Association Between Polymorphism In The Promoter Region Of Lncrna GAS5 And The Risk Of Colorectal Cancer

Recently,the association between abnormal expression of long non-coding RNAs(lncRNAs)and tumorigenesis and development has become a research focus.The growth arrest-specific 5(GAS5)gene of GAS family is a small open reading frame located on the non-coding protein chromosome 1q25.1 in human.The expression of mature GAS5 is abnormal in a variety of tumors and play a role through different ways.Colorectal cancer(CRC)is an important public health problem affecting human health,and its morbidity and mortality are on the rise.Therefore,the early diagnosis and treatment of CRC are significantly.However,there are few studies on the correlation and mechanism between GAS5 and the occurrence and development of CRC,and the evidence is deficient which requires further research.In this study,GAS5 in peripheral blood and tissue samples of CRC patients and controls was tested through case-control design to analyze its correlation with the incidence risk and clinical characteristics of CRC.Through the detection of single nucleotide polymorphisms(SNPs),the relationship between the rs55829688 in GAS5 promoter region and the incidence risk and prognosis of CRC was discussed.Furthermore,the reaction mechanism of GAS5 in CRC was preliminarily explored by detecting the expression difference and distribution of GAS5 in CRC tissues and cells.Then,the transcription factors binding to SNPs in the GAS5 promoter region were analyzed by bioinformatics,and verified by EMSA experiment and dual luciferase reporter gene system experiment.Finally,the molecular mechanism of the association between GAS5promoter region polymorphism and CRC development was further studied by comparing the differences in various cell phenotypes between siRNA cell lines that silenced GAS5 and normal cell lines.Correlation between GAS5 gene polymorphism and the risk of colorectal cancer1.Different lncRNA expression profiles in CRC tissues and adjacent tissues were screened by lncRNA transcription expression profile,and the expression differences of target gene lncRNA GAS5 in CRC were mainly detected and analyzed to determine the regulatory role of GAS5 in CRC.2.The specific position information of GAS5 was obtained from the 1000 Genomes database.The linkage disequilibrium(LD)analysis of the GAS5 gene was performed using Haploview 4.2 software.TagSNPs were selected with the minimum allele frequency(MAF)of the Chinese Han population(CHB)?5%and the threshold r~2 between SNPs in the gene haplotype>0.8.The final SNP rs55829688 was determined by combining UCSC and NCBI to obtain the location information of SNPs sites.3.The case-control design was used to genotype the peripheral blood and tissue DNA of1078 CRC patients and 1175 healthy people in CHB by TaqMan probe method.The association between GAS5 gene polymorphism and CRC risk was analyzed.10%of the samples were extractly randomly for blind repetition to ensure the accuracy of the test results.The basic information of the study population showed that the age and sex of CRC case and control group were matched.Compared with the control group,the proportion of smoking and drinking status of the case group were higher,and the difference was statistically significant(P<0.001,P=0.035).4.The association between GAS5 gene polymorphism and CRC risk was analyzed.The results showed that compared with rs55829688 CC genotype carriers,CT/TT genotype carriers had a higher risk of CRC,rs55829688 was significantly associated with CRC risk(P=0.0016).5.To further assess the impact of various confounding factors on the association between GAS5 gene polymorphism and CRC risk,we performed a stratified analysis.The recessive genetic model showed a significantly increased risk of CRC in patients with CT/TT after adjusting for age and gender.In addition,stratified analysis showed the increased risk in the subgroup of age?56 and female.We also found that the rs55829688 CT/TT genotypes were associated with the increased risk of both colon and rectum cancer and low grade of CRC.However,GAS5 gene polymorphism affects the survival time of CRC patients were didn't find in the survival analysis.Expression and significance of GAS5 in colorectal cancerIn the above study,we identified the association between GAS5 rs55829688 and the risk of CRC.To explore the mechanism of GAS5 response in CRC,we examined the expression and distribution of GAS5 in CRC,providing a basis for subsequent molecular mechanisms.1.The expression difference of GAS5 in CRC tissues and adjacent tissues were analyzed by TCGA database.It was found that GAS5 was higher in CRC tissues than in adjacent tissues.Subsequently,qRT-PCR was used to detect the mRNA levels of GAS5 in 210 pairs of human CRC and adjacent tissues.The results showed that the expression level of GAS5 mRNA in CRC tissues was higher than that in adjacent tissues,and the difference was statistically significant.The results were consistent with the TCGA database results.2.Next,we analyzed the mRNA levels of GAS5 in different genotypes of CRC and adjacent tissues in 210 patients.The results showed that the expression level of GAS5 in rs55829688 CT/TT genotype samples was significantly higher than that in CC genotypes.3.In order to clarify the localization of GAS5 in CRC tissues,we used in situ hybridization(ISH)to detect the distribution of GAS5 in colorectal cancer tissues.The results showed that compared with the adjacent tissue,the expression of GAS5 was higher in the cancer tissue.And GAS5 was mainly located in the nucleus of CRC.No obvious GAS5 expression was found in the adjacent tissues.4.In order to detect the difference of GAS5 expression in CRC cells,the expression of GAS5 in different CRC cells was detected by qRT-PCR.The results showed that the expression of GAS5 in CRC cells was higher than that of normal colorectal cell NCM460.5.In order to clarify the localization of GAS5 in CRC cells,we predicted the location of GAS5 through the GeneCards database primarily.The results showed that GAS5 was distributed in both nucleus and cytoplasm.Next,nucleus cytoplasmic RNA was extracted from CRC cells,and GAS5 expression was detected by qRT-PCR.The results were consistent with the results of GeneCards database.GAS5 was distributed in the nucleus and cytoplasm and mainly distributed in the nucleus.Association between genetic variation of GAS5 promoter region and development of colorectal cancer and its molecular mechanismIn order to fully explore the association between genetic variation of GAS5 promoter region and the development of CRC and its molecular mechanism,we examined the effect of genetic variation of GAS5 promoter region on transcriptional activity primarily,and then used EMSA to detect the binding ability of SNP and transcription factor in GAS5 promoter region.Finally,the GAS5 knockdown cell lines interfered by siRNA were constructed,and the wild type DLD-1 and SW480 cell lines were simultaneously tested for cell phenotype related biological experiments to explore the molecular mechanism of GAS5 rs55829688 affecting the development of CRC.1.Two luciferase reporter gene vectors including rs55829688 T and rs55829688 C were constructed,and the effect of genetic variation in promoter region on transcriptional activity was detected using the dual luciferase reporter gene system.The results showed that the luciferase activity of reporter gene with T allele was higher than that with C allele.2.Given that SNPs in the promoter region of the gene may affect gene transcription and expression by altering the ability to bind to sequence-specific transcription factors,we used AliBaba2 software to predict the DNA of the rs55829688 T>C polymorphic locus in the promoter region of the GAS5 gene.The sequence was predicted by transcription factors,and it was found that rs55829688 T>C alteration may affect the ability of the promoter region to bind to transcription factor YY1.3.The EMSA assay was used to detect the binding ability of SNPs and transcription factor in GAS5 promoter region.It was found that the binding ability of T allele probe to nucleoprotein was stronger than that of C allele probe to nucleoprotein.The rs55829688 T>C polymorphic variation reduced the binding of the transcription factor YY1.4.siRNA was used to perform GAS5 knockdown on DLD-1 and SW480 cell lines,qRT-PCR method was used to detect knockdown efficiency,and siRNA with the highest knockdown efficiency was screened for subsequent transfection experiments.5.Flow cytometry results showed that GAS5 knockdown can promote the apoptosis of CRC cells,and the proportion of cells in S phase and G2-M phase decreased.6.Western blot was used to detect the effect of GAS5 knockdown on apoptosis related proteins,and it was found that GAS5 knockdown enhanced the expression of cleaved PARP and cleaved casepase-3,which were related to apoptosis.7.CCK-8 was used to detect cell viability,and it was found that inhibition of GAS5expression inhibited proliferation of DLD-1and SW480 cells.8.Cell migration and invasion experiments showed that inhibition of GAS5 expression significantly reduced the migration and invasion of DLD-1 and SW480 cells(P<0.01).Taken together,we found that compared with CC genotype,carriers with the rs55829688CT/TT genotype had a significantly increased risk of CRC in our study.Further functional studies indicated that the rs55829688 T>C polymorphism might alter the binding affinity of the transcription factor YY1 to GAS5,resulting in decreased expression of GAS5 and ultimately inhibiting the development of CRC.In addition,flow cytometry,western blot,migration,invasion and CCK-8 showed that GAS5 inhibited apoptosis and promoted the proliferation,invasion and migration of CRC cells.This study demonstrated the association between lncRNA GAS5 gene polymorphism and CRC genetic susceptibility and molecular mechanisms,providing an important basis for early diagnosis and prognosis of CRC.