Effects of circular RNA-SEC31A on the invasion and migration of pancreatic cancer cells and molecular mechanism / 中华胰腺病杂志

Objective:To determine the expression of circular RNA-SEC31A(circSEC31A) in pancreatic cancer and investigate the effects on the invasion and migration of pancreatic cancer cells and the underlying molecular mechanism.Methods:Differentially expressed circRNAs between pancreatic cancer cells (BXPC-3, PANC1, CaPan-2, SW1990) and human normal pancreatic cells (HPDE) were identified by qRT-PCR. Then, two cell lines with high circSEC31A expression were selected to conduct next experiments. According to the sequence of the back-splicing site in circSEC31A, siRNAs for downregulation of circSEC31A were designed and transfected by liposome to silence circSEC31A in pancreatic cancer cells, and grouped as followed siR-circSEC31A#1 and siR-circSEC31A#2. Meanwhile, siR-NC group transfected with non-specific siRNA served as control. Transwell assays and wound healing assays were operated to evaluate the functional role of circSEC31A on the invasion and migration of pancreatic cancer cells. RNA Pull-down assay with circSEC31A probe and oligo control probe was used to screen the miRNA combining with circSEC31A and the effects of miRNA on cell invasion and migration of pancreatic cancer cells were validated. The effect of miR-200c-3p and circSEC31A silencing on the expression of PDK1 mRNA was identified by qRT-PCR. The protein expression of PDK1, downstream Akt and p-Akt after circSEC31A silencing was verified by Western blotting assays.Results:The relative expression level of circSEC31A in HPDE (1.000±0.120) was obviously lower than that in BXPC-3 (1.920±0.130), SW1990 (2.93±0.528), PANC1 (4.557±0.692) and CaPan-2 (5.247±0.194), and all the differences were statistically significant ( P<0.001). Compared with the PANC1 siR-NC group (1301.3±94.6) and CaPan-2 siR-NC group (1835.0±70.1) per 100 high power field, transwell assays showed that the numbers of invasive pancreatic cancer cells was highly decreased in PANC1 siR-circSEC31A#1 group (727.3±92.9), siR-circSEC31A#2 group (792.0±18.1), CaPan-2 siR-circSEC31A#1 group (718.0±90.6), siR-circSEC31A#2 group (692.7±84.8). Wound healing assays showed that silencing circSEC31A decreased the wound healing rate of pancreatic cancer cells in PANC1 siR-circSEC31A#1 group (20.667±3.215)%, siR-circSEC31A#2 group (20.000±4.583)%, CaPan-2 siR-circSEC31A#1 group (28.000±8.185)%, siR-circSEC31A#2 group (29.667±5.686)%, compared with the PANC1 siR-NC group (55.000±4.359)% and CaPan-2 siR-NC group (69.000±3.606)%. RNA Pull-down assays showed that compared with PANC1 oligo probe group (1.000±0.091) and CaPan-2 oligo probe group (1.000±0.153), miR-200c-3p was significantly enriched in the PANC1 circSEC31A probe group (2.237±0.175) and CaPan-2 circSEC31A probe group (2.166±0.156). Compared with PANC1 siR-NC group (939.3±57.0) and CaPan-2 siR-NC group (786.7±51.5) per 100 high power field, the numbers of invasive pancreatic cancer cells were up-regulated in PANC1 siR-miR-200c-3p group (1206.0±99.1) and CaPan-2 siR-miR-200c-3p group (1838.0±105.7), while the low numbers of invasive pancreatic cancer cells were observed in PANC1 siR-miR-200c-3p+ siR-circSEC31A group (932.7±116.4) and CaPan-2 siR-miR-200c-3p+ siR-circSEC31A group (785.3±58.8). Compared with PANC1 siR-NC group (1.000±0.103) and CaPan-2 siR-NC group (1.000±0.107), the relative expression of PDK1 mRNA in PANC1 siR-miR-200c-3p group (1.898±0.159) and CaPan-2 siR-miR-200c-3p group (2.102±0.337) was upregulated. Furthermore, the expression of PDK1 mRNA was decreased in the siR-miR-200c-3p+ siR-circSEC31A group (0.980±0.070, 1.015±0.079). Western blot assays showed that the expression of PDK1 protein in PANC1 siR-NC group, siR-circSEC31A#1 group, siR-circSEC31A#2 group was 0.767±0.086, 0.281±0.191, 0.333±0.062 and in CaPan-2 siR-NC group, siR-circSEC31A#1 group, siR-circSEC31A#2 group was 0.712±0.038, 0.353±0.061, 0.308±0.018. The expression of p-Akt protein in PANC1 siR-NC group and siR-circSEC31A group was 0.741±0.050, 0.114±0.027, 0.139±0.041. In addition, p-Akt protein expression in CaPan-2 siR-NC group and siR-circSEC31A group was 0.823±0.052, 0.141±0.045, 0.280±0.089. PDK1 and p Akt expression in siR circSEC31A group was obviously lower than those in sir NC group. All the differences between either groups above were statistically significant ( P<0.05). Conclusions:circSEC31A is upregulated in pancreatic cancer cells, which facilitates the invasion and metastasis of pancreatic cancer cells via miR-200c-3p/PDK1/Akt signaling pathway, supporting that circSEC31A may function as a new diagnostic and therapeutic target for pancreatic cancer patients.

The effect of lncRNA ADPGK-AS1 on the proliferation and apoptosis of retinoblastoma cells by targeting miR-200b-5p / 中华肿瘤杂志

Objective: To explore the effect of lncRNA ADPGK-AS1 on the proliferation and apoptosis of retinoblastoma cells and its possible mechanism. Methods: The tumor tissues of 31 patients with retinoblastoma admitted to Henan Provincial Eye Hospital from February to June 2020 and their corresponding normal tissues adjacent to the cancer were collected. The expression levels of lncRNA ADPGK-AS1 and miR-200b-5p in retinoblastoma tissues and normal adjacent tissues were detected by real-time fluorescence quantitative polymerase chain reaction (qRT-PCR). Human retinal epithelial cell ARPE-19, human retinoblastoma cell Y-79 and WERI-Rb-1 were cultured in vitro. The expression levels of lncRNA ADPGK-AS1 and miR-200b-5p were detected by qRT-PCR. Y-79 cells were randomly divided into si-con group, si-lncRNA ADPGK-AS1 group, miR con group, miR-200b-5p group, si-lncRNA ADPGK-AS1+ anti-miR con group, and si-lncRNA ADPGK-AS1+ anti-miR-200b-5p group. The proliferation, cloning and apoptosis of cells in each group were detected by tetramethylazol blue method, plate cloning test and flow cytometry, respectively. The targeting relationship between lncRNA ADPGK-AS1 and miR-200b-5p was detected by double luciferase report test, and the expression level of cleaved-caspase-3 protein was detected by western blot. Results: Compared with the adjacent tissues, the expression of lncRNA ADPGK-AS1 in retinoblastoma tissues was increased (P<0.05), while the expression of miR-200b-5p was decreased (P<0.05). Compared with ARPE-19 cells, the expression of lncRNA ADPGK-AS1 in Y-79 and WERI-Rb-1 cells was increased (P<0.05), while the expression of miR-200b-5p was decreased (P<0.05). Compared with the si-con group, the cell viability of the si-lncRNA ADPGK-AS1 group was reduced (1.06±0.09 vs 0.53±0.05, P<0.05), the number of cell clone formation was reduced (114.00±8.03 vs 57.00±4.13, P<0.05), while the apoptosis rate [(7.93±0.68)% vs (25.43±1.94)%] and the protein level of cleaved-caspase-3 were increased (P<0.05). Compared with the miR-con group, the cell viability of the miR-200b-5p group was decreased (1.05±0.08 vs 0.57±0.05, P<0.05), the number of cell clone formation was decreased (118.00±10.02 vs 64.00±5.13, P<0.05), while the apoptosis rate [(7.89±0.71)% vs (23.15±1.62)%] and the protein level of cleaved-caspase-3 were increased (P<0.05). lncRNA ADPGK-AS1 could target the expression of miR-200b-5p. Compared with the si-lncRNA ADPGK-AS1+ anti-miR-con group, cell viability of the si-lncRNA ADPGK-AS1+ anti-miR-200b-5p group was increased (0.53±0.04 vs 1.25±0.10, P<0.05), and the number of cell clones was increased (54.00±4.39 vs 125.00±10.03, P<0.05), while the rate of apoptosis [(25.38±1.53)% vs (9.76±0.71)%] and the protein level of cleaved-caspase-3 were decreased (P<0.05). Conclusion: Interfering with the expression of lncRNA ADPGK-AS1 could inhibit the proliferation and clone formation and induce apoptosis of retinoblastoma cells by targeting the expression of miR-200b-5p.

Research progress of miR-200 family and obesity-related hypertension / 公共卫生与预防医学

Obesity-related hypertension is a common hypertension as well as a common chronic disease with wide distribution and great harm to human health. In recent years, this disease has become one of the hot issues of public health due to the significant increase in prevalence. The pathogenesis and pathway of obesity-related hypertension are not yet clear, and the research on its pathogenesis has received extensive attention. Studies have shown that they are regulated in most biological processes, including differentiation, proliferation, migration, and apoptosis. The miR-200 family is a group of miRNAs, which have been suggested to play a crucial role in obesity-related hypertension and glucolipid metabolism dysfunction in recent years. This paper reviews relevant research results, suggesting that the expression level of miR-200 family in obese patients with hypertension is higher than that in healthy people, which regulates the occurrence and development of hypertension through mediating oxidative stress response and GATA expression level. This review reveals the relationship between miR-200 family and obesity-related hypertension, which offers new clues to explore potential therapeutic targets for obesity-related hypertension.

Sulforaphane Suppressed Stemness Characters of Lung Adenocarcinoma Stem Cells by Down-regulating Methylation of miR-200c Promoter / 肿瘤防治研究

Objective To investigate the effect of sulforaphane (SFN) on the proliferation and self-renewal of lung cancer stem cells and its regulatory mechanism. Methods MTT method was used to detect the effect of SFN on the proliferation of lung adenocarcinoma cell lines H460 and A549; tumor sphere formation experiment was used to detect the ability of tumor sphere formation; Western blot was applied to explore the expression of stemness-related proteins (such as β-catenin, Klf4, c-myc) in lung adenocarcinoma cells before and after SFN treatment; NGS sequencing was used to analyze the effect of SFN on the expression profile of tumor cell miRNAs. qRT-PCR verified the changes in the transcription level of key miRNAs by SFN. Western blot was used to detect the effect of SFN on the expression of DNMTs in tumor cells. We constructed miR-200c promoter-GFP plasmid, and applied IF, methylation PCR and DNA sequencing methods to detect the effect of SFN on the methylation level of tumor spheres and miRNA promoter. Results The miRNAs expression profile of lung adenocarcinoma tumor spheres changed significantly after SFN (5.0μmol/L) treatment, and miRNA-200c increased the most. Compared with the control group, the expression of β-catenin, Klf4, c-myc and Vimentin genes in H460 and A549 cells of SFN-S group decreased, and the protein expression levels of DNMT1 and DNMT3a were also significantly decreased. Compared with the control group, H460 and A549 cells stably expressing pEGFP-R200c plasmid in SFN-S group significantly reduced tumor sphere diameter, while tumor sphere fluorescence intensity increased, and GFP protein expression was up-regulated. There were 9 CpG-rich regions in the miR-200c promoter region in the above-mentioned pEGFP-R200c plasmid cell line, and the methylation levels were 88.9%, 44.4% and 38.8% in the control group, SFN-S group and 5-Aza-dC group, respectively. Conclusion SFN may downregulate the expression of stem-related genes in lung cancer stem cells by epigenetically decreasing the methylation level of miR-200c promoter and promoting the transcription of miR-200c.

lncRNA FLJ26245 regulates the proliferation and migration of prostate cancer PC-3 cells through miR-200a-3p/PTPRG axis / 中国肿瘤生物治疗杂志

@#[摘 要] 目的：探讨lncRNA FLJ26245在前列腺癌组织和细胞中的表达及其对PC-3细胞增殖与迁移能力的影响及其分子机制。方法：选用2017年3月至2019年5月在洛阳中心医院手术切除的52例前列腺癌及相应的癌旁组织标本，以及前列腺细胞系LNCaP、C4-2B、PC-3、DU-145和正常前列腺上皮细胞RWPE-1，用qPCR法检测前列腺癌组织和细胞中FLJ26245的表达水平。分别将FLJ26245 mimic和阴性对照质粒（lncR-NC）转染到PC-3细胞中，用MTT法、细胞划痕愈合实验分别检测细胞的增殖和迁移能力。生物信息学技术预测和双荧光素酶基因报告实验验证FLJ26245与miR-200a-3p、酪氨酸磷酸酶受体G（PTPRG）三者之间的靶向关系。用qPCR和WB法分别检测FLJ26245下游基因及增殖与迁移相关蛋白的表达。结果：FLJ26245在前列腺癌组织和细胞中的表达水平分别显著低于癌旁组织（P<0.01）和RWPE-1细胞（P<0.01或P<0.05），以PC-3细胞中的表达水平为最低（P<0.01）。FLJ26245过表达可明显抑制PC-3细胞的增殖和迁移能力（P<0.05或P<0.01）。FLJ26245可与miR-200a-3p靶向结合，miR-200a-3p可与PTPRG靶向结合（均P<0.01）。FLJ26245过表达的PC-3细胞中miR-200a-3p表达水平显著降低（P<0.01）、PTPRG mRNA和蛋白表达水平均明显升高（均P<0.01），细胞中增殖和迁移相关蛋白cyclin A、CDK2和Twist、N-cadherin均显著降低（均P<0.01）。结论：FLJ26245在前列腺癌组织及细胞中均低表达，其可能通过miR-200a-3p/PTPRG轴调控前列腺癌PC-3细胞的增殖与迁移能力。

miR-200c regulates malignant biological behaviors of triple negative breast cancer MDA-MB-231 cells via targeting cell energy metabolism and multiple signaling pathways / 中国肿瘤生物治疗杂志

@#Objective: To investigate the effects of miR-200c on the proliferation, apoptosis and migration of triple negative breast cancer cell (TNBC) MDA-MB-231 and its metabolism-related molecular mechanism. Methods: miR-200c-231 (MDA-MB-231 overexpressing miR-200c) cells, miR-NC-231 (MDA-MB-231 transfected with miRNA-negative control) and the corresponding transplanted tumor models in nude mice were used as the study subjects. qPCR was used to detect the content of miR-200c and other related genes in cells and transplanted tumor tissues. The number of Ki67 positive cells in tumor tissue was analyzed by immunohistochemistry. The migration and apoptosis of cells were examined by Transwell chamber method and Flow cytometry, respectively. The expressions of proteins associated with proliferation, migration, and metabolism related signaling pathways in cells and tissues were confirmed by Western blotting. The changes of oxygen consumption rate (OCR), extracellular acidification rate (ECAR) and metabolic phenotype were detected by Seahorse energy metabolism detector. UPLC/LTQ-Orbitrap-MStechniquewasusedtoprofilethedifference of metabolites in cells. Results: The content of miR-200c in miR-200c-231cellswassignificantlyhigherthanthatinmiR-NC-231cells.Themass ofmiR-200c-231transplantedtumornotablydecreased,andthenumberofKi67positivecellsintumortissuesalsodecreasedsignificantly. The migration ability of miR-200c-231 cells decreased and the apoptosis rate increased (all P<0.01), accompanied with declined expressionsofZEB1/2,Vimentin,cyclinD1andincreasedexpressionofcleavedPARP(P<0.05orP<0.01),aswellasdecreasedphosphorylation leverofSTAT1/3andNF-κBbutincresedphosphorylationleverofCAMP(allP<0.05).OverexpressionofmiR-200cinMDA-MB-231cells increasedOCRandthecontentof10antitumor metabolites, but decreased ECAR and tryptophan 2,3-plus dioxidase (TDO2) expression (P<0.05 or P<0.01). Conclusion: miR-200c targeting TDO2 elevates the level of intracellular anticancer metabolites in TNBC MDAMB-231 cells, promotes the transformation from glycolysis to aerobic respiration phenotype, and inactivates STAT3 and NF-κB pathyway but activates cAMPpathway TNBC MDA-MB-231 cells, thus affects the malignant biological behaviors of MDA-MB-231 cells.

Detection of expression levels of miR200 family in plasma of breast cancer patients and its significance / 吉林大学学报(医学版)

Objective: To detect the expression levels of microRNA200 (miR200) family in the plasma of the breast cancer patients and the normal controls, and to evaluate their potential values in the screening, progression and prognosis evaluation of breast cancer. Methods: A total of 82 cases of plasma samples of the patients with breast cancer (breast cancer group) and 30 cases of healthy plasma samples (control group) were collected. The microRNAs (miRNAs) were extracted from the plasma samples, the expression levels of miR200 family (miR200a, miR200b, miR200c, miR141 and miR429) were quantified by using real-time PCR technique. The receiver-operating characteristic (ROC) curve was used to assess the diagnostic value of miRNAs, and the associations between the levels of miRNAs and the clinicopathological characteristics were analyzed. Cox proportional hazard mode was used for survival analysis. Results: Compared with control group, the expression level of miR141 in the plasma of the patients in breast cancer group was decreased (P 0 . 05). Concuson: MiR200b and miR141 are likely to become the molecular biological parameters for the diagnosis of breast cancer.

Long Noncoding RNA MALAT1 Regulates Hepatocellular Carcinoma Growth Under Hypoxia via Sponging MicroRNA-200a

PURPOSE: Hepatocellular carcinoma (HCC) is a common cancer worldwide. Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), a long noncoding RNA (lncRNA), has been reported to be aberrantly expressed in hypoxic cancer cells. MALAT1 plays a significant role in many malignancies, including HCC. The aim of this study was to explore the role of MALAT1 in hypoxic HCC cells and its underlying regulatory mechanism. MATERIALS AND METHODS: Quantitative reverse transcription PCR (qRT-PCR) assay was performed to detect the mRNA levels of MALAT1 and microRNA-200a (miR-200a) in HCC cells. Cell invasion and migration ability were evaluated by Transwell assay. Starbase v2.0 and luciferase reporter assay were employed to identify the association between MALAT1 and miR-200a. Cell proliferation and apoptosis were measured by MTT assay and flow cytometry, respectively. RESULTS: MALAT1 levels were significantly upregulated in HCC cells under hypoxia. Hypoxia promoted proliferation, migration, and invasion, and blocked apoptosis in Hep3B cells, which were weakened by knockdown of MALAT1. Starbase v2.0 showed that MALAT1 and miR-200a have a complementarity region, and luciferase reporter assay verified that MALAT1 interacted with miR-200a in Hep3B cells. Moreover, MALAT1 negatively regulated the expression of miR-200a. miR-200a levels were dramatically downregulated in HCC cells under hypoxia. Upregulation of miR-200a inhibited proliferation, migration, and invasion, and induced apoptosis in Hep3B cells under hypoxia. Interestingly, downregulation of miR-200a partially reversed the tumor-suppressive effect of knockdown of MALAT1 on Hep3B cells in hypoxic condition. CONCLUSION: LncRNA MALAT1 was involved in proliferation, migration, invasion, and apoptosis by interacting with miR-200a in hypoxic Hep3B cells, revealing a new mechanism of MALAT1 involved in hypoxic HCC progression.

Effect of Compound Danshen Dropping Pills on Heart of Diabetic Cardiomyopathy Rats by Regulating VEGF/TGF-β1 by miRNA-200b / 中国实验方剂学杂志

Objective: To study the protective effect and mechanism of compound Danshen dropping pills on the heart of rats with type 2 diabetic cardiomyopathy. Method: Seventy-five male SD rats were randomly divided into blank group (15 rats) and model group (60 rats). The model group was fed with high-sugar and high-fat diet, high-sugar and high-fat milk and sucrose water. Six weeks later, rats with foodborne obesity (body mass ≥ the mean body mass of blank control group + 2 times standard deviation) were selected and intraperitoneally injected with 1% streptozotocin (STZ)28 mg·kg-1. After 12 weeks, rats with fasting blood glucose ≥ 11.1 mmol·L-1 and polydipsia, diuresis, polyphagia and weight loss were randomly divided into model group, compound Danshen dropping pills group (0.5 g·kg-1) and metformin group (0.5 g·kg-1) and intervened for 12 weeks. At the end of the experiment, echocardiography was used to evaluate cardiac function. Blood samples were taken to determine the corresponding biochemical indicators of rats. Histopathological changes of myocardium were observed by hematoxylin-eosin (HE) and Masson staining. The ultrastructure of myocardium was observed under electron microscope. Western blot was used to detect the expressions of vascular endothelial growth factor (VEGF), transforming growth factor-beta 1 (TGF-β1) and type Ⅰ collagen (Collagen Ⅰ). Real-time fluorescence quantitative analysis (Real-time PCR) was used to detect the expression of miRNA-200b. Result: Compared with the blank group, the heart weight index (HMI), fasting blood glucose (FBG), glycosylated hemoglobin (HbA1c), fasting insulin (FINS), total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterin(LDL-C) levels in the model group were significantly increased (PPPPPβ1 and Collagen I increased (PPPPPPβ1 and Collagen I was decreased (PPConclusion: Compound Danshen dropping pills may play a protective role in the heart of type 2 diabetic cardiomyopathy rats by lowering lipid, up-regulating the expression of miR-200b and inhibiting the expression levels of VEGF, TGF-β1 and Collagen I.

Effect of miR-200c Regulation of Peptidyl-Prolyl Cis/Trans Isomerase on the Biological Behavior of Hep-2 Cells / 中国医科大学学报

Objective To explore the influence of miR-200 c on the biological behavior of laryngeal carcinoma Hep-2 cells and determine whether miR-200 c exerts its biological function through peptidyl-prolyl cis/trans isomerase (PIN1) in laryngeal carcinoma. Methods A qRT-PCR assay for the expression of miR-200 c was performed in laryngeal carcinoma tissues. Hep-2 cells were transfected with miR-200 c related small RNAs. Transwell assay detected the migration ability of the cells. Immunofluorescence assay was used to detect the abnormal amplification of the centrosome. A dual luciferase reporter gene system was used to detect the binding ability between miR-200 c and PIN1. Western blotting detected the protein expression level of PIN1. Results The expression of miR-200 c in laryngeal carcinoma was significantly increased. miR-200 c inhibited the migration of Hep-2 cells and could weaken the abnormal amplification of centrosome.PIN1 was confirmed as one of the target genes of miR-200 c. miR-200 c inhibited the expression of PIN1 at the translation level and could inhibit Hep-2 cell migration and abnormal centrosome amplification by regulating PIN1. Conclusion miR-200 c can inhibit the migration ability of laryngeal carcinoma cells and abnormal centrosome amplification by regulating PIN1.

MicroRNA-200a Targets Cannabinoid Receptor 1 and Serotonin Transporter to Increase Visceral Hyperalgesia in Diarrhea-predominant Irritable Bowel Syndrome Rats

BACKGROUND/AIMS: MicroRNAs (miRNAs) were reported to be responsible for intestinal permeability in diarrhea-predominant irritable bowel syndrome (IBS-D) rats in our previous study. However, whether and how miRNAs regulate visceral hypersensitivity in IBS-D remains largely unknown. METHODS: We established the IBS-D rat model and evaluated it using the nociceptive visceral hypersensitivity test, myeloperoxidase activity assay, restraint stress-induced defecation, and electromyographic (EMG) activity. The distal colon was subjected to miRNA microarray analysis followed by isolation and culture of colonic epithelial cells (CECs). Bioinformatic analysis and further experiments, including dual luciferase assays, quantitative real-time polymerase chain reaction, western blot, and enzyme-linked immunosorbent assay, were used to detect the expression of miRNAs and how it regulates visceral hypersensitivity in IBS-D rats. RESULTS: The IBS-D rat model was successfully established. A total of 24 miRNAs were differentially expressed in the distal colon of IBS-D rats; 9 were upregulated and 15 were downregulated. Among them, the most significant upregulation was miR-200a, accompanied by downregulation of cannabinoid receptor 1 (CNR1) and serotonin transporter (SERT). MiR-200a mimic markedly inhibited the expression of CNR1/SERT. Bioinformatic analysis and luciferase assay confirmed that CNR1/SERT are direct targets of miR-200a. Rescue experiments that overexpressed CNR1/SERT significantly abolished the inhibitory effect of miR-200a on the IBS-D rats CECs. CONCLUSIONS: This study suggests that miR-200a could induce visceral hyperalgesia by targeting the downregulation of CNR1 and SERT, aggravating or leading to the development and progression of IBS-D. MiR-200a may be a regulator of visceral hypersensitivity, which provides potential targets for the treatment of IBS-D.

Inhibitory effect of miR-200c on epithelial-mesenchymal transiton in triple negative breast cancer and its mechanism / 吉林大学学报(医学版)

Objective: To study the effect of miR-200c on the migration and proliferation abilities of breast cancer MDA-MB-231 and BT-549 cells, and to clarify the mechanism of miR-200c in inhibiting the epithelial-mesenchymal transiton (EMT) of triple negative breast cancer. Methods: The human triple negative breast cancer cell lines (MDA-MB-231 and BT-549) were chosen in this study. The cells were transiently transfected with miR-200c mimics and Lipo2000 (experimental group), miR-200c negative control and Lipo2000 (negative control group), and Lipo2000 alone (reagent control group); at the same time, blank control group was set up. The expression levels of vimentin and (3-catenin mRNA and protein were detected by RT-PCR and Western blotting method. The proliferation rates and migration abilities of MDA-MB-231 cells and BT-549 cells after transfection of miR-200c were analyzed by CCK8 assay and wound healing assay. Results: The RT-PCR and Western blotting showed that the expression levels of vimentin and (3-catenin mRNA and proteins in experimental group were decreased, and the differences were statistically significant compared with blank control group, negative control group and reagent control group (P<0. 05). The CCK8 results showed that the proliferation rates of the cells in experimental group were lower than those in negative control group and reagent control group (P<0. 05). The wound healing assay results showed that the recovery rate of scratch width in experimental group was lower than those in negative control group and reagent control group (P<0. 05). Conclusion: miR-200c might inhibit EMT in triple negative breast cancer by regulating the expressions of (3-catenin and vimentin mRNA and proteins in MDA-MB-231 and BT-549 cells and decreasing the abilities of migration and proliferation of triple negative breast cancer cells.

Suppression on invasion and migration of human tongue squamous cell carcinoma after knocking down EZH2 expression / 中国肿瘤临床

Objective:To explore whether EZH2 can regulate the expression of miR-200b/a/429 and,thus,affect human tongue squamous cell carcinoma(TSCC).Methods:EZH2 was knocked down in TSCC lines SCC15 and UM-1 with siRNA(si-EZH2)method.The expression levels of EZH2 and epithelial mesenchymal transition related proteins were detected by Western blot.qPCR was used to determine the expression level of miR-200b/a/429 after knockdown of EZH2.Transwell and wound-healing assays were employed to detect the invasion and migration ability of tumor cells.The cytoskeleton was observed with an immunofluorescence assay.EZH2 expression in human head and neck squamous cell carcinoma(HNSCC)was detected by an immunofluorescence assay and qPCR.Results:EZH2 was significantly knocked down by siRNA, thus the expression level of miR-200b/a/429 and E-cadherin increased.While the expression of the N-cadherin,Vimentin,MMP2,and MMP9 proteins decreased;the migration and invasion of HNSCC cells in the si-EZH2 group was markedly inhibited.The EZH2 expression level in patients with lymph node metastasis in HNSCC specimens was higher than those without lymph node metastasis(P<0.01).Conclusions:EZH2 inhibits the expression of miR-200b/a/429 and promotes the invasion and migration of TSCC cells.

Expression of MicroRNA-200c in Colorectal Carcinomas and Its Role on Tumor Cell Migration and Invasion / 中山大学学报(医学科学版)

[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.

MiR-200a inhibits migration of NSCLC cell line A549 by targeting Sulfatase 2 / 中华胸心血管外科杂志

Objective To investigate the effect of miR-200a on the migratory ability of NSCLC cells and to explore its possible mechanism.Methods Real-time PCR was performed to analyze the miR-200a expression in NSCLC cell lines A549 and SK-MES-1, and human normal lung bronchial epithelial cell line 16HBE.Hsa-miR-200a mimics, NC mimics, hsa-miR-200a inhibitor and NC inhibitor were transfected into A549 cells using Lipofectamine 2000.Migration of A549 cells was detected by Transwell migration assay.The potential target genes of miR-200a were predicted by bioinformatics software and then verified by dual luciferase reporter gene assay and Western blot.Results MiR-200a was significantly down-regulated in A549 and SK-MES-1 cells(P＜0.05).Exogenous over-expression of miR-200a mimics significantly inhibited migratory a-bility of A549 cells, while over-expression of miR-200a inhibitor generated the opposite effect(P＜0.01).Dual luciferase re-porter assay indicated that miR-200a could directly affect the 3'-UTR of Sulf2 gene to inhibit luciferase activity.Western blot revealed that miR-200a expression could significantly reduce Sulf2 protein expression level in A549 cells.Ectopic expression of Sulf2 protein in miR-200a-overexpressing A549 cells overrode the migration inhibition effect of miR-200a, suggesting that targe-ting Sulf2 represents an important mechanism of the anti-tumour activity of miR-200a in lung cancer.Conclusion MiR-200a inhibits migration of lung cancer cells by targeting Sulf2.

Inhibitory effect of miR-200c on epithelial-mesenchymal transiton in triple negative breast cancer and its mechanism / 吉林大学学报(医学版)

Objective:To study the effect of miR-200c on the migration and proliferation abilities of breast cancer MDA-MB-231 and BT-549 cells,and to clarify the mechanism of miR-200c in inhibiting the epithelial-mesenchymal transiton (EMT) of triple negative breast cancer.Methods:The human triple negative breast cancer cell lines (MDA-MB-231 and BT-549) were chosen in this study.The cells were transiently transfected with miR-200cmimics and Lipo2000 (experimental group),miR-200c negative control and Lipo2000 (negative control group),and Lipo2000 alone (reagent control group);at the same time,blank control group was set up.The expression levels of vimentin and β-catenin mRNA and protein were detected by RT-PCR and Western blotting method.The proliferation rates and migration abilities of MDA-MB-231 cells and BT-549 cells after transfection of miR-200c were analyzed by CCK8 assay and wound healing assay.Results:The RT-PCR and Western blotting showed that the expression levels of vimentin and β-catenin mRNA and proteins in experimental group were decreased,and the differences were statistically significant compared with blank control group,negative control group and reagentcontrol group (P＜0.05).The CCK8 results showed that the proliferation rates of the cells in experimental group were lower than those in negative control group and reagent control group (P＜0.05).The wound healing assay results showed that the recovery rate of scratch width in experimental group was lower than those in negative control group and reagent control group (P＜0.05).Conclusion:miR-200c might inhibit EMT in triple negative breast cancer by regulating the expressions of β-catenin and vimentin mRNA and proteins in MDA-MB-231 and BT-549cells and decreasing the abilities of migration and proliferation of triple negative breast cancer cells.

Effect of miR-200a on the proliferation, migration and apoptosis of lung cancer cell line A549 / 临床与实验病理学杂志

Purpose To investigate the expression of miR200a in different lung cancer cell lines and its effect on proliferation,migration,and apoptosis in A549 cells.Methods The expressions of miR-200a in different lung cancer cell lines were detected by RT-PCR.miR-200a mimics was transfected into A549 cells by Lipofectamine RNAiMax.The change of proliferation ablility of A549 cells was detected by CCK-8 method and plate clone formation assay.Cell migration was examined by Transwell chamber assay.The flow cytometry was used to examine the changes of apoptosis.The possible target genes of miR-200a were forecasted by bioinformatics tools.Results The results of RT-PCR showed that the expression of miR-200a was significantly down-regulated in A549,H23 and H460 cell lines than 16HBE cell line.CCK-8 assay showed that the OD values of the mimics group at 4,and 5 days were significantly lower than those in the negative control (NC) group (P ＜ 0.05).Plate clone formation assay showed rate of colony formation in the mimics group was significantly lower than that in the NC group [(33.13±0.74)％ vs (45.57 ±1.27)％,P＜0.05].Transwell migration assay showed that the cell number of mimics group that passed the Transwell membrane was significantly lower than that of the NC group [(71.60 ± 17.90) vs (140.20 ± 17.88),P ＜0.05].Flow cytometry showed that the apoptosis rate of the mimics group was significantly higher than that of the NC group [(17.80± 1.90)％ vs (11.33 ± 1.96)％,P ＜ 0.05].Tiam1 may be one of the target gene of miR-200a.Conclusion miR-200a can inhibit the proliferation and migration,and promote apoptosis of lung cancer A549 cells,suggesting a potential new therapeutic agent for lung cancer cell.MiR-200a may play a function of regulation of tumor development through target gene Tiam1.

Lineage-specific Expression of miR-200 Family in Human Embryonic Stem Cells during In Vitro Differentiation

Although microRNAs have emerged as key regulators in diverse cellular processes, the roles of microRNAs are poorly understood in human embryonic stem cells (hESCs) during differentiation into specialized cell types. In this study, we used a microRNA array with 799 human microRNA probes to examine the expression profiles of microRNAs in hESCs during differentiation into endodermal and mesodermal lineages in vitro. Among the microRNAs analyzed, 7 and 20 microRNAs were enriched in the developmental process of hESCs into mesodermal and endodermal lineages, respectively. In particular, the expression levels of miR-200 family, which is known to regulate the epithelial to mesenchymal transition (EMT), gradually increased in hESCs during differentiation into hepatocytes while they gradually decreased during differentiation into vascular endothelial cells. Downregulation of ZEB1, a direct target of miR-200 family, and E-CADHERIN, a target protein of ZEB1, was observed in hESCs during differentiation into endodermal and mesodermal lineages, respectively. These results indicate that miR-200 family has an important role in determining the cell fate between endodermal and mesodermal lineages from the pluripotent state.

MiR-200a and miR-200b target PTEN to regulate the endometrial cancer cell growth in vitro

OBJECTIVE@#To study whether miR-200a and miR-200b target PTEN gene expression to regulate the endometrial cancer cell growth in vitro.@*METHODS@#Endometrial cancer cells ECC-1 were cultured and transfected with the miR-200a and miR-200b mimics and inhibitors as well as the negative control mimics and inhibitors, and then the cell proliferation activity as well as the expression of PTEN and downstream genes in cells was determined; after transfection of miR-200a and miR-200b mimics as well as PTEN-3'UTR luciferase report gene plasmids, the fluorescence activity of luciferase reporter gene was determined.@*RESULTS@#12 h, 24 h and 48 h after transfection, the cell proliferation activity of miR-200a mimics group and miR-200b mimics group were significantly higher than those of NC mimics group while the cell proliferation activity of miR-200a inhibitor group and miR-200b inhibitor group were significantly lower than those of NC inhibitor group; 48 h after transfection, PTEN expression in cells and PTEN-3'UTR luciferase reporter gene fluorescence activity of miR-200a mimics group and miR-200b mimics group were significantly lower than those of NC mimics group while p-PI3K and p-Akt expression were significantly higher than those of NC mimics group; PTEN expression in cells and PTEN-3'UTR luciferase reporter gene fluorescence activity of miR-200a inhibitor group and miR-200b inhibitor group were significantly higher than those of NC inhibitor group while p-PI3K and p-Akt expression were significantly lower than those of NC inhibitor group.@*CONCLUSION@#miR-200a and miR-200b can promote the endometrial cancer cell growth in vitro by targeted inhibition of PTEN gene expression.

Effects of miR-200a on the proliferation of lung cancer cells by inhibiting YAP1 / 中国肿瘤临床

Objective: To investigate the effects of miR-200a on the proliferation of lung cancer cells and to identify its direct target genes. Methods:Real-time PCR was performed to analyze the miR-200a expression in 15 paired clinical specimens of non-small cell lung cancer and adjacent noncancerous tissues, human lung cancer cell lines (A549, NCI-H520, and SK-MES-1), and one human normal lung bronchial epithelial cell line (16HBE). The effects of miR-200a on the proliferation of A549 lung cancer cells were detected through CCK-8 method. The candidate target genes of miR-200a were identified by bioinformatics screening and then verified by dual luciferase reporter gene assay, real-time PCR, and Western blot. The effects of YAP1 downregulation on the proliferation of A549 lung cancer cell line were also observed through CCK-8 method. Results:The miR-200a expression in non-small cell lung cancer tissues and lung cancer cell lines was significantly decreased (P<0.01). The upregulation of miR-200a expression could significantly inhibit the pro-liferation of A549 lung cancer cells (P<0.01). Dual luciferase reporter gene indicated that miR-200a could directly affect the 3′-untrans-lated region of the YAP1 gene to inhibit luciferase activity (P<0.01). Real-time PCR and Western blot revealed that the upregulation of miR-200a expression could significantly reduce the mRNA and protein expression levels of YAP1 in A549 lung cancer cells (P<0.01). CCK-8 method indicated that the downregulation of YAP1 could significantly prevent the proliferation of A549 lung cancer cells (P<0.01). Conclusion:MiR-200a inhibits the proliferation of lung cancer cells by targeting YAP1. Thus, miR-200a elicits tumor suppression effects.