Abstract: Objective To screen out microgravity-sensitive miRNAs targeting Runx2,and explore the regulation of miRNAs on Runx2 under simulated microgravity,so as to provide a research target for treating bone loss under microgravity. Methods A bioinformatic tool was used to screen out 4 candidate miRNAs regulating Runx2,and 2D clinostat was used to culture MC3T3-E1 under simulated microgravity condition,and miR-135a-5p mimic or inhibitor were transfected into MC3T3-E1 cells.The expressions of miRNAs and Runx2 were detected by qRT-PCR,and the expression of Runx2 protein was detected by Western blot. Results miR-135a-5p,miR-137-3p,miR-205-5p and miR-217-5p were predicted to regulate Runx2.Among the 4 miRNAs,the expression of miR-135a-5p changed significantly under simulated microgravity condition.Compared with the control group,the expression of miR-135a-5p was up-regulated under simulated microgravity,and showed a steady upward change within 72 hours (P<0.05),while the expression of Runx2 was significantly down-regulated within 72 hours under microgravity (P<0.05).miR-135a-5p affected Runx2 expression at protein level,rather than mRNA level.The protein level of Runx2 was suppressed by overexpression of miR-135a-5p (P<0.01),while it was promoted by knockdown of miR-135a-5p (P<0.01).Transfection of miR-135a-5p inhibitor alleviated the decrease of Runx2 protein expression under microgravity,but it had no effect on mRNA level. Conclusion The expression of miR-135a-5p is up-regulated under microgravity,and is negatively correlated with that of Runx2.miR-135a-5p can regulate the protein expression of Runx2.And we further confirm that miR-135a-5p inhibitor can partially alleviate the decrease of Runx2 in protein expression under microgravity.