| Objective: HPV16 E6/E7 proteins were the main oncogenes and only long-term persistent infection was possible to cause lung cancer.The cancer cells use Warburg effect to consume more glucose to obtain energy by aerobic glycolysis and activated GLUT1 is the main glucose transporter in the process.Our previous studies have shown that HPV16 E6 / E7 protein upregulates the expression of GLUT1 in lung cancer cells.However,whether E6 and E7 protein can promote the glucose uptake of GLUT1 and its molecular mechanism are unclear.Mechanism studies showed that the overexpression of intracellular GLUT1 is not the key point for GLUT1 activation.GLUT1 activation either by stimulating to increase the translocation of GLUT1 from the cytoplasm to the cell membrane,or stimulating the activities of GLUT1 transporters that were already present on the cell membrane.Thus,GLUT1 is translated to the cell membrane and the potential molecular mechanisms involved in the process are the main aims of current study.As a tumor suppressor gene,miR-451 was aberrantly expressed in many cancer cells,and it played an important role in cancer development and metastasis.Studies showed that miR-451 inhibited the growth of cancer cells via down-regulated phosphatidylinositol-3kinase(PI3K)/AKT signaling pathway by directly inhibited its target gene calcium-binding protein 39(CAB39);Gou et al demonstrated that miR-451 negatively regulated GLUT1 by targeting CAB39 in glioma cells.Therefore,studying the relationship between HPV16 E6/E7,miR-451,CAB39 and GLUT1,and whether CAB39 promotes the translocation of GLUT1 to the plasma membrane and the absorption of glucose are the main aims of our current research on HPV-related lung cancer,at the same time,in order to find genes that promote tumor progression and their regulatory relationships,the treatment of HPV-related lung cancer provides a new approach and can provide new research directions for tumor research related fields.Methods: The expression of HPV16 E6 and E7 were first detected by double directional transfection technology in lung cancer cell lines,Western blotting and real-time quantitative polymerase chain reaction(q RT-PCR)experiments to detected CAB39 and GLUT1 proteins and m RNA,the expression of PI3Kp85,P-AKT and AKT protein level only and the m RNA expression level of miR451;then the transfection mimics and inhibitors technology are used to regulate the m RNA expression of miR451 in lung cancer cells,and Western blotting and real-time Quantitative polymerase chain reaction(q RT-PCR)experiments were used to detect the expression levels of CAB39 and GLUT1 protein and m RNA,and the expression levels of PI3Kp85,P-AKT and AKT proteins only;two-way transfection technology was used to regulate the expression of CAB39 in lung cancer cells,and the protein was applied Western blotting and real-time quantitative polymerase chain reaction(q RT-PCR)experiments respectively detected the expression levels of GLUT1 protein and m RNA,and the expression levels of PI3Kp85,P-AKT and AKT proteins;Transfection technology was used to regulate the expression of CAB39 in lung cancer cells,immunofluorescence experiment technology and glucose absorption experiment technology were used respectively,and the plasma membrane distribution of GLUT1 and glucose absorption were detected by confocal microscope and flow cytometry;Co-transfection technology was used to regulate the expression of CAB39 and PI3K/AKT pathways in lung cancer cells,Western blotting was used to detect the expression of PI3Kp85,P-AKT and AKT proteins,and immunofluorescence experiment technology and glucose absorption experiment technology were detected the plasma membrane distribution of GLUT1 and glucose absorption by confocal microscope.Results: 1.The screening of lung cancer cell lines.First,based on our previous results,A549 and LK2 cells were high E6 and E7 expression cell lines,and H460 cells were low E6 and E7 expression cell line.Therefore,we selected the H460 cell line to transfect p EGFP-N1-HPV16 E6 and p EGFP-N1-HPV16 E7 plasmids,and transfect E6 and E7 small si RNA interferors in the A549 and LK2 cell lines.Then,to investigate the roles of miR-451 and CAB39 on regulating GLUT1 translocation to the plasma membrane in lung cancer cell,several lung cancer cell lines were tested and three cell lines were selected(A549/LK2/H460).Real-time quantitative polymerase chain reaction experiment was used to detect the m RNA expression of miR451,Western blotting was used to detect the expression of CAB39 protein,and immunofluorescence and glucose absorption experiments were used to detect the plasma membrane distribution of GLUT1 and glucose absorption,Human bronchial epithelial(HBE)cell line was selected to be used as control for the expression levels of miR-451 and CAB39,higher than HBE was considered as high expression and lower than HBE was considered as low expression.The expression level of miR-451 was high in LK2,but low in A549 & H460.The expression level of CAB39 was high in A549,medium in LK2,and low in H460.Low expression level of GLUT1 on the plasma membrane was observed in all three cell lines.Low glucose uptake(average fluorescence intensity per cell)happened in all three cell lines.According to the above results,design and carry out further measurements.Based on this result,we chose to transfect miRNA-451 mimics in A549 and H460 cell lines,and transfect miRNA-451 inhibitors in LK2 cell lines;chose a plasmid transfected with CAB39 in H460 and LK2 cell lines,Transfection of small si RNA in A549 and LK2 cell lines interfered with the expression of endogenous CAB39.2.HPV16 E6/E7 downregulated the expression of miR-451 but upregulated the expression of CAB39,PI3K(P85?),P-AKT(ser473),and GLUT1.The p EGFP-N1-E6 or E7 vectors were transiently transfected into the low expression H460 cell line,E6 or E7 empty vector and mock transfection as control.The results presented that overexpression of E6 or E7 significantly downregulated the expression of miR-451,but upregulated the expression of CAB39,PI3K(P85?),and P-AKT(ser473)at protein levels only,as well as upregulated the expression of GLUT1 at both protein and m RNA levels.The expression of AKT showed minimal or no change.3.Knocked down of E6 or E7 up-regulated the expression of miR-451,and down-regulated the expression of CAB39,PI3K(P85?),P-AKT(ser473)and GLUT1.To further verify the regulatory roles of both E6 or E7 on miR-451?CAB39,PI3K(P85?),P-AKT(ser473),and GLUT1,we applied E6 or E7-specific si RNA to knockdown the expression of E6 or E7 in the A549 and LK2 cell lines,E6 or E7-nonspecific si RNA and mock specific si RNA were used to serve as the controls.The results indicated that the inhibition of both E6 and E7 upregulated the expression of miR-451 at m RNA levels,but downregulated the expression of CAB39,PI3K(P85?),and P-AKT(ser473)at protein levels only,as well as downregulated the expression of GLUT1 at both protein and m RNA levels.The expression of AKT showed minimal or no change.4.miR-451 down-regulated the expression of CAB39,PI3K(P85?),P-AKT(ser473),and GLUT1.The Hsa-miR-451 was transiently transfected into the low expression H460 and A549 cell lines,miRNA-mimics and mock transfection as control.The overexpression of miR-451 significantly downregulated the expression of CAB39 and GLUT1 at both protein and m RNA levels,and the expression of PI3K(P85?)and P-AKT(ser473)at the protein level only.The expression of AKT showed minimal or no change.5.Knocked down miR-451 up-regulation the expression of CAB39,PI3K(P85?),P-AKT(ser473),and GLUT1.We transfected Hsa-miR-451 inhibitor in high expression cell line LK2,and used miRNA-inhibitors and mock inhibitor as control.The down-expression of miR-451 significantly upregulated the expression of CAB39 and GLUT1 at both protein and m RNA levels,and the expression of PI3K(P85?)and P-AKT(ser473)at the protein level only.The expression of AKT showed minimal or no change.6.CAB39 upregulated the expression of PI3K(P85?),P-AKT(ser473),and GLUT1.The pCMV3-C-Myc-CAB39 vector was transiently transfected into the H460 and LK2 cell lines,pCMV3-C-Myc-empty vector and mock transfection as control.The results showed that overexpression of CAB39 significantly upregulated the expression of GLUT1 at both protein and m RNA levels,and the expression of PI3K(P85?)and P-AKT(ser473)at the protein level only whereas the expression of AKT showed minimal or no change.7.Knocked CAB39 downregulated the expression of PI3K(P85?),P-AKT(ser473)and GLUT1.CAB39-specific si RNA to knockdown the expression of CAB39 in the A549 and LK2 cell lines was performed,CAB39-nonspecific si RNA and mock specific si RNA as control.The results indicated that the inhibition of CAB39 downregulated the expression of GLUT1 at both protein and m RNA levels,and the expression of PI3K(P85?)and P-AKT(ser473)at the protein level only whereas the expression of AKT showed minimal or no change.8.The overexpression of CAB39 significantly promoted both the plasma membrane translocation and the glucose uptake of GLUT1 in the H460 and LK2 cells,The image analysis data showed that GLUT1 expression could only be detected on the plasma membrane transfected with CAB39,but not on the plasma membrane transfected with empty vectors.Compared with the plasm membrane transfected with empty vector,the percentages of GLUT1 expression on the plasma membrane of transfected CAB39 were significantly higher in H460(45.5%)and LK2(67.1%)cells.The flow cytometry data showed that the glucose mean density was 3.3 times higher in H460 cells transfected with CAB39 than that transfected with empty vectors,and 2.0 times higher in LK2 cells transfected with CAB39 than that transfected with empty vectors.9.CAB39 promoted the activation of GLUT1 and the promotion was depended on PI3K/AKT pathway.To further verify whether the promotion of CAB39 was depended on PI3K/ATK pathway,we used specific PI3K/AKT blocker,Miltefosine,to inhibited PI3K/AKT pathway in CAB39 transfected H460 and LK2 cell lines.The results showed that the promotion effects of CAB39 on GLUT1 protein expression,plasma membrane translocation,and glucose uptake were reversed.Conclusion:1.Both E6 and E7 proteins in HPV16 can relieve the inhibitory effect of miR-451 on CAB39 For multiple lung cancer cell lines;2.CAB39 is a key regulatory point for transport to the plasma membrane,up-regulating the expression of GLUT1 protein and m RNA and promoting its membrane localization and glucose absorption;3.The membrane localization of GLUT1 and the increase in glucose uptake induced by CAB39 are achieved through the PI3K/AKT signaling pathway. |
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