Mechanistic Study On The Inhibitory Effect Of Propofol On LPS-induced Aggressiveness Of Non-small Cell Lung Cancer

Part 1 Study on the relationship between HIF-la expression levels and NSCLC prognosisObjective:To determine the relationship between HIF-la expression levels and lymph node metastasis,differentiation status,tumor-node-metastasis(TNM)stage or prognosis of NSCLC.Methods:1.The online software 'Oncomine' was used to analyze HIF-1? mRNA levels of cancer and adjacent tissues in the lung cancer microarray data sets from TCGA(Wachi lung and Stearman lung).2.Immunohistochemical staining was carried out to analyze the clinical relevance of HIF-1 a expression in 187 human NSCLC specimens.3.Kaplan-Meier analysis was performed to study the prognosis of NSCLC patients with different HIF-1? expression.4.The relationship between NSCLC clinicopathologic features and HIF-1? expression levels was analyzed based on IHC scores of HIF-1?.Results:1.In Wachi lung and Stearman lung sets,higher HIF-la mRNA levels were found in lung adenocarcinoma and squamous carcinoma when compared with normal lung tissues.2.The 5-year overall survival(OS)rate of the HIF-1? high expression patients was significantly lower than that of the HIF-1 a low expression patients(18.28 vs.34.04%;P=0.0001).3.HIF-1 a staining was positively correlated with tumor size,lymph node metastasis,differentiation status and tumor-node-metastasis(TNM)stage.Conclusion:Compare with para-tumor tissues,the expression levels of HIF-la were increased in NSCLC tissues,which may be associated with the malignant clinical features and poor prognosis of NSCLC patients.Part 2.Study on the inhibitory effect of propofol on LPS-induced upregulation of HIF-la of non-small cell lung cancerObjective:To investigate the effects of LPS and propofol on the HIF-la expression in NSCLC cellsMethods:1.Different concentrations of LPS(0,1,5,10 ?g/ml)were co-cultured with non-small cell lung cancer A549 cells.The expression of HIF-1 alpha mRNA was detected by fluorescence quantitative PCR(qRT-PCR),the expression of HIF-1 alpha protein was detected by Western blot,and the level of ROS was detected by chemiluminescence quantitative method.2.After treated with 10 ?g/ml LPS,A549 cells were treated with different concentrations of propofol(0?5?10?20 ?g/ml).The expression level of HIF-1 a mRNA in A549 cells was detected by qRT-PCR,the expression of HIF-la protein was detected by Western blot,and the level of oxygen free radical ROS was detected by chemiluminescence quantitative method.3.A549 cells were divided into control group,LPS group(10?g/ml)and propofol group(10?g/ml LPS with 20 ?g/m propofol)for 12 hours.The protein stability of HIF-la was detected by cycloheximide tracing assay(CHX-Chase assay).The subcellular localization of HIF-1? protein was detected by immunofluorescence assay(IF)and the transcriptional activity of HIF-1 was detected by reporter gene assay(reporter assay).Results:1.LPS induced the expression of HIF-1? and ROS generation in A549 cells.2.Propofol suppressed LPS-induced HIF-1? expression and ROS generation.3.LPS-induced protein stability and nuclear accumulation of HIF-1? was attenuated by propofol,which inhibited the transcriptional function of HIF-1.Conclusion:LPS enhanced the transcriptional activity of HIF-1 through increasing expression,protein stability,and nuclear accumulation of HIF-1? in NSCLC cells,while propofol inhibited the effect of LPS on HIF-1?.Part 3.Study on the role of miR-199a-5p on the inhibitory effect of propofol in LPS-induced upregulation of HIF-1? of non-small cell lung cancerObjective:To investigate the role of propofol in HIF-la expression induced by LPS in non-small cell lung cancer(NSCLC)and whether it is regulated by miR-199 and its mechanism.Methods:1.Targetscan and BiBiserv2 were used to predict the putative binding site of miR-199 in HIF-la 3'UTR.2.The levels of HIF-la mRNA and miR-199a/b-5p were examined by quantitative real-time pcr(qRT-PCR).3.The protein level of HIF-1? was examined by Western blot.4.The effect of miR-199a-5p on HIF-1? mRNA was assessed by luciferase reporter assay.5.Bisulfite genomic sequencing(BSP)was used to measure the methylation levels of CpG islands at miR-199a promoter.Results:1.MiR-199a-5p suppressed HIF-la expression via directly targeting the 3'UTR of HIF-la mRNA in A549 cells.2.LPS treatment reduced the level of miR-199a-5p and increased HIF-la expression,which was attenuated by propofol.3.The methylation levels of CpG islands at miR-199a promoter were upregulated by LPS but decreased when administering propofol.4.Eliminating the LPS-induced ROS by catalase reduced the methylation levels of CpG islands at miR-199a promoter,which then increased miR-199a-5p level.5.Suppressing the LPS-induced methylation of miR-199a promoter promoted miR-199a-5p level and inhibited HIF-la expression.Conclusion:Propofol upregulated miR-199a-5p level through inhibiting the LPS-induced methylation of miR-199a promoter,which decreased HIF-1? expression.Part 4.Study on the inhibitory effect of propofol on epithelial mesenchymal transformation(EMT)in non-small cell lung cancer induced by LPSObjective:To investigate the inhibitory effect of propofol on epithelial mesenchymal transformation(EMT)in non-small cell lung cancer induced by LPS.Methods:A549 cells were treated with LPS(10?g/ml)or LPS(10?g/ml)combined with propofol(20?g/ml)?1.The mRNA level of E-cadherin,Vimentin,and MM2/9 was examined by quantitative real-time pcr(qRT-PCR).2.The protein level of E-cadherin,Vimentin,and MM2/9 was examined by Western blot.3.The migratory ability was evaluated by wound healing assay.4.The invasive ability was evaluated by transwell assay.Results:1.LPS induced the downregulation of epithelial marker E-cadherin and downregulation of mesenchymal marker vimentin in A549 cells,which was attenuated by propofol.2.LPS increased the expression of MMP2/9,which was abrogated by propofol.3.In LPS-treated A549 cells,knockdown of HIF-la inhibited the expression of Vimentin and MMP2/9 but increased E-cadherin.4.LPS enhanced migratory and invasive capabilities of A549 cells,while propofol undermined the promotive effect of LPS on the malignant phenotypes of A549 cells.As expected,overexpression of HIF-la antagonized the anti-tumor function of propofol.Conclusion:Propofol suppressed LPS-induced EMT and aggressiveness in NSCLC cells through reducing HIF-1? expression.