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AIM: To investigate the molecular mechanism of lncRNA HOTAIR regulating miR-206 on the proliferation and apoptosis of rheumatoid arthritis synovial cells. METHODS: The synovial tissue from 30 cases of rheumatoid arthritis were collected. Rheumatoid arthritis synovial cells MH7A were cultured. The experiment was divided into si-NC group, si-HOTAIR group, miR-NC group, miR-206 mimic group, si-HOTAIR+NC inhibitor group, si-HOTAIR+miR-206 inhibitor group. Real-time fluorescent quantitative PCR (RT-qPCR) was used to detect the expression levels of HOTAIR and miR-206 in cells. CCK-8 method to detect cell proliferation; flow cytometry to detect cell apoptosis; Western blot to detect cell protein expression of CyclinD1, p21, Bax and Bcl-2; dual luciferase reporter assay to detect HOTAIR and miR-206 targets To combination relationship. RESULTS: Compared with the healthy control group, the expression level of HOTAIR in patients with rheumatoid arthritis was significantly up-regulated, and the expression level of miR-206 was significantly down-regulated (P<0.05). Compared with the si-NC group, the HOTAIR expression level in the si-HOTAIR group was significantly down-regulated, the cell survival rate were significantly down-regulated, and the apoptosis rate were significantly up-regulated (P<0.05). Compared with the miR-NC group, the expression level of miR-206 in the miR-206 mimic group was significantly up-regulated, the cell survival rate were significantly down-regulated, and the apoptosis rate were significantly up-regulated (P<0.05). Compared with the si-HOTAIR + NC inhibitor group, the cell survival rate in the si-HOTAIR+ miR-206 inhibitor group were significantly up-regulated, and the apoptosis rate were significantly decrease (P<0.05). CONCLUSION: Inhibiting the expression of HOTAIR and up-regulating the expression of miR-206 can reduce the proliferation of rheumatoid arthritis synovial cells and promote apoptosis.
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Objective:To investigate the effects and mechanism of miR206 in rat model of denervated muscular atrophy.Methods:From September, 2020 to December, 2020, a total of 40 rats were selected for this study. Denervated muscular atrophy model was established on 16 SPF Sprague-Dawley rats, by removing 1 cm in length of sciatic nerve. The rats were classified into 4 groups according to the sampling time: 0 d, 3 d, 7 d and 14 d(4 rats per group). The other 24 rats were also established into denervated skeletal muscle atrophy models and assigned into 3 groups: denervation add miR206 group, denervation add NC transfection reagent group, and sham-operated group( n=8 in each group). After sampling, the area of cross section of the gastrocnemius muscle and gastrocnemius muscle mass were measured to evaluate muscle atrophy. The mRNA and protein expression of myostatin were determined by real-time PCR and Western blot. Combining with luciferase report to explore the underlying mechanism of miR206, the t-test and oneway ANOVA were used for data analysis used in this study. In one-way ANOVA analysis, if the difference between groups was statistically significant, Bonferroni method would be used for further comparing of all pairs. P<0.05 was considered statistically significant. Results:After excision of a part of sciatic nerve of rat models, gastrocnemius muscle mass of denervation plus miR206 group, denervation plus NC transfection reagent group and sham-operated group were: (0.63±0.04), (0.51±0.02) and (1.05±0.02), respectively. The cross section areas of gastrocnemius muscle in each groups were: (761.30±21.79) μm 2, (640.30±30.31) μm 2 and (1066.00±51.65) μm 2, respectively( P<0.05). Myostatin mRNA expression showed lower in miR206 group than in NC group tested by Western blot, which were(0.57±0.04) in miR206 and (0.81±0.04) in NC group tested by qPCR( P<0.05). The protein expression measured by Western blot test revealed same expression pattern as mRNA expression pattern. The different of relative expression between miR206 group and NC group( P<0.05). Finally, in the mmu-miR206 co-transfected with the MSTN 3'UTR-luciferase sensor group, the relative luciferase activity was measured at 0.26±0.07 and it was significant lower than any other groups( P<0.05). Conclusion:The miR206 can counteract denervated skeletomuscular atrophy through down regulating the myostatin expression. Myostatin is a new discovered target gene of miR206.
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@#[摘 要] 目的：探讨miR-206对雌激素诱导的ER-α36阳性胃癌（gastric cancer, GC）细胞BGC-823增殖和侵袭的影响及其相关机制。方法: 用不同浓度（1、10和100 pmol/L）的雌二醇（estradiol，E2）刺激ER-α36阳性BGC-823细胞后，用qPCR法检测miR-206表达水平，MTT法和Transwell实验分别检测细胞的增殖和侵袭能力，WB法检测细胞中CDK14的表达。将miR-206 mimic、miR-NC、pcDNA-CDK14、pcDNA-vector等转染ER-α36阳性BGC-823细胞，并给予100 pmol/L的E2处理后，用MTT法和Transwell小室法分别检测细胞的增殖和侵袭能力，WB法检测细胞中CDK14的表达。用双荧光素酶报告基因实验验证miR-206与CDK14之间的靶向关系。结果: E2能显著降低ER-α36阳性BGC-823细胞中miR-206表达水平（P<0.05或P<0.01）、增强细胞的增殖和侵袭能力（P<0.05或P<0.01）、上调细胞中CDK14的表达水平（P<0.01）。过表达miR-206能显著降低E2诱导的ER-α36阳性BGC-823细胞的增殖和侵袭能力（均P<0.01）。miR-206通过直接结合CDK14 mRNA的3'-UTR发挥抑制作用，从而负向调节CDK14的表达，进而抑制ER-α阳性BGC-823细胞的增殖和侵袭能力（均P<0.01）。结论: miR-206通过靶向CDK14从而抑制雌激素诱导的ER-α36阳性GC细胞的增殖和侵袭。
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Background: Colorectal cancer (CRC) is currently the third most common cancer type in males and the second most occurring in females. The role of microRNA (miRNA) in the development of colorectal cancer is notfully elucidated. Therefore, understanding the mechanistic interaction between miRNA and their target oncogenes may hold great importance as a possible target for interventional anticancer therapy Aims:To identify miRNAs that are part of the regulating pathway of Monocarboxylate Transporter-4 (MCT4) and Vascular Endothelial Growth Factor (VEGF) oncogenes.Study Design:We used publicly available prediction tools (e.g. TargetScan, MicroCosm, PicTar, and DIANA-microT-CDS) to identify the possible miRNA that target the two oncogenes. Methodology:We used the GeneMania database to visualize the network and verify gene names and remove ambiguity and duplications. Furthermore, we used miRTarBase database to identify experimentally validated targets which we used to further confirm miRNA-oncogene relationships. Finally, we utilized miR-Mfold web-tool to further visualize the circular structures and the simulated miR-1 and miR-206 targeting arrangements.Results:We found two putative miRNA (miR-1 and miR-206) that may downregulate MCT4 coded by SLC16A3gene and VEGF which is coded by VEGF gene. We found relationships between the validated target genes of miR-1 and miR-206 through GeneMania which we extracted from the literature. And we elucidated the proposed structure of these two miRNAs through miR-Mfold web-tool.Conclusion: Our results elucidated a novel regulation pathway in CRC cells and may suggest a potential therapeutic approach for CRC therapy. MiR-1 and miR-206 may help cells go to apoptosis and inhibit the angiogenesis of colorectal cancer cells by down-regulation of MCT4 and VEGF proteins in tumor tissues.
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PURPOSE: This study was undertaken to explore how miR-206 represses osteosarcoma (OS) development. MATERIALS AND METHODS: Expression levels of miR-206, PAX3, and MET mRNA were explored in paired OS and adjacent tissue specimens. A patient-derived OS cell line was established. miR-206 overexpression and knockdown were achieved by lentiviral transduction. PAX3 and MET overexpression were achieved by plasmid transfection. Treatment with hepatocyte growth factor (HGF) was utilized to activate c-Met receptor. Associations between miR-206 and PAX3 or MET mRNA in OS cells were verified by AGO2-RNA immunoprecipitation assay and miRNA pulldown assay. OS cell malignancy was evaluated in vitro by cell proliferation, metastasis, and apoptosis assays. PAX3 and MET gene expression in OS cells was assayed by RT-qPCR and Western blot. Activation of PI3K-AKT and MAPK-ERK in OS cells were assayed by evaluating Akt1 Ser473 phosphorylation and total threonine phosphorylation of Erk1/2, respectively. RESULTS: Expression levels of miR-206 were significantly decreased in OS tissue specimens, compared to adjacent counterparts, and were inversely correlated with expression of PAX3 and MET mRNA. miR-206 directly interacted with PAX3 and MET mRNA in OS cells. miR-206 overexpression significantly reduced PAX3 and MET gene expression in OS cells in vitro, resulting in significant decreases in Akt1 and Erk1/2 activation, cell proliferation, and metastasis, as well as increases in cell apoptosis, while miR-206 knockdown showed the opposite effects. The effects of miR-206 overexpression on OS cells were reversed by PAX3 or MET overexpression, but only partially attenuated by HGF treatment. CONCLUSION: miR-206 reduces OS cell malignancy in vitro by targeting PAX3 and MET gene expression.
Subject(s)
Apoptosis , Blotting, Western , Cell Line , Cell Proliferation , Gene Expression , Hepatocyte Growth Factor , Immunoprecipitation , In Vitro Techniques , MicroRNAs , Neoplasm Metastasis , Osteosarcoma , Phosphorylation , Plasmids , RNA, Messenger , Threonine , TransfectionABSTRACT
Objective To investigate the expression levels of miR-206 in serum and placenta tissues of patients with gestational hypertension and its clinical significance.Methods The expression levels of miR-206 in serum and placental tissue samples from 34 pregnant women with gestational hypertension,26 with mild preeclampsia,27 with severe preeclampsia and 32 normal pregnant women were detected by quantitative real-time PCR (qRT-PCR),and their clinical pathological data were collected for correlation analysis.Results Compared with normal pregnant women,the expression levels of miR-206 in sera and placental tissues of pregnant women with gesta tional hypertension,mild preeclampsia or severe preeclampsia decreased significantly in turn (all P ＜ 0.05),and there was significant difference (F =79.10 and 129.18,respectively,both of P ＜ 0.05).The correlation analysis showed that the expression levels of miR-206 in sera of normal pregnant women and pregnant women with gestational hypertension,mild preeclampsia or severe preeclampsia were positively related to that in placental tissues (r =0.402,0.512,0.416 and 0.397,respectively,all P ＜ 0.05).In addition,the expression levels of miR-206 in serum and placental tissue of pregnant women with gestational hypertension were negatively related to systolic blood pressure,diastolic blood pressure and urinary protein level (P ＜ 0.05),but positively to gestational age,albumin level and neonatal body weight (P ＜ 0.05).Conclusion The expression of miR-206 in serum and placental tissue of pregnant women with gestational hypertension decreases significantly,which may be involved in the development and progression of gestational hypertension.
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Objective To investigate the expression pattern of skeletal muscle specific miR-206,myogenesis related myoD which change with time in dcnervated muscle atrophy rats.Methods From June,2015 to January,2016,40 SPF sprague-dawley rats were equally classified into 5 groups randomly according to standard settled before,5 groups were separately defined as denervated 0d group,denervated 1d group,denervated 7d group,denervated 14d group,and denervated 28d group.Each group contained 8 rats.The rats atrophy models were established by cutting sciatic never on left side.According to the different denervated time,the gastrocnemii on both sides were obtained under anesthesia,respectively.The wet weight ratio of two compared gastrocnemii were measured,and the gastrocnemii transection was observed by HE stain,measured the expression of myoD protein by western blot,obtained the expression of miR-206,myoD mRNA by qPCR.Results According to our study on rats denervated atrophy models,the wet ratio of compared gastrocnemius would decrease rapidly,by HE stain,decease of cross sectional area in muscle fiber was observed as well as degeneration.Collagen fibers hyperplasia appeared and increased with time change.Wet ratio and transaction aera ratio of group Od,1d,7d,14d,28d were 0.99±0.04,0.92±0.07,0.68±0.11,0.39±0.06,0.27±0.07 and 0.99±0.02,0.96±0.04,0.51±0.09,0.34±0.08,0.23±０.03 respectively,difference between experimental groups and control group were statistically significant (P＜ 0.05),the differences between each experimental groups were also statistically significant (P＜ 0.05).After qPCR test of miR-206,myoD mRNA expression,it was found that their expression patterns were similar,miR-206,myoD mRNA increased at first and would reach the expression peak at the 7 th day,after that their contents decreased but still higher at the 14th day when compared with that at the 1 st day.Their expression of group 0d,1 d,7d,14d,28d were 0.24±0.06,0.34±0.04,0.68±0.04,0.49± 0.07,0.25±0.03 and 0.41 ±0.06,0.49±0.09,0.93±0.06,0.66±0.03,0.39±0.04,respectively.All experimental groups were statistically significant different when compared with 0d group except 1d group (P＜ 0.05),the differences between each experimental groups were also statistically significant(P＜ 0.05).The protein expression of myoD was also measured by western blot test,which showed nearly the same expression pattern as the mRNA expression pattern.After injury,the protein expression increased and reached the expression peak at the 7th day.The relative expression of myoD of group 0d,1d,7d,14d,28d measured by grey ratio were 1.03±0.05,1.06±0.06,1.42±0.10,0.66±0.13,0.24±0.07,respectively.The difference between experimental groups and control group were statistically significant (P ＜ 0.05),the differences between each experimental groups were statistically significant (P ＜ 0.05) as well.Conclusion The degree of muscle denervation atrophy was related to the denervated duration in rats.The expression regulation of miR-206 and myoD in gastrocnemius was similar during the muscle denervation atrophy,which suggesting having internal relationship between miR-206 and myoD.
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This study was designed to explore the effect and mechanism of miR-206/miR-613 on the expression of OATP1B1 gene. Bioinformatic analysis was used to predict the potential miRNAs target sites in 3'-untranslated region (3'-UTR) of OATP1B1 mRNA. The expression level of miR-206/miR-613 and OATP1B1 mRNA and protein was determined with RT-qPCR and Western blot, respectively. Luciferase assay was used to explore the exact mechanism of the effect of miR-206/miR-613 on the expression of OATP1B1 mRNA and protein. The results showed that the seed sequences of miR-206/miR-613 has perfect complementary with 3'-UTR of OATP1B1 mRNA in terms of sequence specificity. The secondary structure between miR-206/miR-613 and 3'-UTR of OATP1B1 mRNA was rather stable. The OATP1B1 protein level was down-regulated by 24.7%, 38.8% by overexpression of miR-206/miR-613. The expression was up-regulated by 25%, 38.2% by inhibition of miR-206/miR-613. However, overexpression or inhibition of miR-206/miR-613 had no effect on the expression of OATP1B1 mRNA. The luciferase activity of pMIR/OATP1B1-WT luciferase reporter gene was decreased by 35% and 30% through overexpression of miR-206/miR-613. The expression was increased by 33.1% and 32.5% through inhibition of miR-206/miR-613. When the binding sites in the 3'-UTR of OATP1B1 mRNA complementary with miR-206/miR-613 was mutated, overexpression or inhibition of miR-206/miR-613 had no effect on the luciferase activity. Collectively, miR-206/miR-613 post-transcriptionally regulates the expression of OATP1B1 protein by directly targeting the 3'-UTR of OATP1B1 mRNA.
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Renal cell cancer (RCC) remains one of the most lethal types of cancer in adults. MicroRNAs (miRNAs) play key roles in the pathogenesis of RCC. The role of miR-206 in RCC has not been fully understood. The purpose of this study was to examine the role of miR-206 in the regulation of proliferation and metastasis of RCC and the possible mechanism. miR-206 expression was detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) in RCC cell lines (786-O and OS-RC-2 cells) and clinical samples. MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] method, colony formation and transwell assay were used to detect the tumor-suppressing ability of miR-206 in RCC. Luciferase assay was performed to verify the precise target of miR-206. The results showed that the expression of miR-206 was significantly down-regulated in RCC tissues and cells. The expression level of cyclin G-associated kinase (GAK), a master regulator of tumor proliferation and metastasis, was up-regulated with the decrease in miR-206 in RCC tissues as well as RCC cell lines. In addition, the miR-206 inhibitor promoted the proliferation, migration and invasion of 786-O and OS-RC-2 cells. Bioinformatics combined with luciferase and Western blot assays revealed that miR-206 inhibited the expression of GAK. Moreover, miR-206 regulates RCC cell growth partly through targeting GAK. Our study indicated that miR-206 functions as a tumor suppressor in regulating the proliferation, migration and invasion of RCC by directly targeting GAK, and it holds promises as a potential therapeutic target for RCC.
Subject(s)
Adult , Aged , Female , Humans , Male , Middle Aged , Carcinoma, Renal Cell , Genetics , Metabolism , Pathology , Cell Line, Tumor , Cell Movement , Cell Proliferation , Gene Expression Regulation, Neoplastic , Intracellular Signaling Peptides and Proteins , Genetics , Metabolism , Kidney Neoplasms , Genetics , Metabolism , Pathology , MicroRNAs , Genetics , Protein Serine-Threonine Kinases , Genetics , MetabolismABSTRACT
Objective To investigate the effects of up?regulated miR?206/miR?1 on the proliferation of breast cancer stem cells and the effect mech?anism. Methods Breast cancer stem cells(BCSCs)were isolated from breast cancer cell line MCF?7 by fluorescence?activated cell sorting. Cells in the experiment were divided into the blank control group,the negative control group,the miR?206 group and the miR?1 group. The BCSCs were transfected by negative control mimic,hsa?miR?206mimic and hsa?miR?1mimic in all groups except the blank control group. MiR?206and miR?1 expression levels as well as the transcription factor EVI?1 gene were detected by real time PCR. The expression levels of the transcription factor EVI?1 protein were detected by Western blot. MTT method was used to detect the effects of miR?206 and miR?1 on the proliferation of BCSCs. Results The BCSCs(CD44+/CD24-/low cells)isolated from MCF?7 cell lines were successfully cultured in serum?free medium for subsequent studies. After transfection of hsa?miR?206mimic and hsa?miR?1mimic for 48 hours,miR?206and miR?1relative expression levels increased. EVI?1mRNA ex?pression levels significantly decreased. The results of Western blot and MTT showed that up?regulated expression levels of miR?206 and miR?1 could significantly reduce the expression of EVI?1 protein and inhibited the proliferation of BCSCs. The differences in levels of miR?206,miR?1 and EVI?1 protein were statistically significant(P<0.05). Conclusion Up?regulated miR?206 and miR?1 expression can inhibit the proliferation ability of BCSCs,which may be related to the down?regulation of EVI?1.