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Aim of the Study: The purpose of this study was to identify specific circulating microRNAs (miRNAs) and investigate expression level of their target genes for evaluation of pathogenesis of epithelial ovarian cancer (EOC). Materials and Methods: In this study, we have studied on EOC patients' serum and whole blood, healthy control (HC) serum, and whole blood samples. Sixteen serum samples were collected to compare miRNA expression analysis through microarray. According to microarray results, one of the dysregulated miRNA in serum, hsa-let-7d-3p, was validated by RT-qPCR for discriminate two groups. The hsa-let-7d-3p is one of the tumor suppressive let-7d family members. Let-7d is downregulated in numerous types of cancer, including ovarian cancer and directly targets various oncogenes. We analyzed the let-7d targets, which are High Mobility Group A2 (HMGA2) and (Kirsten Rat Sarcoma Viral Oncogene Homolog), as the oncogenes that are associated with EOC. The relation between target genes of hsa-let-7d-3p and EOC has been examined by Pathway Studio. Twenty serum and whole blood samples collected to analyze expression level of target genes were analyzed by real-time PCR. Results: 31 significantly dysregulated miRNAs were identified by microarray in serum. Hsa-let-7d-3p has been selected for the validation, according to P-value and dysregulated level. RT-qPCR results showed that hsa-let-7d-3p could discriminate EOC patients from HC (P = 0.0484, AUC = 0.7). Furthermore, we identified hsa-let-7d-3p's target genes (HMGA2, KRAS) by bioinformatic analysis. The expression level of genes could discriminate patients with EOC from HC, with a power area under the ROC curves (AUC) of 62 and 64.2, respectively. Conclusion: HMGA2 and KRAS could be translationally downregulated by the hsa-let-7d-3p, and the loss of hsa-let-7d-3p expression led to the progression of EOC related to the tumorigenesis, invasion, and metastasis
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Objective To investigate the expression level of let-7d in osteosarcoma tissues, and to explore the effects of let-7d and its target gene on proliferation, migration and invasion of human osteosarcoma cell line U2OS cells. Methods Tumor and adjacent tumor tissues of 25 patients with osteosarcoma undergoing exairesis in our department were collected from 2010 to 2015. The distance from adjacent tumor tissues to tumor margin was greater than 5 cm. The expressions of let-7d were detected by qPCR in the tumor and adjacent tumor tissues. A U2OS cell model stably overexpressing let-7d was constructed, and the expression of let-7d was confirmed by qPCR. The U2OS cells transfected with pCDH empty virus vector were used as control cells. CCK-8 assay, scratch assay and Transwell assay were used to determine the proliferation, migration and invasion of the cell after transfection, respectively. A downstream target gene of let-7d was identified based on microRNA target gene prediction software and luciferase activity assay, and the expression of the target gene was detected in U2OS cells overexpressing let-7d. Effects of the target gene on the cell proliferation, migration and invasion were analyzed by small interfering RNA technology. Results The expression level of let-7d was significantly lower in the osteosarcoma tissues than that in the adjacent tumor tissues (P<0.01). The expression level of let-7d was significantly lower in U2OS cells compared with human osteoblast cell line hFOB1.19 cells (P<0.01). Compared with the control group, overexpression of let-7d significantly reduced the proliferation, migration and invasion of U2OS cells (all P<0.05). MicroRNA target gene prediction software and luciferase activity assay showed that Rhotekin (RTKN) gene was a direct target gene of let-7d. Compared with the control group, overexpression of let-7d significantly reduced mRNA expression level of RTKN in the U2OS cells (P<0.01). Down-regulation of RTKN significantly inhibited the proliferation, migration and invasion of U2OS cells (all P<0.05). Conclusion Let-7d inhibits proliferation, migration and invasion of osteosarcoma cells by targeting RTKN, which indicates let-7d may be a novel candidate biological therapeutic target for osteosarcoma.
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AIM: To investigate the phenomenon that miR-let-7d regulates the proliferation and invasion abilities of the lung cancer cells through nuclear receptor peroxisome proliferator-activated receptors γ (PPARγ).METHODS: The relation between PPARγ and microRNA was analyzed by bioinformatics.The plasmid reporter assay was used to verify that PPARγ was the target of miR-let-7d.The lung cancer cell line with low expression of PPARγ was selected from different lung cancer cell lines by Western blot.The regulatory role of miR-let-7d in the lung cancer cells was determined by dual luciferase labeling and Western blot.The effect of miR-let-7d on the proliferation ability of lung cancer cells was detected by colony formation assay, the effect of miR-let-7d on the invasive ability of lung cancer cells was detected by Transwell invasion assay.RESULTS: The results of bioinformatic analysis showed that miR-let-7d regulated the expression of PPARγ, and the 3'UTR of PPARγ contained 2 functional miR-let-7d binding sites, indicating that PPARγ is a direct target of miR-let-7d.miR-let-7d was able to directly regulate the expression of PPARγ at mRNA and protein levels.Transfection of miR-let-7d inhibitor promoted the proliferation and invasion abilities of lung cancer cells by increasing the expression of PPARγ.CONCLUSION: miR-let-7d increases the expression of tumor suppressor PPARγ to inhibit the proliferation and invasive abilities of lung cancer cells.
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Aim To examine the role and uderlying mechanisms of Lin28 /let-7d axis in the proliferation of lung fibrobalsts and fibroblasts-into-myfibroblasts tran-sition,and provide novel strategy for the treatment of idiopathic pulmonary fibrosis (IPF).Methods We induced experimental lung fibrosis in mice by intratra-cheally injection of bleomycin (BLM).Ang Ⅱ and TGF-β1 were used to induce fibrogenesis in cultured MRC-5 cells;qRT-PCR and Western blot were applied to determine the changes of Lin28B,collagen 1 α1 and collagen 3α1 ;MTT assay,Edu satining and immun-ofluoresence were used to examine the cell viability, proliferation and fibroblasts-into-myofibroblasts transi-tion in MRC-5 cells.Results Lin28B was increased in the lung of mice with experimental lung fibrosis and in MRC-5 cells treated with AngⅡ or TGF-β1 .Moreo-ver,Lin28B enhanced collagen deposition via inhibi-ting expression of let-7d,which maybe contribute to the progression of IPF.In addition,further studies showed that Lin28B promoted proliferation and fibro-blasts-into-myofibroblasts in MRC-5 cells.Conclusion Lin28B /let-7d axis contributes to fibrogenesis via promotes fibroblasts-into-myofibroblasts transition, which may provide novel approaches for lung fibrosis treatment.
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Objective To analyse the expression and clinical significance of 12 kinds of microRNAs (miR) in patients with ovarian cancer using public gene expression databases. Methods The microRNA expression data were screened in dataset GSE14407 and TCGA database, then 12 kinds of microRNAs were obtained including miR-10B, miR-1244, miR-622, miR-21, miR-503, Let-7D, miR-155, miR-30C, miR-17, miR-101-1, miR-186 and miR-770. The expression data of these 12 kinds of microRNAs were compared and identified to find the differential ones between normal tissue and tumors. Data of 505 ovary cancer patients were divided into two groups by age, tumor grade, clinical stage, disease location, tumor residual and microRNA expression. Kaplan-Meier survival analysis and Cox multivariate analysis were used to compare the overall survival of ovary cancer patients between two groups. Results Compared with ovary cancer, the expression levels of Let-7D and miR-101-1 were higher, but the expression levels of miR-155 and miR-770 were decreased, in adjacent tissue of ovary tumor (P<0.05). The Kaplan-Meier survival analysis result showed that lower survival rates were found in patients with age≥59 years, clinical stage (Ⅲ+Ⅳ) and lower Let-7D expression (P<0.05). The multivariate Cox regression analysis showed that the decreased expression level of Let-7D was the independent risk factor for the prognosis of ovarian cancer. Conclusion The expression of Let-7D is correlated with the prognosis of ovarian cancer, which is the independent biomarker to predict prognosis of ovarian cancer.