The Association Of 5-HT3R Subtype Gene UTR Polymorphism And Diarrhea-predominant Irritable Bowel Syndrome And Mechanism

Irritable bowel syndrome (IBS) is a common functional gastrointestinal disorder. According to the Rome III definition, it is characterized by chronic abdominal discomfort or pain, bloating and changes in bowel function. Studies have reported that IBS is common, with worldwide prevalence ranging from 5% to 15%. Notably, women are twice as likely as men to be affected by IBS, and diarrhea-predominant irritable bowel syndrome (IBS-D) is reportedly the most common subtype of all IBS. IBS-D does not permanently damage the intestines or lead to serious intestinal disease, such as cancer, and however, IBS significantly impacts quality of life. It can affect many aspects of social and professional life, including increased absenteeism from work as well as reduced job opportunities and social interactions. Therefore, IBS places a significant psychological and physical burden on those afflicted with it.The etiology and pathogenesis of IBS remain unclear. Hereditary factors are likely to be involved in the etiology of IBS, with recent studies estimating this influence to be 50% in females. A genetic study in the UK has found that the novel 5-HT3E variant c.*76G>A (rs56109847) and the 5-HT3A variant c.-42C>T (rs1062613) are associated with IBS-D. However, this result for the 5-HT3A variant failed to be replicated in a cohort of German IBS-D patients compared to controls. It might be related to different race and genetic characteristics. But so far, there have not any research focus on whether the two SNP are associated with IBS in Chinese population.The recent breakthrough discovery of a new family of naturally endogenous, small, non-coding but functional RNAs (miRNAs) have been proven to play a role in the development of the wide spectrum of diseases. MiRNAs exerts its function through binding to specific complementary sequences in the 3’untranslated regions (3’UTR) of target mRNAs and negatively regulate target gene transcriptional expression. Recent studies have indicated that single-nucleotide polymorphisms (SNP) that reside in the microRNA (miRNA) target site can affect the binding of miRNA to mRNA, which can affect gene or protein expression and lead to influence the occurrence of certain human diseases.In our study, firstly, we investigated the relationship between the occurrence and symptoms of IBS-D and 5-HT3 A、5-HT3E gene polymorphism in Yangzhou by polymerase chain reaction (PCR)amplification and restriction fragment length polymorphism(RFLP technique. Secondly, we looked for the miRNA with potential combining ability to the SNP rs56109847 in 5-HT3E 3’UTR by bioinformatics prediction and literature. We studied on the function of 5-HT3E 3’UTR SNP rs56109847 through the dual luciferase report gene experiment. Thirdly, we understand the impact of the SNP rs56109847 on the 5-HT3E expression in intestinal by testing colonic mucosa 5-HT3E protein expression in IBS-D patients with different 5-HT3E genotypes. Finally, we studied the TCM syndromes, gynecological events, bowel and psychological symptoms in Chinese women who experienced both IBS-D and premenstrual syndrome (PMS).Our study was divided into five chapters.Chapter I. Association between diarrhea-predominant irritable bowel syndrome and 5-HT3A、5-HT3E gene polymorphism in YangZhou, ChineseObjective:To investigate the relationship between IBS-D and 5-HT3A、5-HT3E gene polymorphism in YangZhou.Methods:Polymerase chain reaction (PCR) amplication and restriction fragment length pdymorphism(RFLP) technique was used to detect the 5’UTR variant c.-42C>T (rs1062613) of 5-HT3A and 3’UTR variant c.*76G>A (rs56109847) of 5-HT3E in 300 healthy subjects and 450 patients with IBS-D.Results:There was significant difference between IBS-D patients and controls in the genotype of c.-42C>T sites, while the frequency of T allele was significantly higher in both female and male patients than that in the controls; The frequency of GA genotype and A allele of c.*76G>A sites was significantly higher in female-patients group than that in the controls, there was no significant difference between male-patients and controls; compared to the CC genetype of rs1062613 site and GG genetype of rs56109847 site, the TT and GA genotype are closely related to the IBS-D.Conclusion:The T allele of 5-HT3A and GA genetype of 5-HT3E may be respectively one of the important predisposing factors of IBS-D and female IBS-D patients in Chinese population.Chapter Ⅱ. Association between the 5-HT3A and 5-HT3E polymorphisms and the symptoms of diarrhea-predominant irritable bowel syndrome among Chinese womenObjective:we aimed to investigate the association between the bowel and psychological symptoms of IBS-D and the 5-HT3A and 5-HT3E polymorphisms in female IBS-D patients not menopause.Methods:Polymerase chain reaction (PCR) amplication and restriction fragment length pdymorphism(RFLP) technique was used to detect the 5’UTR variant c.-42C>T of 5-HT3 A and 3’UTR variant c.*76G>A of 5-HT3E in 104 female IBS-D patients not menopause.Results:Compared to the GG genetype of 5-HT3E, the patients with GA genotype had higher frequency of abdominal distension and even more serious defecate character. Compared to the C allele-carrying patients of 5-HT3A, the patients with TT genotype had higher frequency of feeling of urgency and incomplete defecation. Moreover, the typical form of stool significantly differed between the three 5-HT3A genotypes. The CC genotype of 5-HT3A, compared with the T allele, was associated with increased anxiety symptoms.Conclusion:The TT genotype of 5-HT3A and the GA genotype of 5-HT3E might contribute to the deterioration of the bowel symptoms. The 5-HT3A CC genotype was closely related to the anxiety symptoms.Chapter Ⅲ. The role of miRNA-510 to the 3’UTR of 5-HT3E wil and mut mRNAObjective:Based on the results discovered in chapter Ⅰ and chapter Ⅱ that GA genotype of 5-HT3E might contribute to the occurrence and deterioration of IBS-D, we further study the mechanism.Methods:Based on the bioinformatics prediction and the literature, SNP rs56109847 of 5-HT3E 3’UTR is located in the region of miRNA-510 recognition for binding. Variations in this region could influence miRNA-510 binding to the mRNA of 5-HT3E and change 5-HT3E expression. To test our hypothesis, we first determined whether the SNP could influence 5-HT3E expression by dual luciferase reporter assay. The renilla luciferase recombinant plasmid containing the SNP (pRL-TK-5-HT3E-3’UTR-c.*76A/G) and pGL3-control were co-transfected into cultured cells (HEK293 and HT-29) with the miRNA-510 plasmid or negative control miRNA plasmid. Using a dual luciferase reporter gene assay kit to verify whether or not miR-510 binding to the 3’UTR of 5-HT3E wil and mut mRNA. To investigate whether the SNP without miR-510 could influence 5-HT3E expression, we only transfected pRL-TK-5-HT3E-3’UTR-c.*76A/G and pGL3-control respectively into cultured cells.Results:The c.*76A variant constructs co-transfected with miR-510 showed significantly higher luciferase expression compared to c.*76G allele constructs in HEK293 and HT-29 cells. In both cell lines, no significant luciferase activity differences exist when co-transfecting negative control. When transfected constructs containing the SNP(G/A) without miR-510 or negative control, no difference in luciferase activity was detected in both HEK293 and HT-29 cells.Conclusion:MiR-510 can bind to the 5-HT3E wil mRNA (c.*76G). But the mutant (rs56109847G-A) may escape the normal regulation of miR-510.Chapter Ⅳ. Association between 5-HT3E 3’UTR polymorphisms and the 5-HT3E expression in colonic mucosaObjective:To investigate the 5-HT3E protein expression in colonic mucosa in female IBS-D patients and controls. To understand the association between 5-HT3E 3’UTR polymorphisms and the 5-HT3E protein expression in colonic mucosa.Methods:PCR-RFLP technique was used to detect the 3’UTR variant c.*76G>A of 5-HT3E in 53 female IBS-D patients; 5-HT3E protein was harvested from colon tissues and 5-HT3E expression of IBS-D patients and controls was determined by immunohistochemical and western blot techniques.Results:5-HT3E abundantly expressed around the colonic mucosal glands, a small amount of expression in the stroma. The 5-HT3E protein expression in colonic mucosal tissue in IBS-D patients is higher than in normal colonic mucosal tissue. Compared to 5-HT3E GG genotype patients, the 5-HT3E protein expression in colonic mucosal tissue is higher than that of 5-HT3E GA genotype patients.Conclusion:The 5-HT3E might play an important role in the process of female IBS-D. The SNP rs56109847 is associated with 5-HT3E protein expression.Chapter Ⅴ. The TCM syndromes and psychosomatic status of IBS-D overlapping PMSObjective:The purpose of this study was to investigate TCM syndromes, gynecological events, bowel and psychological symptoms in Chinese women who experienced both IBS-D and premenstrual syndrome (PMS).Methods:The study used the self-reported questionnaire based on previous studies done abroad with the Rome III criteria and SCL-90 Scale. The research was performed on 215 women in China to:(a) understand the association between the TCM syndromes and psychological symptoms of the overlapping patients, (b) compare psychological symptoms between the overlapping group and the IBS-D group, (c) compare gynecological experience of the 2 groups, (d) compare the characteristics of defecation and bowel habits by group.Results:The overlapping patients were divided into four kinds of TCM syndromes and significant difference existed in those who were of liver-related types or non liver-related types on anxiety and depression. The results revealed that compared to simple IBS-D patients, the patients in the overlapping syndromes group (1) had more severe psychological symptoms, (2) had increased gynecological events such as cesarean section history and recurrent miscarriage history, (3) had more severe dysmenorrheal and longer menstrual period, (4) had more severe bowel symptoms.Conclusion:Significant difference existed in the overlapping patients with liver-related types or non liver-related types on anxiety and depression. Compared to simple IBS-D patients, the patients in the overlapping syndromes group had increased gynecological events and more severe bowel and psychological symptoms.In summary, the occurrence and the severity of bowel symptoms of IBS-D in China may be closely related to the SNP rs1062613 and the SNP rs56109847. The possible mechanism is that the SNP 5-HT3E rs56109847G—A upregulates the expression of 5-HT3E protein by escaping the normal regulation of miR-510.