LncRNA Gm4419 Knockdown Ameliorates NF-κB/NLRP3 Inflammasome-mediated Inflammation In Diabetic Nephropathy

Diabetic nephropathy(DN)as the primary cause of end-stage kidney disease is a common complication of diabetes.Recent researches have shown nuclear factor kappa B(NF-κB)and NACHT,LRR and PYD domain-containing protein 3(NLRP3)inflammasome are associated with inflammation in the progression of DN,but the exact mechanism is unclear.Long noncoding RNAs(lnc RNAs)have roles in the development of many diseases including DN.However,the relationship between lnc RNAs and inflammation in DN remains largely unknown.Our previous study has revealed that 14 lnc RNAs are abnormally expressed in DN by RNA sequencing(RNA-seq)and real-time quantitative PCR(q RT-PCR)in the renal tissues of db/db DN mice.In this study,these lnc RNAs were verified their expressions by q RT-PCR in mesangial cells(MCs)cultured under high-and low-glucose conditions.Twelve lnc RNAs displayed the same expressional tendencies in both renal tissues and MCs.In particular,long intergenic noncoding RNA(linc RNA)-Gm4419 was the only one associating with NF-κB among these 12 lnc RNAs by bioinformatics methods.Moreover,Gm4419 knockdown could obviously inhibit the expressions of pro-inflammatory cytokines and renal fibrosis biomarkers,and reduce cell proliferation in MCs under high-glucose condition,whereas overexpression of Gm4419 could increase the inflammation,fibrosis and cell proliferation in MCs under low-glucose condition.Interestingly,our results showed that Gm4419 could activate the NF-κB pathway by directly interacting with p50,the subunit of NF-κB.In addition,we found that p50 could interact with NLRP3 inflammasome in MCs.In conclusion,we provide an experiental evidence to show that linc RNA-Gm4419 may participate in the inflammation,fibrosis and proliferation in MCs under high-glucose condition through NF-κB/NLRP3 inflammasome signaling pathway,and may provide new insights into the regulation of Gm4419 during the progression of DN.Part I The expression and distribution of Gm4419 and the effect of Gm4419 on inflammation,fibrosis and proliferation of mouse glomerulus mesangial cellsObjective: To observe the expression and distribution of Gm4419 and the effect of Gm4419 on inflammation,fibrosis and proliferation in mouse glomerulus mesangial cells cultured with high-and low-glucose conditions.Methods: The expressions of 14 DN-related lnc RNAs in mesangial cells cultured with high-and low-glucose conditions were detected by fluorescence q RT-PCR.Gm4419 was focused on for further study because it was not only the top one upexpressed lnc RNA in DN but also the only lnc RNA that had the potential binding sites with DN inflammation-related gene NF-κB among these 12 lnc RNAs by bioinformatics methods.The location of Gm4419 in mesangial cells was detected by FISH and q RT-PCR.Over-expression plasmid of pc DNA3.1(+)-Gm4419 was constructed,and Gm4419 small interfering RNAs(si RNAs)were designed and synthesized.Then the pc DNA3.1(+)-Gm4419 and Gm4419 si RNAs were transiently transfected respectively into the glomerulus mesangial cells under low-or high-glucose condition.The m RNA levels of proinflammatory cytokices and renal fibrosis markers were detected by q RT-PCR.Western blot and immunofluorescence cytochemistry were employed to detect the protein levels of proinflammatory cytokices and renal fibrosis markers.Ed U assay and flow cytometry were used to detect the proliferation ability of the transfected cells.Results: The expression levels of Gm4419 were significantly higher in high-glucose cultured glomerulus mesangial cells when compared with low-glucose cultured cells(p<0.01).After the over-expression plasmid pc DNA3.1(+)-Gm4419 confirmed by enzyme digestion and DNA sequencing was transfected into the cells cultured under low glucose condition,the inflammation,fibrosis and proliferation ability were enhanced with L-MC mock cells or L-MC pc DNA3.1 group(p<0.05).In addition,after the Gm4419 si RNA with best interfering effect screened by q RT-PCR was transfected into the high-glucose cells,the knockdown resulted in the decrease in the inflammation,fibrosis and proliferation ability in the cells when compare with H-MC mock cells or H-MC si NC cells(p<0.05).Conclusions: lnc RNA-Gm4419 is involved in the regulation of the inflammatory,fibrosis and proliferation of mesangial cells from DN mice.Part II Gm4419 promotes pro-inflammatory cytokines expression through the NF-κB/NLRP3 inflammasome signaling pathway in glomerulus mesangial cellsObjective: To study the activity of NF-κB/NLRP3 inflammasome signaling pathway and the effect of the inflammatory cytokines,fibrosis and proliferation of mesangial cells when the relative expression level of Gm4419 was regulated.Methods: we examined the relative m RNA levels of p50 and p65 by q RT-PCR when over-expression plasmid of pc DNA3.1(+)-Gm4419 was transfected into mouse mesangial cells cultured with low glucose conditions.We examined the relative m RNA levels of p50 and p65 by q RT-PCR when Gm4419 si RNA transfected into mouse mesangial cells cultured with high glucose conditions.The relative protein levels of role factor involved in NF-κB signaling,including IκBα phosphorylation(p-IκBα),IκBα,p50 and p65 were confirmed by western blot when pc DNA3.1(+)-Gm4419 was transfected into mouse mesangial cells cultured with low glucose conditions.We confirmed the relative protein levels of role factor involved in NF-κB signaling,including p-IκBα,IκBα,p50 and p65 by western blot when Gm4419 si RNA was transfected into mouse mesangial cells cultured with high glucose conditions.We examined nuclear translocation of p50 and p65 by immunofluorescence cytochemistry when pc DNA3.1(+)-Gm4419 and Gm4419 si RNA was transfected into mouse mesangial cells.We detected the relative binding levels of p50 and p65 to the promoters of Gm4419 and NLRP3 inflammasome by chromatin immunoprecipitation(Ch IP)assays via regulation of Gm4419 levels.The relative levels of Gm4419 in nucleus and cytoplasm of mesangial cells were examined by q RT-PCR and fluorescence in situ hybridization(FISH)via regulation of p50 levels.The relative m RNA levels of Gm4419,p50 and the relative protein levels of p50 were detected by q RT-PCR,western blot,immunofluorescence cytochemistry and FISH when both Gm4419 and p50 overexpressed or knockdown.The relative m RNA levels of NLRP3 inflammasome were examined q RT-PCR when Gm4419 or p50 was overexpressed.We examined the relative m RNA levels of NLRP3 inflammasome q RT-PCR when Gm4419 or p50 was knockdown.The relative protein levels of NLRP3 inflammasome were detected by western blot and immunofluorescence cytochemistry via regulation of Gm4419 or p50 levels.We examined whether endogenic NLRP3 inflammasome protein was pulled down using anti-p50 antibody by immuoprecipitation.The co-localization of endogenic NLRP3 inflammasome and p50 in MCs was detected immunofluorescence cytochemistry.The relative protein levels of NLRP3 inflammasome and proinflammatory cytokines were detected by western blot when p50 was inhibited by SN50 or(and)p65 was inhibited by JSH-23.Results: pc DNA3.1(+)-Gm4419 resulted in increased the relative m RNA levels of p50 and p65(p<0.01),Gm4419 si RNA resulted in decreased the relative m RNA levels of p50 and p65(p<0.01).pc DNA3.1(+)-Gm4419 resulted in increased phosphorylation and down regulation of IκBα,which corresponded to increased nuclear accumulation of p50 and p65(p<0.01).Gm4419 si RNA significantly decreased phosphorylation and accelerated accumulation of IκBα,which was accompanied by decreased nuclear translocation of p50 and p65(p<0.01).Immunofluorescence cytochemistry assay showed pc DNA3.1(+)-Gm4419 increased nuclear accumulation of p50 and p65 and Gm4419 si RNA decreased nuclear translocation of p50 and p65.Form Ch IP study showed that Gm4419 regulated with the DNA-binding activity of the p50 not p65 element located on the promoters of Gm4419 and NLRP3 inflammasome,then led to change genetic transcriptions of Gm4419 and NLRP3 inflammasome(p<0.01).QRT-PCR and FISH assay showed pc DNA3.1(+)-p50 resulted in increased the relative levels of Gm4419 in nucleus and cytoplasm,p50 si RNA resulted in decreased the relative m RNA levels of Gm4419 in nucleus and cytoplasm.Form q RT-PCR、western blot and FISH study showed that the relative m RNA levels of Gm4419,p50 and the relative protein levels of p50 were significantly up-regulated when Gm4419 and p50 overexpressed,whereas the relative m RNA levels of Gm4419,p50 and the relative protein levels of p50 were significantly down-regulated when Gm4419 and p50 knockdown.pc DNA3.1(+)-Gm4419 or pc DNA3.1(+)-p50 resulted in increased the relative m RNA and protein levels of NLRP3 inflammasome.Gm4419 si RNA or p50 si RNA resulted in decreased the relative m RNA and protein levels of NLRP3 inflammasome.Immuoprecipitation resulted show that endogenic NLRP3 inflammasome protein was pulled down using anti-p50 antibody.Immunofluorescence cytochemistry results showed that endogenic NLRP3 inflammasome and p50 displayed a co-localization in MCs.Western blot resulted displayed that Gm4419 had no effect on the protein expression of NLRP3 inflammasome protein and inflammatory cytokines when p50 was inhibited by SN50 or both p50 was inhibited by SN50 and p65 was inhibited by JSH-23,whereas Gm4419 had significantly effect on the protein expression of NLRP3 inflammasome protein and inflammatory cytokines when p65 was inhibited by JSH-23Conclusions: Gm4419 may participate in the inflammation,fibrosis and proliferation in MCs under high-glucose condition through NF-κB/NLRP3 inflammasome signaling pathway.