| Background and purposeOsteoporosis is a systemic bone disease that has become a global health problem.Its main characteristics are the decrease in bone density and quality of the patient,the deterioration of bone microstructure,and the subsequent decrease in bone strength,bone fragility and increased risk of fractures.Although the drugs for treating osteoporosis have made great progress,the use of these drugs still produces some side effects.Therefore,new treatment methods,especially stem cell-based therapies,have attracted widespread attention from scholars.Bone marrow mesenchymal stem cells(hMSCs)are non-hematopoietic pluripotent stem cells,which mainly differentiate into osteoblasts and adipocytes.Regulating the differentiation of hMSCs into osteoblasts is helpful for the treatment of osteoporosis.Therefore,exploring the molecular mechanism of osteogenic differentiation of hMSCs will help to develop new treatments for osteoporosis.LncRNA is a type of RNA molecule longer than 200 nucleotides that does not code for protein.It can be used as a competitive endogenous RNA(ceRNA)to interact with microRNA(miRNA)and participate in the regulation of target gene expression.Studies have found that lncRNA is inseparable from the occurrence and development of osteoporotic diseases,and is expected to become a potential predictive and therapeutic target for osteoporotic diseases.Literature research has found that IncRNA KCNQ1 overlap transcript 1(KCNQ1OT1)is highly expressed during the osteogenic differentiation of hMSCs and promotes the osteogenic differentiation process.However,its function and molecular mechanism in osteoporotic diseases have not been fully elucidated.Bioinformatics predictions show that miR-320a may be the target gene of KCNQ1OT1.Studies have reported that miR-320a is lowly expressed during the osteogenic differentiation of hMSCs,and miR-320a can inhibit the osteogenic differentiation of hMSCs.In addition,bioinformatics predictions also found that Smad homolog 5(Smad5)may be the direct target gene of miR-320a.Smad5 can play a positive regulatory role during the directed osteogenic differentiation of hMSCs.However,whether KCNQ10T1 can regulate the osteogenic differentiation of hMSCs through the miR-320a/Smad5 molecular axis and participate in the oceurrence and progression of osteoporosis is still unclear.This topic includes the following three parts:Part 1:The expression and function of KCNQ1OT1 in hMSCs osteogenic differentiation;Part 2:miR-320a participates in the osteogenic differentiation of hMSCs mediated by KCNQ10T1;Part 3:KCNQ10T1 through miR-320a/The Smad5 molecular axis mediates the differentiation of hMSCs to osteoblasts.Methods1.Use real-time fluorescent quantitative polymerase chain reaction(qRT-PCR)technology to detect the expression of KCNQ10T1 in the process of osteogenic differentiation of hMSCs,and analyze the localization of KCNQ10T1 by nuclear and cytoplasmic separation experiments;2.qRT-PCR technology to analyze the expression of osteocalcin(OCN),osteopontin(OPN)and Runt-related transcription factor 2(Runx2)in the process of osteogenic differentiation of hMSCs;3.Construct cell lines that knock down or overexpress KCNQ10T1 by transfecting KCNQ10T1 shRNA or overexpression vector plasmids,and test the transfection efficiency of KCNQ1OT1 by qRT-PCR;4.qRT-PCR and Western blot were used to detect the expression of KCNQ1OT1,OCN,OPN and Runx2,the activity of alkaline phosphatase(ALP)was detected by the kit,and the level of cell matrix mineralization was analyzed by alizarin red staining method.;5.Online databases such as bioinformatics starBase v2.0 and TargetScan predict the targeting relationship between KCNQ10T1 and miR-320a,and the dual-luciferase reporter assay and qRT-PCR verify the targeted binding relationship between the two;6.qRT-PCR,Western blot,ALP activity detection and alizarin red staining analysis of the effect of inhibiting or overexpressing miR-320a on the osteogenic differentiation of hMSCs;7.Construct a cell line that knocks down KCNQ10T1 and miR-320a at the same time,qRT-PCR,Western blot,ALP activity detection and Alizarin red staining detect the effect of inhibiting miR-320a on the osteogenic differentiation of hMSCs by knocking down KCNQ1OT1;8.Bioinformatics starBase v2.0 and TargetScan and other online databases predict the targeting relationship between miR-320a and Smad5,and the dual-luciferase reporter assay,qRT-PCR and Western blot verify the targeted combination between the two relationship;9.qRT-PCR,Western blot,ALP activity detection and Alizarin Red staining analysis of the effect of inhibiting or overexpression of Smad5 on the osteogenic differentiation of hMSCs;10.Construct a cell line that simultaneously overexpresses miR-320a and Smad5,qRT-PCR,Western blot,ALP activity detection and Alizarin Red staining to detect the effect of overexpression of Smad5 on the osteogenic differentiation of hMSCs by overexpression of miR-320a;11.qRT-PCR and Western blot analysis whether KCNQ1OT1 affects the expression of OCN,OPN and Runx2 by targeting the miR-320a/Smad5 molecular axis.12.qRT-PCR detects the expression of KCNQ10T1,miR-320a and Smad5 mRNA in the serum of patients with osteoporosis,and analyzes the correlation between their expressions.Results1.KCNQ1OT1 is highly expressed during the osteogenic differentiation of hMSCs,and it gradually increases with the prolongation of osteogenic differentiation induction time.The results of nuclear and cytoplasmic separation experiments show that it is mainly distributed in the cytoplasm;2.In the process of osteogenic differentiation of hMSCs,the expression of osteogenic markers OCN,OPN and Runx2 gradually increased with the prolonged osteogenic differentiation induction time;3.Overexpression of KCNQ1OT1 can promote the expression of OCN,OPN and Runx2,increase ALP activity and the level of matrix mineralization,and knock down KCNQ1OT1 will show the opposite result;4.Bioinformatics and dual-luciferase reporter assays verified that KCNQ1OT1 can specifically bind to miR-320a,and its KCNQ10T1 can negatively regulate the expression of miR-320a;5.The expression level of miR-320a gradually increases with the prolongation of hMSCs osteogenic differentiation induction time.Overexpression of miR-320a can inhibit the osteogenic differentiation of hMSCs,while inhibiting miR-320a can promote the osteogenic differentiation of hMSCs;6.Inhibition of miR-320a can reverse the inhibitory effect of knocking down KCNQ1OT1 on the osteogenic differentiation of hMSCs;7.Bioinformatics and dual-luciferase reporter assays verified that Smad5 is a direct target gene of miR-320a,and miR-320a can negatively regulate the expression of Smad5;8.Overexpression of Smad5 can promote the osteogenic differentiation of hMSCs,while knocking down Smad5 can hinder the differentiation of hMSCs to osteogenic;9.Overexpression of Smad5 can reverse the inhibitory effect of overexpression of miR-320a on the osteogenic differentiation of hMSCs;10.KCNQ1OT1 can affect the expression of Smad5 by targeting miR-320a;11.KCNQ1OT1 can regulate the osteogenic differentiation of hMSCs through the miR-320a/Smad5 molecular axis.12.The expression of KCNQ1OT1 and Smad5 mRNA in the serum of patients with osteoporosis was significantly lower than that of the healthy control group,while the expression of miR-320a was significantly higher than that of the healthy control group;and KCNQ1OT1 and miR-320a,as well as miR-320a and Smad5 mRNA The expression in the serum of osteoporosis patients is negatively correlated.Conclusion1.KCNQ1OT1 is highly expressed during the osteogenic differentiation of hMSCs,and it can promote the osteogenic differentiation of hMSCs.2.KCNQ1OT1 regulates the osteogenic differentiation of hMSCs through sponge adsorption of miR-320a.3.KCNQ1OT1 can regulate the osteogenic differentiation of hMSCs through the miR-320a/Smad5 molecular axis.4.The expression of KCNQ1OT1 and Smad5 mRNA in the serum of patients with osteoporosis decreases,while the expression of miR-320a increases. |
