Cellular function phenotype is regulated by various microRNAs (miRs),
including miR-135a. However, how miR-135a is involved in the calcification in senescent
vascular smooth muscle cells (VSMCs) is not clear yet. In the present study, we first identified the significantly altered
miRNAs in VSMCs, then performed consecutive passage culture of VSMCs and analyzed the expression of miR-
135a and calcification genes in the senescent phase. Next, the effects of the miR-135a inhibition on calcification and calcification
genes were analyzed. The luciferase assay was used to validate the target protein of miR-135a. The western blotting
was used to determine the effects of miR-135a on Krüppel-like factor 4 (KLF4) and signal transducer and activator of
transcription 3 protein (STAT3) expression, as well as the relationship between KLF4 and STAT3. Finally, the quantified
cellular calcification was measured to examine the involvement of miR-135a, KLF4 and STAT3 in VSMCs calcification.
Our results showed that miR-135a was significantly altered in VSMCs. Cell calcification and calcification genes were
greatly altered by miR-135a inhibition. KLF4 was validated as the target RNA of miR-135a. Expression of KLF4 and
STAT3 were both significantly decreased by over expressed miR-135a, while the inhibition of miR-135a and KLF4
siRNA both decreased the STAT3 protein levels. Moreover, the inhibition of miR-135a dramatically increased the calcium
concentration, but co-treatment with KLF4 or STAT3 siRNA both decreased the calcium concentration. The present
study identified miR-135a as a potential osteogenic differentiation suppressor in senescent VSMCs and revealed that
KLF4/STAT3 pathway, at least partially, was involved in the mechanism.