The Association Between Differential Expression Of Circulating LncRNAs And Function Of Pancreas Islet In Type 2 Diabetes Mellitus

BackgroundThe prevalence of type 2 diabetes mellitus (T2DM) has risen dramatically in China. According to a research of JAMA in 2013, the prevalence of T2DM was estimated 11.6% with approximately 114 million individuals, ranking the first worldwide. T2DM has become the third chronic disease which seriously threatens human health together with tumor and cardiovascular disease. Based on the current trends, more than 300 million individuals will suffer from DM by the year of 2020, severely affecting human health and social development. DM is the result of interactions combining genetic and environmental factors which may ultimately lead to the destruction of the pancreatic beta cells function. Due to genetic factors, individuals with genetic susceptibilities to T2DM are easier to develop to DM. It’s unreliable to determine whether the accumulation of related abnormal gene expression has reached the "critical point" through fasting plasma glucose (FPG), postprandial plasma glucose (PPG) or other external indexes. Therefore, further studying the genetic susceptibility, abnormal gene expression and pathogenesis of DM are of great significance for its early diagnosis and individual therapy.Non-coding RNA (ncRNA), known as "dark matter" in life body, has recently become the hotspot in life science. Non-coding RNAs are widely involved in physiology and pathophysiology of human beings. The imbalanced regulations of ncRNA will contribute towards various diseases. In this RNA category, they do not encode any protein but function in cell regulations. Many studies indicate that imbalanced of ncRNAs regulations are closely related to the onset and development of diseases. Long non-coding RNA (lncRNA) are a RNA category that transcript longer than 200 nucleotides. It can regulate gene expressions by combining with target genes as a complex, promoting degradations or inhibiting translations of target mRNA gene. lncRNAs are associated with nearly all physiology and pathology process of human beings by regulating in epigenetic, transcriptional, post-transcriptional and many other levels. Recent studies implicate abnormal expressions of lncRNAs in some complicated diseases, such as tumor, infection, diabetes, cardiovascular and neurological diseases. They can either promote or inhibit the onset of diseases. For example, compared with normal tissues and typical hyperplasia tissues or tumor, lncRNAs have different expressions and those differential expressed lncRNAs can be used to predict tumor. Also, lncRNAs are stable in circulatory system of human and can be detected through peripheral blood. Prostate specific lncRNAs DD3, also called PC A3, have already become a highly specific marker, which is even more specific than the conventional marker--prostate specific antigen (PSA). In recent years, studies of lncRNAs have already made some progress in oncology, however research on its association with pathogenesis of T2DM is still in the early stage. No research was reported to show the lncRNAs expression profiles in peripheral blood of T2DM patients. Some preliminary studies indicate that lncRNAs are closely related to pancreatic beta cell development, proliferation and insulin production. All these results refer it to an important regulation role in pancreatic beta cell dysfunction and the onset of DM. As is well known that it’s hard to get pancreatic tissues from T2DM individuals. So if lncRNAs in peripheral blood can be used as a clinical marker, it may be beneficial for T2DM patients.In conclusion, this research is aimed to construct a circulating lncRNAs expression profile in T2DM patients. Then selecting and identifying the candidated lncRNAs that are closely related to the development and the onset of T2DM through lncRNAs microarray. Function of target genes are analyzed by bioinformatics. Association between aberrantly expressed lncRNAs and pancreatic function of T2DM are further analyzed in cross-sectional studies in order to provide new strategies of early diagnosis and treatment for T2DM patients.This research is divided into two parts:Section 1:Research on identification and function of differentially expressed lncRNAs in T2DM patientsObjectivesThe expression profiles of lncRNAs and mRNA in T2DM group and control group were analyzed by lncRNAs microarray. Clustering analysis and KECG pathway analysis were used to select lncRNAs associated with insulin signaling pathway in T2DM. Additionally, mechanism of differentially expressed lncRNAs in gene regulations were analyzed through cis and trans forecast analysis in order to construct a lncRNAs-mRNA network and explore related key lncRNAs in the process of T2DM.Methods1. Three newly diagnosed T2DM patients and three healthy individuals were involved in this study and they were divided into T2DM group and control group. All of the participants were gender and aged match and their data were clearly recorded. Peripheral blood sample was collected from each participant to extract total RNA.2. LncRNAs microarray was used to detect and select differentially expressed lncRNAs and mRNA in T2DM group compared with control group, based on the standard that fold change (FC value)≥ 2 and p< 0.05.3. Selecting lncRNAs associated with insulin pathway by bioinformatics analysis, and predicting target genes and constructing lncRNAs-mRNA co-expression network. Using Gene set enrich analysis such as GO and KEGG pathway analysis to select lncRNAs related to insulin pathway. Pearson correlation coefficient was used to predict mRNA that was co-expressed with aberrantly expressed lncRNAs. On the basis mentioned above, cis and trans analysis were used to predict target gene. Cis analysis was used to search lncRNAs-mRNA, the length of which was less than 10kb. Similar lncRNAs-mRNA was selected by trans analysis which used Blat tools to make sequence alignments with mRNA (3’UTR).Results1. The results of lncRNAs microarray revealed that compared with control group, 2270 lncRNAs were differentially expressed in T2DM group. Among them,1891 lncRNAs were up-regulated and 379 lncRNAs were down-regulated. In addition, 2194 mRNA were aberrantly expressed in T2DM group when compared with control group, including 653 up-regulated mRNA and 1541 down-regulated mRNA.2. Bioinformatics analysis of differentially expressed genes in peripheral blood of T2DM patients:GO analysis indicated that target genes were associated with the immune process, signal transduction and RNA gene silencing. KEGG pathway analysis indicated that 16 pathways corresponded to up-regulated transcripts and those 10 pathways corresponded to down-regulated transcripts (p<0.01). After selecting the candidated lncRNAs correlated with insulin signaling pathway, further molecular functional annotation analysis shows that these candidated genes was associated with the regulation of cytotoxicity, apoptosis and abnormal signal transduction, which were also the important pathophysiology process of pancreatic beta cell dysfunction. Six most significant differentially expressed genes like Inc-p3134, lnc-p25201, lnc-p34005_v4, Inc-pl8324, Inc-pl2792 and lnc-RNA143599 were selected for further analysis of the association between their expression levels and glucose or lipid metabolism as well as the pancreatic function in T2DM patients.3. With bioinformatics analysis, lnc-p 12792 was found co-expressed with 23 mRNA and these mRNAs were closely related to insulin signaling pathway. Transcription factor 7 like 2 (TCF7L2) gene was found as the regulatory target of lnc-p 12792 by target prediction software. TCF7L2 was a susceptibility gene of T2DM, which was confirmed as the most relevant protein coding gene of glycometabolism dysfunction and it was related to glucose induced insulin secretion. TCF7L2 can also affect the synthesis of glucagon like peptide 1 (GLP-1) to block the insulin signaling pathway and can induce insulin secretion dysfunction and pancreatic beta cell dysfunction.ConclusionAberrantly expressed IncRNAs existed in peripheral blood of T2DM patients, which involved plenty of pathway regulations. Among them, incRNA-p 12792 and its target gene TCF7L2 were related to insulin pathway, which might contribute to the onset and development of T2DM.Section 2:Research on the association between the expression levels of T2DM related lncRNAs and pancreatic beta cell function.ObjectivesInvestigating the circulating expression level of six T2DM related lncRNAs (Inc-p3134, lnc-p25201, lnc-p34005_v4, Inc-p18324, Inc-p12792, lnc-RNA143599) in peripheral blood of T2DM patients and the association between lncRNAs expression level and lipid metabolism, glycometabolism and pancreatic beta cell function, in order to find a new biomarker for early diagnosis and pancreas function evaluation of T2DM patients.Methods1. Thirty newly diagnosed T2DM patients and thirty healthy individuals were involved in this study and they were divided into T2DM group and control group. All data of participants were clearly recorded and peripheral blood sample was collected from each one to extract total RNA.2. Real-time fluorescent PCR (RT-PCR) was used to identify the expression levels of lnc-RNAs. Based on the results of microarrays and bioinformatics analysis, lnc-RNAs related to insulin pathway were detected. Among them, six most significant aberrantly expressed genes were selected. RT-PCR was used to verify the expression levels of aberrantly expressed genes in peripheral blood of T2DM group (n=30) compared with control group (n=30). T-test of independent sample was used to compare the expression levels of aberrantly expressed genes in two groups.3. Body mass index (BMI) and waist-hip ratio (WHR) were detected in two groups and each participant was enrolled in oral glucose tolerance test (OGTT). Fasting plasma glucose (FPG) and 2-hour postprandial plasma glucose (2h-PPG) were detected by glucose oxidase method. Fasting insulin and 2-hour postprandial insulin were detected by chemiluminescent microparticle immunoassay (CMIA). Fasting plasma c-peptide and 2-hour postprandial plasma c-peptide were detected by radioimmunoassay (RIA). Homeostasis model assessment of beta cell (HOMA-P) was used to evaluate the secretion of pancreatic beta cell. Homeostasis model assessment of Insulin Resistance (HOMA-IR) was used to evaluate the insulin resistance. Enzyme-linked immunosorbent assay (ELISA) was used to detect the level of Uric Acid (UA), interleukin 6 (IL-6) and hyper-sensitive C-reactive protein (hsCRP). Automatic biochemistry analyzer was used to detect total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C) and low density lipoprotein cholesterol (LDL-C). Using spearman correlation analysis and multiple stepwise regression analysis to analyze the association between aberrantly expressed lnc-RNA and related metabolic index as well as pancreatic function. Using ROC curve to evaluate the value of diagnosis in T2DM.Results1. Compared with control group, the circulating expression levels of Inc-p3134, lnc-p25201, Inc-p18324, Inc-p12792 and lnc-RNA143599 in T2DM group were significantly up-regulated (P<0.01), while the circulating expression levels of lnc-p34005_v4 was significantly down-regulated (P＜0.01). The results of RT-PCR are consistent with that of microarrays.2. Results of spearman correlation analysis showed that Inc-p3134 was positively correlated to age, WHR, FPG, fasting plasma c-peptide, HOMA-IR, TG, LDL-C and was negatively correlated to HOMA-P (r=-0.412, p< 0.05). It was not correlated to BMI, fasting insulin, TCU HDL-C, UA, IL-6 and hsCRP (p>0.05). Lnc-p 12792 was positively correlated to BMI, WHR, FPG and TC while it was negatively correlated to HOMA-P (r=-0.484, p< 0.05). It was not correlated to age, fasting insulin, fasting plasma c-peptide, HOMA-IR, TG, LDL-C, HDL-C, UA, IL-6 and hsCRP (p>0.05) Lnc-RNA 143599 was positively correlated to age, BMI, WHR, FPG, fasting plasma c-peptide, HOMA-IR, TC, TG and LDL-C while it was negatively correlated to HOMA-p (r=-0.398) and HDL-C (p< 0.05). It was not correlated to fasting insulin, UA, IL-6 and hsCRP (p>0.05). Lnc-p25201 was positively correlated to WHR, FPG, HOMA-IR and TG and it was not correlated to other indexes (p＞0.05). Lnc-p18324 was positively correlated to FPG, HOMA-IR, hsCRP and TG(p< 0.05)and it was not correlated to other indexes(p>0.05). Lnc-p34005_v4 was negatively correlated to age and FPG (p<0.05) and it was not correlated to other indexes (p>0.05).3. Multiple logistic regression analysis found that individuals with higher expression level of lnc-p 12792 in peripheral blood might have higher risk of DM (OR = 1.96). Multiple stepwise regression analysis revealed that FPG(P=0.394, p<0.01), BMI(p=0.379, p<0.01) and HOMA-p(p=-0.391, p<0.01) are the independent factors of the expression level of lnc-p 12792 in peripheral blood. Further stratified analysis by different glucose level show that the circulating expression level of lnc-p 12792 increased with FPG Statistically significant was found in comparison with each group (p<0.05). Using ROC curve to evaluate the diagnostic value of Inc-p12792 in T2DM, its sensitivity was 66.7% and its specificity was 83.3%.ConclusionThe expression level of LncRNA-p 12792 was closely related to FPG level and pancreatic beta cell function. It was positively correlated to the risk of T2DM and might become an alternaltively predictive marker in the early phase of T2DM.