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Background Exposure to arsenic can damage trophoblast cells and thus induce abortion, but the mechanism is not known. Objective To investigate the role of miR-145 and PTEN/AKT/mTOR pathway in arsenic-induced abortion and trophoblast cell damage in rats. Methods In the animal experiment, twenty SD pregnant rats were randomly divided into a normal control group (saline gavage) and an arsenic-induced abortion group (10.65 mg·kg−1 sodium arsenite solution was administered by gavage, and the gavage volume was 10 mL·kg−1), with 10 rats in each group. After the miscarriage occurred in the arsenic-induced abortion group (5-6 d after exposure), placental tissues were collected from the two groups. The mRNA expression levels of microRNA-145 (miR-145), phosphatase and tensin homologue (PTEN), kinase B (AKT), mammalian target of rapamycin (mTOR) were detected by real-time quantitative PCR (RT-PCR), and the protein expression levels of PTEN, AKT, mTOR, p-AKT, and p-mTOR were detected by Western blotting. For the in vitro study with immortalized human trophoblast cell line (HTR-8/SVneo cells), a control group, an arsenic exposure group, an miR-145 overexpression group, and an arsenic exposure+miR-145 overexpression group were prepared and cultured for 72 h with 37 °C and 5% CO2, at cell density of 5×105 cells per well, and the arsenic exposure concentration was 20 μmol·L−1. The MTT method was applied to detect cell viability, crystal violet staining to detect the number of monoclonal formation, flow cytometry to detect the level of apoptosis, Image J Angiogenesis Analyzer 1.8.0 plug-in to evaluate total blood vessel length and total blood vessel number; the detection indexes and methods of genes and proteins were the same as "animal experiment". Results (1) In the animal experiment, compared with the normal control group, the expression level of miR-145 mRNA in the placenta tissues of the arsenic-induced abortion group was increased (P<0.05), and the expression levels of PTEN, AKT, mTOR mRNA and proteins, and p-AKT and p-mTOR proteins were decreased (P<0.05). (2) For the in vitro study, compared with the control group, the cell viability rate, number of monoclonal formation, total vessel length, and total vessel number were decreased, and the apoptosis rate was increased in the arsenic exposure group, the miR-145 overexpression group, and the arsenic exposure+miR-145 overexpression group (P<0.05). Compared with the arsenic exposure group and the miR-145 overexpression group, the cell viability rate, number of monoclonal formation, total vessel length, and vessel number were decreased, and the apoptosis rate was increased in the arsenic exposure+miR-145 overexpression group (P<0.05). Compared with the control group, the levels of miR-145 mRNA in the arsenic exposure group, the miR-145 overexpression group, and the arsenic exposure+miR-145 overexpression group increased (P<0.05), the expression levels of PTEN, AKT, mTOR mRNA and protein and the expression levels of p-AKT and p-mTOR protein were decreased (P<0.05); compared with the arsenic exposure group and the miR-145 overexpression group, the level of miR-145 mRNA in the arsenic exposure+miR-145 overexpression group was increased (P<0.05), and the levels of PTEN, AKT, mTOR mRNA and protein as well as p-AKT and p-mTOR protein were decreased (P<0.05). Conclusion miR-145 might be related to abortion due to arsenic exposure. miR-145 could inhibit the proliferation and angiogenesis of trophoblast HTR-8/SVNEO cells, and promotes their apoptosis; the mechanism may be related to the inhibition of PTEN/AKT/mTOR pathway.
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@#BACKGROUND: Acute respiratory distress syndrome (ARDS) causes substantial mortalities. Alveolar epithelium is one of the main sites of cell injuries in ARDS. As an important kind of microRNAs (miRNAs), microRNA-145 (miR-145) has been studied in various diseases, while its role in ARDS has not been investigated. METHODS: Lipopolysaccharide (LPS) was intratracheally instilled to establish a rat ARDS model. Cytokines from bronchoalveolar lavage fluid (BALF) were measured using rat tumor necrosis factor-α and interleukin-6 enzyme-linked immunosorbent assay kits (R&D Systems), and the pathological structures were evaluated using hematoxylin and eosin (H&E) staining and transmission electron microscope; the lung miR-145 messenger RNA (mRNA) was detected using quantitative polymerase chain reaction. Bioinformatics focused on the target genes and possible pathways of gene regulation. RESULTS: A rat model of LPS-induced ARDS was successfully established. The miR-145 was down-regulated in the LPS-induced ARDS lung, and mitochondrial dysfunction was observed in alveolar epithelial cells, most obviously at 72 hours after LPS. TargetScan and miRDB databases were used to predict the target genes of miR-145. A total of 428 overlapping genes were identified, seven genes were associated with mitochondrial function, and Ogt, Camk2d, Slc8a3, and Slc25a25 were verified. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were enriched in the mitogen-activated protein kinase (MAPK) signaling pathway, and Gene Ontology (GO) biological process was mainly enriched in signal transduction and transcription regulation. CONCLUSIONS: The miR-145 is down-regulated in LPS-induced ARDS, and affects its downstream genes targeting mitochondrial functions.
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Objective To explore the mechanism of microRNA-145 (miR-145) involved in proliferation and apoptosis of breast cancer MCF-7 cells. Methods The immortalized breast cancer cell line MCF-7 cells were cultured in vitro and transfected with miR-145. Real-time PCR and Western blotting were used to detect mRNA and protein levels, respectively. The proliferation level of each group was detected by cell counting kit-8 (CCK-8) method , and the apoptosis level of MCF-7 cells in different treatment groups was detected by flow cytometer. Results Real-time PCR result showed that miR-145 did not affect Caspase-3, proliferating cell nuclear antigen (PCNA) and B cell lymphoma factor 2 (Bcl-2) mRNA levels in MCF-7 cells. Western blotting analysis showed that compared with the control group, transfection of miR-145 for 96 hours significantly increased the expression of Caspase-3 and inhibited the expression of PCNA and Bcl-2. The result of CCK-8 assay showed that the proliferation rate of MCF-7 cells was decreased after overexpression of miR-145 for 72 hours and 96 hours (P<0. 05). The result of flow cytometer showed that the apoptosis rate of MCF-7 cells in overexpression group was significantly higher than that in the control group (P<0. 05). Conclusion MiR-145 can inhibit the proliferation and promote the apoptosis of MCF-7 cells by down-regulating PCNA and Bcl-2 and up-regulating the expression of Caspase-3, which may be a new target for breast cancer treatment.
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Objective MicroRNA-145 (miR-145) is underexpressed in breast cancer. The study aimed to explore the regulatory effect of miR-145 on breast cancer MCF-7 cells by investigating the association of miR-145 with ADAM17 and EGFR. Methods The MCF-7 breast cancer cells were divided into three groups: the transfection group (transfected with microRNA-145 mimics), the control group (without transfection) and the nonsense sequence group (transfected with nonsense microRNA). MTT, transwell and real-time quantitative fluorescence polymerase chain reaction(qPCR) were respectively used to detect the proliferative capacity, invasive ability and expression of MCF-7 breast cancer cells after the transfection of miR-145 in three groups. ADAM17 and EGFR mRNA and protein levels in three groups of breast cancer MCF-7 cells were detected by qPCR and western blot. Results The results of qPCR showed that the relative expression of miR-145 was significantly higher in transfection group (13964.33±1265.30) than those in control group (1.00±0.05) and nonsense sequence group (1.03±0.15) and the difference was statistically significant (P<0.01); the expression of ADAM17 mRNA in transfection group (1.71±0.08) was significantly higher than that in control group (1.00±0.07) and the difference was statistically significant (P<0.01). Compared with the nonsense sequences at 24 h, 48 h, and 72 h, the inhibition rate of MCF-7 in transfection group was significantly increased (P<0.01). The results of transwell invasion showed that the number of transmembrane cells in transfection group [(56.20±2.17)/field] was significantly lower than those in control group [(92.80±3.90)/field] and nonsense sequence group [(91.80±4.97)/field of view ] (P < 0.01). Western blot results showed that the protein content of ADAM17 and EGFR in transfection group was significantly lower than those in the control group and the nonsense sequence group (P<0.01). Conclusion MiR-145 inhibits the proliferation and invasion of breast cancer MCF-7 cell line by acting on the ADAM17-EGFR signaling pathway.
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Objective: To explore the clinic-pathological significance of microRNA-145 expression in human cervical cancer and its ef-fects on Wnt/β-catenin signaling pathway. Methods: Real-time PCR was used to detect the expression of microRNA-145 in 62 cervical cancer samples. The correlation between microRNA-145 expression and the clinic-pathological parameters and its prognostic signifi-cance was analyzed. MicroRNA-145-expressing plasmid and non-sense plasmid were transfected into human cervical cancer HeLa cells, assigned into overexpressed microRNA-145 group and control group. Immunofluorescence staining was performed to detect the expression location of β-catenin. Top Flash luciferase reporter assay was performed to investigate the effects of microRNA-145 on the transcriptional activity of TCF/LEF and direct interactions with Cateninδ-1. Western blot was used to detect the effects of microRNA-145 on the expression of Cateninδ-1, C-MYC, and CyclinD1. Results: The patients with low microRNA-145 expression showed poorer prognosis [(41.28 ± 2.00) months vs . (46.06 ± 0.95) months, P<0.05]. β-catenin immunofluorescence was distributed within the cyto-plasm in the microRNA-145-overexpressed HeLa cells, but mainly within the nucleus and cytoplasm in the control cells. The luciferase reporter system indicated that the transcriptional activity of TCF/LEF was inhibited in the microRNA-145-overexpressed HeLa cells, and validated Cateninδ-1 was a target of miR-145. The expression of Cateninδ-1, C-MYC, and CyclinD1 was decreased in the microRNA-145-overexpressed HeLa cells. Conclusions: microRNA-145 may inhibit cervical cancer progression via Cateninδ-1 and inhibit the Wnt/β-catenin signaling pathway.
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OBJECTIVBE To investigate the intervention of compound Astragalus and Salvia milt-iorrhiza extract (CASE) consisted of astragalosides, astragalus polysaccharides and salvianolic acids on the interaction of microRNA-145/microRNA-21 (miR-145/miR-21) and Smad3C/3L phosphorylation (pSmad3C/pSmad3L) down-stream of transforming growth factor-β (TGF-β)/mitogen activated protein kinase (MAPK) signaling in hepatocellular carcinoma (HCC) progression by in vitro and in vivo experi-ments. METHODS In HepG2 cells and xenografts of nude mice, antagomir/agomir and plasmids of Smad3C/3L phosphorylation site mutation (Smad3 3S-A/Smad3 EPSM) were used to intervene miR-145/miR-21 and pSmad3C/pSmad3L expression respectively,then incorporative CASE treatment. Cell proliferation, migration, apoptosis, tumor growth and histopathologic characteristics of xenografts, relevant proteins of TGF-β/Smad pathway and miR-145/miR-21 were evaluated.RESULTS CASE up-regulated miR-145 while down-regulated miR-21, inhibited cell proliferation,migration and tumor growth, accelerated cell apoptosis in HepG2 cells respectively transfected with Smad3 WT, Smad3 EPSM,Smad3 3S-A plasmids in cultured dishes and xenografts of nude mice,the above effects were more evident in HepG2 cells with increased pSmad3C.In TGF-β1-stimulated HepG2 cells and xenografts of nude mice, CASE antagonized the facilitating effects of miR-145 antagomir/miR-21 agomir on cell migration,proliferation,tumor growth and inhibiting effects of miR-145 antagomir/miR-21 agomir on cell apoptosis; CASE increased miR-145 down-regulated by miR-145 antagomir and decreased miR-21 up-regulated by miR-21 agomir,reduced protein level of pSmad3L and their proteins including TβRⅡ, pERK1/2, pJNK1/2 and pp38 while elevated pSmad3C expression. CONCLUSION These results suggest that pSmad3C/pSmad3L maybe interact with miR-145/miR-21 in HCC progression,which may be one of important molecular mechanisms of CASE's anti-HCC effects.
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AIM:To investigate the molecular mechanism of microRNA(miR)-145 enhancing radiosensitivity of cervical cancer cells.METHODS:Cervical cancer lines HeLa,CaSki,C33A and SiHa were transfected with miR-145-mimic and NC-mimic using Lipofectamine 2000 kit,and the expression levels of miR-145 in the cervical cancer lines and endometrial stromal cells(ESC)were detected by real-time PCR.The transfected cells were exposed to X-ray radiation.At different time points,the cell viability and apoptotic rate were measured by MTT assay and flow cytometry with AnnexinV -FITC/PI staining,respectively.The protein levels of γH2AX and helicase-like transcription factor(HLTF)were monitored by immunofluorescence and Western blot, respectively.RESULTS: High miR-145 levels was found in ESC, while low miR-145 levels were found in HeLa,SiHa,C33A and CaSki cells.The level of miR-145 in the cells transfected with miR-145-mimic was significantly higher than that in the negative control cells(P<0.05).The viability of cervical cancer cells with miR-145 over-expression was notably lower than that of the negative control cells, and the apoptotic rate at 72 h was remarkablely increased(P<0.05)under the same condition.The results of immunofluorescence labeling indicated that the protein level of γH2AX significantly increased in the cervical cancer cells with miR-145 over-expression exposed to radia-tion compared with the negative control cells(P<0.05).The results of Western blot indicated that the protein level of HLTF significantly decreased in the cervical cancer cells with miR-145 over-expression compared with the negative control cells(P<0.05).CONCLUSION:miR-145 was highly expressed in normal endometrial epithelial cells, while low ex-pressed in cervical cancer cells.miR-145 over-expression inhibits growth and significantly enhances radiosensitivity of cer -vical cancer cells.The molecular mechanism is possiblely related to downregulating HLTF expression and inhibiting DNA damage repair,thus resulting in apoptosis.
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Objective To investigate the clinical significance of the expression of serum microRNA-21 (miR-21),microRNA-122(miR-122) and microRNA-145 (miR-145) in patients with primary hepatocellular carcinoma(HCC).Methods A total of 100 patients with HCC who hospitalized in the Central Hospital of Tongchuan Mining Bureau from September 2014 to September 2016 were selected as HCC group,and 100 healthy subjects were selected as the control group.The expression of serum miR-21,miR-122 and miR-145 was detected by real-time quantitative fluorescence polymerase chain reaction.The relationship between the expression of serum miR-21,miR-122,miR-145 and the clinical characteristics of HCC patients was analyzed.Results The expression of serum miR-21 and miR-122 in HCC group was significantly higher than that in the control group(P ＜0.05),and the expression of serum miR-145 in HCC group was significantly lower than that in the control group (P ＜0.05).The expression of serum miR-21,miR-122 and miR-145 in HCC patients was significantly correlated with histological differentiation,clinical stage,tumor size and liver cirrhosis (P ＜ 0.05),but the expression of serum miR-21,miR-122 and miR-145 in HCC patients was not correlated with gender,age,hepatitis B virus antigen and hepatitis C virus antibody(P ＜0.05).The expression of serum miR-21 and miR-122 in the patients with poorly differentiated tissue,high clinical stage,tumor diameter ≥5 cm and liver cirrhosis was significantly higher than that in the patients with medium and high differentiated tissue,low clinical staging,tumor diameter ＜ 5 cm and no cirrhosis (P ＜ 0.05).The expression of serum miR-21 and miR-122 after operation was significantly lower than that before operation(P ＜ 0.05),and the expression of miR-145 after operation was significantly higher than that before operation in HCC patients(P ＜ 0.05).Conclusion The up-regulated expression of miR21 and miR-122 and the down-regulation of miR-145 may be related to the occurrence and development of HCC.The combined detection of serum miR-21,miR-122 and miR-145 is helpful for the diagnosis and evaluation of HCC.
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[Objective]To investigate the role of microRNA-145/Smad interacting protein 1(SIP 1)in VEGF-C-enhanced cervical cancer metastasis.[Methods]Cervical cancer cell line SiHa cells were cultured and treated with VEGF-C to observe its effect on the expression of miR-145 and SIP1. After transfection with specific SIP1 siRNA,the invasion number of cultured cells were calculated by transwell chamber assay.[Results]Treatment with VEGF-C(100 ng/mL)for 12 h,24 h and 48 h all reduced miR-145 expression,with the expression abundance of(82.4±6.4)% (P<0.05),(72.5±7.2)%(P<0.01),and(60.6±9.6)%(P<0.001),respectively,when compared to control.Meanwhile, the same treatment with VEGF-C also increased SIP1 protein expression,with the expression abundance of(142.4 ± 16.5)%(P<0.05),(183.6 ± 11.4)%(P<0.01)and(220.8 ± 15.7)%(P<0.001),respectively. The transfection of miR-145 mimic significantly impaired VEGF-C effect on SIP1 expression. Finally,VEGF-C promoted SiHa cell invasion,which was largely inhibited by the tranfection of SIP siRNA with the inhibitory rate of(56.6±10.3)%(P<0.01).[Conclusion]VEGF-C downregulates miR-145,thus increases SIP1 expression and promotes cervical cancer cell invasion,which may contributes to cervical cancer malignant progression.
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[ ABSTRACT] AIM:To investigate the effects of microRNA145 ( miRNA145 ) on the viability, apoptosis, inva-sion and metastasis of hepatoma HepG2 cells.METHODS: HepG2 cells were randomly allocated into 3 groups: blank control group, empty mimic transfected group and miRNA145 mimic transfected group.Under the induction of Lipofectami-neTM 2000, the recombinant was transfected into HepG2 cells.After transfection, the expression level of miRNA145 was detected by real-time PCR.The protein level of N-cadherin and the mRNA expression levels of miRNA145 and N-cadherin were detected by Western blot and real-time PCR.The cell viability was detected by MTS assay.The cell cycle and apopto-sis were analyzed by flow cytometry.Invasion and metastasis were detected by Transwell assay.RESULTS:Compared with negative control, miRNA145 expression was up-regulated significantly, while the expression of N-cadherin was down-regu-lated significantly.Meanwhile, the cell viability, cell cycle, apoptosis, invasion and metastasis of hepatoma HepG2 cells were all significantly inhibited (P<0.05).CONCLUSION:miRNA145 dramatically inhibits viability, apoptosis, inva-sion and metastasis of hepatoma cells.