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OBJECTIVE@#To predict and verify the target gene of miR-200c-3p and evaluate the inhibitory effect of miR-200c-3p on the proliferation of nephroblastoma cells.@*METHODS@#The putative target genes of miR-200c-3p were predicted by bioinformatics approach. Nephroblastoma cell models with miR-200c-3p overexpression or knockdown were established in SK-NEP-1 and G401 cells with corresponding control groups. The expressions of CCNE2 in SK-NEP-1 and G401 cells in different groups were detected by RT-PCR and Western blotting. A luciferase reporter assay was used to determine the targeting relationship between miR-200c-3p and CCNE2. The effects of miR-200c-3p overexpression or knockdown on cell proliferation was detected by cell counting kit-8 (CCK-8) assay and soft agarose assay.@*RESULTS@#CCNE2 was one of the target genes of miR-200c-3p as predicted by bioinformatics methods. Transfection of the two nephroblastoma cell lines with miR-200c-3p mimic resulted in significantly lowered CCNE2 mRNA and protein expressions ( < 0.05). The results of dual-luciferase assay confirmed that miR-200c-3p bound to the 3'UTR of CCNE2. CCK-8 assay and soft agarose assay demonstrated that overexpression of miR-200c-3p significantly inhibited the proliferation of the nephroblastoma cells ( < 0.01), and knocking down miR-200c-3p in the cells produced the opposite effects.@*CONCLUSIONS@#miR-200c-3p overexpression inhibits the proliferation of nephroblastoma cells by down-regulating its target gene CCNE2.
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Objective To investigate the effect of fluoride exposure on expression of miRNA (miR)-200c and its target in human osteoblast Saos-2 cells.Methods Saos-2 cells were cultured in DMEM/F-12 medium and treated with fluoride (sodium fluoride,NaF).There were two groups including:control group (0 mg/L) and fluoride group (4 mg/L).Cells were harvested after 48 hours of culture with fluoride.The expression of miR-200c,the mRNA of alkaline phosphatase (ALP),osteocalcin (BGP),the target phosphatase and tensin homolog deleted on chromosome ten (PTEN) and dual-specific phosphatase 1 (DUSP1) of miR-200c was detected by qRT-PCR.The protein expression of PTEN and DUSP1 was detected by Western blotting.Results The expressions of ALP,BGP mRNA and miR-200c in Saos-2 cells in the fluoride group (23.60 ± 1.87,9.41 ± 0.94,8.61 ± 0.26) were higher than those in the control group (1.00 ± 0.11,1.00 ± 0.07,1.00 ± 0.12).The differences were statistically significant (t =-24.084,-18.388,-8.687,P ＜ 0.05).The mRNA expressions of PTEN and DUSP1 in the fluoride group (0.63 ± 0.02,0.38 ± 0.02) were lower than those in the control group (1.02 ± 0.24,1.02 ± 0.24).The differences were statistically significant (t =3.327,5.454,P ＜ 0.05).The protein expressions of PTEN and DUSP1 in Saos-2 cells in the fluoride group (1.19 ± 0.10,0.83 ± 0.07) were lower than those in the control group (1.81 ± 0.14,1.44 ± 0.25).The differences were statistically significant (t =6.250,4.171,P ＜ 0.05).Conclusion Exposure to fluorine may increase the expression of miR-200c in Saos-2 cells,and fluorine may act on PTEN and DUSP1 through miR-200c,downregulates the mRNA and protein expression levels of PTEN and DUSP1.
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[Objective] To investigate the expression of microRNA-200c (miR-200c) in colorectal carcinomas (CRC),and analyze its role on tumor cell migration and invasion.[Methods] The expression levels of miR-200c in CRC tissues and adjacent normal mucosa were assessed by real-time quantitative RT-PCR (qRT-PCR).miR-200c mimics were transiently transfected into human colorectal cancer cells,and their roles on cell migration and invasion were analyzed by Transwell assay.Cell proliferation was measured using the Cell Counting kit-8.The expression levels of epithelial and mesenchymal markers as well as related transcription factor ZEB1 were detected by Western blotting.[Results] Lower miR-200c expression was found in primary CRC tissues with lymph node metastasis compared to those without lymph node metastasis and adjacent normal mucosa.Transfection of miR-200c mimics suppressed proliferation,and reduced invasion and migration in SW620 cells.Furthermore,up-regulation of miR-200c inhibited ZEB1,and resulted in increased E-cadherin and reduced Vimentin gene expression.[Conclusion] miR-200c was associated with invasive and metastatic behavior of CRC.These effects may be mediated through regulation of epithelial-mesenchymal transition.
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Objective To identify the expression differences of mircoRNA-29 b (miR-29b),microRNA? 24 (miR-24) and microRNA-200 c (miR-200c) in plasma of infants with primary congenital glaucoma (PCG) and normal,and analyze its clinical significance.Methods The expression quantity of microRNAs (miR-29b,miR-24,miR-200c) in plasma of PCG group (16 cases) and normal control group (49 cases) were detected by RT-PCR,and the relationship between their expression differences and severity of disease were analyzed.The diagnostic value of miRNAs for PCG was evaluated by receiver-operating characteristic curve (ROC).Results The expression quantity of miR-29b,miR-24,miR-200c in PCG group (0.31 ±0.19,0.17 ±0.16,0.55 ±0.18,respectively) were significantly lower than those in normal control group (1.18-±0.52,2.86 ±2.65,1.62 ± 0.76,respectively) (all P ＜ 0.05);The expression quantity of miR-24 and miR-29b in plasma was related to the severity of PCG,the more severe the disease,the lower the expression;ROC curve indicated that miR-24 and miR-29b had a higher diagnostic value for PCG disease than miR-200c.Conclusion Free miRNAs in plasma may be used as a new plasma markers for auxiliary diagnosis of PCG.
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Objective To investigate the impact of miR-200c overexpression on colon cancer cell proliferation ability and the related mechanism.Methods MicroRNAs which may combined with the transcription factor AP-2α were screened and forecasted by the bioinformatics database,while its eukaryotic expression plasmids and specific inhibitor were synthesized.Plasmids PEZX-miR-200c,PEZX-NC,pmirGLO-AP-2α3'UTR,pmir-GLO and the specific inhibitors miR-67-inhibtor,miR-200c-inhibitor were transfected in vitro into colon cancer HCT-116 and SW480 cells and the HEK293T cell by Lipofectamine2000.The expression of AP-2α mRNA and protein in colon cancer cells was analyzed by qRT-PCR,Westem blot and immunocytochemical staining.CCK-8 assay and flow cytometry were adopted to observe the effect of miR-200c on colon cancer cells proliferation and apoptosis.Dual-Luciferase assay experiments were performed to observe the relative luciferase activity induced by miR-200c.Results The proliferation activity was significantly decreased in anti-miR-200c/SW480 group,while in PEZX-miR-200c/HCT-116 group,it was higher than that in PEZX-NC/HCT-116 group.The apoptosis ability was significantly increased in anti-miR-200c/SW480 group [(78±0.7) ％ vs (66±1.1) ％,P ＜ 0.05].The expression of AP-2o both in mRNA and protein levels was decreased in PEZX-miR-200c/HCT-116 group,while the protein level was increased in Anti-miR-200c/SW480 group.The relative luciferase activity inhibited by miR-200c was decreased in HEK-293T cells transfected with PEZX-miR-200c and pmir-GLO-AP-2α3' UTR (0.51±0.09 vs 0.98±0.04,P ＜ 0.01).Conclusion MicroRNA-200c could promote cell proliferation ability by targeting transcriptional factor AP-2α in human colorectal cancer cells.
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Objective To investigate relationship between microRNA-200c and ZEB1 in cervical epithelial mesenchymal transition factor. Methods 50 cases diagnosed with cervical cancer whose tumor tissue were taken to be the experiment group.50 cases diagnosed with uterine myoma whose normal tissue were taken to be the control group.ZEB1 and microRNA-200c of uterine tissue were were detected.Results The average level of ZEB1 in the experimental group was significantly higher than that of the control group (P<0.05), and in cervical cancer, lymph node metastasis and the depth of myometrial invasion had statistically significant (P<0.05).In experimental group, microRNA-200c average was lower than control group (P<0.05), and in cervical cancer, different pathological grade group, lymph node metastasis and deep muscular layer infiltration had statistically significant(P<0.05).In experimental group, ZEB1 and microRNA-200c showed negative correlation(r =-0.270 P =0.001).Conclusion The expression level of ZEB1 in cervical cancer is higher, and the expression level of microRNA-200c in cervical cancer is lower, suggesting that the two factors may play an important role in the invasion and metastasis of cervical cancer.The correlation between the two factors suggests that microRNA-200c may be involved in the process of cervical cancer by ZEB1.