LincRNA 1700020I14Rik Improves The Mesangial Cell Fibrosis And Proliferation Of Diabetic Nephropathy By MiR-34a-5p/Sirt1/HIF-1α Signaling Pathway

Objective To explore the dysexpression and characteristic of candidate linc RNA 1700020I14 Rik in mouse kidney tissuse with diabetic nephropathy and in high glucose medium-cultured mouse renal mesangial cells.Methods Differential expression long non-coding RNAs（lnc RNAs）were obtained from mouse renal tissues with diabetic nephropathy compared with normal mouse using high throughput RNA sequencing（RNA-seq）technique and long intergenic non-coding RNAs（linc RNAs）were screened out from these lnc RNAs.QRT-PCR was used to verify the differential expression linc RNAs both in mouse kidney tissuse with diabetic nephropathy and in high glucose medium-cultured mouse renal mesangial cells.ORF Finder and CAPT online software were used to analysis the potential protein capacity of 1700020I14 Rik.Fluorescence in situ hybridization was performed to explore the expression and location in mouse renal mesangial cells.Results Differential expression lnc RNAs were obtained using RNA-seq technique,and 42 linc RNAs were screened out from these lnc RNAs in which seven linc RNAs were in a consistent expression trend with the results of RNA-seq verified in renal tissues of DN mice and normal control mice by q RT-PCR.Further verification were conducted in high glucose or low glucose medium-cultured mouse renal mesangial cellsusing q RT-PCR and five linc RNAs were finally obtained for the expression of consistent with previous verification results.Then,linc RNA1700020I24Rik（ENSMUSG00000085438;Refseq NR₀27832）was focused on for further study because it was not only significantly down-expressed in DN but also displayed the highest conservative factor on the sequence comparing with their homologous sequence in human and high abundance（FPKM>11）among the final five linc RNAs.Moreover,the coding potential of 1700020I14 Rik was analyzed using ORF Finder and the Coding Potential Assessment Tool（CPAT）,which suggested that1700020I14 Rik tends to be a noncoding RNA.Furthermore,FISH results confirmed 1700020I24 Rik was decreased in high glucose-cultured MCs compared with that in MCs cultured with low glucose medium.Data also showed that since 1700020I24 Rik located in both cytoplasm and nuclear in MCs,it was mainly distributed in the cytoplasm of cells.Conclusion Linc RNA 1700020I14 Rik with no protein-coding ability is down-expressed in DN.It is located in both cytoplasm and nuclear in MCs and mainly distributed in the cytoplasm of cells.Objective To explore the effect of linc RNA 1700020I14 Rik on proliferation and fibrosis in mouse renal mesangial cells.Methods pc DNA3.1（+）-1700020I14 Rik or si RNA against1700020I14 Rik was transfected in MCs to over-expression or knockdown the expression of 1700020I14 Rik with opti-MEM,lipofectamine 2000 and lipofectamine 3000 reagents.The transfection efficacy was measured by q RT-PCR.Cell proliferation was assessed using Cell Counting Kit-8（CCK-8）and a 5-ethynyl-2-deoxyuridine（EDU）labeling/detection kit.Flow cytometry was performed to analyze cell cycle distribution by standard procedure.The effects of 1700020I14 Rik on the expression levels of the well-known renal fibrosis related factors-transforming growth factor（TGF-β1）,Fibronectin（FN）and type 4 collagen（Col-4）were detected by q RT-PCR and Western blot.Results The interfering efficiency of three si RNAs against1700020I14 Rik was measured by q RT-PCR and si RNA-832 emerged the best knockdown efficiency.MCs transfected with pc DNA3.1（+）-1700020I14 Rik or si RNA-832 could effectively increase or decrease the expression of 1700020I14 Rik.The proliferation of MCs transfected with pc DNA3.1（+）-1700020I14 Rik was inhibited and cell cycle analysis by flow cytometry showed that cells were arrested in the S phase whencompared with control cells.Whereas the cell proliferation was enhanced in si RNA-832-transfected cells and the proportion of cells in S phase was observably increased compared with its controls.QRT-PCR data displayed that over-expression of 1700020I14 Rik significantly decreased the m RNA expressions of Col-4,FN and TGF-β1,while 1700020I14 Rik knockdown increased the expressions of Col-4,FN and TGF-β1.Meanwhile,western blot analysis showed the expressions of Col-4,FN and TGF-β1 decreased in MCs transfected with pc DNA3.1（+）-1700020I14 Rik,however,the expressions of Col-4,FN and TGF-β1 were enhanced in si RNA-832 group.Conclusion 1700020I14 Rik knockdown could promote proliferation and fibrosis in MCs under high glucose condition,while Over-expression of 1700020I14 Rik alleviates renal fibrosis.Objective To explore the potential mechanism of the effect of linc RNA 1700020I14 Rik on fibrosis in mouse renal mesangial cells.Methods The candidates of putative targets for 1700020I14 Rik were predicted by online databases（USCS,mi Rbase and Bi Biserv2 software）and mi R-34a-5p was chosen for predicted candidate.QRT-PCR was used to detected the expression level of mi R-34a-5p and relevant expression of1700020I14 Rik in MCs cultured with high glucose or with low glucose.Mi R-34a-5p mimics or inhibitor was transfected with lipofectamine 2000 and the transfection efficacy was measured by q RT-PCR.Dual luciferase reporter assay was performed to determine whether 1700020I14 Rik was directly repressed by mi R-34a-5p.Further,RIP assay was conducted to discuss whether 1700020I14 Rik was regulated by mi R-34a-5p through RNA-induced silencing complex（RISC）in an Ago2 dependent manner.The CCK-8 assay and EDU assay were performed to detect the effect of1700020I14 Rik on Cell proliferation in MCs.Flow cytometry was used to analyze cell cycle distribution.The effects of mi R-34a-5p on the expression levels of hypoxia Inducible Factor 1（HIF-1α）,transforming growth factor（TGF-β1）,Fibronectin（FN）and type 4 collagen（Col-4）were detected by Western blot.Si RNA against Sirt1 was used and co-transfected with mi R-34a-5p inhibitor in cells cultured with low glucose.Western blot assaywas used to measure the protein expression level of HIF-1α,TGF-β1,FN and Col-4.Pc DNA3.1（+）-1700020I14 Rik and mi R-34a-5p mimics were cotransfected in high glucose medium-cultured MCs and si RNA-832 was co-transfected with mi R-34a-5p inhibitor in MCs cultured with low glucose medium.Western blot was used to measure the expression of Sirt1 、HIF-1α、TGF-β1、FN and Col-4.Results Ten candidates of putative targets for 1700020I14 Rik were predicted by online databases,among these,mi R-34a-5p was chosen for predicted candidate for further study.QRT-PCR data showed that mi R-34a-5p mimics or inhibitor was effectively used to up or down the expression of mi R-34a-5p in MCs cultured with high or low glucose.And over-expression of mi R-34a-5p decreased the expression of 1700020I14 Rik in cells with low glucose,while down-expression of mi R-34 a increased the expression of 1700020I14 Rik in cells with high glucose.There is a direct interaction between 1700020I14 Rik and mi R-34a-5p by dual luciferase assays.Additionally,RIP assay was performed and showed that both1700020I14 Rik and mi R-34a-5p were enhanced relative to Ig G control.Over-expression of mi R-34a-5p increased cell proliferation,while inhibiting the expression of mi R-34 a reduced its proliferation.Moreover,silencing of mi R-34a-5p led to an improvement of cell proportion in S phase,while over-expression of mi R-34a-5p reversed the results.Meanwhile,over-expression of mi R-34a-5p enhanced the expressions of HIF-1α,Col-4,FN and TGF-β1,however,inhibition of the mi R-34a-5p down-regulated their expressions.Si RNA against Sirt1 was used and co-transfected with mi R-34a-5p inhibitor and the results showed the down-regulation effect of mi R-34a-5p inhibitor on HIF-1α was abolished by the intervention of si-Sirt1.Furthermore,the effects of mi R-34a-5psilence on increasing the expressions of Col-4,FN and TGF-β1 were impeded by si-Sirt1.Conclusion:1700020I14Rik knockdown could promote proliferation and fibrosis in MCs under high glucose condition,while over-expression of1700020I14 Rik alleviates renal fibrosis.