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@#[摘 要] 目的：探讨miR-125a-5p通过调控Bcl-2相关永生基因4（Bcl-2-associated athanogene 4，BAG4）的表达抑制胃癌细胞迁移和侵袭的分子机制。方法：选用2014年1月至2015年12月兰州大学第一医院手术切除的82例胃癌组织标本及配对的癌旁组织以及人胃癌细胞系MGC803、BGC823、SGC7901、HGC27及人胃黏膜上皮细胞（GES-1），qPCR法检测胃癌组织、癌旁组织及胃癌细胞系中miR-125a-5p的表达水平。分别将miR-125a-5p mimic、miR-125a-5p inhibitor、（si-BAG4）siRNA-BAG4及阴性对照质粒转染至胃癌细胞，划痕愈合实验和Transwell侵袭实验分别检测miR-125a-5p/BAG4信号轴对胃癌细胞迁移和侵袭能力的影响。WB检测胃癌细胞中BAG4蛋白的表达。荧光素酶报告基因实验验证miR-125a-5p和BAG4之间的靶向调控关系。结果：miR-125a-5p在胃癌组织和细胞系中均低表达（均P<0.01）。miR-125a-5p的表达与患者的性别（P=0.953）、年龄（P=0.772）、肿瘤部位(P=0.867)、组织学分级（P=0.745）和肿瘤大小（P=0.088）无相关性，与胃癌患者的T分期（P=0.003）、N分期（P=0.001）、M分期（P=0.027）和TNM分期（P=0.035）显著相关，差异有统计学意义。miR-125a-5p低表达是胃癌患者总生存时间的独立危险因素。过表达miR-125a-5p显著抑制胃癌细胞的迁移和侵袭能力（均P<0.01）。敲降BAG4可逆转miR-125a-5p inhibitor对胃癌细胞迁移和侵袭能力的抑制作用。荧光素酶报告基因实验证实miR-125a-5p可与BAG4 3'非翻译区（untranslated regions，UTR）结合抑制其表达。结论：miR-125a-5p通过靶向下调BAG4的表达水平进而抑制胃癌细胞的迁移和侵袭。
ABSTRACT
@#[Abstract] Objective:ToinvestigatetheroleofmiR-125a-5pininducingthegefitinib(Gef)-resistance of non-small cell lung carcinoma (NSCLC) cells and its possible mechanism. Methods: Human NSCLC drug-resistant cell line A549/GR and NSCLC cell line A549 were chosen for this study. miR-125a-5p mimic, miR-125a-5p inhibitor, pcDNA3.1-APAF1 and empty vector pcDNA3.1 were transfected into A549/GR cells. The expression level of miR-125a-5p in cell lines was detected by qPCR. MTT, Transwell and Flow cytometry were used to detect the effects of Gef on proliferation, migration and apoptosis of cell lines, respectively. The targeting relationship between miR-125a-5p and APAF1 (apoptotic peptidase activating factor 1) was verified by Dual-luciferase reporter gene system. In addition, the expression of APAF1 protein in A549/GR cells was detected by Western blotting. The expression levels of caspase-3 and caspase-9 were assessed by colorimetry. Results: Expression level of miR-125a-5p was upregulated significantly in Gefresistant A549/GR cells (P<0.01). AndtheinfluencesofGefonA549/GRcellswereenhancedby knockdown of miR-125a-5p, including inhibiting cell proliferation and migration (all P<0.05) and inducing apoptosis (P<0.01). Dual luciferase reporter gene assay confirmed that miR-125a-5p targeted APAF1 and negatively regulated its expression. Furthermore, by targetedly downregulating APAF1, miR-125a-5p alleviated the inhibition of proliferation and migration (all P<0.05) and promotion of apoptosis (P<0.05) of A549/GR cells caused by Gef, and attenuated Gef-induced upregulation of apoptosis-related proteins caspase-3 and caspase-9 (all P<0.05). Conclusion: miR-125a-5p promotes Gef-resistance of A549/GR cells, and the underlying mechanisms are promotion of proliferation, migration and inhibition of apoptosis of non-small cell lung cancer cells by targetingAPAF1.
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@# Objective: To investigate the effects of miR-125a-5p targeting signal transducer and activator of transcription-3 (STAT3) on proliferation and invasion of renal cancer cells, and to preliminarily analyze the action mechanism. Methods: During the period from March 2017 to February 2018, 48 pairs of cancer tissues and corresponding normal adjacent tissues (more than 3 cm away from the tumor margin) resected from patients underwent renal cancer surgery at the Department of Urology, the Air Force Hospital of the Northern War Zone were collected for this study. Normal renal HK-2 cells and renal cancer cells (A498, GRC-1, 786-O and ACHN) were cultured in vitro. The expression of miR-125a-5p in above mentioned tissues and cells was detected by qPCR. miR-125a-5p-NC, miR-125a-5p-mimics, pLV-STAT3 and pLV-STAT3 with miR-125a-5p mimics were transfected intoA498 cells, namely NC group (negative control group), miR-125a-5p-mimics group, pLV-STAT3 group and pLV-STAT3+mimics group. The normally cultured A498 cells were used as blank control (Ctrl group). qPCR was performed to detect them RNA expressions of miR-125a-5p and STAT3 in cells of all groups. The bioinformatics prediction software and Dual luciferase assay were performed to analyze the targeting relationship between miR-125a-5p and STAT3. CCK-8, Flow cytometry, Transwell chamber assay were performed to detect cell proliferation activity, apoptosisand invasion, respectively. The expressions of STAT3, Bcl-2, BAX, cleaved cysteinyl aspartate specific proteinase 3 (cl-caspase-3), tumor suppressor gene p21, N-cadherin, E-cadherin, VEGF and HIF-1 in the cells were detected by WB. Results: The expression of miR-125a-5p in renal cancer tissues and cells was significantly lower than that in adjacent normal tissues and normal renal cells (all P<0.05). Compared with NC group, expression of miR-125a-5p in A498 cells transfected with miR-125a-5p-mimics was significantly increased, while expression of STAT3 mRNA was significantly decreased (all P<0.05). STAT3 was the target gene of miR-125a5p. Compared with NC group, cell viability, number of invasive cells, expressions of Bcl-2, N-cadherin, VEGF, HIF-1, and STAT3 as well as its phosphorylation level in miR-125a-5p mimics group were significantly decreased (all P<0.05), while cell apoptosis and expressions of BAX, p21, cl-caspase-3 and E-cadherin were significantly increased (all P<0.05); the cell viability, number of invasive cells, expressions of Bcl-2, N-cadherin, VEGF, HIF-1 and STAT3 as well as its phosphorylation level in pLV-STAT3 group were significantly increased (all P<0.05), while cell apoptosis and expressions of BAX, p21, cl-caspase-3 and E-cadherin were significantly decreased (all P<0.05). Compared with pLV-STAT3 group, cell viability, number of invasive cells, expressions of Bcl-2, N-cadherin, VEGF, HIF-1, and STAT3 as well asits phosphorylation level were significantly decreased in pLV-STAT3 mimics group (all P<0.05), while cell apoptosis, expressions of BAX, p21, cl-caspase-3 and E-cadherin were significantly increased (all P<0.05). Conclusion: miR125a-5p shows low expression in renal cancer tissues and cells, which can inhibit proliferation and invasion of A498 cells and promote cell apoptosis by down-regulating its target gene STAT3.
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Objective To investigate the expression of miR-125a-5p in diffuse large B-cell lymphoma and the relationship among the levels of NF-κB/p65,Bcl2,Caspase3 and Ki-67 protein in clinicopathological features, and the potential prognostic value. Methods A total of 84 cases of DLBCL were enrolled in this study. At the same time,10 cases of reactive hyperplasia tissues were used as the control group. The expression of miR-125a-5p was examined by SYBR Green real-time reverse transcriptase polymerase chain reaction(real-time RT-PCR). The expression of NF-κB/p65,Bcl2,Caspase3,Ki-67 was detected by the immunohistochemical staining assay. The clinical pathological data was collected and some patients were followed up. The clinical data was used for analysis of the prognosis of patients. Results MiR-125a-5p was shown up-regulated in the DLBCL samples ,which was (4.99 ± 8.22)times of that in the reactive hyperplasia samples(P=0.002). The up-regulation of miR-125a-5p in DLBCL was correlated with non-GCB subtype(P=0.027). The up-regulation of miR-125a-5p in DLBCL was sig-nificantly correlated with the expression of NF-κB/p65,Bcl2 and Caspase3(P=0.031,0.030,0.022). There was a significant correlation between non-GCB subtype and NF-κB/p65 level in DLBCL(P=0.002). Kaplan-Mei-er survival analysis showed that the high expression of miR-125a-5p was associated with the prognosis of DLBCL pa-tients(P=0.038). Multivariate Cox regression analysis showed that the high expression of miR-125a-5p may be an independent risk factor for the prognosis of DLBCL(P=0.013). Conclusions MiR-125a-5p may play an im-portant role in the diagnosis of DLBCL. The up-regulation of miR-125a-5p in DLBCL may affect the activation of NFκB/p65,especially in the non-GCB subtype. The up-regulation of miR-125a-5p in DLBCL may influence the de-velopment of tumor via apoptosis pathway. The expression of miR-125a-5p is associated with the poor prognosis of patients,which can be regarded as an independent risk factor for the prognosis of DLBCL.
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Objective To investigate the expression of miR-125a-5p in diffuse large B-cell lymphoma and the relationship among the levels of NF-κB/p65,Bcl2,Caspase3 and Ki-67 protein in clinicopathological features, and the potential prognostic value. Methods A total of 84 cases of DLBCL were enrolled in this study. At the same time,10 cases of reactive hyperplasia tissues were used as the control group. The expression of miR-125a-5p was examined by SYBR Green real-time reverse transcriptase polymerase chain reaction(real-time RT-PCR). The expression of NF-κB/p65,Bcl2,Caspase3,Ki-67 was detected by the immunohistochemical staining assay. The clinical pathological data was collected and some patients were followed up. The clinical data was used for analysis of the prognosis of patients. Results MiR-125a-5p was shown up-regulated in the DLBCL samples ,which was (4.99 ± 8.22)times of that in the reactive hyperplasia samples(P=0.002). The up-regulation of miR-125a-5p in DLBCL was correlated with non-GCB subtype(P=0.027). The up-regulation of miR-125a-5p in DLBCL was sig-nificantly correlated with the expression of NF-κB/p65,Bcl2 and Caspase3(P=0.031,0.030,0.022). There was a significant correlation between non-GCB subtype and NF-κB/p65 level in DLBCL(P=0.002). Kaplan-Mei-er survival analysis showed that the high expression of miR-125a-5p was associated with the prognosis of DLBCL pa-tients(P=0.038). Multivariate Cox regression analysis showed that the high expression of miR-125a-5p may be an independent risk factor for the prognosis of DLBCL(P=0.013). Conclusions MiR-125a-5p may play an im-portant role in the diagnosis of DLBCL. The up-regulation of miR-125a-5p in DLBCL may affect the activation of NFκB/p65,especially in the non-GCB subtype. The up-regulation of miR-125a-5p in DLBCL may influence the de-velopment of tumor via apoptosis pathway. The expression of miR-125a-5p is associated with the poor prognosis of patients,which can be regarded as an independent risk factor for the prognosis of DLBCL.
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Objective To identify the expression level of miR-125a-5p in colorectal cancer (CRC) with various differentiation,and to investigate its diagnostic significance.Methods Real-time polymerase chain reaction was used to analyze the expression level of miR-125a-5p in tumors and paired normal tissues of different differentiated CRC.Receiver-operating-characteristic (ROC) curve was performed to evaluate the specificity and sensitivity of using miR-125a-5p as biomarkers to discriminate CRC patients from normal persons.Results Compared with normal tissues,miR-125a-5p was down-regulated in both high and moderate differentiation,miR-125a-5p could distinguish CRC from normal persons with high sensitivity and specificity,which were 80.00 ％ and 90.00 ％,respectively.Conclusions miR-125a-5p down-regulated in CRC indicates that miR-125a-5p contributes to tumorigenesis,and may be a potential biomarker for CRC.