HPV16 E6 And E7 Lead To The Increase Of GLUT1 Transport Activity In Cervical Cancer Cells By Promoting The Carcinogenic Activity Of P65 Suppressing The Nuclear Output Of HIF-1?

Objective:Cervical cancer is the fourth largest malignant tumor threatening women's health in the world,and it is the leading cause of death for women.Although the screening of cervical cancer is easier than other tumors,the treatment cost of cervical cancer is very high,because its radical operation requires extensive cervical resection and pelvic lymph node clearance,so the operation is traumatic,high risk,more postoperative complications,long recovery time and poor prognosis.Some pathological types or late stage,unsatisfactory surgical results and so on,postoperative radiotherapy or chemotherapy is often needed,so the quality of life is seriously reduced.Therefore,the mechanism of cervical cancer is particularly important.Because after the pathogenesis is clear,blocking the incidence of cervical cancer will greatly reduce the cost of treatment and improve the quality of life.In 2008,German scientist Harald Chur Hausen found that human papilloma virus(HPV)infection leads to cervical cancer and won the Nobel Prize in medicine.Then a large number of studies have shown that HPV infection is related to a variety of human diseases,especially the persistent infection of high-risk HPV is closely related to the occurrence of malignant tumors,and cervical cancer accounts for a large proportion of such malignant tumors.It has been found that HPV is a circular double stranded DNA virus.HPV16 is the most common type of cervical cancer,accounting for over 70% of the total incidence rate of cervical cancer.E6 and E7 proteins in HPV16 are the most important oncogenes in the occurrence and development of cervical cancer.Previous studies have found that E6 protein mainly inhibits cell apoptosis by inhibiting p53 gene,while E7 protein mainly promotes cell proliferation by inhibiting retinoblastoma protein(p Rb).Warburg effect suggests that tumor cells consume more glucose than normal cells by glycolysis even under aerobic conditions.At the same time,the molecular mechanism of E6 and E7 proteins in HPV16 affecting glucose uptake of cervical cancer cells has not been reported.Therefore,although the carcinogenic effect of E6 and E7 proteins has been established,the mechanism of E6 and E7 proteins and the occurrence and development of cervical cancer is still in its infancy,especially the regulation of E6 and E7 proteins on aerobic glycolysis pathway is still blank.Therefore,further study on the molecular mechanism of HPV16 infection in the occurrence of cervical cancer has far-reaching significance and practical value for the prevention,control and early treatment of cervical cancer.Methods: 1.E6 and E7 proteins were transfected into C-33 A cells(HPV negative)respectively;E6 and E7 proteins were interfered in Si Ha cells(HPV16 positive),Western blot and qRT-PCR were used to detect the protein and mRNA expressions of E6,E7 and downstream LKB1,PKM2,p65,HIF-1? and GLUT1 at protein andRNA levels,respectively.2.LKB1 was transfected into Si Ha cells;LKB1 was interfered in C-33 A cells,Western blot and qRT-PCR were used to detect the protein and mRNA expression of LKB1 and downstream PKM2,p65,HIF-1? and GLUT1,respectively.3.PKM2 was interfered in Si Ha cells.Western blot and qRT-PCR were used to detect the protein and mRNA expression of PKM2 and downstream p65,HIF-1? and GLUT1.The expression of p65 and p-p65 protein in nucleus and cytoplasm was detected by nuclear cytoplasmic separation and Western blot,respectively.The subcellular localization of p65 was verified by immunofluorescence technique.4.p65 was interfered in Si Ha cells,Western blot and qRT-PCR were used to detect the protein and mRNA expression of p65,HIF-1?and GLUT1 in Si Ha cells.the protein expression of HIF-1? in nucleus and cytoplasm was detected by nuclear cytoplasmic separation technique and Western blot,respectively.In order to verify the effect of p65 on glucose absorption of cervical cancer cells,glucose absorption assay was used to detect the changes of glucose absorption.5.HIF-1? was interfered in Si Ha cells.Western blot and qRT-PCR were used to detect the protein and mRNA expression of HIF-1? and downstream GLUT1 in Si Ha cells.Glucose absorption test was used to detect the change of glucose absorption and verify the effect of HIF-1?on glucose absorption of cervical cancer cells.6.SPSS software was used to analyze all the data of this experiment,and the data of each group were analyzed by two-tailed t test.Results: 1.Western blot and qRT-PCR results showed that E6 and E7 protein inhibited the expression of LKB1 protein and mRNA,and promoted the expression of PKM2,p65,HIF-1? and GLUT1 protein and mRNA.2.Western blot and qRT-PCR results showed that LKB1 inhibited the protein and mRNA expression of PKM2,p65,HIF-1? and GLUT1.3.Western blot and qRT-PCR results showed that PKM2 promoted the protein and mRNA expression of p65,HIF-1? and GLUT1.The results of nuclear cytoplasmic separation and Western blot showed that the protein expression levels of p65 in the nucleus and cytoplasm were changed slightly;the protein expression level of p-p65 in the nucleus was decreased,while that in the cytoplasm was increased;suggesting that the influencing factors of p65 carcinogenic activity were phosphorylation level and localization in the nucleus.Immunofluorescence assay showed that the fluorescence value of p-p65 in the nucleus decreased after PKM2 interference,suggesting that p65 should be phosohorylated and entry into the nucleus in order to activate its dowmstreams.4.Western blot and qRT-PCR results showed that p65 promoted the protein and mRNA expression of HIF-1? and GLUT1.The results of nuclear cytoplasmic separation and Western blot showed that the protein expression level of HIF-1? in the nucleus was significantly decreased,and the protein expression level of HIF-1? in the cytoplasm was slightly increased,suggesting that p65 promoted the accumulation of HIF-1? in the nucleus.The glucose absorption experiment showed that the glucose absorption decreased after p65 interference.5.Western blot and qRT-PCR results showed that HIF-1? promoted the expression of GLUT1 protein and mRNA.Glucose uptake assay showed that HIF-1? promoted glucose uptake.Conclusion: 1.E6 and E7 are up and down regulated by LKB1,PKM2,p65,HIF-1? and GLUT1.HPV16 infection increases glucose absorption of cervical cancer cells and promotes tumor progression.2.The carcinogenic activity of p65 is determined by phosphorylation and nuclear localization.3.The carcinogenic activity of HIF-1? is determined by the accumulation of HIF-1? in nucleus.4.The transport activity of GLUT1 determines the glucose absorption capacity of cervical cancer cells and represents the potential of tumor development.