Effect Of MicroRNA-29a-3p On Human Corneal Keratocyte

Objective:To investigate the effects of microRNA-29a（microRNA-29a;miR-29a）on human corneal keratocytes myofibroblasts and their proliferation and migration.Methods:In this experiment,human corneal keratocytes were used as experimental objects.Human corneal keratocytes within 6 generations of the growth phase were used to induce myofibroblastic transformation to select transforming growth factorβ1（TGF-β1）.The in vitro culture is divided into the following groups according to the experimental design:control group（Ctrl）,TGF-β1 group,miR-29a control+TGF-β1group（agoNC+TGF-β1）,and overexpression of miR-29a+TGF-β1 group（ago29+TGF-β1）,inhibit expression of miR-29a control+TGF-β1 group（ant NC+TGF-β1）,inhibit expression of miR-29a+TGF-β1 group（ago29+TGF-β1）;The part for detecting cell proliferation and migration ability can be divided into:control group（control,Ctrl）,calf serum group（fetal bovine serum,FBS）,miR-29a control+FBS group（agoNC+FBS）),Overexpression of miR-29a+FBS group（ago29+FBS）,inhibition of expression of miR-29a control+FBS group（ant NC+FBS）,inhibition of expression of miR-29a+FBS group（ago29+FBS）.Cell proliferation and toxicity detection kits（Cell Counting Kit-8,CCK-8）were used to detect cell viability in each group.Real-time fluorescence quantitative polymerase chain reaction（PCR）technology was used to detect the expression of smooth muscle actin（αSMA）,fibronectin（FN）,collagenⅢ,（Col₃）in each group of cells,at the same time,the transfection efficiency of each group of samples is the content of miR-29a was also measured using this method.At the protein level,western blot（WB）was used to detectαSMA,hyaluronan synthase-2（HAS₂）,and matrix metalloproteinase-2（MMP₂）in each group of samples.At the same time,immunofluorescence was used to detect the distribution and expression ofαSMA,secreted protein acidic and rich in cysteine（SPARC,also known as osteonecfin）and FN in human corneal keratocytes.5-Bromo-2-deoxy Uridine（Brdu）was used to detect the proliferation capacity of each group of cells.Wound healing experiments were used to simulate cell migration to detect the migration ability of cells in each group,and Cytation 5cell imaging multi-mode detector was used to collect images of cell migration.Results:Human corneal keratocytes are polygonal,dendritic and arranged regularly.5 ng/ml TGF-β1 was used for cell follow-up treatment for 48 hours.CCK-8 results showed that the concentration of TGF-β1 on human corneal keratocytes for 48 hours could significantly increaseαSMA,FN and Col₃（P<0.05）,but not decrease the cell viability（P<0.01）.At the same time,miR-29a decreased significantly（P<0.05）after treating cells with 5 ng/ml TGF-β1 for 48 hours.Various treatments of human corneal stromal cells during the experiment will not affect cell viability（P>0.5）,real-time fluorescence quantitative PCR results showed that the experimental transfection of miR-29a had good efficiency（P<0.05）,and the comparison between the Ctrl group and the TGF-β1 group showed that TGF-β1 could induceαSMA,FN,and Col in m RNA of human corneal stromal cells Compared with the agoNC+TGF-β1 group and the ago29+TGF-β1 group,the expression ofαSMA,FN,and Col₃ decreased at the m RNA level（P<0.05）.Comparison between the ant NC+TGF-β1 group and the ant29+TGF-β1 group showed that inhibiting the expression of miR-29a in human corneal stromal cells can promote the expression of FN and Col₃ at the m RNA level（P<0.05）.WB results show that the comparison between the Ctrl group and the TGF-β1 group indicates that TGF-β1 can induce a significant increase in protein expression ofαSMA,HAS₂ and MMP₂ in human corneal stromal cells（P<0.05）.Compared with the TGF-β1 group,the expression ofαSMA,HAS2,and MMP2decreased at the protein level after overexpression of miR-29a（P<0.05）.Comparison between the ant NC+TGF-β1 group and the ant29+TGF-β1 group showed inhibition of miR-29a The expression in human corneal stromal cells can promote the expression ofαSMA,HAS₂ and MMP₂ at the protein level（P<0.05）.Immunofluorescence results show that:TGF-β1 can significantly increase the expression of SMA and SPARC in cells（P<0.05）,and after overexpressing miR-29a in cells,the above proteins show a downward trend,otherwise,if miR-29a is inhibited in cells,the expression of the above three proteins showed an increasing trend.The results of Brdu and simulated wound healing experiments do not show that the specific up-regulation of miR-29a in cells can inhibit the proliferation and migration of human corneal keratocytes,nor that the inhibition of the expression of miR-29a in cells can promote the proliferation and migration of cells.Conclusion:TGF-β1 can induce myofibroblast transformation in human corneal keratocytes in vitro.The specific overexpression of miR-29a can inhibit this process.Conversely,inhibiting the expression of miR-29a in cells can promote this process.At the same time,this experiment cannot yet explain that overexpression of miR-29a can inhibit the proliferation and migration of human corneal keratocytes,and that inhibition of miR-29a expression can promote the proliferation and migration of human corneal keratocytes.