| Objective: Osteoporosis is a systemic bone disease characterized by decreased bone mass and damage to bone microstructure,resulting in increased bone brittleness,decreased strength and prone to fractures.With the deepening of China's aging,the incidence of osteoporosis is also increasing year by year.Osteoporosis is mainly divided into primary and secondary osteoporosis,of which primary osteoporosis accounts for a relatively large proportion,mainly seen in post-menopautic women.The main cause of the primary osteoporosis is the decline in estrogen levels caused by the reduction of bone production capacity,accompanied by bone absorption damage,resulting in bone microstructure damage,decreased bone density and bone strength,greatly increasing risk of fractures.At present,the long-term application of anti-osteoporosis drugs is still an important means of osteoporosis treatment.However,it is worth noticing that the current clinical application of drugs are still mainly anti-bone absorption,lack of efficient bonepromoting treatment.As an important source of osteoblasts,bone mesenchymal stem cells are the key cells in bone formation,and their important role in the development of osteoporosis is increasingly recognized by researchers.However,at present,in the pathological state of osteoporosis,it is still unclear how the biological behavior of bone mesenchymal stem cells has changed and the underlying mechanisms there are.In the method of studying various factor changes in a cell in pathological state,RNA sequencing is a more advanced and efficient method in recent years,which can sequence all the RNA expressing in the cells in different states with high volume sequencing.It is able to analyze the changes in the in-cell transcription level quickly,accurately and completely,and at the same time can unearth many unknown RNA that has not been reported,which will fill a large number of gaps in the relevant research field.The exploration of osteoporosis bone mesenchymal stem cells by RNA sequencing can help to reveal the development mechanisms of osteoporosis and find out more efficient treatment options.Methods: A rat model of the ovarian osteoporosis group and the fake surgical group was constructed,and bone marrow interstate charge stem cells were extracted using the whole bone marrow culture method.The osteoporosis model was successfully established by using rat psychic he staining,and bone mesenchymal stem cells were identified from morphology,esophageal identification and differentiation.Collected the third-generation bone mesenchymal stem cells of osteoporosis and false surgical groups for RNA sequencing,PCR verification LNC?000052 high expression in osteoporotic bone mesenchymal stem cells.Construction of LNC?000052 over-expression viruses and knock-down low primer.Using CCK-8 and Ed U experiments to explore the effects of LNC?000052 on bone mesenchymal stem cell proliferation.Using Transwell experiments to explore the impact of LNC?000052 on bone mesenchymal stem cell migration.Using flow cytometers to explore the effect of LNC?000052 on the apoptosis and cell cycle of bone mesenchymal stem cells.Using alizarin red to explore the effect of LNC?000052 on the bone formation ability of bone mesenchymal stem cells.PCR was used to verify the low expression of miR-96-5p in osteoporosis bone mesenchymal stem cells,and PCR and western blot were used to verify the high expression of PIK3R1 in osteoporotic bone mesenchymal stem cells.Using PCR to detect miR-96-5p expression after the adjustment of LNC?000052.Using PCR,western blot,immunofluorescence experiments to detect the changes in the expression of PIK3R1 after regulation of LNC?000052.Build miR-96-5p over-expression and knock-down low protons.Apply PCR,western blot,immunofluorescence experiments to detect thee changes in PIK3R1 expression after miR-96-5p regulation.Using CCK-8 and Ed U experiments to explore the effects of miR-96-5p on bone mesenchymal stem cell proliferation.Using Transwell experiments to explore the impact of miR-96-5p on bone mesenchymal stem cell migration.Using flow cytometers to explore the effect of miR-96-5p on the apoptosis and cell cycle of bone mesenchymal stem cells.Using alizarin red to explore the effect of miR-96-5p on the bone formation ability of bone mesenchymal stem cells.The LNC?000052 and PIK3R1 wild and mutant luciferase vectors were constructed,and the targeted binding between LNC?000052 and miR-96-5p,miR-96-5p and PIK3R1 was verified by double luciferase experiments.The knockdown of miR-96-5p inhibit the function of LNC?000052 on regulating the proliferation,migration,apoptosis,cycle,bone-forming capacity of the bone mesenchymal stem cells,and the downstream PIK3R1 expressio.The bone mesenchymal stem cells,with LNC?000052 knockdown and miR-96-5p overexpression,were transplanted into osteoporosis rats through intravenous injection in the sage,and the therapeutic effect of bone mesenchymal stem cell transplantation on osteoporosis was detected through serological,histological,morphological measurement.Results: HE staining showed that the number of trabecular bone decreased and fat vacuoles increased in ovariectomized osteoporosis group,which proved that the ovariectomized osteoporosis group and sham operation group rat models were successfully constructed.Bone mesenchymal stem cells showed stem cell morphology,positively expressed CD29 and CD90,negatively expressed CD45,had osteogenic and adipogenic differentiation ability,which were in line with the identification criteria.After testing the results of transcription group sequencing,LNC?000052 was highly expressed in osteoporosis bone mesenchymal stem cells,miR-96-5p was low-expressed in osteoporosis bone mesenchymal stem cells,and PIK3R1 was highly expressed in osteoporosis bone mesenchymal stem cells.Over-expressed LNC?000052 inhibits the proliferation,migration and bone-forming capacity of bone mesenchymal stem cells,promotes apoptosis of bone marrow inter-filling stem cells and cell cycle stagnation in G0-G1.Low-expressed LNC?000052 promotes the proliferation,migration and boneforming capacity of bone mesenchymal stem cells,inhibits apoptosis of bone marrow inter-filling stem cells and cell cycle stagnation in G0-G1.PCR and western blot prove that LNC?000052 expression negatively regulates miR-96-5p expression.PCR,western blot and immunofluorescence prove that LNC?000052 positively regulates PIK3R1 expression,and miR-96-5p negatively regulates PIK3R1 expression.The low-expressed miR-96-5p inhibits the proliferation,migration and bone-forming capacity of bone mesenchymal stem cells,promotes apoptosis and the stagnation of cell cycles in G0-G1 of bone mesenchymal stem cells.Over-expressed miR-96-5p promotes the proliferation,migration and bone-forming capacity of bone mesenchymal stem cells,inhibits apoptosis of bone marrow inter-filling stem cells and cell cycle stagnation in G0-G1.The double luciferase experiment shows LNC?000052 shares a targeted binding site with miR-96-5p,and miR-96-5p shares a targeted binding site with PIK3R1.Low-expressed miR-96-5p inhibits the function of low-expressed LNC?000052,which promotes the proliferation,migration,bone-forming ability,and apoptosis and cycle of bone mesenchymal stem cells.In vivo,the treatment effect of osteoporosis is more significant after the transplantation of bone mesenchymal stem cells with low LNC?000052 and overexpressed miR-96-5p.Conclusion: LNC?000052 and PIK3R1 are highly expressed in osteoporosis bone mesenchymal stem cells,and miR-96-5p is low-expressed in osteoporosis bone mesenchymal stem cells.High-expressed LNC?000052 and low-expressed miR-96-5p interfere the normal biological behavior of bone mesenchymal stem cells through PIK3R1.LNC?000052 has a certain targeted binding site with miR-96-5p,and miR-96-5p has a certain targeted binding site with PIK3R1.The knockdown of LNC?000052 and over-expressed miR-96-5p in bone mesenchymal stem cells show a significant positive effect on bone formation and a more significant effect on the treatment of osteoporosis. |
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