Study On The Regulation Of Chemosensitivity Of Breast Cancer By Different Alternative Splicing Patterns Caused By SNP Site Of CCND1 Gene

Objective:On the basis of previous studies,this paper plans to screen the relevant SNPs at the splice site of CCND1 gene through biological databases,aiming to explore the relationship between the differences in splicing patterns caused by SNPs at the splice site of CCND1 gene and the chemosensitivity of breast cancer in Northwest China,and to provide new predictive biomarkers and potential targets for breast cancer patients with clinical chemotherapeutic response.Methods:（1）The correlation between CCND1 gene and breast cancer was analyzed by using the biological database data set related to breast cancer.The GDSC2 dataset in GDSC database was used to analyze CCND1 gene and the resistance of breast cancer cells to anthracyclines（epirubicin）.Mutations in CCND1gene were analyzed according to different classification methods using the relevant mutation data in the Catalogue of Somatic Mutations in Cancer,db SNP and c Bio Portal databases.The mutation data downloaded from Ensembl database were used to analyze and summarize the relevant SNPs at the splice site of CCND1 gene.The selected SNPs were analyzed by gene frequency analysis and literature survey,and SNP with gene frequency>0.01 were screened.（2）Using the method of meta-analysis,the relevant literatures about breast cancer and rs9344 of CCND1 gene in Pub Med and Web of Science databases were searched to extract data from the literatures that met the requirements.Stata 12.0 was used to analyze the correlation between breast cancer and rs9344 of CCND1 gene under fixed effect model or random effect model.Heterogeneity between studies was assessed by Cochran Q test（P value）and I²,and subgroup analysis was performed by ethnicity to explore the source of heterogeneity.Publication bias was assessed by Egger test.（3）Peripheral blood samples of 234 breast cancer patients treated with chemotherapy were collected.Massarray genotyping technology was used for genotyping,and the correlation between rs9344 of CCND1 gene and chemosensitivity of breast cancer was analyzed.In order to verify the accuracy of the typing results,10%of the samples were randomly selected and verified by Sanger sequencing.（4）The collected breast cancer tissue samples were genotyped by Sanger sequencing,and 18 breast cancer tissue samples after neoadjuvant chemotherapy treatment were randomly selected.By semi quantitative RT-PCR and Western blot,the correlation between the difference in the expression of two isoforms of CCND1a and CCND1b caused by rs9344 of CCND1 gene and the chemosensitivity of breast cancer was analyzed.In addition,according to the genotyping results of blood samples,24breast cancer tissue samples after neoadjuvant chemotherapy were randomly selected.The distribution of CCND1a and CCND1b isoforms in tissues was analyzed by immunohistochemical staining.（5）The genotypes of rs9344 in cell lines were analyzed,and the cells carrying wild and mutant genotypes of this site（MCF-7 and MCF-7/ADM）were selected as cell models for subsequent experiments,and the drug resistance of the selected cell models was analyzed.The genotypes of rs9344 were analyzed by Sanger sequencing;The resistance of cells to adriamycin was detected by CCK-8 method.The subcellular localization of SC-35 was analyzed by cellular immunofluorescence to observe whether there were differences in alternative splicing events in cells.The expression of CCND1a and CCND1b isoforms in cells was analyzed by semi quantitative RT-PCR and Western blot;Pymol-2.3.0 and Swiss-model were used for homology modeling to predict the protein structure of CCND1a and CCND1b isoforms,and Zdock was used for docking.The CDK4 molecules on the downstream pathway bound to it were analyzed to explore the relationship between CDK4/Cyclin D1-p RB-E2F1 signaling pathway and chemosensitivity in breast cancer,and the key proteins of CDK4/Cyclin D1-p RB-E2F1 signaling pathway were verified by Western blot.Subsequently,CCND1b specific si RNA was synthesized,and the role of CCND1b in breast cancer chemoresistance was analyzed by RT-PCR,Western blot,CCK-8,and flow cytometry in MCF-7/ADM cells that downregulated CCND1b.Results:（1）According to GEPIA database,the expression of CCND1 in breast cancer samples was significantly higher than that in normal group（P<0.001）.According to c Bio Portal database analysis,about 6%of breast cancer patients have CCND1 gene mutations,and the recurrence free survival rate of patients with CCND1gene mutations is significantly lower than that of patients with CCND1 gene wild-type（P<0.05）.GDSC database showed that CCND1 mutant genome would increase the resistance of cells to anthracyclines（epirubicin）（effect size=0.273;P=0.629;FDR%=58.7）.According to the database,there are about 4000 mutations in the CCND1 gene,including 3643 SNPs.We screened 23 splicing related SNP sites,combined with genotype frequency and literature survey,and finally screened 1 SNP site（rs9344,MAF=0.454）.（2）In this paper,106 articles were screened through the search,and 14 articles were finally included after further elimination,including 7833 breast cancer patients and 8591 healthy controls.Meta analysis showed that there was a significant correlation between rs9344 of CCND1 gene and breast cancer（allelic model,P=0.011;heterozygous model,P=0.013;homozygous model,P=0.016;recessive model,P=0.010）.Subgroup analysis according to different populations showed that carrying the A allele at rs9344 of CCND1 gene could significantly increase the risk of breast cancer in Asian populations.（3）The genotyping results of blood samples were statistically analyzed.The results showed that under the homozygous model,compared with the wild GG type,the distribution of AA genotype between the two groups was statistically different（P=0.021）.Under the allele model,the difference between the A allele and the wild G allele was statistically significant（P=0.014）.These results suggest that carrying the A allele or AA genotype increases the risk of chemotherapy tolerance in breast cancer.Further stratified analysis showed that there was a significant correlation between the rs9344 of CCND1 gene and chemosensitivity of breast cancer in the population aged≥48 years and in the premenopausal state（P<0.05）.（4）In breast cancer chemotherapy tissues,the rs9344 of CCND1 gene was found to be associated with the expression of two isoforms of CCND1a and CCND1b.The results showed that CCND1a and CCND1b were consistently expressed at both protein and m RNA levels.The expression of CCND1a had no significant difference between the two groups（P>0.05）,while the expression of CCND1b had significant statistical significance in both groups（P<0.05）;And CCND1b/a ratio was up-regulated in tolerant tissue samples carrying AA genotype（P<0.05）.CCND1a is mainly distributed in the nucleus,while CCND1b is distributed in both the nucleus and cytoplasm.（5）In vitro cell experiments showed that MCF-7 cells carried the wild-type G allele at rs9344 of CCND1 gene,and MCF-7/ADM cells carried the mutant A allele.Alternative splicing events are more active in MCF-7/ADM,which may be related to the generation of CCND1a and CCND1b isoforms caused by rs9344 of CCND1 gene.In MCF-7/ADM,CCND1a decreased at both m RNA and protein levels（P<0.05）,while CCND1b increased only at m RNA level（P<0.05）.Notably,the ratio of CCND1b/a was significantly up-regulated in MCF-7/ADM cells at the m RNA and protein levels（P<0.05）.Analysis of the downstream pathways revealed that both CCND1a and CCND1b could interact with CDK4 through different binding sites.In addition,E2F1 and p RB were highly expressed in MCF-7/ADM（P<0.05）,but the expression of CDK4 in the two groups was not statistically significant（P>0.05）.Both CCND1a and CCND1b may regulate the sensitivity of breast cancer to chemotherapy through CDK4/Cyclin D1-p RB-E2F1 signaling pathway.Subsequently,the expression of CCND1b isoforms was down regulated.Compared with the control group,the proliferation of MCF-7/ADM cells transfected with si CCND1b was inhibited,and the resistance to ADM was significantly weakened,and the IC₅₀value was also decreased（P<0.05）;Downregulating the expression of CCND1b isoforms will reduce the ratio of CCND1b/a,thus inhibiting the CDK4/Cyclin D1-p RB-E2F1 signaling pathway,preventing the G1/S transition of the cell cycle,leading to cell cycle arrest,thereby improving the chemosensitivity of MCF-7/ADM cells.Conclusion:（1）The rs9344 of CCND1 gene is associated with breast cancer susceptibility;Carrying the A allele and AA genotype increases the risk of breast cancer in Asian populations.（2）The rs9344 of CCND1 gene is associated with chemosensitivity of breast cancer;Carrying the A allele or AA genotype will reduce the risk of chemosensitivity of breast cancer.（3）rs9344 of CCND1 gene is a splice donor site,and its upregulation of CCND1b/a ratio is associated with chemoresistance in breast cancer.（4）The difference of alternative splicing mode caused by rs9344 of CCND1 gene can regulate the sensitivity of breast cancer to chemotherapy through CDK4/Cyclin D1-p RB-E2F1 signaling pathway.Downregulating the expression of CCND1b isoform can inhibit the proliferation of breast cancer cells,delay the cell cycle progression,and reverse the resistance of MCF-7/ADM cells to adriamycin.