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@# Objective: To investigate the mechanism of miR-503 modulates radio-resistance of esophageal squamous cell carcinoma (ESCC) by targeting excision-repair cross-complementing 1 (ERCC1). Methods: The expression level of miR-503 in radio-resistant ESCC tumor tissues and KYSE140 and KYSE140R cells was detected by qPCR. The miR-503 mimic, miR-503 inhibitor or si-ERCC1 was transfected into KYSE140 and KYSE140R cells.After radiation treatment, the colony formation assay and CCK-8 assay were used to detect the proliferation of KYSE140R cells. Flow cytometry was used to detect apoptosis of KYSE140R cells. WB was used to detect changes in protein expression of ERCC1. Dual luciferase reporter gene assay was used to validate the targeting relationship between miR-503 and ERCC1. Results: The expression level of miR-503 was down-regulated in radio-resistant tissues and ESCC cell lines (all P<0.01). Over-expression of miR-503 significantly inhibited cell proliferation and promoted apoptosis of KYSE140R cells (all P<0.01). Dual-luciferase reporter assay validated that ERCC1 was a target gene of miR-503, and miR-503 negatively regulated the expression of ERCC1. Over-expression of miR-503 significantly down-regulated the expression of ERCC1 in KYSE140 and KYSE140R cells (both P<0.01), inhibited cell proliferation (both P<0.01), but significantly increased apoptosis rate (all P<0.01); knockdown of ERCC1 exhibited a similar effect, while knockdown of both ERCC1 and miR-503 reversed the above effects. Conclusion: Over-expression of miR-503 up-regulated the radio-sensitivity of KYSE140R cells by targeting ERCC1.
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Objective: To highlight the relationship between miR-503 and wound healing of diabetic foot ulcer (DFU). Methods: Microarray analysis was used to detect the dysregulated miRNAs between the DFU tissues and normal tissues. The expression of miR-503 in tissues and serum of patients with DFU was detected by qRT-PCR technique. Then, CCK-8 assay was applied to determine the cell proliferation. TUNEL assay was used for assessing the apoptosis of cells after treatment with miR-503. Possible correlation between miR-503 and fbillin1 (FBN1) was predicted according to data accessed on RNA22 website online, and was detected for confirmation by luciferase reporter assay. Results: Microarray analysis showed that miR- 503 was significantly decreased in the DFU tissues compared with normal tissues. While marked increase in the expression of miR-503 in tissues and serum of patients with DFU was confirmed by qRT-PCR technique. Then, CCK-8 assay indicated that transfection of miR- 503 mimic obviously accelerated the cell proliferation. However, TUNEL assays suggested that miR-503 mimic inhibited the apoptosis of cells to improve the survival of fibroblasts. Besides, miR-503 AMO played a role in fibroblasts of DFU tissues exactly countering to miR-503 mimic treatment. It was predicted that MiR-503 is a complementary to the FBN1 by RNA22. Besides, SiRNA-FBN1 promoted the proliferation, but brought down the apoptosis of fibroblasts. Conclusions: MiR-503 regulates the function of fibroblasts and wound healing of patients with DFU by targeting FBN1 directly which provids a novel and critical target for diagnosis and treatment of DFU.
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Objective:To highlight the relationship between miR-503 and wound healing of diabetic foot ulcer (DFU).Methods:Microarray analysis was used to detect the dysregulated miRNAs between the DFU tissues and normal tissues. The expression of miR-503 in tissues and serum of patients with DFU was detected by qRT-PCR technique. Then, CCK-8 assay was applied to determine the cell proliferation. TUNEL assay was used for assessing the apoptosis of cells after treatment with miR-503. Possible correlation between miR-503 and fbillin1 (FBN1) was predicted according to data accessed on RNA22 website online, and was detected for confirmation by luciferase reporter assay.Results:Microarray analysis showed that miR- 503 was significantly decreased in the DFU tissues compared with normal tissues. While marked increase in the expression of miR-503 in tissues and serum of patients with DFU was confirmed by qRT-PCR technique. Then, CCK-8 assay indicated that transfection of miR- 503 mimic obviously accelerated the cell proliferation. However, TUNEL assays suggested that miR-503 mimic inhibited the apoptosis of cells to improve the survival of fibroblasts. Besides, miR-503 AMO played a role in fibroblasts of DFU tissues exactly countering to miR-503 mimic treatment. It was predicted that MiR-503 is a complementary to the FBN1 by RNA22. Besides, SiRNA-FBN1 promoted the proliferation, but brought down the apoptosis of fibroblasts.Conclusions:MiR-503 regulates the function of fibroblasts and wound healing of patients with DFU by targeting FBN1 directly which provids a novel and critical target for diagnosis and treatment of DFU.
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Objective:To investigate effects of miR-503 on cisplatin sensitivity in BEL-7402 cells by targeting of bcl-2.Methods:MiR-503 and bcl-2 mRNA expression levels in hepatocellular carcinoma cells were measured by real-time quantitative (qRT)-PCR;Bcl-protein level was detected by Western blot;miR-503 mimics were transiently transfected to the BEL-7402 cells by liposome transfection;potential target genes of miR-503 were predicted by Bioinformatics software;miR-503 potential targets were validated by dual luciferase activity;and the cell viability was measured by MTT assay.Results:MiR-503 level was down-regulated and Bcl-2 protein expression level was up-regulated in BEL-7402 cells compared with HL-7702 cells.MiR-503 could interact with bcl-2 and inhibit its expression.Cell vitality with miR-503 transfection was significantly reduced compared to that in the negative control.Conclusion:MiR-503 may enhance the sensitivity of BEL-7402 cells to cisplatin and inhibit the cell proliferation by targeting bcl-2.
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Objective:To investigate the mechanism of BCYRN1 regulating miR-503 through Notch1 signaling pathway in the migration and invasion of lung cancer. Methods:The expression of BCYRN1 and miR-503 in different lung cancer cell lines were detected by qPCR. Immunofluorescence and qPCR were used to detect the transfection efficiency of lentivirus BCYRN1 + siRNA transfected lung cancer cells. Double luciferase reporter gene was used to detect the interaction between BCYRN1 and miR-503. Transwell invasion test and scratch test were used to detect the invasion and migration of lung cancer cells after silencing BCYRN1. Western blot was used to detect the expression of Notch1 signaling pathway silence BCYRN1. The effect of silencing BCYRN1 on lung cancer cells in nude mice was measured by subcutaneous tumor formation in nude mice. Results:The expression level of BCYRN1 was the highest in lung cancer cell H1299,and the expression level of miR-503 was relatively high. Immunofluorescence and mRNA levels demonstrated that BCYRN1 + siRNA lentivirus could be effectively transfected into H1299 cells;BCYRN19 binds specifically to the 3′-UTR of miR-503;silencing BCYRN1 could inhibit the invasion and migration of lung cancer H1299 cells;the expression of Notch1 pathway protein was down-regulated after silencing BCYRN1. Compared with NC group,tumor volume and weight of BCYRN1-siRNA group were significantly decreased. Conclusion:BCYRN1 can target the regulation of miR-503 through Notch1 signaling pathway in the invasion and migration of lung cancer H1299 cells.
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Objective:To investigate the mechanism of BCYRN1 regulating miR-503 through Notch1 signaling pathway in the migration and invasion of lung cancer. Methods:The expression of BCYRN1 and miR-503 in different lung cancer cell lines were detected by qPCR. Immunofluorescence and qPCR were used to detect the transfection efficiency of lentivirus BCYRN1 + siRNA transfected lung cancer cells. Double luciferase reporter gene was used to detect the interaction between BCYRN1 and miR-503. Transwell invasion test and scratch test were used to detect the invasion and migration of lung cancer cells after silencing BCYRN1. Western blot was used to detect the expression of Notch1 signaling pathway silence BCYRN1. The effect of silencing BCYRN1 on lung cancer cells in nude mice was measured by subcutaneous tumor formation in nude mice. Results:The expression level of BCYRN1 was the highest in lung cancer cell H1299,and the expression level of miR-503 was relatively high. Immunofluorescence and mRNA levels demonstrated that BCYRN1 + siRNA lentivirus could be effectively transfected into H1299 cells;BCYRN19 binds specifically to the 3′-UTR of miR-503;silencing BCYRN1 could inhibit the invasion and migration of lung cancer H1299 cells;the expression of Notch1 pathway protein was down-regulated after silencing BCYRN1. Compared with NC group,tumor volume and weight of BCYRN1-siRNA group were significantly decreased. Conclusion:BCYRN1 can target the regulation of miR-503 through Notch1 signaling pathway in the invasion and migration of lung cancer H1299 cells.
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Objective To study whether or not CD40 gene is a target of miR-503 in U937 cells,and to observe the regulatory effects of miR-503 on expression of CD40 induced by irradiation in U937cells.Methods The miR-503 sequence was inserted into pcDNA-DEST-47 plasmid to construct the eukaryotic expression vector (pcDNA-DEST-miR-503) and to construct the CD40 gene 3′-UTR luciferase reporter plasmid (psiCHECK2-CD40) at the same time.They were used to transfect U937 cells together for analysis of the regulatory effects of miR-503 on the expression of CD40.Meanwhile the miR-203 and miR-29b were used as controls to observe whether or not CD40 gene was a target of miR-503.The expression change of CD40 after irradiation,and the inhibitory effect of miR-503 on the expression of CD40 was confirmed by Western blot assay.The expression of miR-503 was detected by real-time RT-PCR (qPCR) after irradiation with doses of 5.0 Gy.Results The expression of luciferase in the group of transfected with pcDNA-DEST-miR-503 and psiCHECK2-CD40 plasmids was significantly lower than that in the group transfected with empty plasmid and CD40 gene (t =3.16,P ＜ 0.05).And the miR-203 and miR-29b could not inhibit the activity of CD40 luciferase,but the miR-503 could significantly inhibit the activity of it (t =5.25,P ＜ 0.01 ).The expression of CD40 protein in U937 cells was decreased after the cells were transfected with pcDNA-DEST-miR-503.After irradiation with dose of 5.0 Gy,the expression of miR-503 were increased (t =3.63 - 17.00,P ＜0.01 ) and the expression of CD40 protein decreased.Conclusions The CD40 gene might be the target of miR-503,and miR-503 could regulate the expression of CD40 gene.Irradiation could up-regulate the expression of miR-503 and inhibit the expression of CD40 protein in U937 cells.miR-503 might play a role in the process of regulation of irradiation on expression of CD40 protein in tumor cells.