Association And Mechanism Research Of MALAT1 Gene Polymorphism With Colorectal Cancer Susceptibility

Background and ObjectivesColorectal cancer(CRC)is one of the most common cancers and the second leading cause of cancer-related death worldwide.In China,a total of 376,300 new cases and 191,000 cancerrelated deaths have been reported in 2015.Environmental risk factors,such as smoking,heavy alcohol consumption,high consumption of red and processed meat contribute to the initiation and progression of CRC.Genetic and epigenetic alternations intensify CRC development as well.As the most common form of genomic variation,single nucleotide polymorphism(SNP)refers to a DNA sequence polymorphism caused by a nucleotide variation at the genome level.Long non-coding RNAs(lnc RNAs),as the transcripts greater than 200 nucleotides,are involved in several biological processes.Metastasis associated with lung adenocarcinoma transcript-1(MALAT1)is an evolutionarily highly conserved lnc RNA gene,consisting of more than 8,000 n T and maps to chromosome 11q13.Recent studies demonstrated that MALAT1 not only promotes CRC malignancy,but also affects prognosis.However,there is no consensus on the mechanisms behind MALAT1 exotic expression.It also has been reported that genetic variant in MALAT1 was associated with the risk of various tumors,such as hepatocellular carcinoma(HCC),breast cancer,and lung cancer.However,to date,few studies have focused on the association between the genetic variants in exon region of MALAT1 and risks of CRC.In this study,we assessed whether the selected genetic variants in MALAT1 exon region could contribute to CRC development and disclose their potential mechanisms.Methods Correlation between MALAT1 polymorphism rs664589 and the risk of colorectal cancer1.The location of the MALAT1 were obtained from the LNCipedia.Two SNPs were selected from the Lnc RNASNP database according to the following criteria:(a)minor allele frequency(MAF)> 0.05 in global population;(b)SNPs in MALAT1 gene were predicted to locate in the mi RNA binding sites using Lnc RNASNP database;(c)SNPs may modulate the secondary structure of MALAT1 calculated by RNAfold.The linkage disequilibrium(LD)analysis of the MALAT1 gene polymorphisms was performed by using Haploview 4.2 software.2.The genotypes of selected polymorphisms were determined by Taq Man allelic discrimination method..Pearson’s chi-squared test was used to calculate the frequency distribution of the demographic characteristics and genotype results of the MALAT1 polymorphisms.3.Stratified analysis were performed to further assess the impact of various confounding factors on the association between MALAT1 gene polymorphism and colorectal cancer risk.4.Kaplan-Meier(KM)analysis were used the explore the association between MALAT1rs664589 and overall survival for CRC patients.5.Stratified analysis were performed to further assess the impact of various confounding factors on the association between MALAT1 gene polymorphism and overall survival for CRC patients.Effects of MALAT1 polymorphism rs664589 on the expression of MALAT1 in CRC1.The tissue microarrays(TMAs)(consisting 807 pairs of CRC tissues and adjacent nontumor tissues from 1078 CRC patients)were constructed by the National Engineering Center for Biochip(Shanghai,China).MALAT1 expression levels in CRC cells,fresh paraffinembedded tissues,and TMA were analyzed with the RNAscope 2.0 HD-Brown Manual Assay.2.Kaplan-Meier(KM)analysis were used the explore the association between MALAT1 expression level and overall survival for CRC patients.3.q RT-PCR was used to detected the MALAT1 expression level in 96 pairs of CRC tissues and the corresponding adjacent noncancerous tissue.4.One-way ANOVA was used to assess the relationship between rs664589 genotypes and MALAT1 expression level.Effects of MALAT1 polymorphism rs664589 on biological behavior of human colorectal cancer cell line1.SW480 and SW620 cell lines with indicated genotypes were generated by CRISPR/Cas9 strategy.2.SW480 and SW620 cell lines with different rs664589 genotypes(CC,CG and GG)was used to evaluate the effects of MALAT1 rs664589 on cell proliferation,invasion,and migration.Tumorigenesis of nude mice was performed to evaluate the effect of MALAT1 rs664589 on tumor formation and metastasis capacity of colorectal cancer cells in nude mice.Results Correlation between MALAT1 polymorphism rs664589 and the risk of colorectal cancer1.The results of genotyping showed that compared with the rs664589 CC genotype,CG genotype elevated the risk of CRC(adjusted OR = 1.35,95% CI = 1.08-1.69),and the increased risk was more pronounced in the GG genotype(adjusted OR = 3.57,95% CI = 1.60-7.97).In addition,we found that rs664589 CG/GG genotypes were associated with higher risk of CRC compared with the rs664589 CC genotype in the dominant genetic model(CG/GG vs.CC,adjusted OR = 1.45,95% CI = 1.16–1.80).However,no significant association was observed between rs3200401 genotypes and the risk of CRC(P = 0.1923).2.Stratified analysis showed that CG/GG genotypes profoundly increased the risk of CRC among younger(adjusted OR = 1.48,95% CI = 1.10-1.99)and male subgroups(adjusted OR =1.57,95% CI = 1.19-2.08).Moreover,we found a similar result in subgroups of colon(adjusted OR = 1.48,95% CI = 1.12-1.94),rectum(adjusted OR = 1.43,95% CI = 1.11-1.85),lower grade(adjusted OR = 1.96,95% CI = 1.47-2.61),deepest invasion(adjusted OR = 1.96,95%CI = 1.44-2.67),lymph node metastasis(adjusted OR = 2.41,95% CI = 1.86-3.11),distant metastasis(adjusted OR = 3.63,95% CI = 2.43-5.42),TNM phase III(adjusted OR = 2.12,95%CI = 1.60-2.81),and TNM phase IV(adjusted OR = 3.63,95% CI = 2.43-5.42)patients.3.The results of KM analysis showed that rs664589 CC genotype markedly decreased the overall survival of CRC patients(CG/GG vs.CC,adjusted HR = 2.01,95% CI = 1.68-2.40).4.The result of rs664589 CC genotype markedly decreased the overall survival of CRC patients were also observed in the stratified subgroup,except for depth of invasion at T1,T2,and TNM stage I.Effects of MALAT1 polymorphism rs664589 on the expression of MALAT1 in CRC1.RNAscope assay showed that relative expression of MALAT1 was dramatically accumulated in CRC tumor tissues compared with adjacent tissues(P < 0.001).2.Kaplan-Meier(KM)analysis showed that patients with high expression of MALAT1 gained a significantly shorter overall survival time than those with low levels(log-Rank P <0.001).3.One-way ANOVA analysis showed that individuals with rs664589 CG/GG genotype presented higher MALAT1 levels than those carrying the rs664589 CC genotype in our TMA(consisting 807 pairs of CRC tissues and adjacent non-tumor tissues from 1078 CRC patients)(P < 0.001).Results of q RT-PCR showed that patients with rs664589 CG/GG genotype presented higher MALAT1 m RNA levels than those carrying the rs664589 CC genotype in cancer tissues(P < 0.001),but not in adjacent tissues(P > 0.05).Effects of MALAT1 rs664589 on biological behavior of human colorectal cancer cell line1.Colony formation assay was performed to show that cells with rs664589 CG/GG genotypes presented elevated proliferation capacity in both SW480(P < 0.001)and SW620(P< 0.001)cells.2.Transwell assays showed that cells with rs664589 CG/GG genotypes exhibited increased the migration and invasion capacities in both SW480(P < 0.001)and SW620(P <0.001)cells.3.Tumorigenesis of nude mice assay showed that SW480(P < 0.001)and SW620(P <0.001)cells with G allele developed larger subcutaneous xenografts and more distant metastases(liver and lung tissue)in vivo.Conclusion1.The MALAT1 rs664589 C>G mutation was associated with the increasing risk of colorectal cancer and the poor overall survival for CRC patients.2.The MALAT1 expression level was upregulated in CRC and predicted poor overall survival for CRC patients.2.The MALAT1 rs664589 C>G mutation enhanced the expression level of MALAT1.3.The MALAT1 rs664589 C>G mutation significantly potentiated the aggressiveness phenotypes of CRC and enhanced CRC development.