| Objective: Non Small Lung Cancer(NSCLC)is the major cause of cancer death in the worldwide.More than 80% lung cancer patients were advanced NSCLC patients at the time of diagnosis,and are seriously harmful to our people's health.Although chemotherapy,radiotherapy,and targeted therapy have improved the survival rate,patients with metastatic NSCLC remain have a poor prognosis,the rate of 5 years survival was only 4.2%.Most of advanced NSCLC patients suffered from tumor recurrence and progression,although EGFR-TKI could improve greatly the rate of survival in NSCLC harboring EGFR sensitive mutation,the median progression free survival(PFS)was about 24 months.Therefore,it is necessary and urgent to explore the mechanisms of tumor recurrence and progression in NSCLC.In the recent cancer immunology studies,including tumor immune micro-environment and immune cells ratio imbalance in local tumor niches were significant to tumor recurrence and metastasis.For instance,many researchers suggested tumor expressing highly with immune checkpoints genes(Programmed Death Ligand 1,PD-L1,and Cytotoxic T Lymphocyte Antigen-4,CTLA-4),could integrate with ligands in immune cells,then induce suppression of local anti-tumor immune and influenced worse on chemotherapy overall objective responses(ORRs)and patients' prognosis.Moreover,tumor infiltrating lymphocytes,TIL;regulatory T cell,Treg could also affect ORRs and prognosis.It is quite important to investigate the mechanisms of NSCLC in anti-tumor immune and reverse tumor multidrug resistance,which could help to control tumor recurrence and metastasis,to improve advanced NSCLC patients' prognosis.CBLB(Casitas B-lineage lymphoma)located in chromosome 3q13.11,having 22 exons and the full name is Cbl proto-oncogene B,E3 ubiquitin protein ligase.CBLB belong to Cbl family,including c-Cbl,Cbl-3 and Cbl-b,all of them have some same domain and play important roles in Ubiquitin-proteasome system,UPS.They could involve in many tyrosine kinase negative regulation through substrates degradation selectively.The maintance of immune tolerance is the key point of immune system.All of leukocytes express Cbl-b and keep balance the activation or suppression of the immune cells,therefore conduct anti-tumor immune.Mostly,Cbl-bcan negatively regulate signals activation through antigen or receptor recognition and costimulatory molecules.It is a promising target of immune regulation,for instance,Cbl-b could regulate the threshold of T cell activation throng AKT-2 dependent mechanisms,then decide the fate of Treg.In a paper of Nature in 2014,the findings indicate Cbl-b regulate cancer metastasis via natural killer cells,wich provide a new strategy for cancer immune.In this study,firstly,we investigated CBLB polymorphisms in NSCLC patients and analyzed the associations among SNPs in CBLB and characteristics,prognosis.Furthermore,we testified the candidate SNPs' biology functions in lung cancer cell lines.Furthermore,we studied the associations between CBLB mRNA expression and overall survival(OS).Also,we constructed the CBLB-RNAi lentivirus vector,and transfected into lung cancer cell lines to study the cell migration and invasion,which will assist to furthermore research in the future.Materials and MethodsMethods:393 Caucasian NSCLC patients and 200 Chinese NSCLC patients were enrolled in this study.Blood samples were collected before treatment and CBLB potentially functional SNPs were genotyped by TaqMan methods and first generation sequencing methods in these NSCLC patients.CBLB mRNA levels were determined by RT-PCR.And we verified the biological functions of SNPs by mini-gene system and dual-luciferase reporter gene assay in cell lines.NSCLC patients CBLB mRNA expressions data with clinical survival information were extracted from TCGA database.We analyzed the association between CBLB mRNA expression and overall survival.Lentivirus mediated CBLB RNA interference vector and non-silencing control vector were stably transfected into lung cancer cells.Lung cancer cell lines cell metastasis ability were tested by the transwell migration assay,cell invasion by Matrigel membrane assay and cell proliferation by MTT.In the A549 cells and SW900 transfected with lentivirus mediated CBLB RNA interference vector,expression of PI3 K,p-PI3 K,ERK,p-ERK,Cbl-b,mTOR,p-mTOR,Akt,p-Akt,GSK3?and p-GSK3?proteins were analyzed by Western blot.Statistical analyses were applied with R software and SAS 9.4.OS was determined using univariate log-rank test and multi-variables Cox proportional hazards model(PROC LIFETEST and PROCPHREG were used).Survival analysis was performed by Kaplan-Meier method.All values are expressed as means ± SD.The differences of the results between two groups were evaluated by Student's t-test.P<0.05 was considered to be statistically significant.Results: 1.In the Caucasian and the Chinese NSCLC patients,CBLB potential functional polymorphisms could predict clinical prognosis.Using the screening tool,http://snpinfo.niehs.nih.gov/snpinfo/snpfunc.htm,http://www.regulomedb.org/,potential function domain,MAF > 0.05 and linkage disequilibrium analysis,we identified the three SNPs,rs2305035,rs1042852 and rs7649466 in the Caucasian population;the rs2305035,rs9657904,rs3772534 in Chinese population.The data from Caucasian population in the US was as follow.We identified 393 patients with primary NSCLC who received definitive radiotherapy between March 1998 and February 2009 at a single institution,and genotyped three potentially functional SNPs in CBLB to estimate their associations with local recurrence-free survival(LRFS),distant metastasis-free survival(DMFS),overall survival(OS),and risk of radiation pneumonitis(RP).Our findings suggested compared with those carrying GG genotypes of rs2305035,patients with rs2305035 A variant genotypes(AA and AG)had better LRFS(median 15.8 months vs.15.3 months,adjusted hazard ratio [HR]=0.76,95% confidence interval [CI] 0.60-0.98,P = 0.033),DMFS(median 15.4months vs.14.0 months,adjusted HR=0.74,95% CI 0.57-0.96,P = 0.024)and OS(median 23.5 months vs.22.8 months,adjusted HR=0.72,95% CI 0.56-0.93,P=0.013)times after adjustment in a Cox proportional hazards model.Patients with rs2305035 variant genotypes(GG)had greater risk of developing severe RP(grade ?3 RP)than patients with AA and AG genotypes in an adjusted Cox proportional hazard model.The data from Chinese population was as follow.Initially included 200 patients with advanced NSCLC treated between Oct 2013 and April 2015,from whom DNA samples were available.Blood samples from all enrolled subjects' were collected before treatment.Until to the final visit,116 advanced NSCLC patients received the first-line platinum-based doublet chemotherapy were collected for progression free survival(PFS)analysis and 133 advanced NSCLC patients with overall survival(OS)data for the overall survival(OS)analysis.The used sequencing primers were from Sangon Biotech Co,Ltd Shanghai China.SNP genotyping were applied withABI-3730 XL.PFS and OS were estimated as clinical outcomes.The rs2305035 AA genotype was associated with a worse PFS in female patients and those of non-smokers or light smokers(adjusted HR=3.66,95% CI=1.14-11.81,P=0.030;adjusted HR=3.82,95% CI=1.42-10.24,P=0.008;and adjusted HR=3.72,95%CI=1.39-9.93,P=0.009;respectively),compared with the GG+AA genotypes.We also found that the rs9657904 CC genotype was significantly associated with a worse OS than TT+TC genotypes in male advanced NSCLC patients.Further in silico functional analysis revealed that the rs965704 T allele was significantly associated with lower mRNA expression levels of the CBLB gene.The sample size is small in this study,however the results suggested that the clinical findings with SNPs were different between the US and China,and need larger sample size studies to verify our findings.2.Potential functional SNPs rs2305035 is not testified as the Exonic Splicing Enhancer or Silencer(ESE or ESS)by mini-gene system,rs9657904 is not proved as the transcription factor binding site with Dual-luciferase reporter gene assay.The mutated genotype and wild genotype vector of rs2305035 were constructed by GeneChem Co,Ltd Shanghai China.Transfected to the human renal epithelial cells293 T with 80% fusion in 6 wells dishes by Invitrogen lipofectamine 2000.After 48 hours,we extracted the RNA samples for Real time PCR.The expression levels of CBLB target sequence were the same between the mutated genotype and wild genotype of rs23005035.We got the same result in agarose gel electrophoresis with the reverse transcription product.According to the SNPs functional prediction of transcription factors binding site(TFBS)by http://www.regulomedb.org/,rs9657904 might be as the TFBS of CBLB,and the candidate transcription factor was the Nuclear factor of activated T-cells,cytoplasmic 1(NFATC1).In 293 T cell transfected by NFATC1over-expression plasmid,Cbl-b was up-regulated obviously using western blot test.However,further dual-luciferase reporter gene assay could not testify rs9657904 having effect on the transcriptional activity of CBLB both in 293 T cells and A549 cells.3.We extract the NSCLC patients' survival data and CBLB mRNA expression data from The Cancer Genome Atlas(TCGA)database.The TCGA NSCLC data suggested CBLB mRNA expression levels were not associated with OS in total population,but lower expression of CBLB mRNA was associated with longer OS in squamous lung cancer marginal statistically.Furthermore subgroup analysis found thatlower expression of CBLB mRNA was associated with longer OS in squamous lung cancer and age>65 years.In the adenocarcinoma subgroup,the lower expression of CBLB mRNA was associated with worse OS in the stage III-IV patients marginal statistically;and not in the stage I-II patients.These findings suggested there are difference mechanism of Cbl-b in adenocarcinoma lung cancer and squamous cell lung cancer.4.Lentivirus mediated RNA interference(RNAi)CBLB might inhibit lung cancer cells proliferation through the PI3K-ERK1/2 pathways.The CBLB RNAi target sequences were CCTGATGGGAGGAGTTATA.The vector of Lentivirus mediated RNA interference(RNAi)CBLB was constructed by GeneChem Co,Ltd Shanghai China.After transfected to A549 cells,H1975 cells H1299 cells and SW900 cells,the effect of Cbl-b down-regulation was the best in A549,then the H1975 and SW900.A549,H1975 and SW900 were selected for the migration and invasion research.Compared to the negative control group,A549-RNAi and H1975-RNAi were obviously elevated for the abilities of the migration and invasion by transwell migration assay and Matrigel membrane assay.While the ability of SW900-RNAi was not changed in transwell migration assay,but was suppressed in Matrigel membrane assay.In the western blot experiments of the A549-RNAi,the expression of PI3 K and ERK1/2 was down-regulated,p-PI3 K and p-ERK1/2 were up-regulated,and mTOR were slightly upregulated while p-mTOR was not changed so much;the Akt,p-Akt and GSK3 ? were not changed so much,while p-GSK3 ? was up-regulated,in A549-RNAi group cells.Compared with the SW900-NC group,the expression levels of PI3 K protein in the SW900-KD group were downregulated,but the expression lev-els of p-PI3 K,p-AKT,p-ERK1/2,mTOR,p-mTOR,and p-GSK3?protein were upregulated.There was no significant difference in the expression levels of AKT,GSK3?and ERK1/2 protein between the SW900-NC and SW900-KD groups.Conclusion: 1.In the Caucasian NSCLC patients,genotypes of rs2305035 could predict clinical outcomes;in the Chinese NSCLC patients,rs2305035 and rs9657904 genotypes were associated PFS or OS in the special groups.Biological functions of the two SNPs were not be verified by following experiments.2.The NSCLC data from TCGA database suggested lower expression of CBLB was associated with longer OS in squamous lung cancer marginal statistically,whilewas associated with worse OS in stage III-IV adenocarcinoma lung cancer marginal statistically;which might help to generate hypothesis for difference mechanism of Cbl-b in adenocarcinoma lung cancer and squamous cell lung cancer.3.Lentivirus mediated RNA interference(RNAi)CBLB might promote the abilities of migration and invasion in A549 and H1975 through the PI3K-ERK1/2pathways. |
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