| BackgroudHuman uterine leiomyoma(LYO) are the most common benign neoplasms in reproductive-aged women. The leiomyoma is composed of the proliferation of smooth muscle cells and the average incidence rate of it in reproductive women is about 77%, while the rate of 45-years-old women is more than 60%. It has the ascending rate in incidence rate year by year. The clinical symptoms of LYO are irregular bleeding, amenorrhoea and prolonged bleeding, pelvic pain, recurrent pregnancy loss, and infertility. Hysterectomy is the only effective treatment for LYO. LYO is the leading indication for hysterectomy and is also a serious harm to women’s health. However, the exact mechanisms that contribute to LYO remain unclear now. Environmental factors, estrogen and progesterone, growth factors and changes of extracellular matrix also have been identified to be involved in the initiation and development of LYO.Micro RNAs are a class of small noncoding, single-stranded RNAs, ranging from19 to 24 nucleotides, and serve as regulators of targeted genes expression at the post-transcriptional level. These noncoding RNAs have been implicated as mediators in several physiologic and pathologic processes including inflammation,cell proliferation, differentiation, apoptosis, invasion, migration, drug resistance andangiogenesis. Micro RNA can degrade the m RNA of target gene or inhibit translation of target gene resulting in target gene silencing. This is a fine adjustment of the protein expression of post-transcriptional regulation mechanism which is widely present in cells. Mi RNA involved in the development and progression of various tumors. It is closely related to prognosis, and it is a good biomarker for early diagnosis of tumors. The projects of LYO researches are still few now. Several papers report that there are aberrantly expressed miRNA in LYO and paired myometrium. Targeting to find the aberrantly expressed miRNA in LYO and paired myometrium, then try to explore the mechanisms of the aberrantly expressed miRNA,will lay a good foundation for future research in LYO and miRNA. At present, the mainly research projects of miRNA are Real time-PCR, micro array and the second generation sequencing. Mi RNA array is the most popular and the classic method for screening miRNA of uterine LYO.Part 1: screening of differentially expressed micro RNA in uterine leiomyoma ObjectiveIn this study,we used microarray to screen for different miRNA expression in uterine fibroids and paired uterine flesh layer samples, using chip combined with biological information science to analyze and predict the expression of different miRNA target genes and their biological function.MethodWe collected 10 cases of uterine fibroids and paired tissue of women of childbearing age who took hysterectomy in the Third Affiliated Hospital of Zhengzhou University from November 2012 to January 2013. Using the Agilent human miRNA 8*60K V19.0 chip to screen 3 cases of differential expression of miRNAs in uterine fibroids and paired tissue,the chip results with supportingsoftware of Agilent genespring data preprocessing and variance analysis and cluster analysis was carried out using clustering software Me V. The differential expression of hsa-miR-15b-5p, hsa-miR-21-5p, hsa-miR-34a-3p, hsa-miR-34a-5p were verified by PCR Real-time in 10 cases of uterine leiomyoma and paired muscle tissue. The Mi RDB, Tar Base, targetscan, rna22, Target Miner database of the four miRNA target gene prediction, and the gene ontology Gene Ontology(GO) and Kyoto Encyclopedia of genes and genomes( KEGG) pathway analysis further analysis of target genes may be involved in the biological process and related signaling pathways.Result1.There are 45 differential expression of miRNA in human uterine leiomyoma and normal human uterine tissue.The expression of 19 miRNA in the uterine leiomyoma was increased, and the expression of 26 miRNA decreased in the uterine leiomyoma.2.Verifying miR-15b-5p, miR-34a-5p, miR-34a-3p, miR-21-5p in the uterine leiomyoma tissue showed that expressions of them were significantly increased, and the results of the chip is basically the same.(P <0.0001).3.The target genes of miRNA in the uterine leiomyoma and muscular layer were mainly involved in 30 biological processes, such as morphological development, cell growth and development.4.Differential expression of the miRNA and target genes in Uterine fibroids and muscle layer mainly involved in 25 signal pathway including cancer, MAPK and apoptosis.ConclusionThere were significant differences in the expression of miRNA in the uterine leiomyoma and normal uterine muscle tissuePart 2: the effect of miR-15b-5p over expression and loss of expression on the biological characteristics of uterine leiomyoma cells ObjectiveAccording to the results of the first part of the study, The expression of miR-15b-5p in uterine leiomyoma tissue was significantly increased,and by gene transfection, the miR-15b-5p in various types of leiomyoma cells and myometrial cells over expression and low expression. To observe the effect of low expression of miR-15b-5p on the proliferation of uterine leiomyoma cells, to provide the basis for further research on the biological function of miR-15b-5p and the long-term exploration of its downstream target genes.MethodWe collected 20 cases of uterine fibroids and paired tissue of women of childbearing age who took hysterectomy in the center of reproductive endocrinology at Massachusetts General Hospital from August 2013 to February 2014, and the partial digestion and primary cells were isolated and cultured as uterine leiomyoma cells(p LYO cells) and the uterine muscle cells(p MYO cells). At the same time we cultured the human uterine leiomyoma cell line(cp LYO cells) and myometrial cells(cp MYO cells) in vitro environment.Using Real-time PCR to detect the level of the background expression of miR-15b-5p in different cell. Liposomes in various types of cells were transiently transfected miR-15b-5p simulant(mimic) and inhibitor(inhibitor) and its positive and negative control, by real time PCR was performed to validate the the expression of miR-15b-5p in each group, established high or low expression pattern of miR-15b-5p.Using methylthiazolyldiphenyl-tetrazolium to detect the effect of miR-15b-5p on the proliferation of primary uterine leiomyoma cells.Result1.Background expression of Mi R-15b-5p in various types of cells: the expression level of miR-15b-5p in primary uterine leiomyoma cells was significantly higher than that of primary uterine muscle cell layer; the expression level of miR-15b-5p in uterine flesh tumour cell lines was significantly higher than that of uterine flesh layer cell lines(paired t-test, P < 0.001).2.Mi R-15b-5p mimic was transfected into each cell type for 24 and 48 h, the expression of miR-15b-5p in various types of cells increased significantly.(paired t test, P<0.001).3.Mi R-15b-5p inhibitor was transfected into various types of cells for 24 and48 h, and the expression of miR-15b-5p was significantly decreased in each type of cell.(paired t test, P<0.001).4. In primary uterine leiomyoma cells and uterine leiomyoma cell lines, inhibitor miR-15b-5p group was transfected with different concentrations and compared with the negative control group, the rate of cell proliferation was significantly reduced.(paired t test, P<0.05).ConclusionMi R-15b-5p can promote the proliferation of uterine leiomyoma cellsPart 3: the study of Mi R-15b-5p regulates the biological characteristics of uterine leiomyoma cells downstream target proteins ObjectiveTo predict and verify the downstream target genes and target protein of miR-15b-5p in human uterine leiomyoma cells, and to detect the differential expression of the downstream target protein of miR-15b-5p in uterine leiomyoma andmyometrium, further revealed miR-15b-5p molecular mechanisms which are involved in the pathogenesis of uterine leiomyoma.MethodUsing bioinformatics database Mi RDB, Tar Base, Target Scan, RNA22,Target Miner to predict the target gene of miR-15b-5p,and transfected mimic of miR-15b-5p.Real time PCR was performed to validate the predictions of miR-15b-5p target gene Reck(Reversion-inducing cysteine rich protein with Kazal motifs) in the expression level of m RNA in each cell type; Transfection miR15b-5p inhibitor and the negative control,and using immunoblotting test(Western Blot) verify the expression level of RECK protein in various types of cells;Using cell immune fluorescence(Immunoflunence. If) to detect the expression changes of RECK protein in primary myoma and myometrial cells. Then Western Blot and staining Immunohistochemical(IHC) were used to detect the expression of RECK in leiomyoma and muscular layer and the cell location of RECK protein.Result1. Mimic of miR-15b-5p transfected cells for 24 hours, compared with the blank control group, the expression of m RNA RECK in the cell line and the muscle cell line was decreased(paired t test, P<0.05, P<0.001). Mimic miR-15b-5p transfected cells for 48 hours, compared with the blank control group, expression of m RNA RECK in uterine fibroids cell lines, uterine muscle cell line and the primary generation of uterine leiomyoma cells was decreased.2.Mimic and inhibitor of miR-15b-5p and their negative control cells were transfected with various types of cells for 48 h by 10 n M. Compared with its nagetive control group, in uterine flesh tumour cell lines and primary uterine leiomyoma cell line groups transfected with the mimic miR-15b-5p, the expression of Reck protein were significantly decreased,while in group transfected with miR-15b-5p inhibitor,expression of RECK protein increased significantly, the difference has statistical significance(paired t-test, P<0.001). Compared with its negative control group,inuterine muscle layer cell line transfected miR-15b-5p mimic group, expression of Reck protein were significantly decreased(paired t-test, P<0.001); In transfection Inhibitor group, expression of RECK protein was significantly increased, the difference is statistical significance(paired t-test, P<0.01). The expression of RECK mimic protein was significantly decreased in the primary cultured cells of the uterus.The expression of RECK protein in the transfected inhibitor group was significantly higher, and the difference was statistically significant(paired t test, P<0.05).3.Mi R-15b-5p Inhibitor and its negative control were transfected with Primary uterine leiomyoma cells and primary muscle cells by 40 n M respectively. The expression of RECK protein in transfected group was increased.4.The expression of RECK protein in the uterine muscle layer was significantly higher than that in the paired uterine leiomyoma tissues(paired t test, P<0.001). The expression of RECK protein in uterine muscle cells was significantly higher than that in paired uterine leiomyoma cells(paired t test, P <0.05).5.The expression of RECK protein in the cytoplasm located in the uterine muscle cells, in matched myometrium tissue,expression of RECK protein was significantly increased(non paired t test, P<0.05)ConclusionMi R-15b-5p regulates the pathogenesis of uterine leiomyoma by using RECK as the target gene... |
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