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OBJECTIVES@#Cervical squamous cell carcinoma is the most common cancer in female reproductive system. This study aims to explore the effect of microRNA-9-5p (miR-9-5p) on the migration, invasion, and epithelial-mesenchymal transition (EMT) process of cervical squamous cells.@*METHODS@#Bioinformatics were used to predict the miRNAs that could bind to E-cadherin (E-cad). The Cancer Genome Atlas (TCGA) database was used to analyze and extract significantly differentially expressed miRNAs from part of cervical squamous cell carcinoma tissues and normal cervical tissues, and miR-9-5p was selected as the main research target. The translated regions (UTR) of wild-type E-cad (E-cad-WT 3'-UTR) or the 3'-UTR of mutant E-cad (E-Cad-MUT 3'-UTR) was transfected with miR-9-5p mimic normal control (NC), and miR-9-5p mimic was co-transfected human embryonic kidney cells (293T). The relationship between miR-9-5p and E-cad was detected by double luciferase assay. The expression of miR-9-5p in normal cervical epithelial cell lines (H8) and cervical squamous cell lines (C33A, siha, caski and Me180) were detected by quantitative real-time PCR. Then, the experiments were divided into groups as follows: a block control group, an overexpression control group (mimic-NC group), a miR-95p overexpression group (mimic group), an inhibitory expression control group (inhibitor-NC group), and a miR-9-5p inhibitory expression group (inhibitor group). The changes of migration ability were detected by scratch assay. Transwell invasion assay was used to analyze the changes of invasion ability, and the mRNA and protein changes of E-cad and vimentin were detected by quantitative real-time PCR and Western blotting.@*RESULTS@#MiR-9-5p had a targeting binding relationship with E-cad. Compared with the normal cervical tissue H8 cell line, the miR-9-5p was highly expressed in cervical cancer cell lines (C33A, siha, caski and Me180) (all P<0.05). The luciferase activity of E-cad-MUT was increased compared with that of E-cad-WT in miR-9-5p mimic cells (P<0.05). Compared with the blank control group, the protein and mRNA expressions of E-cad were decreased in the miR-9-5p mimic group (both P<0.05), which were increased in the miR-9-5p inhibitor group (both P<0.05). Compared with H8 cell line, the miR-9-5p was highly expressed in the cervical squamous cell lines (all P<0.05). Compared with the mimic-NC group, the distance of wound healing, the number of caski and Me180 cells invaded below the membrane, and the mRNA and protein expressions of vimentin were all increased in the miR-9-5p mimic group (all P<0.05), while the mRNA and protein of E-cad were decreased (both P<0.05). Compared with the inhibitor-NC group, the distance of wound healing, the number of caski and Me180 cells invading the membrane, and the mRNA and protein expressions of vimentin were decreased in the miR-9-5p inhibitor group (all P<0.05), but the mRNA and protein expressions of E-cad were increased (both P<0.05).@*CONCLUSIONS@#The miR-9-5p is highly expressed in cervical squamous cell carcinoma, which can increase the migration and invasion ability, and promote the EMT process of cancer cells.
Subject(s)
Humans , Female , Cell Line, Tumor , Vimentin/metabolism , Uterine Cervical Neoplasms/genetics , Epithelial-Mesenchymal Transition/genetics , MicroRNAs/metabolism , Carcinoma, Squamous Cell/genetics , Cell Movement/genetics , RNA, Messenger , Cell Proliferation/genetics , Gene Expression Regulation, NeoplasticABSTRACT
The chronic consumption of alcohol causes a worsening of the events that follow the cerebral ischemia. These events are regulated through the expression of several genes and microRNAs. The aimof this work was To analyze and describe the expression profile of PARP and AIF and miRNA-9 proteins in rats submitted to focal cerebral ischemia, associated or not with chronic alcoholism model. Methods: Twenty adult Wistar rats, subdivided into: control; ischemic; alcoholic and ischemic / alcoholized for immunohistochemical analysis and miRNA-9 gene expression. Results: There was a reduction in the protein expression of PARP-1 and a positive marking for AIF in the ischemic / alcoholized group. The miRNA-9 did not obtain significant expression. The association of ischemia with chronic alcohol use promoted a tendency to low expression of miRNA-9, low expression of PARP-1 and high expression of AIF, indicating an interference in the protective effect of miRNA-9 be observed in the other groups.
El consumo crónico de alcohol provoca un empeoramiento de los eventos que siguen a la isquemia cerebral. Estos eventos están regulados a través de la expresión de varios genes y microRNA. El objetivo de este trabajo fue analizar y describir el perfil de expresión de las proteínas PARP y AIF y microRNA-9 en ratas sometidas a isquemia cerebral focal, asociadas o no, con el modelo de alcoholismo crónico. Veinte ratas Wistar adultas se dividieron en: grupo control, isquémico alcohólico, e isquémico / alcoholizado para análisis inmunohistoquímico y expresión de genes microRNA-9. Resultados: Hubo una reducción en la expresión de proteínas de PARP-1 y un marcado positivo para AIF en el grupo isquémico / alcoholizado. No se observó una expresión significativa en el microRNA-9. La asociación de la isquemia con el consumo crónico de alcohol promovió una tendencia a la baja expresión de microRNA-9, baja expresión de PARP1 y alta expresión de AIF, lo que indica una interferencia en el efecto protector de microRNA-9 en los otros grupos.
Subject(s)
Animals , Rats , Brain Ischemia/metabolism , Alcoholism/metabolism , Immunohistochemistry , Brain Ischemia/genetics , Rats, Wistar , MicroRNAs/metabolism , Disease Models, Animal , Alcoholism/genetics , Apoptosis Inducing Factor/metabolism , Poly (ADP-Ribose) Polymerase-1/metabolismABSTRACT
Background:MicroRNA-9 (miR-9)is involved in the modulation of a variety of physiological process such as organ development and self-renewal and multipotential differentiation of cells. Moreover,its aberrant expression is closely associated with several types of malignant tumors. Aims:To investigate the effect of miR-9 on biological behavior of human gastric cancer cells. Methods:Expression of miR-9 in normal human gastric epithelial cell line GES-1 and human gastric cancer cell line BGC-823 and SGC-7901 was detected by real-time PCR. Then the two gastric cancer cell lines were transiently transfected with miR-9 mimic,miR-9 inhibitor and empty vector,respectively;the efficiency of transfection was assessed by real-time PCR. CCK-8 assay,Transwell assay and wound healing assay were used to examine the cell proliferation and migration capacities. Results:The expression level of miR-9 was significantly higher in gastric cancer cells than in normal gastric epithelial cells (P＜0. 05). Compared with cells transfected with empty vector,overexpression of miR-9 occurred in cells transfected with miR-9 mimic and effectively promoted the proliferation and migration of BGC-823 and SGC-7901 cells (P ＜ 0. 05 ). Conversely,transfection with miR-9 inhibitor significantly suppressed the proliferation and migration of gastric cancer cells (P＜0. 05). Conclusions:Up-regulating miR-9 expression promotes the proliferation and migration of gastric cancer cells,which indicates that miR-9 overexpression may be associated with progression of gastric cancer.
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AIM: To investigate the inhibitory effect of microRNA-9 (miR-9) on epithelial-mesenchymal transition (EMT) in the gastric cancer SGC-7901 cells and its mechanism.METHODS: The gastric cancer cell line SGC-7901 was transfected with miR-9 mimics or negative control mimic (NCM), as miR-9 or NCM group, respectively.The SGC-7901 cells without transfection were used as control group.The expression level of miR-9 in each group was detected by RT-qPCR.The migration and invasion abilities of the SGC-7901 cells in the 3 groups were detected by Transwell assay.The protein expression of N-cadherin, E-cadherin, α-catenin and neuropilin-1 (NRP1) was determined by Western blot.Antagonistic effect of NRP1 over-expression on miR-9 inhibition of EMT was detected by Western blot.The relationship between miR-9 and NRP1 was analyzed by dual luciferase assay.RESULTS: The expression level of miR-9 in miR-9 group was significantly up-regulated, which was 538 times higher than that in control group (P<0.05).The number of migratory cells in miR-9 group was significantly lower than that in control group (P<0.05).Compared with control group, the protein expression of N-cadherin and NRP1 in miR-9 group was significantly decreased, while the protein expression of E-cadherin and α-catenin protein was significantly increased.Over-expression of NRP1 resulted in the increase in the protein expression of N-cadherin in the gastric cancer cells of miR-9 group, and the decrease in the protein expression of E-cadherin and α-catenin significantly.The result of dual luciferase assay showed that NRP1 was a downstream target gene of miR-9 (P<0.05).CONCLUSION: miR-9 may inhibit the expression of EMT-related proteins through the downstream target gene NRP1, thus inhibiting the EMT of gastric cancer SGC-7901 cells.
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Objective:To investigate the role of MicroRNA-9-1 in inducing epidermal stem cells(ESCs) differentiation into neurons.Methods:The lentiviral of MicroRNA-9-1 was constructed and transfected into rats epidermal stem cells.The experiment was divided into transfected group,non-transfected group and the negative control group.The β-mercaptoethanol was as an inducer for triggering the ESCs to differentiate into neurons.The GFP fluorescence expression of epidermal stem cells after transfection was observed under inverted fluorescence microscope.The protein and mRNA expression level of microtuble-associated protein 2 (MAP-2) was detected by immunocytochemical method and RT-PCR,respectively.Results:The result of Positive clone PCR confirmed successful construction of MicroRNA-9-1 in rats.Transfection after 48 h,the expressing of GFP fluorescence at peak in transfected group,and transfection efficiency reached (85.6+1.9)％.Most ESCs differentiated into neurons in transfected group after β-mercaptoethanol induction 7 h,and the effect was significantly better than non-transfected group and the negative control group.The protein ((87.3± 0.6)％) and mRNA (about twice over) expression levels of MAP-2 in transfected group was higher than those in non-transfected group and the negative control group (P＜0.05).Conclusion:The lentiviral of MicroRNA-9-1 has high transfection efficiency in rats ESCs,and could promoted ESCs differentiate into neurons under β-mercaptoethanol induced.
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@#Objective To investigate the effect of electroacupuncture (EA) at Quchi (LI11) and Zusanli (ST36) acupoints on rats with focal cerebral ischemia, and whether it was mediated by microRNA- 9 (miR- 9) regulating neural stem cells proliferation in subventricular zone. Methods Fifty-two Sprague-Dawley rats were randomly assigned into sham group (n=12), model group (n=12), EA group (n=12), EA+dimethylsulfoxide (DMSO) group (n=8) and EA+miR-9 mimics group (n=8). The middle cerebral artery occlusion model was induced. DMSO and miR-9 mimics were performed by intracerebroventricular injection 30 minutes before modeling. The EA group, EA+ DMSO group and EA+miR-9 mimics group were electroacupunctured at Quchi and Zusanli acupoints the next day after modeling. All the groups were intraperitoneally injected with bromodeoxyuridine (BrdU). The BrdU co-localized with Nestin was observed by immunofluorescence. The expression of miR-9 in subventricular zone was detected by real time qPCR. Results The neurological deficit score was lower (t=9.600, P=0.006), and the infarct volume was smaller (t=14.080, P=0.024) in the electroacupuncture group than in the model group. The expression of Nestin and BrdU co-localizated with Nestin increased; while the expression of miR-9 decreased in subventricular zone (P<0.05) in the electoacupuncture group. However, the expression of Nestin, BrdU and BrdU co-localizated with Nestin was lower in the EA+miR-9 mimics group than in the EA+ DMSO group. Conclusion Electroacupuncture at Quchi and Zusanli acupoints may promote the neural stem cells proliferation in subventricular zone by downregulating the miR-9 expression.
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PURPOSE: This study was designed to investigate the role of taurine-upregulated gene 1 (TUG1) in MCF-7 breast cancer cells and the molecular mechanism involved in the regulation of microRNA-9 (miR-9). METHODS: The expression of TUG1 in breast cancer tissues and cells was evaluated using quantitative reverse transcription polymerase chain reaction. Cell viability was examined using a 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay; cell cycle progression and apoptosis were analyzed using flow cytometry. A dual luciferase reporter assay was used to detect the relationship between TUG1 and miR-9. The expression of methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) was measured by western blot. RESULTS: Higher expression of TUG1 was observed in breast cancer tissues and cell lines than in the corresponding controls. TUG1 knockdown reduced proliferation, suppressed cell cycle progression, and promoted apoptosis of MCF-7 cells. The dual luciferase reporter assay showed that TUG1 could negatively regulate the expression of miR-9. MiR-9 inhibition abrogated the effect of TUG1 knockdown on the proliferation, cell cycle progression, and apoptosis of MCF-7 cells. TUG1 positively regulated the expression of MTHFD2 in breast cancer cells. CONCLUSION: TUG1 knockdown was significantly associated with decreased cell proliferation and it promoted apoptosis of breast cancer cells through the regulation of miR-9.