| Objective: As we all know,lung cancer is one of the most common malignant tumors in the world,which is a serious threat to human health and quality of life.In our country,the incidence and mortality of lung cancer are showing a gradual upward trend.At the same time,the cancer burden of lung cancer in China is relatively heavy,accounting for 36.98% of the total number of cases and 39.21% of the total number of deaths in the world.The hidden symptoms of early lung cancer may lead to the loss of surgical opportunity in early diagnosis.Therefore,chemotherapy or radiotherapy has become the main treatment for patients with advanced lung cancer.Despite the progress in terms of diagnosis and treatment of lung cancer,but the 5-year survival of patients with lung cancer has been less than satisfactory.The occurrence and development of lung cancer are closely related to the abnormal expression of many genes,such as oncogenes and tumor suppressor genes.Therefore,in-depth exploration of the molecular mechanisms of lung cancer progression will help to develop new lung cancer prevention and treatment targets.Micro RNAs(mi RNAs)are a class of endogenous(18-24 nucleotides)non coding single stranded RNA molecules,which play an important role in different biological processes such as metabolism,growth,cell differentiation and development,apoptosis,and cell signal transduction.By matching the complementary sequence of mi RNA seed region(2-8)with the target 3'UTR and encoding region,m RNA target was identified and m RNA degradation and translation inhibition were used as endogenous inhibitors of gene expression.At the same time,they also play an important role in post transcriptional regulation of gene expression.A large number of studies have proved that whether the single nucleotide gene polymorphisms in pri-mi RNA and pre-mi RNA that affect mi RNA biogenesis and change the expression level of mature mi RNA,or the single nucleotide polymorphisms that affect the ability of mi RNA to recognize its m RNA target sequence or the formation of mi RNA targeted double strand,they may affect human phenotypic variation and disease susceptibility by modifying mi RNA regulation.Previous studies have shown that mi R-143 plays a major role as a tumor suppressor gene in a variety of human cancers.In this paper,we studied the relationship between the single nucleotide polymorphism in the promoter region of mi R-143 and the risk of lung cancer,the relationship between the CBFB polymorphism of mi R-143 target gene and the risk of lung cancer,the expression of mi R-143 and CBFB of lung adenocarcinoma tissues from TCGA database,and the effect of mi R-143 on the biological behavior of lung cancer cells.Four aspects of behavioral influence are preliminary exploration of the mechanism of miR-143 and CBFB in lung cancer.Methods:First,we browsed the literature and screened the results of the NCBI db SNP database,and at the same time integrated the SNP function prediction website,we selected the mi R-143/mi R-145 promoter region rs3733845 and rs3733846 sites to be studied for the next step Research.Our study is a hospital-based case-control study to explore the relationship between the rs3733845 and rs3733846 polymorphisms in the mi R-143/145 promoter region and the risk of lung cancer in 575 non-smokers and 575 cancer-free controls.Our cases came from three hospitals,they are the First Affiliated Hospital of China Medical University,the Fourth Affiliated Hospital of China Medical University and Liaoning Cancer Hospital.All research subjects signed an informed consent form.This study was approved by the Ethics Committee of China Medical University.We used the Taq Man probe method to genotype two single nucleotide polymorphisms(SNPs)in the mi R-143/145 promoter region of 575 patients and 575 controls.Logistic regression analysis was used to evaluate the relationship between the polymorphism of mi R-143/mi R-145 promoter and the risk of lung cancer in women.A cross-analysis method was used to explore the interaction between two SNPs and environmental risk factors(cooking oil fume exposure and passive smoking exposure).In the second part of the study,we studied and explored the relationship between the rs12598154 and rs17768165 polymorphisms of the CBFB gene in non-smoking women and the risk of lung cancer.This part is a hospital-based case-control study.Our research subjects include 556 cases and 395 controls,and all have signed informed consent.This study was approved by the Ethics Committee of China Medical University.Professionally trained interviewers collect demographic data and information on environmental exposure(cooking oil fume and passive smoking exposure).The Taq Man probe method was used to genotype the genomic DNA of all subjects.Logistic regression analysis was used to evaluate the relationship between rs12598154 and rs17768165 of CBFB gene and the risk of lung cancer in non-smoking women.A crossanalysis method was used to explore the interaction between two SNPs and environmental risk factors(cooking oil fume exposure and passive smoking exposure).The third part of the study: We firstly downloaded and analyzed lung adenocarcinoma data from the TCGA database,and used R software to analyze and explore the expression,prognosis,and possible biological pathways of mi R-143-3p in lung adenocarcinoma.Using biological information websites(Targetscan,mi RWalk,mi RDB,mi RDIP),the target gene CBFB of mi R-143-3p was predicted.Secondly,we downloaded and analyzed lung adenocarcinoma related data from the TCGA database,and used R software to analyze and explore the expression,prognosis and possible biological pathways of CBFB in lung adenocarcinoma.Part 4: Firstly,the expression of mi R-143-3p in lung adenocarcinoma cell lines A549 and H1299 and human normal lung epithelial cell line BEAS-2B was detected by qRTPCR,and mi R was transfected into A549 and H1299 cell lines-143-3p mimics,inhibitor and NC plasmids change the expression of mi R-143-3p.The change of lung adenocarcinoma cell proliferation ability was detected by MTS experiment;the change of lung adenocarcinoma cell migration ability was detected by Transwell experiment;Annexin 7-AAD/APC detected changes in the apoptotic ability of lung adenocarcinoma cells.The dual luciferase experiment and the recovery experiment verified the effect of mi R-143-3p on cell proliferation,migration,and apoptosis by inhibiting the expression of CBFB.In this study,we constructed CBFB over-expression plasmids,lowexpression plasmids,and negative control plasmids to change the expression of CBFB in A549 and H1299 cells.The MTS experiment was used to detect the changes in the proliferation of lung adenocarcinoma cells;the transwell experiment was used to detect the migration ability of lung adenocarcinoma cells;The changes in lung adenocarcinoma cell apoptosis ability were detected by Annexin 7-AAD/APC.Results: Part 1: The relationship between rs3733845 and rs3733846 and lung cancer risk is not statistically significant.However,in the stratified analysis,the rs3733845 T allele increased the risk of lung adenocarcinoma.Subjects carrying the AG genotype of rs3733846 showed a higher risk of lung adenocarcinoma compared with the AA genotype.At the same time,compared with the AA genotype,the risk of lung adenocarcinoma associated with carriers of the G allele of rs3733846 is significantly higher in the dominant model.There is no statistically significant association between rs3733845 and rs3733846 and lung squamous cell carcinoma and small cell lung cancer.The interaction between rs3733845 and rs3733846 risk genotypes and cooking oil fume exposure is statistically significant on a multiplicative scale.The interaction between rs3733845 and rs3733846 loci and passive smoking exposure is not statistically significant in association with lung cancer susceptibility.Part 2: The rs12598154 and rs17768165 sites of CBFB have no statistically significant association with the risk of lung cancer.The results of stratified analysis showed that in non-smoking females,the rs12598154 and rs17768165 sites of CBFB were not statistically associated with the susceptibility of lung adenocarcinoma,lung squamous cell carcinoma,and lung small cell carcinoma.In the non-smoking female population,the interaction between the rs12598154 and rs17768165 loci of CBFB and cooking oil fume exposure was not statistically significant in association with lung cancer susceptibility.The interaction between rs12598154 and rs17768165 sites of CBFB and passive smoking exposure is not statistically significant in association with lung cancer susceptibility.Part 3: The expression of mi R-143-3p in the lung adenocarcinoma dataset in the TCGA database was significantly down-regulated and had a good diagnostic performance.Bioinformatics predicts that mi R-143-3p may participate in the p53 signaling pathway.Using Targetscan and other bioinformatics websites were used to predict that CBFB may be a downstream target gene of mi R-143-3p,and the expression of CBFB and mi R-143-3p in the lung adenocarcinoma dataset of the TCGA database was negatively correlated.In the lung adenocarcinoma dataset of the TCGA database,the expression of CBFB was significantly up-regulated,and the diagnostic performance was good,and the differential expression of CBFB is significantly related to the prognosis of patients with lung adenocarcinoma.The results of GSEA predict that CBFB may participate in the p53 signaling pathway.Part 4: Results showed that mir-143-3p was significantly lower expressed in lung adenocarcinoma cells.Compared with negative control group,the low expression of mir-143-3p could significantly promote the proliferation and migration of lung adenocarcinoma cells,and significantly inhibit the apoptosis of lung adenocarcinoma cells.Upregulation of mi R-143-3p expression can significantly inhibit the proliferation and migration of lung adenocarcinoma cells,and enhance the apoptosis ability of lung adenocarcinoma cells.Results showed that mi R-143-3p expression was significantly up-regulated in p53 overexpression group.QRT-PCR and Western Blot results showed that mi R-143-3p could significantly inhibit the expression of CBFB.The results of cellular functional recovery assay showed that mi R-143-3p could regulate the proliferation,migration and apoptosis of lung adenocarcinoma cells by significantly inhibiting the expression of CBFB.Results showed that CBFB was highly expressed in lung adenocarcinoma cells.Compared with the negative control group,the high expression of CBFB could significantly promote the proliferation and migration of lung adenocarcinoma cells,and significantly inhibit the apoptosis of lung adenocarcinoma cells;on the contrary,the low expression of CBFB could significantly inhibit the proliferation and migration of lung adenocarcinoma cells,and significantly enhance the apoptosis of lung adenocarcinoma cells.Western Blot results showed that the expression of p53 protein was significantly up-regulated in CBFB overexpression group,on the contrary,the expression of p53 protein was significantly down regulated in CBFB low expression group.Conclusion: 1.In nonsmoking females,rs3733845 and rs3733846 were related to the susceptibility of lung adenocarcinoma.There was no statistically significant association between rs3733845 and rs3733846 and lung squamous cell carcinoma and small cell lung cancer.Moreover,the interactions between rs3733845 and rs3733846 risk genotypes and cooking oil fume exposure were statistically significant on a multiplicative scale.2.The rs12598154 and rs17768165 sites of CBFB had no statistically significant association with the risk of lung cancer.3.In the TCGA database,the expression of mi R-143-3p was down-regulated in lung adenocarcinoma,while the expression of CBFB was up-regulated,both of which have good diagnostic performance.Bioinformatics predicted that mi R-143-3p and CBFB may participate in the p53 signaling pathway.4.Hsa-mi R-143-3p could have a tumor suppressor effect in lung adenocarcinoma.It could inhibit the proliferation and migration of lung adenocarcinoma cells by inhibiting the expression of CBFB,and promote the apoptosis of cancer cells. |
PDF Full Text Request