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Objective:To detect the expression level of long non-coding RNA (lncRNA) nuclear receptor subfamily 2 group F member 2-antisense RNA 1 (NR2F2-AS1) and microRNA-129-5p (miR-129-5p) in glioma tissues and to explore its clinical significance.Methods:The glioma tissues of 103 patients with glioma who underwent surgical treatment in the neurosurgery department of Shanxi Provincial People′s Hospital from January 2015 to September 2016 and 50 normal brain tissues removed due to craniocerebral surgery in the same period were selected as the research objects. Real time fluorescence quantitative polymerase chain reaction (qRT-PCR) was used to detect the expression levels of lncRNA NR2F2-AS1 and miR-129-5p in tissue samples. The correlation between the two indexes and the relationship between the expression changes of the two and clinicopathological parameters of glioma patients were analyzed. Kaplan Meier curve was used to analyze the 5-year cumulative survival rate of glioma patients with different expression levels of lncRNA NR2F2-AS1 and miR-129-5p. Cox regression analysis was used to analyze the factors affecting the poor prognosis of glioma patients.Results:Compared with normal brain tissue, the relative expression of lncRNA NR2F2-AS1 in glioma was higher and the relative expression of miR-129-5p was lower (all P<0.05). There was significant negative relationship between the relative expression of lncRNA NR2F2-AS1 and miR-129-5p in glioma ( r=-0.756, P<0.05). The expression of lncRNA NR2F2-AS1 and miR-129-5p in glioma was related to World Health Organization (WHO) grade and tumor length (all P<0.05). The 5-year cumulative survival rate of patients in lncRNA NR2F2-AS1<2.89 group was higher than that in lncRNA NR2F2-AS1≥2.89 group, and the 5-year cumulative survival rate of patients in miR-129-5p<0.55 group was lower than that in miR-129-5p≥0.55 group (all P<0.05). Multivariate analysis showed that high expression of lncRNA NR2F2-AS1, low expression of miR-129-5p, WHO grade Ⅲ-Ⅳ and tumor length were the risk factors affecting the prognosis of patients with glioma (all P<0.05). Conclusions:The increased expression of lncRNA NR2F2-AS1 and the decreased expression of miR-129-5p in glioma tissues are involved in the clinical biological progress of glioma and are closely related to the prognosis of patients.
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Objective To investigate the effects of miR-129-5p on the proliferation and migration of osteosarcoma cells and the regulation of HMGB1 gene. Methods The expression of miR-129-5p and HMGB1 in osteosarcoma cell line MG-63, Saos-2 and osteoblast hFOB1.19 were detected by RT-PCR and Western blot. Bioinformatics methods were used to predict whether there were binding sites between mir-129-5p and HMGB1 gene. Double luciferase reporter gene system was used to verify the interaction between miR-129-5p and the target gene HMGB1. miR-129-5p mimic and inhibitor were transfected into osteosarcoma cell lines with low and high miR-129-5p expression, respectively, and the transfection efficiency was detected by RT-PCR. After successful transfection, the proliferation and migration of osteosarcoma cell lines were detected by CCK-8 assay, scratch assay and Transwell migration assay, respectively, and Western blot was used to detect the expression of HMGB1 in the transfected osteosarcoma cell lines. Results Expression of miR-129-5p in osteosarcoma cells was lower than that in normal osteoblasts (P < 0.05), and the expression of HMGB1 in osteosarcoma cell lines was higher than that in normal osteoblasts (P < 0.05). There were binding sites between miR-129-5p and HMGB1 genes, and the luciferase activity of HMGB1-WT plasmid group was down-regulated after transfection with miR-129-5p mimic (P < 0.05). Transfection of miR-129-5p mimic significantly increased the expression of miR-129-5p in MG-63 cells (P < 0.05), inhibited the proliferation and migration of MG-63 cells (P < 0.05), and decreased the expression level of HMGB1. After transfection with miR-129-5p inhibitor, the expression of miR-129-5p in Saos-2 cells was significantly decreased (P < 0.05), the proliferation and migration abilities of Saos-2 cells were enhanced (P < 0.05), and the expression level of HMGB1 was also increased. Conclusion miR-129-5p may inhibit the proliferation and migration of osteosarcoma cells through HMGB1 gene.
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@#[Abstract] Objective: To observe the expression of long-chain non-coding RNA (lncRNA) TPTEP1 in bladder cancer tissues and cells, and to observe its effect on the proliferation and invasion of bladder cancer cells and its molecular mechanism. Methods: From August 2017 to October 2019, 43 cases of bladder cancer tissues and paracancer tissues from the patients treated by surgery in the Department Urology, People's Hospital of Dongxihu Distric of Wuhan City. Real-time fluorescence quantitative polymerase chain reaction (qPCR) was used to detect the expression of lncRNA TPTEP1 in bladder cancer tissues and bladder cancer cell lines (T24, BIU-87, 5637, J82, UM-UC-3). The bladder cancer cells with the lowest lncRNA TPTEP1 expression were selected as the experimental object, and transfected with the negative control plasmid (the control group) and lncRNA TPTEP1 over-expression plasmid (the experimental group), respectively. The effect of lncRNA TPTEP1 upregulation on cell proliferation and invasion was detected by MTT method and Transwell experiment. Bioinformatics techniques were used to predict the possible target molecules of lncRNA TPTEP1. qPCR and WB were used to detect the expression levels of lncRNA TPTEP1 downstream molecules. Results: Compared with adjacent tissues, the expression of lncRNA TPTEP1 in bladder cancer tissues was down-regulated (P<0.01). Compared with normal bladder epithelial cells, the expression of lncRNA TPTEP1 in bladder cancer cell lines was down-regulated (P<0.05), and its expression in T24 cells was the lowest (P<0.01). Up-regulation of lncRNA TPTEP1 could inhibit the proliferation (P<0.05) and invasion (P<0.01) of T24 cells. Bioinformatics technology showed that lncRNA TPTEP1 could bind with miR-129-5p, and miR-129-5p could bind with EMP3; up-regulating lncRNA TPTEP1 could inhibit the expression of miR-129-5p in T24 cells (P<0.01), and indirectly promote the mRNA and protein expressions of EMP3 (P<0.01) in T24 cells. The expression of MAPK/ERK signaling pathway related proteins such as p-MEK, p-ERK1/2, p-AKT and p-PI3K decreased (P<0.01). Conclusion: Up-regulating the low-expressed lncRNA TPTEP1 in bladder cancer cell lines can inhibit the proliferation and invasion of bladder cancer T24 cells, and its mechanism is related to indirect promotion of EMP3 gene expression by down-regulating the expression of miR-129-5p.
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Abstract Deregulation of miRNA expressions was identified as a novel feature of tumor biology in Ewing sarcoma (EWS). The aim was to evaluate the regulatory role of miR-129-2-3p in EWS cell lines and human EWS tissue samples. EWS cell lines TC-71, TC-106, and CHLA-99 were used in the study and real-time PCR was utilized to investigate the functional role of tumor suppressor mir-129-2-3p and miR-129-2-3p levels in the cells. Proliferation, migration, invasion and apoptosis assays were carried out within the scope of functional in vitro studies. Expression levels of CDK6 and SOX4, which are miR-129-2-3p target genes, were examined. Moreover, the change in expression levels of miR-129-2-3p in EWS tumor tissues was also examined. It was determined that miR-129-2-3p expression markedly diminished in all the studied cell lines. In addition, miR-129-3p was found to decrease in proliferation, migration, invasion and apoptosis assays in all EWS cell lines. CDK6 and SOX4 levels were also decreased in miR-129-2-3p transfected cell lines. It was found that miR-129-2-3p levels were significantly decreased in EWS tumor tissue samples compared to the corresponding adjacent normal tissue samples. In line with the results of our current study, where the possible function of miR‐129-2-3p in EWS cell lines was examined, for the first time in the literature miR-129-2-3p was shown to have low expression level in EWS lines and EWS tumor tissue samples, and to provide a tumor suppressor effect.
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OBJECTIVE@#To explore the pathogenesis of gastric cancer through a bioinformatic approach to provide evidence for the prevention and treatment of gastric cancer.@*METHODS@#The differentially expressed genes (DEGs) in gastric cancer and normal gastric mucosa in GSE79973 dataset were analyzed using GEO2R online tool. GO analysis and KEGG pathway enrichment analysis of the DEGs in DAVID database were performed. The protein interaction network was constructed using STRING database, and the key genes (Hub genes) were screened and their functional modules were analyzed using Cytoscape software. The GEPIA database was used to validate the Hub genes, and the Target Scan database was used to predict the microRNAs that regulate the target genes; OncomiR was used to analyze the expressions of the microRNAs in gastric cancer tissues and their relationship with the survival outcomes of the patients.@*RESULTS@#A total of 181 DEGs were identified in gastric cancer, and 10 hub genes were screened by the protein- protein interaction network. Functional analysis showed that these DEGs were involved mainly in protein digestion and absorption, PI3K-Akt signaling pathway, ECM-receptor interaction and platelet activation signal pathway. GEPIA database validation showed that COL1A1 was highly expressed in gastric cancer tissues and was associated with a poor prognosis of patients with gastric cancer. MiR-129-5p was found to bind to the 3'UTR of COL1A1 mRNA, and compared with that in normal tissues, miR-129-5p expression was obviously down-regulated in gastric cancer tissues, and was correlated with the prognosis of the patients.@*CONCLUSIONS@#COL1A1 under regulation by MiR-129-5p is a potential therapeutic target for gastric cancer.
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Humans , Collagen Type I , Computational Biology , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , MicroRNAs , Therapeutic Uses , Phosphatidylinositol 3-Kinases , Stomach Neoplasms , Drug TherapyABSTRACT
Objective: To investigate the effects of microRNA-129-5p (miR-129-5p) on the proliferation and apoptosis of osteosarcoma cells, and to explore the possible molecular mechanism. Methods: The expression of miR-129-5p in osteosarcoma MG63 cells and osteoblasts hFOB1.19 cells was determined by real-time fluorescent quantitative PCR (RFQ-PCR). After miR-129-5p mimics were transfected into osteosarcoma MG63 cells, the expression of miR-129-5p was detected by RFQ-PCR to verify the transfection efficiency. CCK-8 assay and plate clony formation experiment were used to detect the cell proliferative ability, while FCM was performed to analyze the cell apoptosis. Double luciferase reporter gene system was used to verify the interaction between miR-129-5p and the target gene p21-activated kinase 5 (PAK 5). The expressions of PAK5 mRNA and protein in MG63 cells with miR-129-5p overexpression were tested by RFQ-PCR and Western blotting, while the expression levels of phospho-glycogen synthase kinase-3β (p-GSK3β) and β-catenin were detected by Western blotting. After siRNA-PAK5, PAK5 overexpressed plasmids and miR-129-5p mimics＋ PAK5 overexpressed plasmids were separately transfected into osteosarcoma MG63 cells, the above methods were used to further verify the molecular mechanism of miR-129-5p regulating the proliferation and apoptosis of osteosarcoma cells. Results: The level of miR-129-5p in MG63 cells was lower than that in hFOB1.19 cells (P < 0.05). After the transfection with miR-129-5p mimics, the proliferation and clony formation abilities of MG63 cells were decreased (P < 0.001, P < 0.01), while the apoptosis rate was increased (P < 0.05). miR-129-5p regulated negatively the expression of PAK 5 gene (P < 0.05). After the transfection with miR-129-5p mimics or siRNA-PCK5, the expression levels of PAK5 mRNA and protein were decreased (all P < 0.01), while the expressions of p-GSK-3β and β-catenin proteins were also decreased (both P < 0.05, both P < 0.01). The overexpression of PAK5 could restore the effects of miR-129-5p on proliferation and apoptosis of osteosarcoma cells (both P < 0.05). Conclusion: miR-129-5p can inhibit the proliferation ability of osteosarcoma cells, and promote apoptosis by down-regulating the expression of PAK 5 gene. The mechanism may be associated with the expression regulation of p-GSK-3β and β-catenin proteins.
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@# Objective: To investigate the effects and mechanisms of miR-129-5p on invasion, migration and epithelial-mesenchymal transition (EMT) of cervical cancer HeLa cells. Methods:Cervical cancer HeLa cells were selected. The target gene of miR-129-5p was screened by bioinformatics prediction software, and the targeting relationship between miR-129-5p and MAPK1 was verified by dual luciferase reporter gene assay. HeLa cells were transfected with miR-129-5p mimic, miR-129-5p inhibitor and pcDNA-MAPK1 alone or in combination.The expressions of miR-129-5p and MAPK1 in HeLa cells were detected by qPCR; the invasion and migration ability of HeLa cells were detected by Transwell and scratch-healing experiments, respectively; and the expressions of E-cadherin, N-cadherin, MAPK1, STAT3 and Bcl-xL were detected by WB. The subcutaneous xenograft model of HeLa cells in nude mice was constructed to observe the effect of miR-129-5p over-expression on the growth of transplanted tumors. The expressions of EMT and MAPK1 pathwayrelated proteins in transplanted tumor tissues were detected by WB. Results: miR-129-5p could bind with the 3'UTR region of MAPK1, and over-expression of miR-129-5p targetedly inhibited the expression of MAPK1 (P<0.01). Compared with the control group, the number of invasive cells in the miR-129-5p mimic group decreased (P<0.01), the scratch healing rate decreased (all P<0.01); The expression of E-cadherin was up-regulated, and the expressions of N-cadherin, MAPK1, STAT3 and Bcl-xL were down-regulated (all P< 0.01), while co-transfection of MAPK1 reversed the above phenomenon.The nude mice HeLa cell xenograft model was successfully established. Compared with the control group, the tumor mass of the miR-128-3p mimic group was reduced; the expression of E-cadherin was up-regulated in tumor tissues, while the expressions of N-cadherin, MAPK1, STAT3 and Bcl-xL were down-regulated (all P<0.01). Conclusion: Over-expression of miR-129-5p inhibits invasion, migration and epithelial-mesenchymal transition of cervical cancer HeLa cells by targeting MAPK1.
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Objective To explore whether MiR-129-5p participates in radiosensitivity of medullary thyroid cell MZ-CRC-1 by inhibiting the gene expression of high mobility group protein B1 ( HMGB1) . Methods The radioresistant cell line MZ-CRC-1/R was established from MZ-CRC-1. Cell survival fraction was analyzed by colony formation assay. The expressions of miR-129-5p in MZ-CRC-1 and MZ-CRC-1/R cells were detected by qRT-PCR. Cell viability was determined by MTT assay. Cell apoptosis was measured by flow cytometry. Dual-luciferase reporter assay was performed to confirm the relationship between miR-129-5p and HMGB1. Besides, the protein expressions of HMGB1 and p-AKt were evaluated by western blot. Results Compared with that of MZ-CRC-1 cells, the survival fraction of MZ-CRC-1/R cells was significantly increased (t=3. 038, 4. 330, 4. 885, 4. 568, P<0. 05), the cell viability of MZ-CRC-1/R cells was also increased ( t=3. 637, 7. 734, 11. 896, 14. 522, P<0. 05) , and the expression of miR-129-5p(0.26±0.03) was significantly decreased in MZ-CRC-1/R cells(1.00±0.06) (t=19. 107, P<0. 05) . Compared with miR-NC-inhibitor group, cell viability was promoted and cell apoptosis was blocked in the miR-129-5p-inhibitor group ( t=5. 156, 6. 005, 9. 649, 8. 659, P<0. 05) . Moreover, miR-129-5p mimic suppressed cell viability and enhanced cell apoptosis after irradiation ( t=3. 118, 5. 034, 6. 005, 7. 488, 6. 362, P<0. 05) . Overexpression of miR-129-5p inhibited the protein expressions of HMGB1 and p-AKt (t=9. 325, 10. 614, P<0. 05). In addition, HMGB1 depletion rescued cell apoptosis that was reduced by miR-129-5p inhibitor in MZ-CRC-1 cells ( t=6. 700, P<0. 05) , while HMGB1 overexpression attenuated the effect of miR-129-5p upregulation on MZ-CRC-1/R cells ( t=7. 073,P<0. 05) . Conclusions miR-129-5p increased the radiosensitivity of medullary thyroid-like cell MZ-CRC-1 by inhibiting HMGB1.
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Objective The aim of this study was to investigate the effect of miR-129 on the proliferative activity and apop-tosis of osteosarcoma MG-63 cells. Methods Thirty cases of osteosarcoma and its adjacent paracancerous tissues were collected. RT-PCR was used to detect the expressions of miR-129 mRNA and SEPHS1 mRNA. Western blot was used to detect the protein level of SEPHS1. After MiR-129 was over-expressed and knocked down,the cell proliferation was detected in MG-63 cells by CCK-8,and apoptosis was detected in MG-63 cells by Westerm blot and hoechest staining. Results In this study,the expression of miR-129 in osteosarcoma tissues was significantly higher than that in para-cancerous tissues(P<0. 05). Through the established model in vitro of miR-129 overexpression or knockdown,it was determined that the miR-129 mimetic and inhibitor concentrations were optimal for overexpression and knockdown at 50 nM and 200 nM, respectively. The possibility of binding between miR -129 and SEPHS1 was predicted by software,and the luciferase reporter assay further confirmed the binding relationship between miR-129 and SEPHS1. Knockdown of miR-129 significantly inhibited the proliferation of MG-63 cells and accelerated the apoptosis of MG-63 cells. Conclusion miR -129 plays a key role in regulating the proliferation and apoptosis of osteosarcoma cells by binding to SEPHS1.
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Although Taxol has improved the survival of cancer patients as a first-line chemotherapeutic agent, an increasing number of patients develop resistance to Taxol after prolonged treatment. The potential mechanisms of cancer cell resistance to Taxol are not completely clear. It has been reported that microRNAs (miRNAs) are involved in regulating the sensitivity of cancer cells to various chemotherapeutic agents. In this study, we aimed to explore the role of miR-129-5p in regulating the sensitivity of breast cancer cells to Taxol. Cell apoptosis and autophagy, and the sensitivity of MCF-7 cells to Taxol were assessed with a series of in vitro assays. Our results showed that the inhibition of autophagy increased the Taxol-induced apoptosis and the sensitivity of MCF-7 cells to Taxol. Up-regulation of miR-129-5p also inhibited autophagy and induced apoptosis. Furthermore, miR-129-5p overexpression increased the sensitivity of MCF-7 cells to Taxol. High mobility group box 1 (HMGB1), a target gene of miR-129-5p and a regulator of autophagy, was negatively regulated by miR-129-5p. We found that interference of HMGB1 enhanced the chemosensitivity of Taxol by inhibiting autophagy and inducing apoptosis in MCF-7 cells. Taken together, our findings suggested that miR-129-5p increased the chemosensitivity of MCF-7 cells to Taxol through suppressing autophagy and enhancing apoptosis by inhibiting HMGB1. Using miR-129-5p/HMGB1/autophagy-based therapeutic strategies may be a potential treatment for overcoming Taxol resistance in breast cancer.
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Humans , Female , Breast Neoplasms/metabolism , Paclitaxel/metabolism , HMGB1 Protein/metabolism , MicroRNAs/metabolism , MCF-7 Cells/metabolism , Antineoplastic Agents, Phytogenic/metabolism , Autophagy/genetics , Breast Neoplasms/genetics , Breast Neoplasms/drug therapy , Gene Expression Regulation, Neoplastic/genetics , Up-Regulation/genetics , Paclitaxel/therapeutic use , Apoptosis/genetics , Drug Resistance, Neoplasm/genetics , HMGB1 Protein/genetics , MicroRNAs/genetics , Antineoplastic Agents, Phytogenic/therapeutic useABSTRACT
Objective To investigate the expression of RNA binding protein KSRP in multiple acute myeloid leuke-mia(AML) patients. Methods KSRP expression among AML patients and normal controls was analyzed in gene expression omnibus (GEO) datasets and the cancer genome atlas(TCGA) database. KSRP expression was inhibi-ted by lentivirus-mediated gene transduction in THP-1 cells,cell proliferation and apoptosis were analyzed. Results KSRP has lower expression in t(15;17) acute promyelocytic leukemia while has higher expression in mixed lineage leukemia(MLL) translocated acute monocytic leukemia or acute myelomonocytic leukemia. KSRP knock-down sig-nificantly promoted apoptosis (P<0.01),as well as suppressed cell proliferation (P<0.01). In addition, overex-pression of miR-129 increased cell proliferation(P<0.05). Conclusions KSRP may regulate AML-M5 cell growth through promoting miR-129 processing.
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OBJECTIVE To investigate the effect of microRNA-129(miR-129)expression on malignant phenotypes of esophageal squamous cell cancer(ESCC) cells and its possible molecular mechanisms. METHODS The constructed miR-129-overexpressed vector (pGCMV/EGFP/miR-129) and negative control vector (pGCMV/EGFP/miR-NC) were stably transfected into ESCC cell lines (Eca109 and EC9706),respectively. Quantitative real-time PCR(qRT-PCR)was performed to detect the expression of miR-129. MTT and flow cytometry(FCM)assays were performed to analyze the effects of miR-129 on proliferation, cell cycle and apoptosis of ESCC cells. Furthermore,a luciferase reporter vector with the putative B-cell lymphoma-2(Bcl-2)3′-untranslated region(pLUC/Bcl-2-3′-UTR-wt and pLUC/Bcl-2-3′-UTR-mut)was constructed to explore whether Bcl-2 was a direct target gene of miR-129 by detecting luciferase activity. Next,Western blotting was performed to detect the expression of Bcl-2, cleaved caspase 3 and total caspase 3 proteins. RESULTS Overexpression of miR-129 significantly inhibited proliferation(P<0.01),induced cell arrest in G0/G1 phase(P<0.05)and enhanced apoptosis (P<0.05)in ESCC cells. Luciferase reporter assay indicated that Bcl-2 was identified as a direct target gene of miR-129. Results of Western blotting showed that overexpression of miR-129 significantly reduced the expression of Bcl-2 protein and increased the expression of cleaved caspase 3 protein,but induced no changes in total caspase 3 protein in ESCC cells. CONCLUSION miR-129 functions as a tumor suppressor in ESCC cells by targeting Bcl-2 gene. Therefore,miR-129 will be a potential molecular target for the treatment of human ESCC.
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Objective To investigate the role of microRNA-129-5p (miR-129-5p) in the regulation of epithelial-mesenchymal transition (EMT) of human peritoneal mesothelial cells (HPMCs) isolated from peritoneal dialysate effluents and TGF-β1 induced HPMCs line.Methods The isolated cells were cultured from peritoneal dialysate effluents overnight of 10 patients just started PD and 12 patients with PD over 6 months.Taqman PCR assay was used to determine the expression of miR-129-5p in the HPMCs.Moreover,the expression of miR-129-5p in HPMCs induced by 5 μg/L TGF-β1 for 0-72 h was also detected by Taqman PCR.HPMCs were pre-transfected with miR-129-5p precursor (pre-mir-129-5p) to overexpress miR-129-5p,then incubated with TGF-β1 for 48 h,and the expression of EMT associated gene and protein was detected by real-time PCR,Western blotting and immunofluorescence,respectively.Furthermore,the effect of TGF-β1 on the expression of Smad interacting protein-1 (SIP1) and the regulation of pre-miR-129-5p on the SIP1 expression also were investigated.Results MiR-129-5p expression significantly down-regulated in the HPMCs isolated from PD patients over 6 months than from PD start patients(P ＜ 0.01).Similarly,TGF-β1 remarkably decreased miR-129-5p in HPMCs lines on time-dependent manner (P ＜ 0.01).Pre-mir-129-5p dramatically restored the expression of epithelial marker E-cadherin,while inhibited the expression of Vimentin,a mesenchymal marker,in HPMCs induced by TGF-β1 (all P ＜ 0.01).In addition,TGF-β1 increased SIP1 expression in HPMCs time dependently,while the high level of SIP1 protein was obviously repressed after transfected of pre-miR-129-5p (P ＜ 0.01),but there was no obvious change of its mRNA expression.Conclusion MiR-129-5p modulates EMT formation of HPMCs in PD process,possibly by posttranscriptional inhibition of SIP1.Targeting miR-129-5p/SIP1 may provide a new approach for the prevention and treatment of peritoneal fibrosis during PD.