Association Study And Functional Analysis Of SNPs In Long Noncoding RNA CDKN2B-AS1 With Breast Cancer Susceptibility

Breast cancer is the top diagnosed cancer in the world,and the fifth leading cause of cancer death.Previous research showed that multiple biological and social factors might increase the risk of breast cancer.With the continuous attention to long non-coding RNA（lnc RNA）,the discussion about the influence of single nucleotide polymorphisms（SNPs）on the secondary structure and function of lnc RNA has gradually increased.Previous studies have found that the expression level of CDKN2B-AS1 in triple-negative breast cancer was much higher than that in normal tissues,but the association between CDKN2B-AS1 genetic variant SNPs and breast cancer susceptibility has not been reported.ObjectiveThe aim of the study was to analysis the relationship between the genetic variation of lnc RNA CDKN2B-AS1 SNPs and the metastasis mechanism of breast cancer in women,and to explore the regulation of the genetic variation of CDKN2B-AS1 on the invasion and metastasis of breast cancer,which provides a theoretical basis in early screening,individualized treatment and treatment for breast cancer patients.Methods（1）SNPs screening and function prediction:The CDKN2B-AS1 gene polymorphic SNPs was screened combining bioinformatics and database,and a total of 13 SNPs was selected（rs2518723,rs7866783,rs10965215,rs3217992,rs77792598,rs3217986,rs2069418,rs4977753,rs75917766,rs74925072,rs7859727,rs74772332,rs78545330）.The secondary structure of CDKN2B-AS1 was predicted in RNAfold website,lnc RNASNP2 and DIANA were used to predict the targete mi RNAs of CDKN2B-AS1 SNPs and further potential biological functions.（2）Genotyping:The case-control study was carried out based on（age±2）frequency matching.PASS 15.0 software was used to calculate the sample size,a total of 504 cases and 505 controls were included.The genotyping of CDKN2B-AS1 SNPs by SNPscan TM Multityping Kit.（3）Real-time fluorescent quantitative PCR（q RT-PCR）:SYBR method q RT-PCR experiment was conducted to detect the relative expression of CDKN2B-AS1 functional SNPs rs10965215 and rs2518723 in plasma of different genotypes.The analysis result was expressed byΔCt.（4）Double luciferase reporter gene experiment:Dual luciferase experiments was conducted in MDA-MB-231 and MCF-7 breast cancer cell lines to verify the effect of rs10965215 A>G mutation on the binding ability of CDKN2B-AS1 and mi R-4440.SYBR method q RT-PCR test was performed to compare the relative expression of mi R-4440 in normal breast cells（MCF10A）and breast cancer cell lines（MDA-MB-231）,the analysis result was expressed by 2^-ΔΔCt.（5）Effect of Mi R-4440 on breast cancer cell function:By constructing mi R-4440overexpression and negative control（NC）lentiviral vectors,screening stable transgenic strains in MDA-MB-231 breast cancer cells.CCK8,scratch experiments and Transwell experiment were performed to explore the effect of mi R-4440 overexpression on breast cancer cell proliferation,migration and invasion.（6）Statistical Analysis:The t-test andχ² test of two independent samples was conducted to describe the basic information of the research object and calculate whether each SNP met the Hardy-Weinberg equilibrium in the control group.Unconditional Logistic regression analysis was performed to explore the association between CDKN2B-AS1 SNPs and breast cancer susceptibility,adjusting for age,age at menarche,menopausal status,number of pregnancy,number of abortion,history of breastfeeding and family history of first-level tumors.The SNPs haplotype analysis was conducted in SHEsis online software.Gene-environment-interaction analysis was calculated by multi-factor dimensionality reduction（MDR）.The relative expression of the target gene and the dual luciferase report experiment both used independent sample t-test.In the CCK8 experiment,the difference OD values between the mi R-4440overexpression group and negative control（NC）were calculated in the independent sample t test.The Transwell experiment results acquired by counting the stained cells,and the t test was used analysis the difference in the number of migrated or invaded cells between different groups.The scratch experiment used the Image J software to calculate the scratch area,and then the t test was used to analyze the difference in scratch healing rate.Results（1）The individuals with CDKN2B-AS1 SNPs rs2518723 C>T mutation（OR:1.393,95%CI:1.043-1.861）,rs10965215 A>G mutation（OR:1.662,95%CI:1.267-2.180）,rs77792598 C>G mutation（OR:1.268,95%CI:0.999-2.987）,rs4977753 T>C mutation（OR:1.397,95%CI:1.071-1.822）,rs75917766 C>T mutation（OR:1.645,95%CI:1.150-2.353）and rs78545330 C>G mutations（OR:1.374,95%CI:1.051-1.795）might increase the risk of breast cancer.The stratification results showed that CDKN2B-AS1 SNP rs75917766 C>T mutation（OR:1.833,95%CI:1.071-3.138）and the C>T mutation of rs74925072（OR:2.308,95%CI:1.354-3.935）might increase the risk of ER-positive breast cancer.rs3217992 C>T mutation（OR:1.670,95%CI:1.084-2.574）was associated with an increased risk of Her-2 positive breast cancer,while the T>G mutation of rs3217986（OR:0.522,95%CI:0.303-0.899）might reduce the risk.The C>G mutation at site rs77792598 might increase ER-positive（OR:1.776,95%CI:1.211-2.603）and Luminal breast cancer（OR:0.466,95%CI:0.236-0.924）,while for triple-negative breast cancer,rs77792598 C>G mutation might be a protect factor（OR:0.466,95%CI:0.236-0.942）.Rs74772332 G>A mutation could not only increase the risk of ER-positive breast cancer（OR:2.490,95%CI:1.615-3.838）,but also PR-positive（OR:1.708,95%CI:1.158-2.520）and Luminal breast cancer（OR:1.782,95%CI:1.198-2.651）.While for triple-negative breast cancer,the GA genotype of rs74772332 was a protective factor（OR:0.268,95%CI:0.115-0.624）.Similarly,rs78545330 T>A mutation was a risk factor for ER-positive（OR:2.001,95%CI:1.333-3.005）and Luminal breast cancer（OR:1.603,95%CI:1.104-2.329）,but it could reduce triple-negative breast cancer（OR:0.434,95%CI:0.219-0.856）risk.（2）Haplotype analysis results found C_rs2518723T_rs78545330T_rs3217992T_rs3217986 C_rs2069418Ars10965215Trs4977753 Crs75917766Crs77792598Grs7866783Crs74925072Trs7859727Grs74772332 haplotype might increase the risk of breast cancer（OR:1.254,95%CI:1.025-1.534）,whileTrs2518723Ars78545330Crs3217992Trs3217986Crs2069418Grs10965215Crs4977753Trs75917766 Grs77792598 Grs7866783C_rs74925072T_rs7859727A_rs74772332haplotype had a protect effect（OR:0.678,95%CI:0.472-0.974）.The result of CDKN2B-AS1 SNPs-environment interaction analysis revealed that there was an interaction between CDKN2B-AS1 rs10965215 G genotype,age at menarche,and number of miscarriages,and this interaction model could increase the risk of breast cancer.The incidence of breast cancer is 3.279 times than that of the control group（OR:3.279,95%CI:2.534-4.244）.（3）q RT-PCR result displayed that the relative expression of CDKN2B-AS1 in rs2518723 homozygous wild CC genotype（10.03±0.62）（P=0.028）and heterozygous TC genotype（9.827±0.62）（P=0.026）individuals were both lower than homozygous mutant TT genotype（12.23±0.68）.In rs10965215,the individual with homozygous wild AA genotype CDKN2B-AS1 relative expression（8.88±3.43）was lower than heterozygous GA genotype（11.08±2.90）（P=0.011）and homozygous mutant GG gene type（11.31±2.90）（P=0.030）.（4）The results of dual luciferase reporting experiments in MDA-MB-231 and MCF-7 breast cancer cells showed that when rs10965215 wild A genotype was carried,there was an interaction between CDKN2B-AS1 and mi R-4440,while the interaction between CDKN2B-AS1 and mi R-4440 has not been observed yet when happened rs10965215 A>G mutation.（5）The q RT-PCR was carried out to detect of mi R-4440 expression level and found that the expression level of mi R-4440 in MDA-MB-231 cells（1.38±0.06）was higher than MCF10A cells（1.00±0.04）（P=0.002）.（6）The CCK8 experiment results in MDA-MB-231 cells showed the proliferation ability of mi R-4440 over expression group at 24 h（P<0.001）,48 h（P=0.049）,72 h（P<0.001）and 96 h（P=0.014）were all higher than NC group.（7）Transwell migration results showed that the number of MDA-MB-231cells in the mi R-4440 overexpression group passing through the chamber was 366,the NC group was 248,and there was a statistical difference between the two groups（P=0.043）.The scratch test results showed that the scratch healing rate of MDA-MB-231 cells in the mi R-4440 overexpression group was higher than that in the NC group（P=0.007）.What’s more,Transwell invasion experiment found that in mi R-4440overexpression group,the number of MDA-MB-231 cells passed through the chamber was 570,while the number of cells in the NC group was 425,the cell invasion ability of the mi R-4440 overexpression group was higher than that of the NC group（P=0.046）.Conclusions（1）Individuals with CDKN2B-AS1 SNPs rs2518723 C>T,rs10965215 G>A,rs77792598 C>G,rs4977753 T>C,rs75917766 C>T and rs78545330 T>A mutation may increase the risk of breast cancer.（2）Haplotype Crs2518723Trs78545330Trs3217992Trs3217986 Crs2069418Ars10965215Trs4977753 Crs75917766C_rs77792598G_rs7866783C_rs74925072T_rs7859727G_rs74772332 is a risk factor for breast cancer risk.And the haplotype Trs2518723Ars78545330Crs3217992Trs3217986Crs2069418Grs10965215Crs4977753Trs75917766 Grs77792598G_rs7866783C_rs74925072T_rs7859727A_rs74772332can reduce the risk of breast cancer.Further more,CDKN2B-AS1 rs10965215,age at menarche and the number of miscarriages have an interaction,and can increase the risk of breast cancer;（3）The relative expression of CDKN2B-AS1 in plasma of different genotypes of CDKN2B-AS1 rs2518723 C>T and rs10965215 A>G mutations is different.（4）CDKN2B-AS1 rs10965215 A>G mutation may mediate the interaction between CDKN2B-AS1 and mi R-4440,regulate the expression of mi R-4440,and then affect the proliferation,invasion and migration of breast cancer cells.