Effecfs Of Endothelium 27nt-miRNA On Phenotypic Changes,proliferation And Apoptosis Of Vascular Smooth Muscle Cells And Related Mechanisms

Objectives 1 To elucidate the phenotypic changes of aortic vascular smooth muscle cells by endothelium 27nt-mi RNA and related molecular mechanisms.2 To investigate the effects of endothelium 27nt-mi RNA on proliferation,migration and apoptosis of aortic vascular smooth muscle cells.Methods 1 Lentivirus construction,transfection and establishment of VSMCs dedifferentiation model Shanghai Jikai Gene Company constructs lentivirus according to the corresponding sequenceand and analyze whether the vector was successfully constructed by PCR and DNA sequencing.Finally,the corresponding vector was packaged with lentivirus to 27nt-mi RNA,anti-27nt-mi RNA and negative control NC lentiviral.The lentiviruses were transfected into VSMCs,the green fluorescent expression intensity of the cells was observed under a fluorescence microscope to determine the establishment of 27nt-mi RNA cell lines.2 To detect the effect of 27nt-mi RNA on phenotype switching of VSMCs According to the experimental design and requirements,VSMCs were randomly divided into normal group,PDGF-BB group,mimic 27nt-mi RNA+PDGF-BB group,Anti-27nt-mi RNA+PDGF-BB group,and NC+PDGF-BB group.The normal group was culture with no treatment,and the PDGF-BB group was co-cultured with 10 ng/m L PDGF-BB lasted 48 h to establish the dedifferentiation model.The other three groups of VSMCs were transfected with mimic 27nt-mi RNA lentivirus,Anti-27nt-mi RNA lentivirus and negative control lentivirus firstly,and then treated with 10 ng/m L PDGF-BB lasted 48 h to establish a dedifferentiation model.The m RNA levels of SMA and SM22? were detected by RT-PCR.The protein levels of SMA and SM22? were detected by western blotting and immunocytochemistry.3 To detect the effects of 27nt-mi RNA on proliferation,migration and apoptosis of VSMCs According to the experimental requirements,VSMCs were randomly divided into control group,mimic 27nt-mi RNA group and Anti-27nt-mi RNA group.Then cells were transfected with negative control sequence,27nt-mi RNA overexpression,and Anti-27nt-mi RNA lentiviral vector respectively.The proliferative ability of VSMCs in each group was detected by MTT assay and Cell counting;the migrated ability of VSMCs in each group was detected by scratch test;Hoechst 33258 staining for detecting cell apoptos is and the apoptosis rate of VSMCs in each group was detected by flow cytometry.Results 1 Under the fluorescence microscope,the green fluorescence of the transfected cells was strong,The lentiviral transfection efficiency is more than 90%,and the cell state is well,indicating that the cell lines were constructed.2 In the phenotypic transformation experiment,compared with the normal group,the expression of SMA and SM22? m RNA and protein in PDGF-BB group were significantly decreased(P<0.05).The expression of SMA and SM22? m RNA and protein in mimic 27nt-mi RNA+PDGF-BB group was up-regulated significantly while compared with NC+PDGF-BB group and Anti-27nt-mi RNA+PDGF-BB group(P<0.05).3 In the proliferation,migration and apoptosis experiments,compared with the negative control group,the proliferative ability and migration ability of VSMCs in mimic 27nt-mi RNA group were inhibited(P<0.05),and the apoptotic cell increase and the apoptosis rate was increased(P<0.05).The proliferative ability and migratied ability of VSMCs in anti-27nt-mi RNA group were increased(P<0.05),while the apoptosis rate have no significant differences(P>0.05).Conclusions 1 27nt-mi RNA promotes the expression of contractile-associated genes SMA and SM22? at the transcriptional level,and inhibited PDGF-BB-induced phenotypic transformation.2 27nt-mi RNA inhibites the proliferation and migration of VSMCs significantly while promotes the apoptosis rate of VSMCs.