| Objective To investigate the effect of lnc RNA Gm2115 on migration of bone marrow mesenchymal stem cells in diabetic nephropathy and its potential mechanism.Methods RNA sequencing(RNA-seq)was used to detect the differential expression of lnc RNAs in the renal tissues of normal and diabetic nephropathy mice.q RT-PCR was used to detect the expression of the candidated lnc RNAs in the renal tissues of normal and diabetic nephropathy mice.The femur and tibia of C57 BL / 6J male mice were used to extract the original generation of bone marrow mese nchymal stem cells(BMSCs),flow cytometry was used to evaluate the CD phenotypes on the surface of BMSCs.Adipogenic and osteogenic differentiations of BMSCs were detected by fluorescence microscopy.DN-like microenvironment(high glucose,high LPS,high AGEs)was cultured for BMSCs,and DN BMSCs model was constructed.The migration ability of DN and normal BMSCs was detected by wound healing test.The candidate DN-related lnc RNAs were detected by q RT-PCR,and it was found that the differential expression trend of lnc RNA Gm2115 was only consistent with the results of RNA-seq and q RT-PCR in mice renal tissue.Therefore,lnc RNA Gm2115 was selected as the research object.The expression and distribution of lnc RNA Gm2115 in the nucleus and cytoplasm of BMSCs was examined by q RT-PCR and fluorescent in situ hybridization(FISH).ORF finder was used to predict lnc RNA Gm2115 protein-coding ability,ORFs on NCBI finder predicted amino acid sequence were compared and analyzed lnc RNA Gm2115 coding ability,and added a flag after most likely coding ORF,and builded an expression vector transfection tool to cells,western blot was used to detect the flag label expression to determine lnc RNA Gm2115 coding ability.Lnc RNA Gm2115 overexpressed lentivirus vector was constructed to determine the optimal MOI infection value of BMSCs,and lnc RNA Gm2115 si RNAs were designed and synthesized.q RT-PCR was used to verify overexpression and knock down effect.The migration ability of BMSCs after overexpression and knockdown of lnc RNA Gm2115 was detected by wound healing test and Transwell migration test.Western blot was used to detect the expression of BMSCs migration factor p21-activated kinase 1(PAK1)and matrix metal loproteinases 9(MMP9)after overexpression and knockdown of lnc RNA Gm2115.In order to further explore the potential mechanism of lnc RNA Gm2115 regulating BMSCs migration,online prediction software Diana tools was used to predict t he potential mi RNA target genes of lnc RNA Gm2115.Furthermore,the binding sites and binding energies of these mi RNAs and lnc RNA Gm2115 were further detected by software RNA V22.The binding of lnc RNA Gm2115 and mi R-29b-1-5p was further verified by dual luciferase assay.The expression of mi R-29b-1-5p in normal and DN BMSCs was detected by q RT-PCR.Furthermore,q RT-PCR was used to detect the expression of mi R-29b-1-5p when lnc RNA Gm2115 was overexpressed or knocked down.In order to further study the effect of mi R-29b-1-5p on BMSCs migration ability,we designed and synthesized mi R-29b-1-5p mimics and inhibitor in vitro and transfected them into BMSCs.The transfection efficiency was detected by q RT-PCR,and the expression of lnc RNA Gm2115 was detected by q RT-PCR when mi R-29b-1-5p was overexpressed or knocked down.The migration ability of BMSCs after overexpression and knocking down mi R-29b-1-5p was detected by wound healing test and Transwell migration test.Western blot was used to detect the expression of BMSCs migration factors p21-activated kinase 1(PAK1)and matrix metal loproteinases 9(MMP9)after overexpression and knockdown of mi R-29b-1-5p.The target genes of mi R-29b-1-5p was detected by mi RTar Base database.The promoter binding sites was detected by Mat Inspector 7.2.Furthermore,the signaling pathways related to BMSCs migration were sought.Further,the expression of Akt1?p-Akt1 was detected by western blot when mi R-29b-1-5p was overexpressed or knocked down.Western blot was used to detect the expression of BMSCs migration factor PAK1 and MMP9 after lnc RNA Gm2115 and mi R-29b-1-5p were overexpressed in normal BMSCs and after lnc RNA Gm2115 and mi R-29b-1-5p were knocked down in DN BMSCs.Results The expression of lnc RNA Gm2115 in mice renal tissues and BMSCs cultured in DN micro environment was significantly up-regulated,suggested that lnc RNA Gm2115 may be an important factor involved in the regulation of the biological behavior of DN BMSCs.Flow cytometry was used to detect the expression of C D29 and CD45,and the results showed that BMSCs expressed positively the CD29,with a positive expression rate of 99.91%,and negatively CD45(7.71%),and had the potency of differentiating into fat,bone cells.The above results indicated that BMSCs model was successfully constructed.The migration ability of BMSCs and normal BMSCs cultured in DN microenvironment was detected by wound healing test,and the migration ability of DN BMSCs was significantly decreased after 48 h of culture.The results of FISH and q RT-PCR indicated that lnc RNA Gm2115 was distributed in both the nucleus and cytoplasm of BMSCs,and was mainly located in the cytoplasm of BMSCs.ORF Finder analyzed the protein encoding capacity of lnc RNA Gm2115.Flag tags were added after the most li kely encoding ORF1 and transfected into tool cells.Western blot results showed that there was no protein expression at flag tags,indicating that lnc RNA Gm2115 had no protein encoding capacity.Lnc RNA Gm2115 overexpressed lentivirus vector was transfected into normal BMSCs.The optimal MOI value of BMSCs was 75 when observed under fluorescence microscope.q RT-PCR results showed that it had a good overexpression effect.The si RNAs were transfected into BMSCs cultured in DN microenvironment,q RT-PCR detected the knock down effiency and the results showed that si Gm2115.1,si Gm2115.2,and si Gm2115.3 all had knocked down effects,but si Gm2115.1 and si Gm2115.3 had the best knock down effects,so they were used as subsequent experimental tools.The results of wound healing test and Transwell migration experiment suggested that lnc RNA Gm2115 inhibited BMSCs migration in BMSCs cultured in DN microenvironment.Western blot results showed that knocking down lnc RNA Gm2115 could up regulate the expression of migration factors PAK1 and MMP9 of DN BMSCs.Further bioinformatics prediction suggested that there was a binding site between lnc RNA Gm2115 and mi R-29b-1-5p.The results of dual luciferase assay confirmed that lnc RNA Gm2115 was bound to mi R-29b-1-5p.The results of q RT-PCR showed that the expression of mi R-29b-1-5p was inhibited after the overexpression of lnc RNA Gm2115,while knockdown of lnc RNA Gm2115 could promote the expression of mi R-29b-1-5p.The expression of lnc RNA Gm2115 was inhibited after up regulation of mi R-29b-1-5p,while the expression of lnc RNA Gm2115 was inhibited after up regulation of mi R-29b-1-5p,suggesting that both lnc RNA Gm2115 and mi R-29b-1-5p have a negative feedback effect and may participate in the regulation of migration of DN BMSCs.The results of wound healing test and Transwell migration experiment suggested that mi R-29b-1-5p could promote BMSCs migration in DN microenvironment.Western blot results showed that the up-regulated expression of mi R-29b-1-5p could significantly up regulate the expression of DN BMSCs migration factors PAK1 and MMP9.Mi RTar Base database and RNA V22 software showed that mi R-29b-1-5p had the highest binding capacity with Glioma associated oncogene homolog 1(Gli1).Literature reported that Gli1 could regulate Akt1 expression in BMSCs migration related signal pathways.Mat Inspector 7.2 confirmed the presence of binding sites for Gli1 and Akt1 promoters.Further western blot results confirmed that lnc RNA Gm2115 may play a role in the migration of DN BMSCs through mi R-29b-1-5p/Gli1/Akt1 pathway.Conclusion lnc RNA Gm2115 is significantly highly expressed in renal tissue of DN mice and BMSCs cultured in DN microenvironment,and the expression of lnc RNA Gm2115 inhibits the migration of DN BMSCs,while the target gene of lnc RNA Gm2115,mi R-29b-1-5p,promotes the migration of DN BMSCs.mi R-29b-1-5p may regulate Akt1 signaling pathways which related to BMSCs migration through target gene Gli1,Moreover,lnc RNA Gm2115 may play a role in the migration of DN BMSCs through mi R-29b-1-5p/Gli1/Akt1 pathway.They could be used as a new molecular target to promote the migration of DN BMSCs,providing a new idea for the prevention and treatment of DN. |
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