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AIM: To investigate the effect of the expression of miR-375 on the proliferation and invasion of choroidal melanoma(CM)MUM-2B cells.METHODS: MUM-2B cells were cultured and were transfected with miR-375 mimic sequence(mimic group), miR-375 inhibitor sequence(inhibitor group), negative control group and no treatment(blank group). The qRT-PCR, CCK-8, apoptosis and Transwell experiments were used respectively to detect the expression of miR-375, cell proliferation activity, apoptosis, cell migration and invasion.RESULTS: Compared with the negative control group(1.01±0.10)and the blank group(1.03±0.07), the expression level of miR-375 in the cells of the mimic group(2.65±0.15)was increased, while the expression level of miR-375 in the cells of the inhibitor group(0.28±0.06)was decreased(P<0.05). Compared with the blank group and negative control group, the OD values of the cells in the mimic group at 24, 48, 72, and 96h were decreased(P<0.05), while the OD values of the cells in the inhibitor group at 24, 48, 72, and 96h were increased(P<0.05). Compared with the apoptosis rates in the blank group and negative control group, which were(20.54±4.01)% and(22.80±4.28)%, the apoptosis rate in the mimic group(39.11±3.37)% was increased(P<0.05), while it was decreased in the inhibitor group(10.13±2.17)%(P<0.05). Compared with the blank group and negative control group, the number of migration cell and the number of invasion cell in the mimic group were decreased(P<0.05), while the number of migration cell and the number of invasion cell in the inhibitor group were increased(P<0.05). CONCLUSIONS: Up-regulating the expression of miR-375 in MUM-2B cells can reduce cell proliferation activity, accelerate cell apoptosis, and inhibit cell migration and invasion, while down-regulating the expression of miR-375 has the opposite effect. It indicates that miR-375 may play the function of tumor suppressor in the course of CM.
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Abstract Objective: This study aims to explore the effect and mechanism of miR-375 in Laryngeal Squamous Cell Carcinoma (LSCC) cell progression. Methods: LSCC cells (LSC-1 and TU177) were transfected with miR-375-mimic, miR-375-inhibitor or miR-375-mimic + oe-CST1. The expression of miR-375, CST1, MMP-2, and MMP-9 was measured. The effect of miR-375-mimic, miR-375-inhibitor or miR-375-mimic + oe-CST1 on cell biological functions, including cell proliferation, migration, invasion, and apoptosis, was also assessed. The potential relationship between CST1 and miR-375 was predicted by Jefferson software and validated by dual luciferase reporter gene assay. Results: Downregulated miR-375 expression was found in LSCC cells. Overexpression of miR-375 inhibited the viability and migration and promoted apoptosis of LSCC cells. Jefferson database and dual luciferase reporter gene assay confirmed that miR-375 directly targeted CST1. Over-expression of CST1 could reverse the anti-cancer effect of miR-375 overexpression in LSCC cells. Conclusion: Collected evidence showed that miR-375/CST1 axis was implicated in LSCC progression. Level of evidence: Level 3
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Objective:To investigate the effect of miR-375-3p on DNA damage repair and radioresistance of colorectal cancer cells.Methods:After overexpression of miR-375-3p in HCT116 and HT29, cell proliferation ability was detected by CCK-8 assay, clone formation ability was detected by clone formation assay, apoptosis was detected by Annexin V-FITC/PI double staining method and cell cycle distribution was detected by flow cytometry, and the formation of γ-H2AX foci were used to analyze homologous recombination (HR) repair efficiency. Bioinformatics was used to predict the downstream target genes of miR-375-3p in the HR repair pathway. A dual luciferase reporter gene assay was used to validate the regulation effect of miR-375-3p on Rad51 gene. The expression of miR-375-3p in HCT116 cells irradiated with 60Co γ-rays at 2 and 6 Gy was measured by RT-qPCR. The inhibition effect of miR-375-3p on the radiosensitivity of HCT116 cells was analyzed after irradiation with different doses of 0, 1, 2, 4 and 6 Gy. Results:Overexpression of miR-375-3p inhibited the proliferation and colony formation ability, induced G1 phase cycle arrest and cell apoptosis of colorectal cancer cells, enhanced DSBs formation, inhibited Rad51 expression, and significantly decreased HR repair efficiency ( t = 10.055, P < 0.05). Dual luciferase reporter gene assay demonstrated that miR-375-3p bound to Rad51 3′UTR region ( t = 5.013, P< 0.05). In addition, irradiation increased miR-375-3p expression, and inhibition of miR-375-3p expression reduced radiosensitivity of colorectal cancer cells ( t=6.460, 5.619, 10.150, P<0.05). Conclusions:miR-375-3p inhibited the homologous recombination repair efficiency of DSBs and enhanced the radiosensitivity of colorectal cancer cells.
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This study was designed to prepare a novel lipid bilayer coated hollow mesoporous silica nanocarrier for co-delivery of gene drugs and chemotherapeutic drugs to enhance the inhibitory activity of antitumor drugs in hepatoma cells. Hollow mesoporous silica was synthesized by modified StÖber method. Lipid-fusion principle was used to prepare lipid-hollow co-loaded doxorubicin (DOX) and miR-375 (LHMSN-DOX/miR-375). Meanwhile, the morphology, particle size, surface potential, drug loading and release were characterized in vitro. The inhibition of cell proliferation, cell migration and invasion was then evaluated. The results indicated that the core-shell structure of LHMSN-DOX/miR-375 was clear with an intact outer lipid membrane and an ordered internal HMSN mesoporous structure. The drug release amount was pH responsive while the drug was rapidly released under simulated intracellular acidic conditions relative to normal physiological environment. Compared with free DOX, LHMSN-DOX/miR-375 can deliver DOX and miR-375 to liver cancer cells and inhibit the proliferation, migration and invasion of cells more effectively.
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Objective:Abnormal angiogenesis is an important hallmark of HCC. Ectopic miR-375 overexpression led to repression of proliferation, migration, invasion, and colony formation, and it induced apoptosis in hepatoma cells as well. In this study, we explored the effect of miR-375 on HCC angiogenesis. Methods:We evaluated the antiangiogenic effects of miR-375 using human umbilical vein endothelial cells, tube formation assays, rat aortic ring sprouting assays, and chicken chorioallantoic membrane assays. Bioinformatics software was used to predict the downstream target gene of miR-375. MiR-375 regulation to target genes was explored by overexpres-sion and knockdown of miR-375 in hepatoma cells. Luciferase assay was performed to confirm its molecular mechanism. Rescue assay of target gene was further used to prove that miR-375 inhibited HCC angiogenesis by directly regulating its target gene. Results:MiR-375 inhibited HCC angiogenesis. Platelet-derived growth factor-C (PDGFC) was a potential target gene of miR-375. MiR-375 inhibited PDGFC expression in hepatoma cells by targeting its 3′-UTR. MiR-375 exerted its antiangiogenic effect partially by PDGFC inhibition. Conclusion:MiR-375 repressed tumor angiogenesis by targeting PDGFC in HCC.
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Objective: To investigate the role of microRNA-375 (miR-375) in the occurrence of trastuzumab-resistance in human epidermal growth factor receptor 2 (HER2)-positive breast cancer cells through regulating epithelial-mesenchymal transition (EMT). Methods: The sensitivities of HER2-positive breast cancer cell lines SK-BR-3 (a parental sensitive cell line) and SK-BR-3R (a trastuzumab-resistant cell line) to trastuzumab were detected by MTT assay. The expression levels of miR-375 and EMT-associated proteins E-cadherin and vimentin in the two cell lines were detected by real-time fluorescent quantitative PCR and Western blotting, respectively. After miR-375 mimic was transfected into SK-BR-3R cells, the changes of miR-375, E-cadherin and vimentin expression levels were detected by real-time fluorescent quantitative PCR and Western blotting, and the change of trastuzumab sensitivity of SK-BR-3R cells was detected by MTT method. The potential target genes of miR-375 were predicted by bioinformatics software, and metadherin (MTDH) was selected as one of target genes. Luciferase reporter assay was conducted to verify the role of miR-375 in regulation of MTDH gene transcription. In addition, the changes of E-cadherin and vimentin expression levels in SK-BR-3R cells after transfection with MTDH siRNA were detected by Western blotting. Results: Compared to the sensitive SK-BR-3 cells, the sensitivity of SK-BR-3R cells to trastuzumab decreased significantly (P < 0.001), and the expression levels of miR-375 (P < 0.001) and vimentin (P < 0.05) were significantly down-regulated, but the level of EMT marker E-cadherin was significantly up-regulated (P < 0.05). After transfection with miR-375 mimic, the expression levels of miR-375 (P < 0.001) and vimentin (P < 0.01) were up-regulated, while the level of E-cadherin was down-regulated (P < 0.001); the trastuzumab sensitivity of SK-BR-3R cells was increased (P < 0.001). A negative regulatory effect of miR-375 on the transcription of MTDH gene was observed (P < 0.001). After MTDH-siRNA transfection, the expressions of MTDH and vimentin were down-regulated (both P < 0.001), while the expression of E-cadherin was up-regulated (P < 0.001). Conclusion: miR-375 plays a role in drug-resistance of HER2-positive breast cancer cells to trastuzumab through regulating EMT, which is related to the target regulation of MTDH gene expression.
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Objective MiR-375 is lowly expressed in esophageal squamous cancer cells and the downstream target gene of miR-375 remains unclear .This paper discusses the role of miR-375 in regulating the expression of short stature homobox 2 ( SHOX2) in human esophageal squamous cancer cells . Methods The bioinformatics software TargetScan , miRanda, PicTar, miRTarget2, and PITA were used to predict the assumptive targets of miR-375 in SHOX2.Then, two recombinant luciferase gene report plasmids containing wild pSHOX2 3′UTR ( pSHOX2-375-WT ) and mutant pSHOX2 3′UTR ( pSHOX2-375-mut ) were constructed , sequenced , and identified.Human esophageal squamous cancer cells were co-transfected with miR-375 mimics and pSHOX2-375-WT or pSHOX2-375-mut, respectively , and divided into 7 groups: pmiR, pSHOX2-375-WT, pSHOX2-375-WT +miR-375, pSHOX2-375-WT +miR-NC, pSHOX2-375-mut, pSHOX2-375-mut+miR-375, and pSHOX2-375-mut+miR-NC, each subjected to the measurement of luciferase activity .The expressions of SHOX 2 mRNA and protein were de-termined after transfection of the esophageal squamous cancer cells with miR-375 mimics, and so were the expressions of miR-375 and SHOX2 in the esophageal squamous carcinoma tissue samples obtained postoperatively . Results Prediction with the five software showed only one conserved function site of miR-375 in SHOX2 3′UTR at 1156-1170 bp.Luciferase activity was significantly lower in the pSHOX2-375-WT+miR-375 group (0.261 ±0.036) than in the pmiR (1.818 ±0.061), pSHOX2-375-WT (1.820 ±0.086), pSHOX2-375-WT+miR-NC (1.851 ±0.094), pSHOX2-375-mut (1.861 ±0.059), pSHOX2-375-mut +miR-375 (1.896 ± 0.048), and pSHOX2-375-mut+miR-NC group (1.760 ±0.062) ( P<0.01).SHOX2 mRNA and protein expressions were sup-pressed by the overexpression of miR-375 in the EC9706 cells.The expression of miR-375 was decreased, while that of SHOX2 in-creased in the esophageal squamous carcinoma tissue as compared with the normal esophageal tissue . Conclusion MiR-375 regu-lates the expression of the SHOX 2 gen in esophageal squamous cancer cells .
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Objective To investigate the effect of miR-375 inhibited by 2'-O-me-375 on lipoapoptosis of NIT-1 pancreatic β cells. Methods NIT-1 cells were divided and treated according to the optimal condition: mock (without lipofectamine) ,lipofectamine( transfected only with lipofectamine) ,NC-miRNA (transfected with negative control miRNA) ,and 2'-O-me-375( transfected with 2'-O-me-375) groups. 72 hours later, all cells in each group were cultured with 500 μmol/L palmitate for 48 h. The percentage of apoptotic cells was detected by Hochest33342 staining and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL). The protein expression of myotrophin ( V1 ) , a target gene of miR-375, was detected by Western blotting. Results Compared to the other three groups,the cell apoptosis rate of 2'-O-me-375 group was the lowest (P<0.01) .along with the highest VI expression level(P<0. 01). Conclusion Inhibition of miR-375 decreases pancreatic (3-cell lipoapoptosis.