A Novel CeRNA Regulatory Network Involving The Long Noncoding RNA NEAT1,miRNA-466f-3p And Its MRNA Target In Osteoblast Autophagy And Proliferation In Osteoporosis

Research BackgroundWith the aging of the population in China,the number of primary and secondary osteoporosis patients in China has increased year by year.Osteoporosis is a common systemic metabolic disease bone disease,which is more common in the elderly.However,the mechanism and treatment of osteoporosis are not satisfactory.In addition,autophagy can regulate the occurrence and progression of osteoporosis.However,the enhancement or reduction of autophagy activity in patients with osteoporosis is still a major controversial issue.It has been found that lncRNA NEAT1 can act as a key switch for adipogenesis and osteogenesis in bone marrow mesenchymal stem cells.However,the specific mechanism of lncRNA NEAT1 in bone metabolism,especially how to regulate autophagy and cell activity of osteoblasts is still unknown.Objective1.To study the mechanism of autophagy regulation,the changes of mRNA and nc RNA(miRNA,lncRNA)in MC3T3-E1 cells during FSS reaction,to provide a theoretical basis for the pathogenesis of osteoporosis.2.In vitro,to reveale how the lncRNA NEAT1/miRNA-466f-3p/HK2signaling pathway regulates the autophagy and cell activity of osteoblasts in the aspects of function and mechanism.3.To determine the effect of si-lncRNA NEAT1 on bone metabolism and autophagy level of bone tissue in vivo,and to establish potential strategies for the treatment of osteoporosis.Methods1.Load MC3T3-E1 cells with 12dyne/cm~2 FSS for 45 minutes.Western blot was used to detect the expression of LC3 and P62,and the autophagosome was observed under transmission electron microscopy.Then the cells were loaded with FSS for transcriptome sequencing.The whole transcriptome sequencing technique was used to analyze the changes of mRNA and nc RNA(miRNA,lncRNA)in MC3T3-E1 cells during FSS reaction.Through GO and KEGG signaling pathways,the biological functions and potential mechanisms of mRNA and miRNA were further explored.Based on the theory of complementary base pairing and competitive endogenous RNA,construct lncRNA mRNA,miRNA mRNA,and lncRNA miRNA mRNA regulatory networks.2.Cell function assay:Western blot assay,transmission electron microscopy(TEM),immunofluorescence(IF)staining,quantitative real-time PCR analysis to verify the biological functions of NEAT1,miR-466f-3p and HK2 respectively.In the mechanism verification part,the relationship between NEAT1,miR-466f-3p and HK2 was predicted and verified through bioinformatics analysis,FISH and double luciferase reporter gene experiment analysis.In addition,the regulation network of NEAT1/miR-466f-3p/HK2 signal pathway was clarified by osteoblast co-transfection and rescue experiments.3.Through in vivo transfection of si lncRNA NEAT1 into the distal femoral cortex of ovariectomized osteoporosis mice,we analyzed the effects of si-NEAT1on bone metabolism and autophagy level of bone tissue by serological bone turnover indicators,micro-CT and hematoxylin eosin(HE)staining,Masson(C)trichrome staining,alizarin red staining,immunohistochemistry and other methods,and established potential strategies for treating osteoporosis.Results1.Under 12dyne/cm~2 FSS conditions,MC3T3-E1 osteoblasts showed upregulation of LC3 expression and downregulation of P62 at the protein level after 45 minutes of intervention.TEM revealed that more autophagosomes were generated in FSS group,which confirmed that FSS can induce autophagy in MC3T3-E1 cells.Compared with MC3T3-E1 cells without FSS,MC3T3-E1 cells loaded with FSS identified 412 autophagy related differentially expressed lncRNAs(DElncRNAs),88 DE miRNAs,and 19 DEmRNAs.At the same time,the ceRNA regulatory network was constructed.2.Knockdown of lncRNA NEAT1 in osteoblasts can cause a decrease in protein expression levels of LC3,BECN1,and ATG5,while an increase in protein expression levels of P62 and PCNA.The lncRNA NEAT1 and miR-466f-3p expression in the cytoplasm is higher than that in the nucleus.miR-466f-3p is a direct target of lncRNA NEAT1.miR-466f-3p can directly bind to the 3′UTR of HK2.Knocking down lncRNA NEAT1 significantly increased the expression of miR-466f-3p,while overexpression of miR-466f-3p significantly reduced the mRNA and protein levels of HK2.After lncRNA NEAT1 silencing,the mRNA and protein levels of HK2 were significantly reduced.Overexpression of HK2 can not only reverse the decreased LC3 protein level and upregulated p62 protein level after lncRNA NEAT1 downregulation,but also reverse the increase in MC3T3-E1cell viability mediated by lncRNA NEAT1 downregulation.3.In the OVX induced osteoporosis mouse group,lncRNA NEAT1expression was upregulated.Bone microstructure analysis showed that compared with the NC group and PBS group,the bone density,bone volume fraction,number and thickness of trabeculae in the si-NEAT1 group were significantly increased,while the level of trabecular separation was significantly reduced(P<0.05).The bone mass of the si-NEAT1 group increased.si-NEAT1 can increase the volume of bone trabeculae and promote osteogenesis.HE staining of the distal femur in mice showed a reduction in the deterioration of bone microstructure in the si-NEAT1 group.ARS and Masson staining showed significant bone formation in si-NEAT1.IHC staining of femurs showed that the expression of LC3 and HK2 in the si-NEAT1 group was lower than that in the si-NC group and blank group.Conclusions1.FSS can upregulate the autophagy level of MC3T3-E1 cells.This provides new information for further research on the molecular mechanisms of osteoporosis.2.lncRNA NEAT1 upregulates the expression of HK2 through sponge adsorption of miRNA-466f-3p,thus regulating the autophagy and cell activity of osteoblasts.3.Knocking down NEAT1 can inhibit autophagy level of bone tissue and increase bone mass in OVX induced osteoporosis mice.This study enriches and improves the regulatory mechanism of autophagy and osteoporosis,and is expected to provide a novel target of menopause induced OP.