Studies On Colorectal Cancer Related Genes CD44,Twist1,and MALAT1

Background: Colorectal cancer(CRC)is one of the most common tumors in digestive system.Its morbidity and mortality are second only to gastric cancer and esophageal cancer,and are the third highest in gastrointestinal malignancies,which is a serious threat to human health.With the improvement of p eople's living standard,the diet habits and structure have changed,and the intake of high-fat and high-protein food has increased,which makes the incidence of colon cancer rise year by year[1][2].According to statistics in the past decade,there are about 1.2 million new cases of colon cancer in the world every year.The proportion of developing countries is increasing year by year.China has about 400000 new cases every year,accounting for about one-third of the world's total.[3] Some progress has been made in the development and progression of colon cancer and targeted therapy in recent years,the prognosis of patients has been improved,but the mortality rate is still high.It is reported that the 5-year survival rate of patients in Asia in the last five years is only 40%,especially in China and India[4].Colon cancer is a kind of cancer with polygenic mutation,accompanied by activation of many proto-oncogenes and inactivation of anti-on cogenes.The molecular mechanism of its occurrence and development is so complex that it has not yet been studied clearly.Therefore,it is of great significance for the diagnosis and treatment of colon cancer to deeply study the molecular mechanism of its occurrence and development.Tumor heterogeneity refers to the presence of different genetic genomes or subtypes of cloned cells in the tumor focus.It includes gene phenotypic differences among tumors in the same pathological grade of the same patient as well as between cancer cells in different parts of the same lesion in the same patient.As a result,there are different processes of proliferation,i nvasion,migration,apoptosis,treatment and recurrence in different subtypes of tumo rs[5].Colon cancer is one of the most heterogeneity of common tumors,its characteristics determine the large number of colon cancer typing.Therefore,it is of great importance to analyze the heterogeneity of gene expression in colon cancer and to identify and study the new subtypes in order to study the molecular mechanism of carcinogenesis and development of colon cancer and tu mor therapy.CD44 is a glycoprotein widely expressed on the cell membrane,binding to a variety of ligand molecules and involving in tight cell-cell or cell-matrix junctions.In addition,it is highly expressed in a variety of malignant cancers,involved in the occurrence and development of cancer and metastasis[6][7];CD44 is a multifu nctional transmembrane glycoprotein,which plays an important role in tumor migration,invasion and angiogenesis.It has been found that in human chondrosarcoma cell lines,tumor cells recognize hyaluronic acid by dimerization of CD44 and promote adhesion between tumor cells [8].The Twist family consists of two genes,Twist1 and Twist2.Twist1 is able to inhibit the expression of E-cadherin and decrease the adhesion between cells,which induces EMT,in order to enhance the invasiveness of tumor.Cancer cells can become fusiform after high expression of Twist1,which is beneficial to metastasis of cancer cells[9].Tumor neovascularization is the formation of vascular ducts without vascular endothelial cells following a plastic change in tumor cells in a speci fic microenvironment.Blood circulates through these vascular ducts,through which tumor cells obtain nutrients needed for growth,invasion,and metastasis[10][11][12][13].MALAT1 is a kind of lnc RNA with 8.5kb,located at 11q13 and characterized by Ji et al[14].in the study of early non-small cell lung cancer(NSCLC).MALAT1(also known as ? gene)is named for its clinical significance in predicting early metastasis and survival of NSCLC.In a subsequent study,it was showed that MALAT1 is widely expressed in normal tissues and conserved in other mammalian species,suggesting that MALAT1 plays a potentially important role in development and evolution[15].Recently,it has been shown that MALAT1 shows a vital role not only in clinical but also in various physiological and pathological processes,such as cell proliferation,cell death,cell cycle,cell migration,invasion,immunity,and so on.It is involved in angiogenesis through signaling pathways,making it become possible biomarkers and drug targets[16][17][18][19][20].Previously,it had been shown that single nucleotide polymorphisms(SNPs)in lncRNA contributed to coronary heart diseases[21][22].Up to now,SNPs in MALAT1 has been studied in lung cancer[23][24],HBV associated liver cancer[25],pulmonary hypertension [26].Hence,it is hypothesized that SNPs in MALAT1 may be associated with individual susceptibility to colon cancer.To this end,a two-stage case-control study was conducted to analyze the relationship between SNPs in MALAT1 and colon cancer risk in Chinese Han population.In this study,the three genes mentioned above were selected to explore the relationship between CD44,Twist1 and the occurrence and development of colon cancer,as well as the association between MALAT1 and susceptibility to colon cancer.Thus the high-risk and susceptible population of colorectal cancer can be identified in order to provide a new idea for the individualized prevention and treatment of colon cancer.Objective: 1.The expression of CD44 and Twist1 in human colon cancer cells was analyzed by tumor database retrieval,and the expression in normal colonic epithelial cells and colon cancer cell lines was studied.2.Stable silencing CD44 and Twist1 cell lines were constructed to investigate the effects of CD44 and Twist1 on the proliferation,apoptosis,migration and infiltrat ion of colon cancer at the cellular level and at the animal laboratory level as a whole.3.To investigate the relationship between SNPs of lnc RNA MALAT1 gene and susceptibility to colorectal cancer.Methods: Part1 Exploring the function and mechanism of CD44 and Twist1 in colon cancer treatment at the cellular level 1.Using the Oncomine and TCGA data samples of colon cancer clinical information,analyze the expression of target genes in colon cancer patients.2 Using Real time PCR and western blot method of detection of target genes in normal colon epithelial cells and the expression level in colon cancer cell line.3.Stable silencing of CD44 and Twist1 in colon cancer cells SW480 and HT-29.4.After transfecting the synthesized target gene si RNA into colon cancer cells,the proliferation activity of colon cancer cells was detected by MTT assay.5.After transfecting the synthesized target gene siRNA into colon cancer cells,Transwell cell migration and invasion assays were used to detect the migration and invasion ability of colon cancer cells.6.Western Blot was used to detect the effect of knockdown target gene on apoptosis-related protein caspase9 and migration and invasion-related protein E-cadherin in colon cancer cell proliferation-related protein cyclin D1.7.Through the nude mouse model of subcutaneous tumor formation,the colon cancer cell line HT-29 knockdown target gene and the colon cancer cell SW480 overexpressing the target gene were inoculated subcutaneously in nude mice to study the tumor gene formation and metastasis of the target gene.influences.8.Western blot was used to detect the apoptosis-related protein caspase9 and the migration and invasion-related protein E-cadherin in nude mice.Part 2 Relationship between lnc RNA MALAT1 gene polymorphism and susceptibility to colon cancer 1.In this study,966 patients with CRC and 988 healthy controls were enrolled.Inclusion criteria are histopathologically confirmed CRC without chemotherapy or radiotherapy.Cancer-free controls came from the same hospital at the same time,matched by sex and age(±5 years).Face-to-face interviews were conducted by trained interviewers and a questionnaire is distributed on demographic characteristics and lifestyle factors.At the same time,5m L peripheral blood was collected from each individual and stored in sodium citrate anticoagulant tube at-80 ? for DNA separation.2.Filtering tag SNPs in GVS Database,linkage disequilibrium value R2 should be less than 0.8.The allele frequency(MAF)of SNP in(CHB)population of Chinese Han population should be above 0.05.3.The genotypes of taqman probe alleles were determined by SNPs screening.Non-conditional Logistic regression model was used to analyze the association among age,sex,alcohol consumption,smoking,family genetic history and CRC susceptibility.Results: Part1.Exploring the function and mechanism of CD44 and Twist1 in colon cancer treatment at the cellular level 1 Through the analysis of the colon cancer clinical samples,found that CD44 and Twist1 in colon cancer patients were significantly high expression.2.Real time PCR and western blot test found that CD44 and Twist1 m RNA and protein levels in the normal colonic epithelium in colon cancer cell lines were significantly higher expression.3 Successfully design and synthesize CD44 and Twist1 si RNA using bioinformatics software.4.The proliferation activity of colon cancer cells SW480 and HT-29 after si-CD44 and si-Twist1 was significantly inhibited by MTT assay,and the statistical difference was significant(p<0.05).5.Transwell cell migration and invasion experiments showed that the migration and invasion abilities of colon cancer cells SW480 and HT-29 after si-CD44 and si-Twist1 were significantly decreased,and the statistical difference was significant(p<0.05).6.Western Blot assay showed that the expression of proliferation-related protein cyclin D1 was significantly decreased in si-CD44 and si-Twist1 colon cancer cells SW480 and HT-29,and the expression of apoptosis-related protein caspase9 was significantly increased,while migration and invasion-related protein E-The expression of cadherin increased significantly(p<0.05).7.Through the subcutaneous tumor model of nude mice,it was found that the tumor formation ability of colon cancer cells SW480 and HT-29 was significantly weakened after stable silencing of CD44 and Twist1,and the subcutaneous tumor formation volume was significantly smaller,and the statistical difference was sign ificant(p<0.05)..8.Western Blot and immunofluorescence assay showed that the expression of proliferation-related protein cyclin D1 was significantly decreased in si-CD44 and siTwist1 colon cancer cells SW480 and HT-29(p<0.05),and the expression of apoptosis-related protein caspase9 was significantly increased(p< 0.05),while the expression of migration and invasion-related protein E-cadherin was significantly increased(p<0.05).Part 2 Relationship between lnc RNA MALAT1 gene polymorphism and susceptibility to colon cancer 1.Statistical analysis showed that there was no significant difference in age,sex,smoking and drinking between the patients and the control group.However,the distribution of "cancer family history" in colorectal patients was significantly higher than that in control group(P < 0.001).2.Three tag SNPs,were selected by using GVS database: rs11227209,rs619586 and rs3200401.Another SNP(rs1194338)in the promoter region of MALAT1,has been reported to be associated with CRC susceptibility.3.The results of the first stage showed that the distribution of rs619586 and rs1194338 alleles was statistically significant,while that of rs11227209 and rs3200401 was not,which was verified in the samples of the second stage.It was found that compared with rs619586-AA genotype,the CRC risk in patients with AG genotype and GG genotype was significantly reduced(P < 0.05).Compared with rs1194338-CC genotype,the CRC risk of AC genotype and AA genotype was significantly reduced(P <0.05).Conclusion: 1.CD44 and Twist1 in samples of colon cancer and colon cancer cells were present significant high expression.2.CD44 and Twist1 can promote colon cancer cell proliferation,migration and invasion ability,and inhibit the apoptosis of colon cancer cells and promote the growth of colon cancer cells to subcutaneous tumor,prompt CD44 and Twist1 in colon cancer development play an important role in the process,can be used as a target gene for the treatment of colon cancer.3.CD44 and Twist1 related proteins can be adjusted by proliferation cyclin D1,apoptosis related proteins caspase9 and the expression of E-cadherin protein involved in migration and invasion to influence the colon cancer cell proliferation,apoptosis,migration and invasion.4.Genetic variation in lnc RNA MALAT1 may have an effect on the risk of CRC,in which rs619586 and rs1194338 are significantly associated with CRC susce ptibility.5.The risk of different genotypes of rs619586 and rs1194338 was different.The probability of CRC risk of rs619586-AA and rs1194338-CC genotypes was sig nificantly increased.