed that inhibition of E6/E7 expression significantly up-regulated the expression of NBR2 m RNA,AMPK m RNA and p-AMPK(Thr-172)protein,while down-regulated the expression of GLUT1 protein and m RNA,and the protein expression of AMPK showed little or no change.5.NBR2 up-regulated p-AMPK(Thr-172)and down-regulated GLUT1 expression.NBR2 mimics were transiently transfected into low expression A549 and H1299 cell lines,with empty NBR2 vectors and simulated transfection as controls.The transfection efficiency and the expression of AMPK,p-AMPK(Thr-172)and GLUT1 were detected by Western blotting and Real-time PCR.The results showed that the overexpression of NBR2 significantly up-regulated the expression of AMPK m RNA and p-AMPK(Thr-172)protein,while down-regulated the expression of GLUT1 protein and m RNA,and showed little or no change in the protein expression of AMPK.6.NBR2 knockout down-regulated p-AMPK(Thr-172)and up-regulated GLUT1 expression.The cell line SK with high expression of NBR2 was selected to use NBR2-specific si RNA to knock out the expression of NBR2,and NBR2 non-specific si RNA and simulated specific si RNA were used as controls.The transfection efficiency and the expression of AMPK,p-AMPK(Thr-172)and GLUT1 were detected by Western blotting and Real-time PCR.The results showed that inhibition of NBR2 expression significantly down-regulated the expression of AMPK m RNA and p-AMPK(Thr-172)protein,while up-regulated the expression of GLUT1 protein and m RNA,and showed little or no change in the protein expression of AMPK.7.AMPK was knocked out and GLUT1 expression was up-regulated.Cell lines(A549,H1299,SK)with high expression of p-AMPK(Thr-172)were selected and the expression of p-AMPK(Thr-172)was knocked out by AMPK-specific si RNA.Non-specific si RNA and simulated specific si RNA were used as controls.Western blotting and real-time PCR were used to detect the expression of AMPK,p-AMPK(Thr-172)and GLUT1,respectively.The results showed that inhibition of P-AMPK(Thr-172)significantly upregulated the expression of GLUT1 protein and m RNA,and the protein expression of AMPK showed little or no change.8.AMPK knockdown significantly promoted GLUT1 plasma membrane translocation and glucose uptake in A549 cells.AMPK-specific si RNA was transfected into A549 cells and non-specific si RNA was used as control.The results of Western blotting showed that the expression of p-AMPK(Thr-172)protein was decreased,while the expression of GLUT1 protein was increased.The expression of GLUT1 in cell membrane was significantly increased and that of GLUT1 in cytoplasm was significantly decreased by immunofluorescence technique under confocal microscope.After AMPK was knocked out,the glucose uptake assay showed an increase in glucose uptake.Conclusion:1.Accurate identification of the ciliary and endplate of benign cells,as well as the nuclear enlargement,arrangement and escape status of adenocarcinoma cells,is of great significance for clinical cytological diagnosis.2.Both E6 and E7 proteins in HPV16 can down-regulate the expression of NBR2 m RNA.3.NBR2 promotes the phosphorylation of AMPK.4.Inhibition of AMPK promotes GLUT1 plasma membrane translocation and glucose uptake.