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Changes in expression of miR-337-3p and PTEN in human osteoarthritic cartilageOBJECTIVE:To determine the expression of miR-337-3p and PTEN in human osteoarthritic cartilage tissue and to analyze the correlation between miR-337-3p and PTEN and OA.Methods:Normal cartilage tissue and OA cartilage tissue were taken.The mRNA levels of miR-337-3p and PTEN were determined by qRT-PCR.The expression of PTEN protein was determined by Western blot and statistical analysis was performed.RESULTS:Compared with normal chondrocytes,the expression of miR-337-3p in OA chondrocytes was significantly decreased,and the expression of PTEN mRNA was significantly increased,p<0.01.Correlation analysis showed a negative correlation between miR-337-3p and PTEN mRNA levels.The quantitative results of PTEN protein showed that the expression of PTEN protein in OA chondrocytes was significantly increased,p<0.01.Conclusion:The expression of miR,337-3p is decreased and the expression of PTEN mRNA is up-regulated in OA chondrocytes.The expression of PTEN protein 1s increased,and miR-337-3p may be involved in the regulation of OA.Part ? Overexpression of miR-337-3p promotes proliferation of OA chondrocytes and inhibits apoptosisOBJECTIVE:To determine the effect of miR-337-3p on chondrocyte proliferation by measuring the expression of OA chondrocyte proliferation after overexpression of miR-337-3p.METHODS:OA chondrocytes were overexpressed with miR-337-3p mimic and an increase in chondrocyte density was observed under a fluorescent inverted microscope.Cell viability was measured by MTT assay,and the results showed that the activity of OA chondrocytes overexpressing miR-337-3p was increased.Cell cycle,apoptosis rate and Ki-67 expression were detected by flow cytometry,and the proliferation of chondrocytes was determined by the index of cell proliferation activity.In addition,we used qRT-PCR to detect PTEN mRNA expression,Western blot to detect PTEN protein expression and downstream effector protein expression levels,to determine the regulation of miR-337-3p on PTEN and the effect on chondrocyte proliferation.Results:The density of OA chondrocytes increased after overexpression of miR-337-3p.Cells detected by MTT assay showed enhanced activity of OA chondrocytes.Flow cytometry showed that OA chondrocytes were arrested in G0/G1 phase,and miR-337-3p overexpression relieved this blocking and promoted DNA replication and cell proliferation activity.The apoptosis rate of OA chondrocytes(10.75±1.38),the overexpression of miR-337-3p,the apoptosis rate of OA chondrocytes(4.38±0.97),the apoptosis rate decreased significantly,p<0.01.Simultaneously,the number of Ki-67(+)OA chondrocytes increased significantly after miR-337-3p overexpression by flow cytometry,p<0.01.In addition,qRT-PCR showed that the relative expression level of PTEN mRNA in OA chondrocytes(5.13±0.40),and the relative expression level of PTEN mRNA(2.43±0.43)after overexpression of miR-337-3p decreased significantly.Western blot analysis showed that the expression of PTEN protein was decreased in OA chondrocytes after overexpression of miR-337-3p,the phosphorylated AKT(pAKT)was increased,and pAKT/AKT was elevated.Conclusion:miR-337-3p overexpression inhibits PTEN mRNA.And PTEN protein expression promotes phosphorylation of effector downstream of PTEN/AKT pathway,promotes chondrocyte proliferation,enhances chondrocyte activity,and inhibits chondrocyte apoptosis.Part? miR-337-3p regulates PTEN expressionOBJECTIVE:To identify the regulatory effect of miR-337-3p on PTEN by detecting the relative expression of PTEN mRNA and PTEN protein by overexpression or expression inhibition of OA chondrocytes miR-337-3p,and identify miR-337-3p and PTEN binding sites and regulatory relationships by luciferase reporter gene.METHODS:OA chondrocytes were over-expressed or expressed in miR-337-3p.The relative expression of PTEN mRNA in OA chondrocytes was detected by qRT-PCR.The OA chondrocytes were transfected with miRNC for control.The relative expression level of PTEN protein was detected by Western blot and statistical analysis was performed.The luciferase reporter gene verified the binding site of miR-337-3p and PTEN,and detected the OA chondrocytes after overexpression or expression inhibition of miR-337-3p,and compared the relative activity of luciferase.RESULTS:OA chondrocytes proliferated after overexpression of miR-337-3p.miR-337-3p binds to the 109th-116th nucleotide of PTEN 3'UTR,and the relative expression of PTEN mRNA and PTEN protein in chondrocytes is decreased after overexpression of miR-337-3p.miR-337-3p and PTEN showed a negative regulatory relationship.Conclusion:Overexpression of miR-337-3p promotes the proliferation of OA chondrocytes.miR-337-3p reduces PTEN expression by binding to PTEN,thereby activating the PI3K/AKT pathway and promoting downstream AKT phosphorylation,thereby promoting chondrocyte proliferation.Part IV PTEN regulates chondrocyte growth via PI3K/AKT signalingpathwayOBJECTIVE:To transfect OA chondrocytes with Si-PTEN(PTEN siRNA)and analyze the proliferation and apoptosis of OA chondrocytes by transfection with Si-NC,and to detect the changes of expression levels of downstream effectors,and to clarify the PTEN pair.The regulation of PI3K/AKT pathway and its effect on the growth of OA chondrocytes.Methods:Flow cytometry was used to analyze cell viability,cell cycle,apoptosis and relative count of Ki-67 positive cells.The expression of PTEN mRNA after transfection of Si-PTEN was detected by qRT-PCR.Western blot was used to detect the expression levels of downstream AKT and pAKT proteins.RESULTS:By transfecting Si-PTEN,PTEN expression in OA chondrocytes was reduced,and PI3K/AKT signaling pathway was activated,which enhanced the phosphorylation of downstream effector molecule AKT and promoted the proliferation of chondrocytes.Conclusion:PTEN is an important regulator of PI3K/AKT pathway,which is negatively regulated.It inhibits PTEN expression and activates PI3K/AKT pathway,promotes downstream AKT phosphorylation,and enhances OA chondrocyte proliferation. |
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