| Part One Analysis of Differentially Expressed Genes in Endoplasmic Reticulum Stress of INS-1-3 Cells Using Digital Gene Expression Profiling(DGE)TechnologyObjective: Hyperglycemia,hyperlipidemia and other metabolic disorders induce endoplasmic reticulum stress(ERS)in pancreatic beta cell.Then they activate the downstream pathway of endoplasmic reticulum stress and induce apoptosis and cell dysfunction.This study aims to investigate whether common differentially expressed genes and pathways exist in the ERS induced by various chemicals and high levels of glucose and palmitate in INS-1-3 β cells.Method: INS-1-3 cells were cultured and ERS was induced by exposure cells to thapsigargin(TG,0.1μmol/L,16 hours),tunicamycin(TM,2.5μg/ml,16 hours)or palmitic acid(PA,0.3mmol/L,12 hours)+high glucose(HG,16.7mmol/L,12 hours).Then the cells were collected to extract RNA.Digital gene expression(DGE)profiling technique was used to detect differentially expressed genes.Then Gene Ontology enrichment analysis and KEGG pathway analysis of differentially expressed genes were performed among differentially expressed genes.Real time reverse transcription polymerase chain reaction(RT-PCR)and Weatern Blot(WB)was used to verify the expression changes of target genes.Results : 1.Evaluate the model of endoplasmic reticulum stress.GRP78/Bip and CHOP are known as endoplasmic reticulum stress markers.In this study,TM,TG and HG+PA were used to culture cells and the m RNA levels of GRP78 and CHOP were detected by RT-PCR.After exposing cells to TM,TG and HG + PA,m RNA levels of GRP78 were significantly increased by 4.92 folds,6.96 folds and 6.96 folds(P < 0.05),CHOP m RNA level was also significantly increased 5.92 folds,5.27 folds and 6.51 folds(P < 0.05),the results showed that TM,TG and HG+PA could induce endoplasmic reticulum stress in INS-1-3 cells.2.Digital gene expression and quality assessment of reads.By base calling,the original image data produced by the sequencer is transferred into sequences,which are defined as "raw reads"(or "raw data").As the raw reads may contain low quality reads and or adaptor sequences,preprocessing is necessary before starting further analysis.Normally,data cleaning(or data filtering)is performed to obtain "clean reads"(or "clean data")for further analysis.The numbers of raw reads of control groups were 11968330,12351978,12559884,and clean reads accounted for 99.53%,99.53%,99.52%.In TG groups,the numbers of raw reads were 12227268,12048856,12421592,and clean reads accounted for 9.53%,99.53%,99.54%.In HG+PA groups,the numbers of raw reads were 12572491,12148256,12074804,and clean reads accounted for 99.53%,99.53%,99.53%.3.Screening of differentially expressed genes.We apply NOIseq method coming from an article published in Genome Research 2011[6],to screen differentially expressed genes between two groups.As described in this article,NOISeq method shows a good performance when comparing it to other differential expression methods,like Fisher’s Exact....Test(FET),edge R,DESeq and bay Seq.NOISeq maintains good True Positive and False Positive rates when increasing sequencing depth,while most other methods show poor performance.What’s more,NOISeq models the noise distribution from the actual data,so it can better adapt to the size of the data set,and is more effective in controlling the rate of false discoveries.Compared to control groups,there were 135 differentially expressed genes in TM groups,57 differentially expressed genes in TG groups and 74 differentially expressed genes in HG+PA groups.4.Gene ontology enrichment analysis of differentially expressed genes.Gene Ontology(GO)is an international standardized gene functional classification system which offers a dynamic-updated controlled vocabulary and a strictly defined concept to comprehensively describe properties of genes and their products in any organism.GO covers three domains: cellular component,molecular function and biological process.In TM groups,distribution of gene ontology enrichment was: cellular component 40%,molecular function 20%,biological process 40%.In TG groups,distribution of gene ontology enrichment was: cellular component 43%,molecular function 18%,biological process 39%.In HG+PA groups,distribution of gene ontology enrichment was: cellular component 55.6%,biological process 44.4%.The molecular function annotation indicated that these differentially expressed genes were mostly related to oxidative stress.The annotated results of biological pathway indicated that these genes were mainly involved in the process and presentation of cell antigen,protein metabolism,and signal transduction of cell homeostasis.5.Pathway enrichment analysis of differentially expressed genes.Genes usually interact with each other to play roles in certain biological functions.Pathway-based analysis helps to further understand genes biological functions.KEGG is the major public pathway-related database.Pathway enrichment analysis identifies significantly enriched metabolic pathways or signal transduction pathways in DEGs comparing with the whole genome background.In TM groups,the 120 differentially expressed genes were related to 120 signal pathways.In TG groups,the 57 differentially expressed genes were related to 72 signal pathways.In HG+PA groups,21 signal pathways were enriched.Genes that were differentially expressed were enriched to endoplasmic reticulum stress,antigen processing and presentation,protein export,and most of all,the maturity onset diabetes of the young(MODY)pathway.In MODY signal pathway,the expressions of motor neuron and pancreas homeobox 1(Mnx1)were declined to 45%,59%,35% and the expressions of NK6 homeobox 1(Nkx6.1)were declined to 55%,46%,54%.6.Verification of digital gene expression results.Expression of Nkx6.1,Mnx1 and Bhlha15 in TM and TG groups were detected by r RT-PCR and Weatern Blot.After exposing to TM and TG,the m RNA and protein levels of Nkx6.1 and Mnx1 decreased significantly(P < 0.05),m RNA and protein levels of Bhlha15 increased(P < 0.05).The trend is the same as the digital gene expression profile.The results of the digital gene expression profile indicated that the m RNA levels of Nkx6.1 and Mnx1 in TM group decreased to 55% and 45% respectively,while the m RNA levels of Bhlha15 increased to 3.84 folds.In the TG group,the m RNA levels of Nkx6.1 and Mnx1 decreased to 46% and 59% respectively,while the m RNA levels of Bhlha15 increased to 3.7 folds.RT-PCR results indicated that the m RNA levels Nkx6.1 and Mnx1 in TM group decreased to 81% and 80% respectively,while the m RNA levels of Bhlha15 increased to 5.77 folds.In the TG group,the m RNA levels of Nkx6.1 and Mnx1 decreased to 68% and 51% respectively,while the m RNA levels of Bhlha15 increased to 5.51 folds.Therefore,RT-PCR and Weatern Blot have confirmed the DGE results.Part Two The Function of Nkx6.1 in Endoplasmic Reticulum Stress Induced by Glucolipotoxicity in INS-1-3 CellsObjective: The homeodomain transcription factor Nkx6.1 plays an important role in β-cell differentiation,development and mature β-cell survival,function.DGE results showed that glucotoxicity is associated with the inhibited expression of Nkx6.1.This study aims to establish Nkx6.1 over-expression INS-1-3 cells by lentivirus and induce ERS by high glucose and palmitic acid.And investigate the protective effect of Nkx6.1 on glucolipotoxicity by comparing cell proliferation,apoptosis and insulin secretion of different experimental groupsMethod: A control group was cultured in normal medium.Exposure to thapsigargin(TG,0.1μmol/L,16 hours),tunicamycin(TM,2.5μg/ml,16 hours)or palmitic acid(PA,0.3mmol/L,12 hours)+high glucose(HG,16.7mmol/L,12 hours)was used to induce ER stress.Nkx6.1 overexpression was established in INS-1-3 cell lines by lentivirus infection.Then the cells were collected to extract RNA.Real time polymerase chain reaction(RT-PCR)was used to verify the expression changes of ERS markers,GRP78/Bip and CHOP.Cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT)assay.And the degree of apoptosis was detected by flow cytometry.Glucose stimulates insulin secretion(GSIS)was used to detect the insulin secretion of INS-1-3 cells and ELISA was used to detect insulin concentrations.Results: 1.Nkx6.1 overexpression validation.Among the three ERS models induced by TM,TG or HG+PA,the expressions of Nkx6.1 were significantly decline,indicating the transcription factor might play a role in the process of endoplasmic reticulum stress in INS-1-3 cells.In this study,Nkx6.1 overexpression cells were obtained by lentivirus transfection.In this study,transfected cells were cultured in normal or HG+PA medium,the m RNA and protein levels of Nkx6.1 were detected.The INS-1-3 cells treated with lentiviruses contained Nkx6.1 exhibited a significant increase in Nkx6.1 m RNA level at 8.1 folds(P<0.05)and an increase in Nkx6.1 protein level at 1.35 folds(P < 0.05)relative to uninfected cells,suggesting that overexpression of Nkx6.1 in INS-1-3 cells was successfully established.In HG+PA culture condition,the m RNA levels of Nkx6.1 declined by 32.0%,14.2%,38.0%(P>0.05).After exposed to HG+PA,the protein levels of untransfected and Nkx6.1 overexpression INS-1-3 cells exhibited declines of 19.6%,18.5% respectively(P<0.05).Therefore,high glucose and palmitic acid could reduce the m RNA and protein levels of Nkx6.1 in INS-1-3 cells.2.Nkx6.1 overexpression alleviates ERS induced by high glucose and palmitic acid in INS-1-3 cells.After exposed to TM,TG,and HG plus PA,Bip/GRP78 m RNA levels were significantly increased by 4.92 folds,6.96 folds and 6.14 folds respectively(P < 0.01).And the protein levels of Bip/GRP78 increaesd by 1.60 folds,1.59 folds and 1.78 folds.Meanwhile,TM,TG and HG plus PA significantly increased CHOP m RNA and protein levels(P<0.01).Therefore,glucoliopotoxicity could cause ER stress similar to TM and TG in INS-1-3 cells.Despite the m RNA level of Nkx6.1 significantly increased after being exposed to HG plus PA,but the m RNA levels of the ERS markers was significantly lower than untransfected and GFP expression INS-1-3 cells(P < 0.01).3.The proliferation of INS-1-3 cells.In the light of the importance of Nkx6.1 gene in the development of pancreatic β cells and the suppression of Nkx6.1 gene to ER stress,overexpression of Nkx6.1 might attenuate the ERS induced by glucolipotoxicity.To test this speculation,we measured the INS-1-3 cell viability by using MTT with different experimental manipulations.There were no significant differences among INS-1-3 cells,GFP-expressing and Nkx6.1-expressing INS-1-3 cells incubated in normal culture medium(P>0.05).As the cells exposed to high glucose plus palmitic acid,the proliferation of both INS-1-3 cells and GFP-expressing cells decreased by about 45% and 50%,respectively(P < 0.01).In contrast,treatment with high glucose plus palmitic acid did not significantly inhibit the cell proliferation in Nkx6.1 over-expressing cells(P>0.05).It suggested overexpression of Nkx6.1 gene could alleviate impairment induced by chronic exposure to elevated concentrations of glucose and palmitic acid.4.The apoptosis of INS-1-3 cells.We next explored the role of Nkx6.1 gene in maintaining cell viability.The flow cytometry analysis showed that there were no significant differences of apoptosis markers between Nkx6.1-expressing cells and un-transfected cells in normal culture medium.After 24 hours exposure to high level of glucose plus palmitic acid,the levels of marker for early apoptosis,late apoptosis and total apoptosis were significantly elevated by 174.9%,54.2% and 91.3% in INS-1-3 cells,respectively(P<0.05).In contrast,exposure Nkx6.1 over-expressing INS-1-3 cells to high glucose plus palmitic acid did not change the early and late stage of apoptosis markers(P > 0.05).Consequently,these apoptosis markers significantly increased in INS-1-3 cells compared to Nkx6.1 expressing INS-1-3 cells after exposure to in HG plus PA(P < 0.01).These data suggested that Nkx6.1 played a protective role towards glucolipotoxicity.5.Nkx6.1 over-expression attenuated the impairment of insulin secretion in glucolipotoxicity in INS-1-3 cells.Overexpressing Nkx6.1 was used to investigate the impact of Nkx6.1 on insulin secretion of INS-1-3 cells.The basal insulin secretion(BIS)and the glucose stimulated insulin secretion(GSIS)were not different between Nkx6.1 over-expressing and GFP expressing INS-1-3 cells(P>0.05)in normal culture.After chronic exposure GFP expressing cells to high levels of glucose plus palmitic acid,BIS and GSIS decreased by 32.3% and 56.0%,respectively(P < 0.05).Nkx6.1 over-expressing group demonstrated only a 5.8% decrease of GSIS by(P>0.05),It suggested over expression of Nkx6.1 protected INS-1-3 cells from glucolipotoxicity.Part Three Liraglutide Alleviates Endoplasmic Reticulum Stress in INS-1-3 Cells via Transcription Factor Nkx6.1Objective : The glucagon-like peptide-1(GLP-1)mitigates the endoplasmic reticulum(ER)stress in β cells.In previous studies,TM,TG and other chenmicals could induce ERS and change gene expression levels in MODY signal pathway.Nkx6.1 could partially alleviate the toxicity effects induced by glucolipotoxicity in INS-1-3 cells.This study aims to investigate the relation of Nkx6.1 and liraglutide in the protection effect of endoplasmic reticulum stress in INS-1-3 cells.Method: INS-1-3 cells were incubated for 24 hours and divided into five groups for treatment: control group(normal medium),0.5 μmol/L thapsigargin(TG,16 hours)-treated group,TG and GLP-1 co-treated group.Knockdown of Nkx6.1 was established in INS-1-3 cell lines by si RNA.Real time reverse transcription polymerase chain reaction(RT-PCR)was used to verify the expression changes of Nkx6.1 and two ERS markers.And the protein levels of PERK,IRE1α,ATF-6 and the downstream molecules were detected.Cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT)assay.Flow cytometry analysis was used to detect apoptosis rate.Glucose stimulates insulin secretion(GSIS)was used to detect the insulin secretion of INS-1-3 cells and ELISA was used to detect insulin concentrations.Results: 1.m RNA and protein levels of Nkx6.1 in INS-1-3 cells.After exposed to TG,the m RNA and protein levels of Nkx6.1 were significantly decreased by 43.3% and 51.2%,respectively.Cells subjected to liraglutide exhibited significant increase in Nkx6.1 m RNA level by 4.8 folds compared to TG group.Meanwhile,the protein levels of Nkx6.1 increased by 2.5 folds.Treatment of INS-1-3 cells with Ad-si CTRL caused no significant changes in both m RNA and protein levels,whereas Ad-si Nkx6.1 treatment caused 40.6% and 55.4% decrease,respectively.2.The apoptosis and proliferation of INS-1-3 cells.Compared to untreated cells,TG-treated cells showed significantly decreased cell viability(47.6% of the control,P<0.05).Liraglutide attenuated TG-induced cell death.But after being treated with Ad-si Nkx6.1,the cells showed decreased cell viability by 24.7%,compared to Ad-si CTRL-treated cells(P<0.05).We detected the cell proliferation by flow cytometry.After prolonged exposure to TG,the early apoptosis,late apoptosis and total apoptosis were significantly elevated by 3.19 folds,2.59 folds and 2.78 folds in INS-1-3 cells,respectively(P<0.05).Furthermore,compared with TG-treated group,co-treatment with liraglutide and TG decreased cells apoptosis(P < 0.05).But when Ad-si Nkx6.1 was added into the media,the cell apoptosis went back up(1.55 folds,1.10 folds and 1.21 folds of the TG+L+si CTRL group).3.Liraglutide attenuates the impairments of insulin secretion against ER stress in INS-1-3 cells.Suppression of insulin secretion was observed in TG-treated group with both basal and 20 m M glucose media(P<0.05).What’s more,then TG-treated cells were cultured with liraglutide,accompanied with the increased-expression of Nkx6.1,the cells secreted 7.19 ng/m L/h and 17.53 ng/m L/h,demonstrated an increase of 2.39 folds and 1.89 folds in insulin secretion with 2.5m M and 20 m M glucose,respectively.However,the treatment with Ad-si Nkx6.1 partially abolished the effect of liraglutide on GSIS(P<0.05).4、Liraglutide decreased the expression of ER stress markers in INS-1-3 cells.Bip/GRP78 and Calnexin m RNA were expressed at low levels in control group,but their expressions were augmented with TG treatment.Furthermore,the protein levels of Bip/GRP78 and Calnexin were assessed by Western Blot analysis.In accordance with the changes in m RNA expression,TG significantly increased the protein expression of Bip/GRP78 and Calnexin by 2.69 folds and 2.50 folds compared with that in the control group(P<0.05).Liraglutide-treatment significantly decreased the m RNA levels of Bip/GRP78 and Calnexin,indicating alleviated ER stress(P<0.05).After using the small interfering(si)RNA to silence Nkx6.1,it was further identified that ER stress responses were fortified,as indicated by an increase in the m RNA and protein levels of Bip/GRP78.Though a trend toward increased Calnexin was observed after Nkx6.1 being silenced,it showed no significant effect on the m RNA and protein levels of Calnexin(P>0.05).5、Liraglutide partially alleviated TG-induced ER stress via Nkx6.1.In order to explore the effect of liraglutide and Nkx6.1 on ER stress,we further investigated the three signaling transducers in ER stress.An exposure to TG significantly increased p-PERK in Nkx6.1 expressing cells compared to normal cultivated group(P < 0.05).Phosphorylated e IF2α(p-e IF2α),a downstream molecule in the PERK pathway,was increased in TG-treated cells(P < 0.01),indicating that the PERK pathway was activated.Liraglutide-treatment decreased the protein levels of p-PERK and p-e IF2α by 37.4% and 39.1%,respectively(P<0.05).But after Ad-si Nkx6.1 treatment,both the expression of p-PERK and p-e IF2α tended to increase,only changes in p-e IF2α had significant difference.Since IRE1α,a homolog of IRE1,is constitutively expressed in all cells and tissues,we used IRE1α to evaluate IRE1 pathway in this study.The data showed that TG-treatment increased the expressions of IRE1α and p-JNK by 2.63 folds,5.38 folds and liraglutide reduced the protein levels by 34.3%,60.0%,respectively(P < 0.01).Interestingly,Ad-si Nkx6.1 treatment significantly increased the levels of IRE1α and p-JNK(1.82 and 1.48 folds compared to the Ad-si CTRL-treated cells,respectively,P < 0.05)in TG and liraglutide co-treated β cells.ATF-6 protein was expressed at low level in control group,but the expression increased significantly after TG treatment.Liraglutide treatment caused a 44.2% reduction,whereas knockdown of Nkx6.1 was able to reverse the liraglutide-mediated protective effects in INS-1-3 cells.In INS-1-3 cells,there was a 3.52-fold increase of CHOP(P< 0.01)compared with the control,while liraglutide-treatment decreased CHOP(66.8% reduction).And INS-1-3 cells treated with Ad-si Nkx6.1 exhibited a 1.79-fold induction in CHOP protein levels(P< 0.01).Conclusion: 1.Thapsigargin(TG),tunicamycin(TM)or palmitic acid(PA)+high glucose(HG)cause increased apoptosis and dysfunction in INS-1-3 cells.2.By using digital gene expression profiling(DGE)technology,the differentially expressed genes were in MODY signal pathway among the three ERS models.The common gene changes in this pathway were Nkx6.1 and Mnx1 3.Over-expression of Nkx6.1 could alleviate the increased apoptosis,decreased proliferation and insulin secretion induced by glucolipotoxicity.4.Liraglutide protected the pancreatic INS-1-3 cells from TG-induced ER stress partially via transcription factor Nkx6.1.5.Transcription factor Nkx6.1 protective effects on ER stress was partially through the suppressing of PERK,IRE1 and ATF-6 pathways. |
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