Single Nucleotide Polymorphism Of CYP24A1 Gene And Lung Cancer Risk And Prognosis In A Female Population As Well As Its Mechanism

Objective:Worldwide,malignant tumors are now the leading cause of death.Among them lung cancer poses a great threat to human health and life span.The occurrence of lung cancer is the result of the combination of genetic factors and environmental factors.Smoking currently is the biggest risk factors of lung cancer.But it is estimated that the incidence of 15%male lung cancer patients and 53%female lung cancer patients are not caused by smoking.More and more studies have shown that genetic factors play very important roles in the occurrence and development of lung cancer.But the exact mechanism has not been fully clarified.Therefore,it is necessary to further explore the genetic mechanism affecting the occurrence and development of lung cancer to improve the prediction of the risk of lung cancer.Single nucleotide polymorphism is the most extensive type of variation in the genome.SNPs play different roles in biological processes depending on their location in different functional regions of the genome.1,25（OH）₂D₃ is the active form of vitamin D₃ in the human body.In addition to regulating calcium homeostasis and bone salt deposition,it also plays an important role in cell proliferation and differentiation.Studies have shown that mesenchymal transformation of epithelial cells in non-small cell lung cancer is related to the body’s sensitivity to vitamin D.The active form of vitamin D-1,25（OH）₂D₃ at a certain dose has shown differentiation induction and anti-malignant cell proliferation effect in some tumor cells.25-hydroxyvitamin D3-24-hydroxylase（CYP24A1）is a member of the cytochrome P450 enzyme family and the expression of CYP24A1 is a 1,25（OH）₂D₃-dependent process.Its main function is to inactivate 1,25（OH）₂D₃.Studies have shown that lower expression of CYP24A1 in human normal lung bronchial epithelial cells.Evidence suggests that CYP24A1 in human breast,prostate and lung cancer tumor cells were found high expression.Part of the occurrence of tumor and prognosis of conditions and the level of serum vitamin D₃ and CYP24A1mRNA expression levels have close relations.Studies showed that CYP24A1 SNPs can influence 1,25（OH）₂D₃ activity in prostate cancer.Some studies have indicated that CYP24A1 is a potential predictive marker of 1,25（OH）₂D₃ in NSCLC and its expression was affected by its single nucleotide polymorphism.By studying the abnormal regulation of CYP24A1 and its effect on tumor,it is likely to clarify the reasons for inhibiting CYP24A1.So as to exert the anti-tumor effect of 1,25（OH）₂D₃and improve the prognosis and survival time of tumor patients.With the discovery of more single nucleotide polymorphic sites on CYP24A1,the correlation between these sites and tumor risks has also attracted attention.However,due to the function of its metabolism 1,25（OH）₂D₃,more studies have focused on hormone-related diseases,such as prostate cancer and breast cancer.Few susceptibility analyses related to lung cancer,and relatively few researches have focused on the influence of single nucleotide polymorphism on lung cancer function.Therefore,in order to clear CYP24A1 on the relationship between single nucleotide polymorphisms and lung cancer risk and prognosis among non-smoking women in China,we firstly carried out this case-control study and survival study.Then,the effects of 1,25（OH）₂D₃ on the biological behavior of lung cancer and the expressioin of CYP24A1 mRNA were explored.The possible effects of target sites on the biological behavior of lung cancer with 1,25（OH）₂D₃.At last,we carried out a meta-analysis to explore the relation between cancer risk and SNPs in CYP24A1.Methods:In the first part,studies related to cancer on CYP24A1 single nucleotide polymorphism loci were collected.We selected the literatures on CYP24A1polymorphism loci associated with cancer,and then we apply Haploview linkage disequilibrium loci,the application of existence SNPinfo affect gene function for predicting the single nucleotide polymorphism loci,then a RegulomeDB website to select loci functional scores.The selected sites were used as targets to analyze the relationship between CYP24A1 single nucleotide polymorphisms and lung cancer susceptibility of Chinese non-smoking women.Information of observation objects was collected.The genotyping of biological samples were carried out by Taqman method and we carried out stratification analysis and gene-environment interaction analysis.Then the patients were followed up regularly.The prognosis was studied with lung cancer death as the outcome event.The study aimed to find the relationship between polymorphism of CYP24A1 gene and survival time of non-smoking women with lung cancer.In the second part,we analysised the lung expression data from TCGA and GEO by GEPIA.The anti-proliferation effect of 1,25（OH）₂D₃ on different types to lung cancer cell lines was studied and the effect of CYP24A1 mRNA expression in different types of lung cancer cell lines was investigated.High,medium and low concentrations of 1,25（OH）₂D₃ were prepared.Lung A549 adenocarcinoma cell lines and LK-2 lung squamous cell lines were added for cell culture.The cell morphology was observed at 24h,48h after culture,and the absorbance was detected by microplate assay.Then we studied the effect of rs6068816 on the biological behavior of lung cancer.Transient transfer of wild-type（C allele）and mutant（T allele）plasmid were taken into lung adenocarcinoma A549 cell line.Cultured at the optimum concentration of 1,25（OH）₂D₃ and CCK-8 assay was used to detect the changes in cell proliferation.The cells were stained with fluorescent dyes using7-aad and Annexin v-apc dyes,and apoptosis was detected by flow cytometry.After the cells were fixed,the DNA in the cells was stained with fluorescent dye by PI dye,and the cell cycle was detected by flow cytometry,so as to determine the influence of different genotypes of rs6068816 on the biological behavior of lung cancer.In the third part,we studied the application of online database resources such as PubMed,Web of Science,CNKI,Wanfang database and bibliographic index to analysis of single nucleotide polymorphism loci in genes.Heterogeneity test,bias test and sensitivity analysis were performed on the selected literature.At the same time,the data of the first part of this study were added to enlarge the sample size in order to further discover the relationship between single nucleotide polymorphism loci and cancer risk.The statistical hypothesis tests used in this study were both bilateral tests.It was considered that P<0.05 was the standard with statistical differences.All statistical analysis was carried out by SPSS software.Ratio Ratio and 95%CI by Logistic Regression Survival curve was made by Kaplan-Meier method.Log-rank was used to test the differences among different survival curves.The risk ratio and95%confidence interval were calculated by using Cox regression model.Results:According to the results of bioinformatics analysis,rs6068816 located in the CYP24A1 coding region may have the effect of selective splicing,and the function score is the highest,so rs6068816 is selected as the research target.By Taqman genotyping results found that rs6068816 locus on the T allele and reduce the risk of lung cancer in nonsmoking women related,in lung adenocarcinoma and middle-late stage lung cancer,the site of the T allele can reduce the risk of lung cancer in nonsmoking women in China,also found in exposure to lampblack,rs6068816 dot on the T allele was also a protective factor.The result of GEPIA showed that the expression of CYP24A1 in lung adenocarcinoma tissues and lung squamous cell carcinoma were higher than that in normal lung tissues expression.A549 lung adenocarcinoma cell lines and LK-2lung squamous cell lines were cultured in high,medium and low concentrations of1,25（OH）₂D₃ for 24h,48h respectively.OD value and CYP24A1 mRNA content were measured.The results showed that at each time point,the expression level of CYP24A1 mRNA in A549 and LK-2 cells were higher than nagtive controls group.The difference was statistically significant.The absorbance of A549 and LK-2 cell lines in the medium concentration group and the high concentration group of1,25（OH）₂D₃,after 48h culture,A549 and LK-2 cell line showed significantly lower absorbance than NC group.Transient transfection of A549 lung cancer cells was conducted to construct rs6068816 wild-type and mutant cell models.Relevant functional experiments were conducted after cultured according to experimental requirements.The results showed that compared with the wild type,rs6068816mutant could inhibit the proliferation of A549 cells.Apoptosis was promoted and the proportion of apoptotic cells increased.Compared with the control group without1,25（OH）₂D₃ treatment,both the wild-type group and the mutant group treated with100nM also showed inhibitory effect on the proliferation of A549 cell lines and promoted apoptosis.The results of meta-analysis showed that mutant homozygous genotype at rs6068816 locus were associated with reduced overall cancer risk.The mutated heterozygous AG genotype at rs4809957 locus is associated with an increased overall risk of cancer.The homozygous genotype of rs2296241 mutation is associated with an increased overall cancer risk in Caucasian.At the same time,the results of allele analysis also suggest that G allele at rs2296241 locus is associated with increased overall cancer risk in Caucasian population.After stratification by cancer types,we found that homozygous GG genotype at rs2296241 locus was associated with an increased risk of prostate cancer in the population as a whole.Allele analysis also suggests that G allele is associated with a higher risk of prostate cancer in the general population.The literature included in this study did not find the relationship between rs2762939 models and cancer risk.No publication bias was found in this study.Conclusions:Rs6068816 is associated with the lung cancer risk of developing non-smoking women in and T allele is associated with reducing the risk of developing lung adenocarcinoma and advanced lung cancer in non-smoking women.No associations were found between survival time of non-smoking women and rs6068816.1,25（OH）₂D₃ could induce the increase of CYP24A1 expression and inhibit the proliferation of LK-2 and A549 cell lines.1,25（OH）₂D₃ can promote apoptosis of A549 cell line.Compared with the wild type,the mutant at rs6068816can promote the apoptosis of lung adenocarcinoma cell lines and inhibit the proliferation of lung adenocarcinoma cell lines.1,25（OH）₂D₃ can promote the apoptosis of A549 cell line.Rs6068816,rs4809957 and rs2296241 were associated with cancer risk.