| Recent studies have shown that proliferation and migration of vascular smooth muscle cells(VSMC)are the pathological basis of vascular remodeling and related diseases,such as atherosclerosis and restenosis after PTCA.Phenotypic switching of VSMCs plays an important role in VSMC proliferation and migration.It has been well known that VSMC phenotype can be divided into more differentiated contractile(differentiated)and less differentiated secreted(synthetic or dedifferentiated)phenotype.Under the influence of vascular injury factors,contractile VSMCs are transformed into synthetic ones,which undergo proliferation,migration,and a large number of synthesis and secretion of extracellular matrix,leading to the formation of atherosclerotic plaques and angiogenesis.Krüppel-like factor 4(KLF4)is a transcription factor with zinc finger structure,which plays an important role in cell growth,proliferation,differentiation and apoptosis by regulating the expression of genes rich in GC/GT sequence in the promoter region.In the cardiovascular system,vascular endothelial cells,VSMC and macrophages all express KLF4,which is regulated by shear stress,transforming growth factor-beta(TGF-?)and platelet-derived growth factor BB(PDGF-BB),and participates in the occurrence and development of cardiovascular disease.Platelet-derived growth factor(PDGF)is a polypeptide growth factor synthesized and released by a variety of cells,which can stimulate the proliferation of many kinds of cells,such as VSMC,fibroblasts and so on.Under the stimulation of vascular injury or inflammatory factors,VSMC,macrophages,platelets,infiltrating inflammatory cells and damaged endothelial cells can all secrete PDGF.PDGF acts on VSMC,in an autocrine and paracrine manner to promote phenotypic transformation and abnormal proliferation of VSMC.Circular RNA(circ RNA)is a kind of covalently closed circular non-coding RNA formed by pre-m RNA reverse splicing.So far,thousands have been identified in humans circ RNA,many of which are expressed in a tissue-specific or disease-specific manner.Although the biological function of most circ RNAs is still unknown,more and more research results indicate that circ RNA can regulate the expression of genes by adsorbing micro RNA and interacting with RNA binding proteins,and even some circ RNAs can be translated to produce polypeptides.Some research results show that circ RNA is involved in the development of cardiovascular disease.Early in our laboratory,circ ACTA2 up-regulates the expression of ?-actin by releasing mi R-548f-5p inhibition of ?-actin gene expression by adsorbing mi R-548f-5p.However,it has not been reported whether KLF4 and PDGF regulate VSMC phenotype and the proliferation and differentiation are related to the circ RNA induced by them.Objective: To investigate the role and underlying mechanism of KLF4-induced circ FAM53B-1/2 in PDGF-BB repressed differentiation of vascular smooth muscle cells.Methods:1.Human vascular smooth muscle cell(HVSMC),cultured in vitro was infected with KLF4 adenovirus expression vector(Ad-KLF4)and analyzed by circ RNA microarray.By comparing the differential expression between KLF4 overexpression and control cells,circ RNA,regulated by KLF4 was screened and verified by real-time quantitative polymerase chain reaction(PCR).2.VSMCs were treated with PDGF-BB at different concentrations for different times to determine the optimal concentration and duration of PDGF-BB treated VSMCs.Real-time quantitative PCR(q RT-PCR)was used to detect and analyze KLF4,circ FAM53B-1,and circ FAM53B-2 expression levels,and Western blot was used to analyze KLF4 protein levels.After VSMCs were infected with Ad-KLF4 for 12 hours,then PDGF-BB(10 ng/m L)was given to stimulate cell proliferation.Cells were collected,and the expression of circ FAM53B-1 and circ FAM53B-2 was analyzed by q RT-PCR.3.The expressing vectors for circ FAM53B-1,and circ FAM53B-2 were constructed and were used to transfect VSMCs.After 24 h,cells were collected,and the expression of the two circ RNAs was detected by real-time PCR.At the same time,the expression of VSMC differentiation markers smooth muscle actin(SMA)and smooth muscle protein 22 alpha(SM22?)was detected by Western blot analysis.4.VSMCs were transfected with circ FAM53B-1 and circ FAM53B-2 to overexpress circ FAM53B-1 and circ FAM53B-2 in VSMCs,and serum response factor(SRF)and Myocardin expression was detected by Western blot analysis.Results:1.circ FAM53B-1 and circ FAM53B-2 are expressed in VSMCs.Thirty-one circ RNAs(fold change>4)up-regulated by KLF4,which w-ere detected by circ RNA chip analysis,were selected end validated by rea-l-time PCR.As a result,20 circ RNAs were significantly up-regulated by KLF4.Then circ FAM53B-1 and circ FAM53B-2 were identified by agarose gel electrophoresis and sanger sequencing.The results showed that the mol-ecular weights and nucleotide sequences of both circ RNAs were correct.2.KLF4 up-regulates the expression of circ FAM53B-1 and circ FAM53B-2 in VSMCs.VSMCs were infected with KLF4-expressing adenoviruses(Ad-KLF4),circ FAM53B-1 and circ FAM53B-2 expression was determined by real-time PCR.The results showed that KLF4 overexpression significantly induced the formation of circ FAM53B-1 and circ FAM53B-2.At the same time,KLF4 substantially increased FAM53 B linear m RNA expression.3.PDGF-BB promotes circ FAM53B-1 and circ FAM53B-2 formation through up-regulating the expression of KLF4.After VSMC was stimulated with 10 ng/m L PDGF-BB for different time and VSMC with different concentrations of PDGF-BB for 24 h,the results of q RT-PCR and Western blot showed that PDGF-BB promoted the expression of KLF4 in a concentration and time-dependent manner,and up-regulated the expression of circ FAM53B-1 and circ FAM53B-2 in a time-dependent manner.More importantly,when VSMC was infected with Ad-KLF4 for 12 hours and then stimulated by PDGF-BB,the expression levels of circ FAM53B-1 and circ FAM53B-2 were significantly higher than those of transfected empty vector.The results showed that PDGF-BB promoted the formation of circ FAM53B-1 and circ FAM53B-2 by inducing the expression of KLF4.4.circ FAM53B-1 and circ FAM53B-2 down-regulate the expression of VSMC differentiation marker genes SMA and SM22? by inhibiting the expression of SRF,and then inhibit the differentiation of VSMC.circ FAM53B-1 and circ FAM53B-2 expression vectors infected VSMC.On the basis of the successful expression of two kinds of circ RNA in VSMC confirmed by real-time PCR,the expression of VSMC differentiation marker genes SMA and SM22? was detected by Western blot.The results showed that the overexpression of circ FAM53B-1 or circ FAM53B-2 significantly down-regulated the expression of SMA and SM22?.Because the expression of VSMC differentiation marker gene is regulated by SRF,we further examine the effect of overexpression of circ FAM53B-1 and circ FAM53B-2 on SRF expression.Western blot results showed that overexpression of circ FAM53B-1 and circ FAM53B-2 in VSMC significantly inhibited the expression of SRF.These results suggest that circ FAM53B-1 and circ FAM53B-2 inhibit the differentiation of VSMC by inhibiting the expression of transcription factor SRF.Conclusions:1.In VSMC,KLF4 up-regulates the expression of circ FAM53B-1 and circ FAM53B-2.2.PDGF-BB promotes the formation of circ FAM53B-1 and circ FAM53B-2 by up-regulating KLF4 expression.3.circ FAM53B-1 and circ FAM53B-2 down-regulate the expression of VSMC differentiation marker genes SMA and SM22? by inhibiting the expression of SRF,thus inhibiting the differentiation of VSMC. |
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