| Background and objectiveWith the changes in life quality and un-healthy life style, the gradual increasingly incidence of coronary heart disease (CHD) around world, which have beening threatened people’s health. At present, except medicine in treating CHD, the treatments of CHD also include percutaneous coronary interention (PCI), coronary artery bypass grafting (CABG) and so on, which greatly improve people’s quality of life and prognosis;whereas, neointimal hyperplasia (NIH) which occars in post-operation, inevitably leads to vascular restenosis and limits the long-term therapeutic effect. It has been confirmed that, abnormal proliferation and migration of vascular smooth muscle cells (VSMCs) play an important role in the development and progression of NIH. Inhibition of abnormal function in VSMCs could be a potential mesaurement to alleviate progression of NIH.MicroRNAs (miRNAs) are endogenous,short(about 18 to 22 nucleotides), single noncoding RNA and serve as regulatory factors by way of selectively binding with 3’untranslated region (3’UTR) of targeted messenger RNA (mRNA), which cause mRNA degradation or inhibit translation. As regulatory gene, miRNAs not only are widely involved in normal physiological processes such as proliferation, differentiation, apoptosis;also participated in many pathological processes such as tumor, cardiovascular diseases, neurological disorders.Recently, increasing research indicate that, miRNAs are aberrant expressed in vascular diseases and regulate abnormal proliferation and migration of VSMCs and NIH. Previous studies have showed that miR-146a, miR-31, miR-424/322 could directly promote the VSMCs proliferation;and miR-1, miR-133 and miR-365 have the function of inhibiting VSMCs proliferation,meanwhile affect the neointimal formation in balloon injury model. Above studies show that the abnormal miRNA expression in vascular injury lesions, participate in the regulation of VSMCs functional change, and influence the formation of NIH. However, at present, the investigations of miRNAs inregulating VSMCs are confined to arteries, few literatures were reported on venous SMCs.While a characteristic of miRNAs is specific expression in tissue and cell.Therefore,it’s believed that miRNAs can also regulate biological characteristics of venous SMCs.Referring to previous reports, miR-136 and miR-23b are involved in regulating cell biological function.For example,miR-136,which is abnormal expression in glioma, breast cancer, non-small cell lung cancer and other types of tumors, also in coronary artery atheromatous plaque, is implicated in the regulating cell proliferation, migration and apoptosis. miR-23b belongs to the family of miR-23/24/27, is highly conserved and located in the sequence of human chromosome 9. Previous study demonstrate that miR-23b is abnormal expression in a variety of tumors,such as colon cancer, prostate cancer, breast cancer, renal clear cell carcinoma,even in endothelial cells, participating in regulating cell proliferation, migration and apoptosis. Whether miR-136, miR-23b regulate abnormal venous SMCs functional change, so far there exist no related literature.Therefore, the role and mechanism mediated by miR-136, miR-23b in venous SMCs and NIH in grafted veins still remain unknown and is worthy to investigate deeply.In our study, vein graft models in rat and venous SMCs in vitro were chosen as research object. The objective is to ascertain characteristic expression of miR-136, miR-23b in grafted veins and venous SMCs,to explore the influence of miR-136,miR-23b on proliferation and migration in venous SMCs,further to elucidate the molecular mechanism of miR-23b mediated biological functions,which can lay a foundation for deeply understanding the molecular mechanism of NIH after vein transplantation and searching for a potential molecular therapeutic targets for NIH.Methods1. Expression analysis of miR-136 and miR-23b in grafted veins and venous SMCsTo establish vein transplantation model, external jugular vein was transplanted to common carotid artery in rats.1 week,2 weeks,4 weeks after transplantation,grafted veins were harvested, ipsilateral un-transplanted veins were harvested as control. miR-136 and miR-23b expression level in grafted veins was measured by Real-time PCR. In vitro,venous SMCs were cultured and induced by serum (10%FBS) and PDGF-BB (20ng/mL). Cells stimulated for different time points were harvested and detected the expression of miR-136 and miR-23b respectively by Real-time PCR.2. The biological function of miR-136 and miR-23b in venous SMCs1) To overexpression, venous SMCs were transient transfected respectively with different concentrations of miR-136 or miR-23b mimics by using siRNA-MateTM. Meanwhile, cells were transfected with meaningless sequences as a negative control (NC). This experiment was divided into Group NC, Group NC+serum, Group mimic+serum.48 hours after transfection, transfective efficiency and appropriate concentration were dertermined by Real-time PCR.2) The effect of miR-136 and miR-23b overexpression on venous SMCs proliferation:this experiment was divided into Group NC,Group NC+serum, Group mimic+serum. Cell proliferation was detected by CCK-8 and EDU assay at 48 hours after transfection.3) The effect of miR-136 and miR-23b overexpression on venous SMCs migration:this experiment was divided into Group NC,Group NC+ serum, Group mimic+serum. Cell migration was detected by Wound-Healing and Transwell assay at 48 hours after transfection. 3. Screen and validation of miR-23b targeted gene1) Downstream target genes of miR-23b were screened by way of bioinformatics website, such as miRanda et al.2) Construction of dual-luciferase reporter genes:Wild or mutant type of targeted gene 3’UTR, respectively was cloned into dual-luciferase reporter plasmid:pmiR-GLO. Thus, wild type and mutuant type of recombinant plasmid were constructedand and then validated by colony PCR and identification of enzyme reaction, further to ascertain by plasmid sequencing.3) Measurement of dual-luciferase activity:miR-293T cells were co-transfected by using X-tremeGENE siRNA transfection reagent.This experiment was divided into two groups:miR-293T cells were co-transfected with recombinant plasmid (WT or MU type) respectively and NC in Group control; miR-293T cells were co-transfected with recombinant plasmid (WT or MU type) respectively and miR-23b mimic in Group treatment.48 hours after transfection, relative luciferase activity was detected using the dual-luciferase report gene detection kit, which further verified whether the miR-23b could combine with predicted targeted gene.4. Regulation between miR-23b and targeted gene expressionTargeted gene expression was measured by Real-time PCR in transplantated vein at 1 week,2 weeks,4 weeks. Cell experiment was divided into Group NC, Group NC+ serum, Group mimic+serum, mRNA and protein expression in candidate targets were detected in venous SMCs by Real-time PCR and Western Blot.5. The effect of miR-23b targeted gene on venous SMCs biological characteristics1) By means of transient transfection to interfer expression of miR-23b targeted gene, venous SMCs were transfected with different concentrations of siRNA using siRNA-MateTM transfection reagent. Meanwhile, cells were transfected with meaningless sequences as a negative control. This experiment was divided into Group NC+serum, Group siRNA+serum.48 hours after transfection, efficiency and concentration of transfection were dertermined by Real-time PCR and Western Blot.2) The influence of miR-23b targeted gene on cell proliferation and migration:the experiment was divided into Group NC+serum, Group siRNA+serum; cell proliferation was measured by CCK-8 assay and cell migration was measured by Wound-Healing assay.Results1. miR-136 and miR-23b respectively is down-regulated in grafted veins and venous SMCs1) 1 week,2 weeks after vein transplantation, miR-136 expression level is significantly down-regulated (P<0.01,P<0.05), but up-regulated at 4 weeks (P>0.05); the expression of miR-136 was also decreased in cells induced by serum or PDGF-BB for 24 hours (P< 0.05).2) Compared with un-transplanted veins, miR-23b expression level was significantly down-regulated in grafted veins at 1 week,2 weeks,4 weeks (P<0.0l, P<0.001,P<0.01),and minimum expression at 2 weeks. In vitro, miR-23b expression was significantly down-regulated and decreased time-dependently in cells induced by serum or PDGF-BB, reaching the lowest level at 24 hours (P< 0.01).2. The effect of miR-136 and miR-23b overexpression on venous SMCs proliferation and migration2.1 miR-136 overexpression inhibits venous SMCs proliferation and migration1) CCK-8 experimental results showed, optical density (OD) value was significantly higher in Group NC+serum than that in Group NC (P＜0.05); but in Group mimic+serum, OD value decreased significantly after transfection with miR-136 mimic, compared with Group NC+serum (P<0.05). EDU assay demonstrated:in Group NC+serum, the percentage of EDU labelled positive cells was higher than that in Group NC and Group mimic+serum (P＜0.001, P＜0.01). These results indicated that miR-136 overexpression inhibits venous SMCs proliferation.2) Wound-Healing assay results showed, cell migration rate at 24 hours was significantly higher in Group NC+serum than that in Group NC(P<0.001); but in Group mimic+serum, migration rate decreased significantly in cells transfected with miR-136 mimiC,Compared with Group NC+serum (P<0.01).Transwell assay further verified the results of Wound-Healing assay:the amount of migrated cells in Group NC+serum were higher than that in Group NC and Group mimic+serum (both P<0.05).These results indicated that miR-136 overexpression inhibits venous SMCs migration.2.2 miR-23b overexpression inhibits venous SMCs proliferation and migration1) EDU assay results showed, the percentage of EDU labelled positive cells was significantly higher in Group NC+serum than that in Group NC(P<0.001); but in Group mimic+serum transfected with miR-23b mimic, the percentage of positive cells decreased significantly,compared with Group NC+serum (P＜0.01). EDU assay show:in Group NC+serum, OD values was higher than that in Group NC and Group mimic+serum (both P<0.01).These results indicated that miR-23b overexpression inhibits venous SMCs proliferation.2) Wound-Healing assay results showed, cell migration rate at 24 hours was significantly higher in Group NC+serum than that in Group NC (P＜0.01), but cell migration rate decreased significantly in Group mimic+serum transfected with miR-136 mimic, compared with Group NC+serum (P<0.01).Transwell assay was further verified, as showed by Wound-Healing assay:the amount of migrated cells in Group NC+serum were higher significantly than that in Group NC and Group mimic+serum (both P＜0.05). These results indicated that miR-23b overexpression inhibits venous SMCs migration.3. RBX1 is a directly target of miR-23b1) Based on the analysis of predicated results, RBX1 may be a directly downstream target of miR-23b.2) Dual-luciferase reporter genes (pmiR-GLO WT-3’UTR RBX1 and pmiR-GLO MU-RBX13’UTR) were constructed. Recombinant plasmids, which contain wild type or mutant type RBX1 mRNA binding sites of miR-23b were confirmed by colony PCR, enzyme reaction identification and plasmid sequence.3) Dual-luciferase report gene assay showed, compared with Group control, relative luciferase activity reduced significantly (.P<0.01) in Group treatment co-transfected with wild type recombinant plasmid and miR-23b mimic. Whereas, in Group treatment co-transfected with mutant type recombinant plasmid and miR-23b mimic, there existed no statistical difference comparing with the Group control (P> 0.05). The results suggest that miR-23b can specificly bind the 3’UTR of targeted gene RBX1.4. miR-23b regulates RBX1 expressionRBX1 mRNA expression was significantly up-regulated in grafted veins at 1 week, 2 weeks,4 weeks (all P＜0.01), and maximum expression at 2 weeks. In vitro, cell experiments showed,compared with Group NC, RBX1 mRNA and protein level raised significantly in Group NC+serum (both P<0.05); however, in Group mimic+serum transfected with miR-23b mime, RBX1 mRNA and protein level decreased significantly, compared with Group NC+serum (P<0.05,P<0.01). The results demonstrate that miR-23b can regulate the mRNA and protein expression of RBX1.5. Knock-down level of RBX1 inhibits venous SMCs proliferation and migrationCCK-8 assay results demonstrated, compared with Group control, OD value was decreased significantly in Group si-RBX1+serum (P<0.05), indicating that lower expression of RBX1 inhibits cell proliferation. Wound-Healing assay showed, cell migration rate in 24 hours of Group si-RBX1+serum reduced significantly, compared with Group control (P<0.05), suggesting that inhibition expression of RBX1 can suppress cell migration. The results show that knock-down expression of RBX1 inhibits venous SMCs proliferation and migrationConclusion1. miR-136 and miR-23b is down-regulated in grafted veins and venous smooth muscle cells.2. miR-136 and miR-23b overexpression can inhibit proliferation and migration of venous smooth muscle cells.3. RBX1 is a direct target of miR-23b in venous smooth muscle cells; knocked-down expression of RBX1 inhibits cell proliferation and migration. |
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