The Functional Study Of Let-7d MicroRNA In Vascular Smooth Muscle Cell Proliferation

Background: MiRNAs(miRNAs)are single-stranded small molecular non-coding RNA containing about 19-25 bases,which are highly conservative in evolution and regulate the gene expression at the transcriptional level. Posttranscriptional regulation by miRNAs is important for many aspects of development, cell differentiation, proliferation and apoptosis, hormone secretion and tumor formation.The let-7 miRNA was originally discovered in the nematode Caenorhabditis elegans, where it regulates cell proliferation and differentiation, but subsequent work has shown that both its equence and its function are highly conserved in mammals. Recent results have now linked decreased let-7 expression to increased tumorigenicity and poor patient prognosis. It is found that let-7 regulates'stemness'by repressing self-renewal and promoting differentiation in both normal development and cancer. The most important possible targets of let-7 are RAS and HMGA2. Recently several reports have indicated the expression level of let-7 miRNA was reduced in human lung cancer, and it was shown to be the anticancer miRNA that represses RAS and/or c-MYC expression at the translational level. There are three kinds of RAS in human, including NRAS, KRAS, HRAS. It is confirmed that let-7 has complementary sites in human RAS 3'UTR which suggests that let-7 has relationship with RAS in mammalian animals. It is possible that let-7 miRNA family negatively regulates RAS to inhibit the tumor proliferation. On the other hand, RAS is also the key factor in the progress of VSMCs proliferation, migration and hypertrophy as well as the development of hypertension.In this study, we mainly focus on the following aspects: First, using microassay, we tried to find the different expression of miRNAs between SHR and WKY VSMCs which have different growth ability, and chose let-7d as the subject of our further study. Secondly, It was predicted that RAS was one promising target of let-7d through bioinformatics and published literature. Thirdly, we did the transfection of let-7d mimic into the SHR/WKY VSMCs and HCASMCs to test whether RAS was regulated by let-7d. And comparison was made between transfected cells and negative control to figure out the impact on the proliferation and cell cycle.The significance of this study is: To detect if let-7d can regulate VSMCs proliferation and test the potential target gene. Accordingly, it provides theoretical gist for studying the vascular physiological function, exploring the pathogenesis of vascular diseases, as well as the screening of new therapeutic targets.Part I Cell culture of SHR/WKY VSMCs and analysis of miRNAs expression between themObject: Analyze the difference of miRNAs expression between SHR and WKY VSMCs.Methods: (1) Rat aortic smooth muscle cells were isolated from the medial layer of thoracic aorta obtained from female SHR and WKY rats (10 weeks old) and cultured in Dulbecco's modified Eagles medium supplemented with 10% fetal bovine serum, penicillin, streptomycin. The cells of passage 3 to 6 were used. The animal experiment was performed in accordance with the US National Institutes of Health guidelines and approved by the animal ethical committee of the Second Military Medical University. HCASMCs were cultured in HCASMC PROLIF (M231/SMGS) with the same treatment mentioned above. (2) Total RNA of SHR-VSMCs and WKY-VSMCs were harvested using RNeasy mini kit according to the manufacturer's instructions. After RNA quantitative measurement on the Nanodrop instrument and confirmation by gel electrophoresis, the samples were labeled using the miRCURY? Hy3?/Hy5? Power labeling kit and hybridized on the miRCURY? LNA Array (v.11.0). Scanning was performed using the Axon GenePix 4000B microarray and the GenePix pro V6.0 was used to read and quantify the raw intensity of the image. Validation of candidate gene(s) from the miRNA microarray expression analysis is performed by using Real-time PCR with TaqMan? MicroRNA Assays. Results: microassay and RT-PCR showed that the miRNAs expression was different between SHR and WKY VSMCs: the main up-regulated miRNAs in SHR VSMCs compared with WKY VSMCs were mir-92b , mir-378 , mir-219-2-3p ; and the down-regulated miRNAs were let-7d, let-7f,mir-98,mir-1,mir-500,let-7a,;let-7i,mir-34b.Conclusion: the miRNAs expression of SHR-VSMCs is different from WKY-VSMCs. The most significant down-regulated miRNA in SHR-VSMCs is let-7d. Given the growth ability is different between SHR and WKY VSMCs, we suppose that let-7d might play an important role in VSMCs proliferation and probably participate the pathology of some vascular diseases. Part II The prediction of let-7d target geneObject: predict the target genes of let-7d through bioinformatics and published literature.Methods: Using the following software such as miRanda, MiRBase, TargetScan, Pictar and BibiServ , as well as published literatures, we chose KRAS as our target gene for the next study.Results: (1) There are several target genes of let-7d after screening, and finally we choose KRAS which is associated with the cardiovascular diseases to be the target of our next research. (2) According to some literatures which have high impact factor, it is found that KRAS is regulated by let-7 in mammalian animals, especially in tumor cells.Conclusion: The prediction of bioinformatics suggests that KRAS is a corresponding target gene of let-7d which probably repress the mRNA translation by combining with the 3'UTR of KRAS.Part III The relationship between let-7d and KRAS in VSMCsObject: Analyze the relationship between the expression of let-7d and KRAS in VSMCs.Methods: SHR-VSMCs, WKY-VSMCs and HCASMCs were transfected by let-7d mimics and negative control. Cells were harvested 72 hours after transfection. Total protein was isolated and western blot was used to detect the relationship between let-7d and KRAS.Results: in vitro, transfection of let-7d in VSMCs can inhibit the expression of KRAS in protein level.Conclusion: over-expression of let-7d in VSMCs can inhibit the expression of KRAS in protein level, which indicates that let-7d may take part in the VSMCs proliferation related diseases by binding to the 3'UTR of KRAS.Part IV The relationship between let-7d and VSMCs proliferationObject: Analyze the relationship between the let-7d and cell proliferation in SHR-VSMCs, WKY-VSMCs and HCASMCs.Methods: (1) Transfectants (SHR-VSMC/NC, SHR-VSMC/let-7d, WKY-VSMC/NC, WKY-VSMC/let-7d, HCASMC/NC and HCASMC/let-7d) were resuspended in 200 ul of cell culture medium and seeded in 96-well microtiter plates at 2.5 x 10^-4 cells/ml, and incubated for 48 h or 72 h. 10μl CCK-8 reagent (Dojindo,Japan) was added to each well 2 h before the end of incubation. Optical density value (OD) of each sample was measured at a wave length of 450 nm on a microplate reader. (2) Transfectants were typsinized, washed with PBS twice, and then fixed with 70% ethanol on ice for 1 h. The fixed cells were spun down, resuspended in PBS at 1×106 cells/ml. After incubation with ribonuclease (RNase A) at a final concentration of 3000 units/ml at 37 degrees Celsius for 30 min, trypsinized cells were filtered through a nylon mesh. The cell suspension was stained with propidium idide (PI) before analysis on a FACSAria flow cytometer.Results: (1) Over-expression of let-7d in VMSCs (SHR/WKY-VSMCs, HCASMCs) can inhibit the cell proliferation. (2) transfection of let-7d in VSMCs can increase the percentage of cells at G1 phase and decrease the percentage of cells at S phase in SHR/WKY-VSMCs and HCASMCs.Conclusion: Over-expression of let-7d in VSMCs can inhibit the cell proliferation and modulate cell cycles.