| BackgroundType 2 diabetes mellitus(T2DM)is a group of metabolic diseases characterized by extensive insulin resistance and pancreatic ?-cell dysfunction.It is widespread worldwide and has become one of the most serious chronic non-communicable diseases.Traditional methods of diabetes treatment include dietary habits,lifestyle adjustments,and drug interventions,but these methods are difficult to control the development of T2DM and related complications.In recent years,a large number of studies have shown that bariatric surgery could not only reduce weight,but also has a significant therapeutic effect on T2DM.Sleeve gastrectomy(SG)is currently the most widely performed weight loss surgery in the world.Although the clinical and basic research on the improvement of T2DM by SG is gradually deepening,the underlying mechanism has not yet been fully elucidated.As the central regulator of glucose homeostasis,liver could store or release glucose depending on the metabolic needs.The hepatic glucose and lipid metabolism disorders play an important role in the development of insulin resistance or diabetes.At the same time,long-term exposure to high glucose can trigger the endoplasmic reticulum stress of liver cells,which in turn induces apoptosis,and excessive apoptosis will further aggravate the body's insulin resistance state.Circular RNA is a special type of non-coding RNA with a covalent ring stucture.It is characterised with stable structure,high abundance and tissue-specific expression,and plays an important role in a variety of physiological processes.Circular RNA can play a molecular sponge function by targeting and binding microRNA(miRNA),thereby regulating the expression of parental genes.It has been reported that miRNA plays an important role in the remission of diabetes after bariatric surgery,and the role of circular RNA is not clear.It is worth noting that some circular RNAs have been confirmed to be related to the pathogenesis of diabetes.Based on this,in-depth exploration of the role of circular RNA in the remission of T2DM after SG is of far-reaching significance for the identification of the underlying mechanism of bariatric surgery and the diagnosis and treatment of diabetes.In this study,a rat model of obesity combined with T2DM was constructed,and the improvment on postoperative insulin resistance and T2DM were observed after SG surgery and sham surgery.Using circular RNA sequencing to detect the expression of circular RNA in the liver tissues of rats after surgery,we preliminarily clarify the changes in the expression profile of circular RNA in liver tissues of rats after SG,and explore the ideals molecular markers that can be used to monitor the effect of surgery and the degree of diabetic relief.At the same time,we screened out the differentially expressed circular RNADOCK7(CircDOCK7),and further clarified the role of circDOCK7 in the relief of diabetes after bariatric surgery and its potential molecular mechanism through functional experiments,clarifying the role of circular RNA in bariatric surgery treatment and providing a theoretical basis of the development of diabetic diagnosis and treatment technology based on circular RNA.Part ? Profiling of differentially expressed circular RNAs in diabetic rats after sleeve gastrectomyObjectiveHigh-fat feeding and low-dose streptozotocin(STZ)injection were used to construct obese rats with T2DM.SG and sham operations were performed on successfully constructed rats.To explore the improvement of SG on body weight,glucose and lipid metabolism in rats,as well as the changes in the expression profile of circular RNA in rat liver after surgery,and further detect the circularization characteristics of circDOCK7 and its expression in liver tissues after surgery.Methods1.To study the T2DM rat model induced by high-fat diet combined with low-dose STZ intraperitoneal injection.The rats that reached the successful model were randomly divided into two groups(SG group and SHAM group),with 10 rats in each group,and underwent SG operation and sham operation respectively.After the operation,the rats' body weight,food intake,glucose homeostasis,insulin sensitivity,liver function,and lipid metabolism were monitored regularly to evaluate the effect of the operation.2.The livers of 5 rats in the SG group were collected at 12 weeks after the operation.The livers of the rats in the SHAM group were used as controls for circular RNA sequencing.The differentially expressed circular RNAs after SG surgery were screened out,and the RNA sequencing results were verified by qRT-PCR.3.Bioinformatics analysis was performed on the differentially expressed circular RNAs selected,and select the ideal circular RNA molecule(circDOCK7)to explore its role in improving the body's metabolism after SG.4.After extracting genome DNA(gDNA)and total RNA of rat liver cells,reverse transcription to obtain cDNA,then perform polymerase chain reaction(PCR)amplification.Sanger sequencing was then performed to verify the sequence of the target circular RNA and the back splicing site.Divergent primers and convergent primers were designed respectively,and cDNA and gDNA were used as templates for amplification.The amplified products were subjected to agarose gel electrophoresis to verify the circularization properties of circular RNA.5.Treatmnt with RNase R and actinomycin to test the stability of circDOCK7.Results1.Food intake and weight:Due to perioperative stress and food restriction,the weight and food intake of the two groups showed a downward trend within 1 week after surgery,and then gradually increased.Compared with the SHAM group,the rats in the SG group gained weight and significantly reduced their food intake since the fourth week after surgery.2.Glucose homeostasis:From the second week after the operation,compared with the SHAM group,the fasting blood glucose of the SG group showed a significant decrease.Oral glucose tolerance test(OGTT)was performed at the 2nd and 12th weeks after the operation.The results showed that the AUCGTT of the SG group was significantly reduced,indicating that the glucose homeostasis of the rats after SG surgery was significantly improved.3.Insulin sensitivity:Insulin tolerance experiments and homeostasis model assessment of insulin resistance index in the 2nd and 12th weeks after surgery showed that SG surgery can improve insulin sensitivity in type 2 diabetic rats.4.Changes in indicators of liver function and lipid metabolism:Staining of liver tissue sections showed that the number of fatty degeneration cells in the liver of the SG group was reduced,and the number of lipid droplets in the liver cells was reduced,which is consistent with the results of tissue homogenate testing showing that the content of triglycerides in the liver of the SG group was reduced.5.Liver circular RNA expression profile:In 10 liver samples,a total of more than 20,000 circular RNA expression abundances were detected.Taking P<0.05 and differential expression multiple>1.0 time as the improved differential screening criteria,a total of 113 differentially expressed circular RNA molecules were screened,including 71 high-expressed and 42 low-expressed molecules.Among them,33 circular RNA molecules that were specifically expressed only after SG were screened.The qRT-PCR detection of rat liver tissue showed that it was consistent with the sequencing results.6.With the improvement of insulin sensitivity and diabetes status,the expression of circDOCK7 in the liver tissue of rats after SG was significantly increased.The molecular sequence and reverse splicing site of circDOCK7 were confirmed by Sanger sequencing of the PCR amplified products.circDOCK7 is derived from the DOCK7 gene(chr5:117742776-117743766)and consists of three exons with a length of 429 nucleotides.The results of agarose gel electrophoresis showed that circDOCK7 can be amplified by convergent and divergent primers in cDNA,but only by convergent primers in gDNA.However,the divergent primer of the linear molecule beta-actin used as a control showed no amplification products in cDNA and gDNA.After adding RNase R,the expression of circDOCK7 hardly changed,while the expression of linear mRNA decreased significantly.After adding the cell transcription inhibitor Actinomycin D,the expression of circDOCK7 did not decrease significantly within 24 hours compared with linear mRNA.Conclusions1.SG surgery could significantly reduce body weight,improve glucose homeostasis and liver glucose and lipid metabolism.2.Compared with the SHAM group,the liver of diabetic rats in the SG group had significant differential expression of circular RNA.Part of the circular RNA is only specifically expressed after SG,which is expected to be an ideal biomarker for monitoring the prognosis of bariatric surgery.3.circDOCK7 is an RNA molecule with a closed loop structure,with strong stability,and its expression in the liver after SG is significantly increased.Part ? The role and mechanism of circRNA DOCK7 in the remission of type 2 diabetes mellitus after sleeve gastrectomyObjectiveCellular experiments were conducted to clarify the role of circDOCK7 in the regulation of glucose and lipid metabolism,and to explore the effects of circDOCK7 on liver glycolipid after SG from two aspects,the regulation of glucose metabolism and insulin signaling pathway,and the liver cell apoptosis induced by diabetes with high glucose and insulin resistance.Methods1.Constructing a small interfering RNA targeting the circularization site to down-regulate the expression of circDOCK7.The glucose absorption of liver cells was detected by the glucose colorimetric method and the fluorescent glucose analog method.2.Using qRT-PCR to detect the expression levels of key molecular genes in the glycometabolism pathway of liver cells after circDOCK7 knockdown.3.Using western blot to detect the expression changes of key proteins in the insulin sensitivity pathway of liver cells after knockdown of circDOCK7.4.Using high glucose combined with insulin treatment to construct a rat hepatic cell insulin resistance model.The circDOCK7 overexpression plasmid was constructed,and the glucose uptake of liver cells was detected by overexpression of circDOCK7 in the liver cells of insulin resistant rats.5.Using CCK-8 and EdU proliferation experiments to detect the regulatory effect of circDOCK7 knockdown on rat liver cell proliferation.The apoptotic cells were labeled with fluorescein isothiocyanate and propidium iodide to detect the effect of circDOCK7 knockdown on rat liver cell apoptosis.6.The intracellular sub-localization of circDOCK7 was verified by RNA fluorescence in situ hybridization,and bioinformatics analysis was used to predict that it might bind to the regulated miRNA molecule(miR-139-3p),and qRT-PCR was used to detect the postoperative growth of circDOCK7.Mouse liver tissue and the expression level of this miRNA in rat liver cells after knocking down circDOCK7 to verify its correlation.7.Through transfection of miR-139-3p analogues and co-transfection of circDOCK7 small interference and miR-139-3p inhibitor to explore the process of miR-139-3p involved in circDOCK7 regulating cell apoptosis.8.Verifying the intracellular localization of circDOCK7 and miRNA through RNA FISH co-localization experiment,and verify whether circDOCK7 and target miRNA have the potential to bind.9.Predict the binding target of miR-139-3p through bioinformatics,and explore the expression changes of target genes by transfection of miR-139-3p analogs and co-transfection of circDOCK7 small interference and miR-139-3p inhibitors,explore whether it is the role of circDOCK7 in regulating cell apoptosis.Results1.After knocking down the expression of circDOCK7 in rat hepatocytes with small interfering RNA,the glucose uptake ability of rat hepatocytes is weakened.The measurement of glycometabolism-related pathways found that the mRNA levels of gluconeogenesis-related genes in rat liver cells were significantly up-regulated,while glycolysis and glycogen synthesis pathways were not significantly changed.After detecting the expression of proteins related to the insulin signaling pathway,it was found that the phosphorylation level of insulin receptor substrate 1,PI3K,and AKT decreased.2.Overexpression of circDOCK7 in the hepatocyte model of insulin resistance rats significantly improved the impaired glucose absorption capacity of the hepatocytes.3.CCK-8 experiment showed that knocking down circDOCK7 inhibited the proliferation of hepatocytes.Similarly,EdU proliferation experiments showed that,compared with the control group,the proportion of EdU stained cells decreased significantly after circDOCK7 was knocked out.Flow cytometry detected the effect of circDOCK7 on cell apoptosis.The results showed that compared with the control group,the proportion of apoptosis in the transfection group increased in the early and late stages.4.The results of RNA-FISH experiments show that circDOCK7 is mainly located in the cytoplasm.Bioinformatics predictions show that miR-139-3p is a potential binding target of circDOCK7.In addition,in the SG group and SHAM group,the expression of miR-139-3p was confirmed to be negatively correlated with circDOCK7.In rat hepatocytes,down-regulation of circDOCK7 can significantly increase the relative expression level of miR-139-3p.5.Transfection of miR-139-3p analogs to rat hepatocytes can promote cell apoptosis,showing a similar effect to knocking down circDOCK7.At the same time,co-transfection of rat hepatocytes with si-circDOCK7 and miR-139-3p inhibitor showed that inhibiting the expression of miR-139-3p can significantly improve the apoptosis caused by circDOCK7 silencing.6.RNA FISH detection showed that circDOCK7 and miR-139-3p co-localized in the cytoplasm.7.Bioinformatics predicts that the apoptosis-related gene MCM3 is the potential binding site of miR-139-3p.Western blot was used to detect the protein expression levels of apoptosis-related target genes after transfection of miR-139-3p analogs and co-transfection of circDOCK7 small interference and miR-139-3p inhibitors.The results showed that the expression of MCM3 was down-regulated after transfection of miR-139-3p analogues,which was consistent with the results after knocking down circDOCK7.After co-transfection of circDOCK7 small interference and miR-139-3p inhibitor,the inhibition of target protein expression was improved.Conclusions1.circDOCK7 can significantly regulate the glucose uptake of liver cells and the activity of insulin signaling pathway.2.Knockdown of circDOCK7 can promote the expression of gluconeogenesis-related genes,but has no significant effect on glycolysis and glycogen synthesis pathways.3.Knockdown of circDOCK7 can inhibit the proliferation of hepatocytes and induce apoptosis.4.circDOCK7 can improve its inhibitory effect on apoptosis-related gene MCM3 by adsorbing miR-139-3p,thereby regulating liver cell apoptosis. |
PDF Full Text Request