A Study On The Roles And Interventions Of LncRNA MALAT1 Regulated MiR-17 In The Dysfunction Of Islet β Cells Induced By Cigarette Smoking

Diabetes mellitus is a metabolic disease associated with many factors such as the environment and diet include type I diabetes with absolute insulin deficiency,type II diabetes with insulin resistance and progressive loss of beta-cell,and other special types of gestational diabetes.Islet β cell dysfunction is a common pathological basis for type I and type II diabetes.Smoking is one of the leading risk factors for diabetes.Smoking could cause an increase in pro-inflammatory factors in the serum.By causing inflammation and infiltration of tissues such as the pancreas,the inflammation is further amplified and the protease is released,thereby causing apoptosis of the islet β cells,impairing its function,and causing abnormal insulin secretion.Although smoking is an independent risk factor for islet β cells dysfunction based on extensive epidemiological investigations,the exact pathogenesis is still unclear.In addition,the number of diabetic patients has increased year by year due to chronic inflammation caused by smoking,but effective interventions are still lacking.Non-coding RNAs(nc RNAs),such as long-chain non-coding RNAs(lncRNAs)and micro RNAs(mi RNAs),play important regulatory roles in the development of many human diseases.lnc RNA can affect target gene function by adsorbing mi RNA.Although several studies have shown that smoking affects islet nc RNA expression by causing inflammatory infiltration and plays an important role in the development and progression of diabetes,its exact function still need to be further studied.Andrographolide could inhibit inflammation,and it has a promising future for the intervention of islet β cell dysfunction and diabetes caused by smoking.Therefore,to study the role of nc RNA and evaluate the intervention effect of andrographolide in smoking-induced islet β cell dysfunction,it is helpful to elucidate the molecular mechanisms of smoking-induced diabetes and find new therapeutic targets.Part I The roles of lnc RNA MALAT1 regulated mi R-17 in the dysfunction of islet β CellsObjectiveTo investigate the effects of cigarette smoke extract(CSE)on the secretion of inflammatory factors,glucose-stimulated insulin secretion and insulin synthesis related genes in MIN6 cells.MethodsBy using(1)Mice islet β(MIN6)cells were treated with 0,20,40 or 80 μg/m L CSE for 24 h.(2)After pretreatment of MIN6 cells with 50 n M mi R-17 mimic or mi R-NC for 24 h,MIN6 cells were treated with 40 μg/m L CSE for 24 h.(3)The pre-treated MIN6 cells were transfected with si RNA or Con si RNA of 100 n M lnc RNA MALAT1 for 24 h or and then treated with 40 μg/m L CSE for 24 h.(4)The pre-treated MIN6 cells were co-transfected with mi R-17 mimic or mi R-NC with si RNA or Con si RNA of 100 n M lnc RNA MALAT1 for 24 h,and then treated with 40 μg/m L CSE for 24 h and other processing methods.To establish an in vitro exposure model of CSE-induced islet β cell dysfunction model.Enzyme-Linked Immunosorbent Assays(ELISA)was used to observe the secretion levels of IL-6 and IL-1β in MIN6 cells.ELISA was used to observe glucose-stimulated insulin secretion(GSIS);Western Blots to detect the expression levels of insulin synthesis related proteins maf A,TXNIP;q RT-PCR detection of cell lnc RNA MALAT1 and mi R-17 expression;luciferase reporter gene detection mi R-17 and lnc RNA MALAT1 binding Using mi R-17 inhibitor or mimic,lnc RNA MALAT1 si RNA and other interference techniques to explore the role of lnc RNA MALAT1 in the regulation of mi R-17 in CSE-induced islet β cell dysfunction,respectively.Results1.The effect of CSE on insulin secretion,synthesis and inflammatory factors in MIN6 cellsIt was found that the secretion levels of IL-6 and IL-8 in MIN6 cells,glucose-stimulated insulin secretion and insulin synthesis inhibitory factor TXNIP expression gradually increased with CSE treatment dose.The expression of insulin transcription factor maf A in MIN6 cells gradually decreased with increasing dose of CSE treatment.It is suggested that CSE can cause the decrease of insulin secretion and synthesis in MIN6 cells and increase the release of inflammatory factors.2.The effect of CSE on mi R-17 and lnc RNA MALAT1 levels in MIN6 cellsIt was found that the expression of lnc RNA MALAT1 were increased and the expression of mi R-17 were decreased in MIN6 cells,and both had a dose-effect relationship with CSE treated dose.It has been predicted that lnc RNA MALAT1 has a binding site with mi R-17.It is suggested that lnc RNA and mi RNA play an important role in smoking-induced islet β cell dysfunction.3.The roles of mi R-17 in the damage of islet β cell function induced by CSEmicro RNA target gene prediction websites revealed the presence of a binding site for mi R-17 in the 3’-UTR of TXNIP.It was found that overexpression of mi R-17 can block the glucose secretion-stimulated insulin secretion of MIN6 cells induced by CSE;mi R-17 has a stable binding site with the negatively regulated insulin synthesis gene TXNIP,and mi R-17 mimic can be used to verify that mi R-17 can affect the expression level of TXNIP.mi R-17 mimic can increase the TXNIP of MIN6 cells and decreases the expression of maf A.The results suggest that mi R-17 plays a role in CSE-induced islet β cell dysfunction.4.The role of lnc RNAMALAT1 in the damage of islet β cell function induced by CSEThe results showed that lnc RNA MALAT1 and mi R-17 have stable binding sites.It was found that mi R-17 mimic and PGL3-MALAT1-WT co-transfection combined with 40 μg/m L CSE treatment of MIN6 cells significantly lower luciferase activity than CSE treatment alone.The reduction of lnc RNA MALAT1 expression caused a increase in free mi R-17;Inhibition of lnc RNA MALAT1 expression significantly blocked CSEinduced glucose-stimulated insulin secretion and decreased insulin synthesis-related protein levels in MIN6 cells.It is suggested that CSE can affect islet β cell function through lnc RNA MALAT1.5.The roles of lnc RNA MALAT1 regulated mi R-17 in the dysfunction of islet β CellsThe results showed that lnc RNA MALAT1 si RNA combined with mi R-17 inhibitor interaction experiment demonstrated that CSE can up-regulate the target gene TXNIP by causing high expression of lnc RNA MALAT1,which leads to a decrease in insulin synthesis and impaired islet β cell function.ConclusionCSE can cause elevated levels of inflammatory factors in MIN6 cells,induce an increase in the level of lnc RNA MALAT1,thereby causing a decrease in mi R-17 levels,and the inhibition of TXNIP is released,further causing a decrease in maf A and inhibition of insulin synthesis.Part Ⅱ Intervention of andrographolide on CSE-induced β cell dysfunctionObjectiveTo evaluate the intervention effect of andrographolide on CSE-induced islet β cell dysfunction,and to explore the possible mechanisms.MethodsMIN6 cells were pre-treated with 0.05% DMOS or 1,2.5,5 μM andrographolide for 24 h,and MIN6 cells were treated with 40 μg/m L CSE for 24 h to establish a cell model of andrographolide.The secretion level of inflammatory factors IL-6,IL-1β and GSIS in MIN6 cells were observed by ELISA;Western blots detection of insulin synthesis related proteins expression levels of maf A,TXNIP and expression of apoptosis-related proteins Caspase-3,Cleaved Caspase-3 at the cellular level.To investigate the effect of anti-inflammatory drug andrographolide on CSE-induced islet β cell damage.Results1.The effect of andrographolide on the secretion of inflammatory factors in MIN6 cells induced by CSEThe results showed that the andrographolide treatment alone did not affect the secretion levels of inflammatory factors IL-6 and IL-1β in MIN6 cells compared with the CSEtreated group.The andrographolide pretreatment combined with 40 μg/m L CSE-treated compared with the CSE-treated group,the secretion levels of IL-6 and IL-1β in MIN6 cells were decreased,and the effect on IL-6 secretion level had a significant dose-effect relationship.The results suggest that andrographolide can block the increase of inflammatory factors in MIN6 cells induced by CSE.2.The effect of Andrographis on the synthesis and secretion of insulin in MIN6 cells induced by CSEIt was found that the andrographolide treatment alone did not affect the GSIS ability of MIN6 cells compared with the CSE-treated group.The andrographolide pretreatment combined with 40 μg/m L CSE-treated compared with the CSE-treated group,GSIS ability rebound and the expression level of TXNIP is decreased and the expression level of maf A is increased and it has a dose-effect relationship.The results suggest that andrographolide can block the decrease of insulin synthesis in MIN6 cells induced by CSE.3.The effects of Andrographis on the increase of apoptosis-related proteins in MIN6 cells induced by CSEThe results showed that the andrographolide treatment alone did not affect the expression of Cleaved Caspase-3 in MIN6 cells compared with the CSE-treated group.The andrographolide pretreatment combined with 40 μg/m L CSE-treated compared with the CSE-treated group,the expression level of cleaved Caspase-3 is decreased in MIN6 cells,and it has a dose-effect relationship.The results suggest that andrographolide can block the apoptosis of MIN6 cells induced by CSE.ConclusionCSE-induced inflammatory factor release from MIN6 cells and impaired islet β cell function(reduced insulin synthesis and increased apoptosis)can be effectively blocked by andrographolide.The results showed that the anti-inflammatory drug andrographolide interfered with the damage of islet β cell function induced by CSE.Part III The effects of cigarette smoke exposure on the function of mouse islet β cells and the intervention of andrographolideObjectiveFurther confirmation the effect of cigarette smoke exposure in mice islet β cell function caused by inflammatory infiltration and release of inflammatory factors,and the intervention of andrographolide at the animal level.MethodsHealthy C57BL/6 mice at 4 weeks of age(about 20 g body weight)were randomly divided into 3 groups: control group,cigarette smoke exposure group,cigarette smoke combined with andrographolide treatment group.To establish a model of islet β cell function damage induced by cigarette smoke exposure and an intervention model of andrographolide.Mice were placed in the cigarettes exposure system for 5 days a week,exposed to 1 mg of cigarette smoke of 1 mg of total particulate matter for 1 h,and exposed repeatedly for 4 h for 12 weeks.The mouse of the andrographolide intervention group were intraperitoneally injected at a dose of 100 mg/kg three times a week for one day.Peritoneal glucose injection test(IPGTT),fasting blood glucose was used to evaluate glucose metabolism in mice.ELISA was used to detect serum insulin,IL-6 and IL-1β secretion levels.Immunohistochemistry was used to detect islet inflammatory infiltration and insulin levels.TUNEL staining was used to detect islet β cells apoptosis level;Western Blots detection of islet Caspase-3 and Cleaved Caspase-3 expression levels.To study the effects of cigarette smoke exposure on islet β cell function by causing inflammatory infiltration and release of inflammatory factors,and toinvestigate the intervention of andrographolide at the animal level.Results1.The effects of cigarette smoke exposure on glucose metabolism disorders in mice and the intervention of andrographolideIt was found that cigarette smoke and andrographolide did not affect the body weight of the mice.However,cigarette smoke caused an increase in fasting blood glucose levels,which was further discovered by IPGTT experiments.Cigarette smoke impaired the ability of glucose clearance in mice;compared with the cigarette smoke group,andrographolide effectively inhibited the increase in fasting blood glucose and the decrease in glucose clearance in mice caused by cigarette smoke.It suggests that andrographolide can antagonize hyperglycemia caused by exposure to cigarette smoke.2.The the effects of cigarette smoke exposure on inflammatory infiltration,release of inflammatory factors and the intervention of andrographolideIt was found that cigarette smoke caused an increase in the surface marker CD68 in the islet suggesting macrophage infiltration.Cigarette smoke also caused a significant increase in serum levels of inflammatory factors IL-6,IL-1β and TNF-α.In mice treated with andrographolide combined with cigarette smoke,islet inflammatory cell infiltration and serum inflammatory factor release were inhibited.It suggests that andrographolide can antagonize the inflammatory infiltration and release of inflammatory factors caused by exposure to cigarette smoke.3.The effects of cigarette smoke exposure on mouse islet β cells function and intervention of andrographolideIt was found that cigarette smoke caused a decrease in serum insulin,islet area,and insulin-positive β cells,and an increase in islet apoptosis,Cleaved Caspase-3 level.In the mice treated with andrographolide combined with cigarette smoke,the above effects were inhibited to some extent.It suggests that andrographolide can antagonize the decrease of insulin synthesis in islet islet β cells and the apoptosis of islet β cells induced by exposure to cigarette smoke,and has a protective effect on islet β cell function.ConclusionCigarette smoke exposure can cause reduced islet β cell insulin synthesis,increased islet β cell apoptosis,and β cell dysfunction.In the mice treated with andrographolide combined with cigarette smoke,the above effects were inhibited to some extent.The results provided a basis for islet β cell dysfunction by cigarette smoke exposure.It was further suggested that the intervention of anti-inflammatory drug andrographolide in islet β cell function damage caused by cigarette smoke.Part IV The levels and significances of serum lnc RNA MALAT1 and mi R-17 in people with different smoking levelsObjectiveTo investigate the levels of serum IL-6,IL-8 and other inflammatory factors,the levels of lnc RNA MALAT1 and mi R-17 in people with different smoking levels.To explore the effects of smoking on fasting blood glucose,glycosylated hemoglobin(Hb A1c),insulin sensitivity index(HOMA-IR)and beta cell function index(HOMA-β)%.MethodsIn the established cohort,smokers of different degrees were divided into non-smoking group,mild smoking group,medium smoking group and heavy smoking group according to the number of years of smoking(average number of packages/day × number of years of smoking).Epidemiological investigations collected basic data and diabetes related indicators,calculated HOMA-IR and HOMA-β;BD-CBA human serum inflammatory factor test kit was used to detect the expression of serum inflammatory factors;q RT-PCR was used to detect serum lnc RNA MALAT1 and mi R-17 level.Results1.The impact of smoking on diabetes related indicatorsA survey of diabetes-related indicators in smokers of different degrees found that smokers’ fasting blood glucose and Hb A1 c were significantly higher than those of nonsmokers,and the mean was higher than the diagnostic criteria.As the number of years of smoking has increased,a lower HOMA-β value is presented.It is suggested that a large amount of smoking leads to abnormal glucose metabolism and weakens β cell function.2.The effect of smoking on the secretion level of serum inflammatory factorsA survey of different levels of smoking found that compared with non-smokers,the level of serum inflammatory factor TNF-α was significantly increased with the increase of smoking years,and the levels of IL-6,IL-8 and IL-1β in heavy smokers were significant increase.It is suggested that smoking may cause the secretion of serum inflammatory factors IL-8,L-1β,IL-6 and TNF-α to increase,resulting in chronic inflammation.3.The impact of smoking on the expression of lnc RNA MALAT1 and miR-17A survey in smokers with different smoking degreration found that smoking caused upregulation of lnc RNA MALAT1 and down-regulation of mi R-17 levels in smokers’ serum,respectively.Linear regression analysis showed a positive correlation between serum lnc RNA MALAT1 levels and mi R-17 levels were correlated with fasting blood glucose.It is suggested that the changes of serum lnc RNA MALAT1 and mi R-17 levels may be related to smoking-induced islet β cell damage,which may be used as a biomarker for diabetes-assisted diagnosis of diabetes.ConclusionSmoking causes abnormal glucose metabolism and weakens β cell function,causing up-regulation of serum lnc RNA MALAT1 and down-regulation of mi R-17 and is associated with elevated fasting blood glucose,resulting in increased secretion of serum inflammatory factors and chronic inflammation.In summary,the novel findings of this study are as follows:1.CSE can cause elevated levels of inflammatory factors in MIN6 cells,induce an increase in the level of lnc RNA MALAT1,thereby causing a decrease in mi R-17 levels,and the inhibition of TXNIP is released,further causing a decrease in maf A and inhibition of insulin synthesis.2.CSE-induced inflammatory factor release from MIN6 cells and impaired islet β cell function(reduced insulin synthesis and increased apoptosis)can be effectively blocked by andrographolide.The results showed that the anti-inflammatory drug andrographolide interfered with the damage of islet β cell function induced by CSE.3.12 weeks of cigarette smoke exposure can cause elevated fasting blood glucose in mice,impaired glucose tolerance;increased inflammatory cell infiltration in islets,increased release of serum inflammatory factors IL-6,IL-1β and TNF-α;reduced islet β cell insulin synthesis,increased islet β cell apoptosis,and β cell dysfunction.In the mice treated with andrographolide combined with cigarette smoke,the above effects were inhibited to some extent.The results provided a basis for islet β cell dysfunction by cigarette smoke exposure.It was further suggested that the intervention of antiinflammatory drug andrographolide suggests that cigarette smoke exposure may cause islet β cell function damage and cause diabetes by causing tissue inflammation.4.Smoking causes abnormal glucose metabolism and weakens β cell function,causing up-regulation of serum lnc RNA MALAT1 and down-regulation of mi R-17 and is associated with elevated fasting blood glucose,resulting in increased secretion of serum inflammatory factors and chronic inflammation.