The Study Of Selection And Mechanism Of Key LncRNA Associated With Hypertriglyceridemia In The Population With Abnormal Glucose Metabolism

Abnormal glucose metabolism is a growing public health concern worldwide.In addition to the health risks posed by itself,abnormal glucose metabolism can also cause other diseases in the body.Hypertriglyceridemia is a major comorbidity of abnormal glucose metabolism,and abnormal glucose metabolism combined with hypertriglyceridemia is also a high-risk factor for the deterioration of the organism.Therefore,it is important to investigate the biomarkers in this specific population and to elucidate the molecular mechanisms that regulate hypertriglyceridemia.Previous studies have shown that long non-coding RNAs(lncRNAs)can be used as diagnostic markers or potentially therapeutic targets for diseases,the specific mechanism of lncRNAs in hypertriglyceridemia combined with abnormal glucose metabolism remains to be investigated.ObjectiveThis study was intended to find the key lncRNA for hypertriglyceridemia in a population with abnormal glucose metabolism by bioinformatic methods,in order to take it as a biomarker for the population with hypertriglyceridemia and abnormal glucose metabolism.In vitro experiments were conducted to investigate the molecular regulation of this lncRNA in the development of hypertriglyceridemia,thus providing a theoretical basis for the pathogenesis and targeted therapy of hypertriglyceridemia in the population with abnormal glucose metabolism.Methods1.Transcriptome sequencing was performed to construct lncRNA expression matrices using peripheral blood samples of patients with combined hypertriglyceridemia(n=12)and those with normal triglycerides(n=12)in the population with abnormal glucose metabolism.2.The gene modules significantly associated with triglycerides in the population with abnormal glucose metabolism were screened by WGCNA method,and the genes within the modules were analyzed by GO,KEGG and GSEA enrichment to derive the lncRNAs corresponding to the key mRNAs for the next screening verification.3.The key lncRNA was screened from the lncRNAs obtained in step 2 by the GSE130991 dataset in the GEO database,and the relative expression of this key lncRNA was measured by RT-q PCR in whole blood samples from patients with combined hypertriglyceridemia in the abnormal glucose metabolism population(n=80)and those with normal triglycerides in the abnormal glucose metabolism population(n=80).4.The correlation between the relative expression of this key lncRNA and triglyceride levels was analyzed,and the potentially clinical value of the relative expression of this key lncRNA for hypertriglyceridemia in people with abnormal glucose metabolism was investigated by the receiver operating characteristic curve(ROC).5.The localization of this key lncRNA in HepG2 cells was determined by Fluorescence in situ hybridization(FISH).6.The silent expression model of the target lncRNA was constructed using small interfering RNA(si RNA)-transfected cells,and the overexpression model of the target lncRNA was constructed using overexpression plasmid-transfected cells;the target lncRNA and mRNA co-transfected cell model was constructed using si RNA cotransfection technique.HepG2-IR model was constructed by insulin induction of HepG2 cells.The effects of this target lncRNA on the cell proliferation and apoptosis ability of HepG2-IR model were determined by CCK-8 assay for cell proliferation and flow cytometry assay for detecting apoptosis.The effects of the target lncRNA and mRNA on the TG levels of HepG2-IR cells were determined by GPO-PAP enzyme assay;the effects of the target lncRNA and mRNA on the expression levels of transcription factors in HepG2-IR cells were detected by RT-q PCR assay;the effects of the target lncRNA and mRNA on the expression levels of lipid metabolism-related enzymes and HepG2-IR cells were investigated by Western Blot assay.Results1.Screening of the key lncRNA in people with abnormal glucose metabolism combined with hypertriglyceridemiaThe whole-transcriptome sequencing was performed on people with abnormal glucose metabolism and hypertriglyceridemia.After constructing a lncRNA expression matrix,genes in modules significantly related to TG were obtained through WGCNA method,then GO,KEGG,and GSEA enrichment analysis were conducted to obtain the key mRNA TKFC in population with abnormal glucose metabolism and hypertriglyceridemia.There were 7 lncRNAs related to TKFC,and the key lncRNA LNC317.5 was screened by the GSE 130991 dataset in the GEO database.2.Expression of LNC317.5 in peripheral blood of population with abnormal glucose metabolism combined with hypertriglyceridemiaRT-qPCR validation of peripheral blood samples collected from 80 patients with hypertriglyceridemia combined with abnormal glucose metabolism and 80 patients with normal triglycerides combined with abnormal glucose metabolism revealed that the relative expression level of LNC317.5 in the population with abnormal glucose metabolism and hypertriglyceridemia was lower than that of its control group(0.48 vs1.00,t=-5.393,P<0.001).The relative expression of LNC317.5 showed a significant negative correlation with TG in the total study sample(r=-0.218,P=0.006).In addition,the relative expression of LNC317.5 had an area under the ROC curve of 0.688(95%CI: 0.604-0.772,P<0.001)for TG levels in all study samples,suggesting that LNC317.5might be a potential biomarker for patients with abnormal glucose metabolism combined with hypertriglyceridemia.3.Effects of LNC317.5 on the biological functions of HepG2-IR cellsSilent-expression cell model and overexpression cell model of LNC317.5 were established,and the HepG2-IR model was induced by 0.1 μmmol/L insulin for 48 h to investigate the effects of LNC317.5 on the TG level and biological functions of HepG2-IR cells.(1)RNA FISH experiments showed that LNC317.5 was expressed in both nucleus and cytoplasm of HepG2 cells.(2)The silencing of LNC317.5 expression significantly increased TG accumulation in HepG2-IR cells,while the overexpression of LNC317.5 significantly decreased TG levels in HepG2-IR(P<0.05).(3)LNC317.5had no significant effect on the proliferation profile of HepG2-IR cells(P>0.05).(4)The silencing of LNC317.5 expression significantly increased the relative apoptosis rate of HepG2-IR cells,while the overexpression of LNC317.5 significantly decreased the relative apoptosis rate of HepG2-IR(P<0.05).(5)In HepG2-IR cells,the silencing of LNC317.5 expression significantly increased the relative expression of its target gene TKFC at the molecular level(P<0.01)and protein level(P<0.01),while the overexpression of LNC317.5 significantly decreased the relative expression of its target gene TKFC at the molecular level(P<0.01)and protein level(P<0.05).(6)In HepG2-IR cells,the silencing of LNC317.5 expression significantly reduced the relative expression of transcription factors PPARα(P<0.01)and PPARγ(P<0.001)and significantly increased the relative expression of SREBP-1c(P<0.001).The overexpression of LNC317.5 significantly increased the relative expression of transcription factors PPARα and PPARγ and significantly decreased the relative expression of SREBP-1c(P<0.01).(7)In HepG2-IR cells,the silencing of LNC317.5expression significantly reduced the relative expression levels of ACADM and CPT1A(P<0.05)and significantly increased the relative expression levels of FAS and ACC1(P<0.05);the overexpression of LNC317.5 significantly increased the relative expression levels of ACADM(P<0.05)and significantly decreased the relative expression level of FAS and ACC1(P<0.05),but no significant effect was observed on the relative expression level of CPT1A(P>0.05).4.Joint effects of LNC317.5 and TKFC on biological functions of HepG2-IR cellsHepG2-IR cotransfection models were established by simultaneous silencing of LNC317.5 and TKFC expression to explore the co-impact of LNC317.5 and TKFC on the biology of HepG2-IR cells,and the experimental grouping for this part was designed as the following four groups: a.lncRNA-NC + mRNA-NC b.lncRNA-NC + si-mRNA c.si-lncRNA + mRNA-NC d.si-lncRNA + si-mRNA.Compared with the control group,the cotransfection of LNC317.5 and TKFC significantly reduced the TG level,relative apoptosis rate,relative expression of transcription factor PPARα and relative expression level of ACC1 in HepG2-IR cells(P<0.05);it significantly increased the relative expression of the transcription factor PPARγ and the relative expression level of CPT1A(P<0.05);no significant effect of it was seen on the proliferation profile of HepG2-IR cells,the relative expression of the transcription factor SREBP-1c,and the relative expression levels of ACADM and FAS(P<0.05).Conclusions1.The relative expression of LNC317.5 in whole blood samples of the population with abnormal glucose metabolism combined with hypertriglyceridemia was lower than that of the population with normal triglycerides and abnormal glucose metabolism,and this significant difference still existed in different age subgroups and existed between different sexes.2.LNC317.5 expressed both in the nucleus and cytoplasm of HepG2 cells.3.LNC317.5 had an inhibitory effect on the apoptosis of HepG2 IR cells,and it could regulate the level of TG in HepG2-IR cells,which might be due to its effects on the expression levels of transcription factors(PPARα,PPARγ,SREBP-1c)and related enzymes(ACADM,CPT1 A,FAS,ACC1).4.LNC317.5 was negatively correlated with the expression of TKFC.LNC317.5and TKFC might together affect the expression levels of TG in HepG2-IR cells.LNC317.5 might be a potential biomarker for abnormal glucose metabolism combined with hypertriglyceridemia.