miR-135a Suppresses Calcification in Senescent VSMCs by Regulating KLF4/STAT3 Pathway

Abstract

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.

MicroRNA-17-5p mediates hypoxia-induced autophagy and inhibits apoptosis by targeting signal transducer and activator of transcription 3 in vascular smooth muscle cells

“…In VSMCs, STAT3 is associated with cell senescence and calcification, and STAT3 expression is significantly decreased in response to miR-135a overexpression (24). Recent in vitro and in vivo research has demonstrated that miR-17-5p overexpression promotes cardiomyocyte apoptosis, which is induced by oxidative stress via the targeting of STAT3 (16).…”

Hydroxytyrosol supplementation modulates the expression of miRNAs in rodents and in humans

“…miR-483-3p has been also associated with the capacity of adipocytes to store lipids and dysregulation of its expression could directly inhibit adipose tissue expandability and lipid storage, which in turn affect other tissues by stimulating ectopic triglyceride storage and lipotoxicity [36]. Regarding the other miRNAs and their function, miR-196b has been associated with inflammatory bowel disease [37], insulin [38] and cancer [39]; miR-346 has been implicated in rheumatoid arthritis [40], osteogenic differentiation [41] and cancer [42]; miR-1247 was implicated in cancer [43,44]; miR135a was implicated in proliferation [45], inflammation [46] and calcification [47]; and miR-193a-5p was associated with cancer [48,49]. Although their effects in the intestine need to be evaluated, it is clear that HT could specifically modulate their expression and exert biological effects through this mechanism.…”

Function, Role, and Clinical Application of MicroRNAs in Vascular Aging

“…For example, miR-135a acts as a potential osteogenic differentiation suppressor in senescent VSMCs by targeting both KLF4 and STAT3 [137]. Increased calcium deposition was observed in the combined treatment with mimics of miR-221 and miR-222 [135].…”