HPV16 E6/E7 Upregulated The MRNA Expression And The Amplification Of HTERC By Releasing LKB1 To Promote Phosphorylation Of SP1 In Lung Cancer Cells

Objective:Lung cancer is the most common malignant tumor of the respiratory system,the incidence and mortality of lung cancer has reached the first place in all system tumors.Current researches indicate that the occurrence and progress of lung cancer is not only related to the signal pathways thay related to the abnormal initiation of cell proliferation,invasion and metastasis,but also closely related to the abnormal increase of telomerase activity.Therefore,comprehensive understanding of the regulation mechanism of telomerase activity in lung cancer cells can provide new directions for the development of new tumor targeting drugs and provide new ideas for clinical diagnosis and treatment.HPV(human papilloma virus)is a small DNA virus.Large numbers of epidemiological evidences show that a variety of human benign and malignant diseases are closely related to the infection of HPV,and long-term persistent high-risk HPV infection may induce cervical cancer.HPV16 is a common high-risk HPV,E6 and E7 are the two main oncoprotein of HPV16.It is reported that HPV16 is closely related to the occurrence and progression of non-small cell lung cancer(NSCLC),However,the mechanism of E6/E7 in the occurrence and progression of NSCLC is not clear.My previous studie have found that E6/E7 have the ability to inactivate LKB1,and LKB1 reduce the expression of hTERT via downregulating transcription activity of transcription factor SP1.Although we have found a possible pathway for E6/E7 to promote cancer development and progression,the specific mechanism hidden behind this phenomenon is not clear yet.Fan et al.reported that the amplification ratio of the RNA template of telomerase hTERC gene in the cell was significantly higher in the NSCLC than in benign cells.Hopman et al.also reported that the amplification of hTERC copy number is significantly related to the integration of HPV gene in lung cancer.But the specific mechanism of the above phenomenon is not clear yet.We speculate that E6/E7 may up regulate the transcriptional activity of SP1 by inhibiting LKB1 and eventually up-regulation the expression of hTERC.The purpose of this study is to reveal the specific regulatory mechanisms among HPV16 E6/E7,LKB1,SP1 and hTERC in lung cancer cells,and to provide new ideas for early diagnosis and treatment of HPV16 related NSCLC.Methods: Patients and specimens.The study was conducted according to the guidelines of the institutional review boards at the First Affiliated Hospital of China Medical University,we have obtained internal review board approval and/or patients informed consent for this study.A total of 106 brushing cells of patients with lung cancer who attended the laboratory of cytopathology at the First Affiliated Hospital of China Medical University during the period 2013-2014 were included in the study.There were 88 males(83.0%)and 18 females(17.0%),with a mean age of 64.3 years(range 40�79).Of the malignant cells,there were 20 adenocarcinomas(AC)and 86 squamous cell carcinomas(SCC).These 106 patients,as well as 68 randomly selected patients without lung cancer(58 with inflammation and 10 with endobronchial tuberculosis),were included as study subjects and control subjects,respectively.These all had biopsies,resections or clinical follow-up negative for malignancy.All bronchoscopies were performed by two experienced bronchoscopists.Forceps biopsies and brushings were obtained from all subjects,including three to four forceps biopsies and two endobronchial brushings.In order to obtain sufficient numbers of cells in the brushings,these were taken before the forceps biopsies.Endobronchial brushings were transferred to a small vial containing SurePathTM preservative fluid(BD Tripath,Burlington NC,USA).A mucolytic agent(1 m L,BD Tripath)was added to the brushings in SurePath TM preservative fluid,incubated and vortexed.Additional mucolytic agent was added to the mixture until the mucus was completely lysed.The mixture was transferred and centrifuged.The supernatant was removed and the pellet was resuspended in distilled water.This suspension was vortexed again and centrifuged.The supernatant was removed and the pellet was vortexed and transferred to a AutoCyte PREP system(BD Tripath)for automatic preparation and staining of slides.Cytology was recorded as positive if malignant cells were reported.Specimens that were reported as �atypical' have been included as �negative' in the present study,because the patients with uncertain results cannot be treated as malignant in clinical practice.The residual material was used for RNA extraction.Cell culture.The H1299,H460 and A549 cell lines were obtained from the Shanghai Cell Bank.The LK2 cell line was a gift from Dr.Hiroshi Kijima.We emplyed qRT-PCR(quantitative real-time polymerase chain reaction)for detection of E6/E7,LKB1,SP1 and hTERC in bronchial brushing cells mRNA expression and the correlation among each other,FISH(fluorescence in situ hybridization)was used to detect the abnormal amplification level of hTERC and the association of which with the mRNA expression levels of E6/E7,LKB1 and hTERC.At first,we genetically manipulated the expression of E6/E7 in lung cancer cell lines,using Western blot and qRT-PCR,luciferase and FISH,detected the protein and mRNA expression of E6/E7 and LKB1,the protein,mRNA expression and transcriptional activity of SP1 and the mRNA expression,amplification of hTERC.Then we genetically manipulated the expression of LKB1 in the same four lung cancer cell lines,using Western blot and qRT-PCR,luciferase and FISH,detected the protein and mRNA expression of LKB1,the protein,mRNA expression,transcriptional activity and the phosphorylation level changes of threonine 739 and threonine 453 sites of SP1,and the mRNA expression,abnormal amplification of hTERC.Finally,we interfered the expression of SP1 by small interfering RNA(siRNA),detected the mRNA expression of hTERC,and also using ch-IP(chromatin immunoprecipitation)to detecte the binding of SP1 to hTERC promoter.Results: 1.E6 mRNA,E7 mRNA,SP1 mRNA and hTERC mRNA and the amplication of hTERC were more significantly increase in the malignant group compared with the benign group,whereas LKB1 mRNA was significantly decreased in the malignant group(P < 0.01).In the malignant group,SP1,hTERC mRNA and the amplification of hTERC was strongly positively correlated with E6,E7 m RNA(P < 0.01),whereas LKB1 mRNA was negatively correlated with E6,E7,SP1,hTERC mRNA and the amplification of hTERC(P < 0.01).2.Compared with the control groups,overexpression of E6/E7 significantly decreased the protein,mRNA expression level of LKB1 but upregulated the protein,mRNA expression level,transcriptional activity of SP1,as well as the mRNA expression level and the amplification of of hTERC.In contrast,E6/E7 knockdown in A549 and LK2 cells,increased the protein,mRNA expression level of LKB1,but downregulated the protein,mRNA expression level,transcriptional activity of SP1 as well as the mRNA expression level and the amplification of hTERC compared with the control groups.3.Compared with the control groups,transfection of LKB1 significantly decreased the protein,mRNA expression level,transcriptional activity of SP1,as well as the mRNA expression level of hTERC.In contrast,LKB1 knockdown in H1299/NCI-H460 cells increased the mRNA,protein expression level,transcriptional activity of SP1,as well as the mRNA expression level of hTERC.And overexpression of LKB1 significantly enhanced the phosphorylation of SP1 at Thr739 and Thr453.In contrast,LKB1 knockdown inhibited the phosphorylation of SP1.4.With the delay of time,the expression of E6/E7 increased,LKB1 gradually reduced to none,and the phosphorylation of SP1 at Thr739 and Thr453 was gradually reduced.5.SP1 knockdown in H1299/A549 cells significantly decreased the mRNA expression of hTERC and SP1 could be directly bind to the hTERC promoter region.Conclusion: 1.The mRNA expression and amplification of hTERC were positively correlated with the mRNA expression of E6/E7,and negatively correlated with the mRNA expression of LKB1.2.In lung cancer cells,both E6 and E7 have inhibition effect on the protein and mRNA expression of LKB1.3.HPV16 E6/E7 inactivate LKB1 and relieves the phosphorylation of LKB1 on SP1,upregulate the mRNA expression and amplification of hTERC,therefore play a key role in the occurrence of lung cancer.