Long Non-coding RNA Rpph1 Promotes Inflammation And Proliferation Of Mesangial Cells In Diabetic Nephropathy Via An Interaction With Gal-3

Objective:Diabetic nephropathy(DN)is one of the serious complications of diabetes,its pathogenesis involves inflammation,oxidative stress and other factors.In recent years,studies have shown that renal inflammation is crucial to promote the development of DN,and inflammation may be a key factor to stimulate the known biochemical and metabolic disorders in DN.Although inflammation has been considered as an important link in the occurrence and development of DN,the specific mechanism is still inconclusive,and the current diagnosis and treatment methods are still not ideal.In recent years,many long non-coding RNAs(lncRNAs)have been identified by whole transcriptome sequencing technology,with a length of over 200 nucleotides and a lack of protein-coding ability.More and more evidences show that long non-coding RNA can participate in many physiological or pathological processes by interacting with proteins or RNA.Abnormal expression of long non-coding RNA is associated with many diseases,including diabetic nephropathy.We found differential expression of lncRNAs in diabetic nephropathy by RNA sequencing,in these lncRNAs,seven dnrelated lncRNAs,including the significantly expressed lncRNA Rpph1,were identified by quantitative real-time PCR(qRT-PCR)in vivo and in vitro.In addition,we investigated the expression level of LncRNA Rpph1 in renal tissues of DN mice or normal mice and in mesangial cells cultured with high or low glucose.And detected the subcellular distribution of Rpph1 in mesangial cells cultured under high or low glucose condition,predicted the noncoding properties of long non-coding RNA Rpph1.The results showed that after overexpression or down-regulation of Rpph1,the expression of inflammatory factors and the proliferation in mesangial cells(MCs)could be regulated.RNA-pulldown and mass spectrometry showed that Rpph1 interacting directly with the DN related factor galectin-3(Gal-3),and in low-glucose cultured mesangial cells,overexpression of Rpph1 promoted MCs inflammation and cell proliferation through theGal-3 /MEK/ERK signaling pathway.Whereas down-regulation of Rpph1 inhibited MCs inflammatory response and cell proliferation through the Gal-3 /MEK/ERK pathway in mesangial cells cultured with low glucose.In general,these results provide new insights into the relationship between Rpph1 and Gal-3 /MEK/ERK signaling path ways for the occurrence and development of DN.Methods: RNA sequencing(RNA-seq)was used to detect the differential expression of lncRNAs in the renal tissues of diabetic nephropathy mice and normal mice.There were95 differentially expressed lncRNAs,among which 46 were up-regulated and 49 downregulated.According to FPKM>150 and FC>2,7 lncRNAs with the most significant expression difference were screened out.qRT-PCR was used to detect the differential expression of these 7 lncRNAs in renal tissues of diabetic nephropathy mice and mesangial cells cultured under high or low glucose conditions.It was found that lncRNA Rpph1 had the highest conserved degree and the highest differential expression in renal tissue and mesangial cells of diabetic nephropathy mice.Therefore,lncRNA Rpph1 was selected as the object of this study.The expression and distribution of lncRNA Rpph1 in the cytoplasm and nucleus of renal tissue and mesangial cells of diabetic nephropathy mice were detected by qRT-PCR and FISH.Bioinformatics website ORF finder(www.ncbi.nlm.nih.gov/orffinder/)and CPAT(Coding-Potential Assessment Tool)(http://lilab.research.bcm.edu/cpat/)were used to predict the protein Coding potential of Rpph1.pcDNA3.1(+)-rpph1 overexpressed plasmid was constructed,and Rpph1 siRNAs were designed and synthesized.Immunohistochemistry,qRT-PCR,western blot,ELISA and immunofluorescence were used to detect the expression of Tnf-α and Mcp-1 in renal tissues and mesangial cells of diabetic nephropathy mice.The effect of overexpression and knockdown of Rpph1 on the expression of inflammation-related factors Tnf-and McP-1 in renal tissue and mesangial cells cultured with high and low glucose in diabetic nephropathy mice was also investigated.The proliferation of mesenchymal cells was detected by EdU after overexpression and knockdown of Rpph1.In order to further explore how lncRNA Rpph1 regulates inflammatory cytokines and cell proliferation in diabetic renal mesangial cells,the proteins interacting with Rpph1 were detected by rna-pulldown and mass spectrometry,and it was found that Rpph1 and dn-related factor galectin-3 bind each other.Gal-3 siRNAs were designed and synthesized,Gal-3 overexpressed plasmids were constructed,and the effects of transfection of Gal-3 siRNA andoverexpressed plasmids on proliferation of mesenchymal cells were detected.Western blot was used to detect the expression levels of MEK,p-mek,ERK,p-erk,c-jun,p-c-jun proteins related to the MEK/ERK pathway after transfection of gal-3 siRNA and overexpressed plasmids.The expression of inflammatory cytokines Tnf-and Mcp-1 was detected by ELISA,and the effect of Gal-3 on proliferation of mesangial cells was detected by EdU.In addition,after transfection with Rpph1 overexpressed plasmid and siRNA,western blot was used to detect the expression of MEK/ERK pathway related proteins.Transfection of Rpph1 overexpressed plasmids and gal-3 siRNA was performed in mesenchymal cells cultured with low glucose,and transfection of Rpph1 siRNA and gal-3 overexpressed plasmids were performed in mesenchymal cells cultured with high glucose.Western blot analysis of MEK/ERK pathway related protein expression,ELISA detection of inflammatory cytokines Tnf-α,McP-1 expression and EdE detection on the proliferation of mesangial cells.Morever,MEK specific inhibitor U0126 was transfected into mesangial cells cultured with low glucose,and the expression of MEK/ERK pathway protein was detected by western blot.Furthermore,siRNAs of Mek1 and Mek2 were designed and synthesized,and the MEK/ERK signaling pathway protein expression was detected by western blot after transfection in high-glucose cultured mesenchymal cells.Results: The expression of lncRNA Rpph1 in renal tissue of diabetic nephropathy mice and mesangial cells cultured under high glucose condition was significantly upregulated compared with that of normal renal tissue and mesangial cells cultured under low glucose condition.The results of qRT-PCR and FISH experiments showed that lncRNA Rpph1 was distributed in the cytoplasm and nucleus of mesangial cells.However,it was mainly expressed in the cytoplasm,and the expression was higher in the kidney tissue of diabetic mice than that of normal mice.ORF finder and CPAT software were used to analyze Rpph1’s protein-coding ability,it turns out that Rpph1doesn’t code for proteins.Three siRNAs of Rpph1 were transfected into mesenchymal cells cultured with high glucose,and Rpph1 overexpressed plasmids were transfected into mesenchymal cells cultured with low glucose.The siRNA with the best interference efficiency was screened by qRT-PCR,in which Rpph1 siRNA3 had the best interference efficiency,and the results showed that pcDNA3.1(+)-Rpph1 overexpression plasmid could significantly improve the expression of Rpph1.Immunohistochemistry and results showed that the expression of inflammatorycytokines Tnf-and Mcp-1 in the kidney tissue of diabetic nephropathy mice was significantly higher than that of normal mice.The results of qRT-PCR suggested that the expression of Tnf-α and Mcp-1 in the mesangial cells cultured with high glucose was up-regulated compared with that in the mesangial cells cultured with low glucose.Western blot,cellular immunofluorescence and ELISA showed that Rpph1 significantly up-regulated the expression of Tnf-α and Mcp-1,and EdU showed that Rpph1 promoted mesangial cell proliferation.In addition,Rpph1 and gal-3 bind to each other by rnapulldown and mass spectrometry,and promote the expression of inflammatory cytokines and proliferation of mesangial cells in diabetic kidneys by MEK/ERK signaling pathway.After transfection of Rpph1 overexpressed plasmids and gal-3siRNA in low-glucose cultured mesenchymal cells,western blot,EdU and ELISA results showed that gal-3 siRNA could reverse the role of Rpph1 in promoting the expression and proliferation of inflammatory cytokines in mesenchymal cells.Similarly,after transfection of Rpph1 siRNA and gal-3 overexpressed plasmids in high-glucose cultured mesangial cells,the results showed that gal-3 overexpression could reverse the inhibitory effect of Rpph1 siRNA on the expression and proliferation of inflammatory factors in mesangial cells.In addition,after transfection with MEK specific inhibitors,it blocked the effects of Rpph1 and gal-3 on the expression of Tnf-and McP-1,as well as the proliferation of mesangial cells.Conclusions: LncRNA Rpph1 is significantly highly expressed in renal tissue of diabetic nephropathy mice and in mesangial cells cultured with high glucose,and Rpph1 can regulate the proliferation of renal mesangial cells and the expression of inflammatory factors in diabetic nephropathy through MEK/ERK signaling pathway.It can be inferred that lncRNA Rpph1 may be involved in the occurrence and development of diabetic nephropathy,providing an interesting direction for the inflammation and proliferation of diabetic nephropathy mesangial cells.