Growth inhibitory effects of three miR-129 family members on gastric cancer

Reduced expression of microRNA-129 (miR-129) has been reported in several types of tumor cell lines as well as in primary tumor tissues. However, little is known about how miR-129 affects cell proliferation in gastric cancer. Here, we show that all miR-129 family members, miR-129-1-3p, miR-129-2-3p, and miR-129-5p, are down-regulated in gastric cancer cell lines compared with normal gastric epithelial cells. Furthermore, using the real-time cell analyzer assay to observe the growth effects of miR-129 on gastric cancer cells, we found that all three mature products of miR-129 showed tumor suppressor activities. To elucidate the molecular mechanisms underlying down-regulation of miR-129 in gastric cancer, we analyzed the effects of miR-129 mimics on the cell cycle. We found that increased miR-129 levels in gastric cancer cells resulted in significant G₀/G₁ phase arrest. Interestingly, we showed that cyclin dependent kinase 6 (CDK6), a cell cycle-associated protein involved in G₁-S transition, was a target of miR-129. We also found that expression of the sex determining region Y-box 4 (SOX4) was inversely associated with that of miR-129-2-3p and miR-129-5p but not of miR-129-1-3p. Together, our data indicate that all miR-129 family members, not only miR-129-5p, as previously thought, play an important role in regulating cell proliferation in gastric cancer.     

微小RNA-129在肿瘤发生中的作用机制

微小RNA-129（microRNA-129, miR-129）是一种能产生miR-129-5p、miR-129-1-3p和miR-129-2-3p等3种成熟产物的miRNA,它们在细胞生长、发育、癌变等生命过程中有重要作用. miR-129在多种常见肿瘤中呈现高表达（如：胃癌和肺癌）或低表达（如：乳腺癌和食管鳞状细胞癌）的多样现象,而且这种异常表达与肿瘤的发生发展有密切联系. miR-129通过作用于性别决定区Y框蛋白4（sex-determining-region Y-box 4, SOX4）、细胞周期蛋白依赖激酶6（cyclin-dependent kinase 6, Cdk6）、含缬酪肽蛋白（valosin containing protein, VCP）和磷脂酰肌醇蛋白聚糖-3（glypican-3, GP3）等多种靶基因而在肿瘤发生、发展过程中起着重要作用.     

MiR-143-3p suppresses cell proliferation,migration, and invasion by targeting Melanoma-Associated Antigen A9 in laryngeal squamous cell carcinoma

Previously we found that melanoma-associated antigen-A9 (MAGE-A9) was a significantly upregulated biomarker in laryngeal squamous cell carcinoma (LSCC). A high expression of MAGE-A9 indicates an unfavorable survival outcome, and the MAGE-A9 expression level is an independent prognostic factor of LSCC. To explore the mechanism of MAGE-A9 upregulation, several predicted regulatory microRNAs were screened and validated in LSCC cells. In the current study, we found that miR-143-3p (MAGE-A9 related miRNAs) expression levels correlated negatively with the MAGE-A9 protein expression in LSCC tissues. Dual-luciferase reporter assays and Western blot analysis revealed MAGE-A9 to be a direct target of miR-143-3p. Furthermore, a series of in vitro gain- and loss-of-function assays revealed that miR-143-3p inhibited LSCC cell proliferation, migration, and invasion. Also, miR-143-3p suppressed LSCC tumorigenesis in vivo. These effects were clinically relevant, as a lower expression of miR-143-3p occurred in severer clinical stages and represented poor overall survival in patients with LSCC. Taken together, these results suggest that downregulation of miR-143-3p contributes to tumor progression through upregulation of MAGE-A9. The expression level of these two key molecules maintained LSCC progression, thus, highlighting the potential of miR-143-3p as a therapeutic target for human LSCC.     

VCP/p97, down-regulated by microRNA-129-5p, could regulate the progression of hepatocellular carcinoma

Valosin containing protein (VCP)/p97 plays various important roles in cells. Moreover, elevated expression of VCP in hepatocellular carcinoma (HCC) is correlated with increased incidence of recurrence. But the role of VCP in HCC progression in vitro and in vivo is unclear. And there are few reports about the regulation mechanism on the expression of VCP in HCC. In this study, it was identified that the level of VCP was frequently increased in human HCC tissues. In addition, down-regulation of VCP with siRNAs could dramatically suppress the genesis and progression of tumor in vivo. It was found that miR-129-5p directly inhibited the expression of VCP in several HCC cell lines. Meanwhile, the level of VCP in HCC tissues was negatively associated with the level of miR-129-5p. Our further investigation showed that the enhanced expression of miR-129-5p also suppressed tumor growth in vivo. Moreover, it was revealed that miR-129-5p could inhibit the degradation of IκBα and increase the apoptosis and reduce the migration of HCC cells by suppressing the expression of VCP. Our results revealed that the expression of VCP was directly regulated by miR-129-5p and this regulation played an important role in the progression of HCC.     

Inhibition of microRNA-129-5p expression ameliorates ultraviolet ray-induced corneal epithelial cell injury via upregulation of EGFR

Existing evidence has highlighted the effect of ultraviolet light radiation leading to corneal epithelium impairment. During this study, we aim to investigate the effect of microRNA-129-5p (miR-129-5p) on the wound healing process of corneal epithelial cells (CECs) induced by ultraviolet rays in mice by targeting epidermal growth factor receptor (EGFR). First, mouse models of ultraviolet ray-induced CEC injury were established and intrastromally injected with different mimic, inhibitor, and short interfering RNA (siRNA) to detect the effect of miR-129-5p on CEC injury. Subsequently, the corneal tissues were obtained to detect the antioxidant ability and EGFR-positive expression rate. The dual-luciferase reporter gene assay was used to test whether EGFR could directly target miR-129-5p. To further investigate the specific mechanism of miR-129-5p and EGFR in CEC injury, CECs were cultured and transfected with miR-129-5p mimic, miR-129-5p inhibitor, siRNA-EGFR, and miR-129-5p inhibitor + siRNA-EGFR. miR-129-5p has been proven to directly target EGFR. Inhibition of miR-129-5p is able to increase the antioxidant capacity, EGFR-positive rate and the expressions of EGFR, B-cell lymphoma-2, zonula occluden-1, occludin, and keratinocyte growth factor-2, but decrease the expression of vascular endothelial growth factor, BCL2-associated X protein, interleukin (IL)-1β, and IL-4. Inhibition of miR-129-5p arrests cells at the S and G2 phases and decreases apoptosis. Our study provides evidence stating that inhibiting miR-129-5p and upregulating EGFR could aid in the repair of mice CEC injury induced by ultraviolet radiation. Therefore, inhibition of miR-129-5p might provide a basic theory in the repair of CEC injury caused by ultraviolet rays.     

miR-129-5p and -3p co-target WWP1 to suppress gastric cancer proliferation and migration

Gastric cancer (GC) is a worldwide health problem. Uncovering the underlining molecular mechanisms of GC is of vital significance. Here, we identified a novel oncogene WW domain-containing E3 ubiquitin protein ligase 1 (WWP1) in GC. WWP1 could promote GC cell proliferation and migration in vitro and expedite GC growth in vivo. We also found out two microRNAs (miRNAs): miR-129-5p and -3p could both target WWP1. Interestingly, miR-129-5p bound to the CDS region of WWP1 mRNA. The miR-129 pairs (miR-129-5p and -3p) play pivotal roles in GC to suppress its proliferation and migration in vitro and slow down GC growth in vivo by repressing WWP1. In summary, we identified two tumor suppressive miRNAs which share the same precursor that could regulate the same oncogene WWP1 in GC. Our finding would add new route for GC research and treatment.     

miR-129-5p inhibits proliferation,migration, and invasion in rectal adenocarcinoma cells through targeting E2F7

microRNAs (miRNAs), a kind of small noncoding RNAs, are considered able to regulate expression of genes and mediate RNA silencing. miR-129-5p was shown to be a cancer-related miRNA. However, the influence of miR-129-5p in rectal adenocarcinoma (READ) development remains to be determined. Based on the TCGA data, downregulation of miR-129-5p in READ samples was observed. Manual restoration of the miR-129-5p in SW1463 and SW480 cell lines significantly inhibited invasion, migration, and proliferation of READ cell lines, while the apoptosis ability was enhanced. Meanwhile, we found E2F7 acted as a potential target of miR-129-5p and was upregulated in READ samples. E2F7 upregulation reversed the repression of miR-129-5p on READ development. Finally, in vivo experiments showed that inhibition of tumor growth in nude mice was achieved through upregulating miR-129-5p. Overall, our findings suggest increasing of miR-129-5p leads to the suppression of READ progression through regulating the expression of E2F7, which may provide novel insights into the treatment of READ.     

MicroRNA-129-5p inhibits 3T3-L1 preadipocyte proliferation by targeting G3BP1

MicroRNAs have been regarded to play a crucial role in the proliferation of different cell types including preadipocytes. In our study, we observed that miR-129-5p was down-regulated during 3T3-L1 preadipocyte proliferation, while the expression of G3BP1 showed a contrary tendency. 5-Ethynyl-2′-deoxyuridine (EdU) incorporation assay and flow cytometry showed that overexpression of miR-129-5p could bring about a reduction in S-phase cells and G2-phase arrest. Additional study indicated that miR-129-5p impaired cell cycle-related genes in 3T3-L1 preadipocytes. Importantly, it showed that miR-129-5p directly targeted the 3′UTR of G3BP1 and the expression of G3BP1 was inhibited by miR-129-5p mimic. Moreover, miR-129-5p mimic activated the p38 signaling pathway through up-regulating p38 and the phosphorylation level of p38. In a word, results in our study revealed that miR-129-5p suppressed preadipocyte proliferation via targeting G3BP1 and activating the p38 signaling pathway.     

miR-129-5p调节小鼠乳腺上皮细胞内Igf-1的表达

MicroRNAs(miRNAs)是一类大约22个核苷酸的非编码RNA.它能通过调控生长因子表达,引发肿瘤形成、细胞增殖和组织器官发育.本文通过构建miR-129-5p靶基因序列的双荧光素酶报告载体分析了miR-129-5p与靶基因之间的关系,应用脂质体转染技术和实时荧光定量技术观察了miR-129-5p在小鼠乳腺上皮细胞中的表达量及其变化,通过CASY细胞活力仪检测转染后的细胞增殖与活力变化,采用Western 印迹方法检Igf-1的变化.结果表明：miR-129-5p在小鼠乳腺青春期表达最高,成功构建了Igf-1基因 3′UTR荧光素酶报告载体, miR-129-5p抑制其荧光素酶活性（P <0.01）,转染抑制子后miR-129-5p表达降低（P < 0.01）,胰岛素样生长因子（Igf-1)表达增强（P <0.05）,细胞增殖和活力增强（P <0.01）,结果提示：miR-129-5p可能通过抑制靶基因蛋白Igf 1的表达,进而抑制小鼠乳腺上皮细胞增殖和活力.     

Silencing of long noncoding RNA MEG3 enhances cerebral protection of dexmedetomidine against hypoxic-ischemic brain damage in neonatal mice by binding to miR-129-5p

Hypoxic-ischemic brain damage (HIBD) is a leading cause of neonatal acute mortality and chronic nervous system injury. Recently, it has been found that long noncoding RNAs (lncRNAs) play a significant role in the neurodevelopment and etiopathogenesis of HIBD. Here, the researchers aimed to determine the role of lncRNA maternally expressed gene (MEG3) in the therapeutic effect of dexmedetomidine (DEX) in neonatal mice with HIBD through the regulation of microRNA-129-5p (miR-129-5p). HIBD models were established in C57/BL6 neonatal mice. Subsequently, the target relationship between MEG3 and miR-129-5p was predicted and verified. The neonatal mice were injected with DEX, ad-shMEG3, and mimics and inhibitors of miR-129-5p to identify roles of MEG3 and miR-129-5p in therapeutic effects of DEX on neuronal apoptosis and injury, cerebral atrophy, and learning and memory ability of neonatal mice with HIBD. MEG3 directly targeted and inhibited the expression of miR-129-5p. Silencing of MEG3 or upregulation of miR-129-5p effectively promoted the therapeutic effect of DEX on neonatal mice with HIBD. Silencing of MEG3 or upregulation of miR-129-5p reduced the neuronal apoptosis rate and degree of cerebral atrophy, and also enhanced the learning and memory ability of HIBD neonatal mice. Collectively, the key findings obtained from the present study support the notion that MEG3 silencing enhances the therapeutic effect of DEX on neonatal mice with HIBD by binding to miR-129-5p.     

Downregulation of microRNA-129-5p increases the risk of intervertebral disc degeneration by promoting the apoptosis of nucleus pulposus cells via targeting BMP2

miR-129-5p is implicated in many diseases, such as laryngeal cancer and breast cancer. In this study, we studied the mechanism underlying the role of BMP2 in intervertebral disc degeneration (IDD). We used a luciferase assay system to determine the relationship between BMP2 and miR-129-5 expression. In addition, Western blot and real-time PCR were used to confirm the regulatory relationship between miR-129-5p and its targets, while flow cytometry was used to evaluate the effect of miR-129-5p on the apoptosis of neural progenitor cells (NPCs). BMP2 was confirmed as a direct target of miR-129-5p. Furthermore, the expression of miR-129 was downregulated along with upregulated BMP2 expression in IDD patients. Meanwhile, BMP2 was validated as the target of miR-129-5p in cells transfected with miR-129-5p and BMP2 siRNA. Also, compared with NPCs transfected with blank/scramble controls or miR-129-5p inhibitors, the NPCs treated with miR-129-5p mimics or BMP2 siRNA exhibited evidently elevated viability and inhibited apoptosis. The data demonstrated that miR-129-5p was poorly expressed in IDD patients, and the dysregulation of miR-129-5p might contribute to the development of IDD by targeting BMP2 expression.     

Interferon-β Induced microRNA-129-5p Down-Regulates HPV-18 E6 and E7 Viral Gene Expression by Targeting SP1 in Cervical Cancer Cells

Infection by human papillomavirus (HPV) can cause cervical intraepithelial neoplasia (CIN) and cancer. Down-regulation of E6 and E7 expression may be responsible for the positive clinical outcomes observed with IFN treatment, but the molecular basis has not been well determined. As miRNAs play an important role in HPV induced cervical carcinogenesis, we hypothesize that IFN-β can regulate the expressions of specific miRNAs in cervical cancer cells, and that these miRNAs can mediate E6 and E7 expression, thus modulate their oncogenic potential. In this study, we found that miR-129-5p to be a candidate IFN-β inducible miRNA. MiR-129-5p levels gradually decrease with the development of cervical intraepithelial lesions. Manipulation of miR-129-5p expression in Hela cells modulates HPV-18 E6 and E7 viral gene expression. Exogenous miR-129-5p inhibits cell proliferation in Hela cells, promotes apoptosis and blocks cell cycle progression in Hela cells. SP1 is a direct target of miR-129-5p in Hela cells. This study is the first report of a cellular miRNA with anti-HPV activity and provides new insights into regulatory mechanisms between the HPV and the IFN system in host cells at the miRNA level.     

Retracted: microRNA-129-5p involved in the neuroprotective effect of dexmedetomidine on hypoxic-ischemic brain injury by targeting COL3A1 through the Wnt/β-catenin signaling pathway in neonatal rats

Our study aims to elucidate the mechanisms how microRNA-129-5p (miR-129-5p) involved in the neuroprotective effect of dexmedetomidine (DEX) on hypoxic-ischemic brain injury (HIBI) by targeting the type III procollagen gene (COL3A1) through the Wnt/β-catenin signaling pathway in neonatal rats. A total of 120 rats were obtained, among which 15 rats were selected as sham group and rest rats as model, DEX, DEX + negative control (DEX + NC), DEX + miR-129-5p mimics, DEX + miR-129-5p inhibitors, DEX + XAV-939, and DEX + miR-129-5p inhibitors + XAV-939 groups. A dual-luciferase reporter assay was performed for the target relationship between miR-129-5p and COL3A1. Weight rate and water content of cerebral hemisphere were detected. Quantitative real-time polymerase chain reaction and Western blot analysis were conducted to detect miR-129-5p expression and expressions of COL3A1, E-cadherin, T-cell factor (TCF)− 4, and β-catenin. The DEX, DEX + miR-129-5p mimics, DEX + XAV-939 groups had increased weight rate of the cerebral hemisphere, but decreased water content of left cerebral hemisphere, levels of COL3A1, β-catenin, TCF-4, and E-cadherin in the hippocampus compared with the model and DEX + miR-129-5p inhibitors groups. COL3A1 was verified as the target gene of the miR-129-5p. Compared with the DEX + NC and DEX + miR-129-5p inhibitors + XAV-939 groups, the DEX + XAV-939 and DEX + miR-129-5p mimics groups had elevated weight rate of the cerebral hemisphere, but reduced water content of left cerebral hemisphere, levels of COL3A1, β-catenin, TCF-4, and E-cadherin in the hippocampus. Our findings demonstrate that miR-129-5p improves the neuroprotective role of DEX in HIBI by targeting COL3A1 through the Wnt/β-catenin signaling pathway in neonatal rats.     

MiR-129-5p is down-regulated and involved in the growth,apoptosis and migration of medullary thyroid carcinoma cells through targeting RET

Dysregulation of the REarranged during Transfection proto-oncogene (RET) pathway and microRNA (miRNAs) are crucial for the development of medullary thyroid carcinomas (MTC). Here we demonstrate that miR-129-5p is down-regulated in MTC tissues and cell lines and inhibits RET expression by directly binding its 3′ untranslated regions. Ectopic expression of miR-129-5p significantly decreases cell growth, induces apoptosis and suppresses migration ability in MTC cells through decreasing the phosphorylated AKT, thus functioning as a tumor suppressor. These findings give new clues for understanding MTC carcinogenesis and may help in developing a therapeutic approach for the treatment of RET-activated MTC.     

血清外泌体lncRNA PCGEM1、miR-129-5p与非小细胞肺癌患者临床病理特征及预后的关系

摘要 目的：研究血清外泌体长链非编码核糖核酸（lncRNA）前列腺癌基因表达标记1（PCGEM1）、微小核糖核酸（miR）-129-5p与非小细胞肺癌（NSCLC）患者临床病理特征及预后的关系。方法：选取2016年2月-2018年1月南京脑科医院收治的125例NSCLC患者作为NSCLC组,同期选取体检的70例健康人群作为健康组。采集两组静脉血,提取血清外泌体;采用实时定量聚合酶链式反应（qRT-PCR）检测血清外泌体lncRNA PCGEM1、miR-129-5p表达情况;采用Pearson相关性分析lncRNA PCGEM1与miR-129-5p的关系。并分析血清外泌体lncRNA PCGEM1、miR-129-5p与NSCLC患者临床病理特征的关系。对NSCLC患者行5年随访,绘制Kaplan-Meier曲线分析预后情况,多因素Cox比例风险回归模型分析预后不良危险因素,受试者工作特征（ROC）曲线分析lncRNA PCGEM1、miR-129-5p对NSCLC预后的预测价值。结果：：NSCLC组lncRNA PCGEM1相对表达量高于健康组,miR-129-5p相对表达量低于健康组（P＜0.05）。血清外泌体lncRNA PCGEM1相对表达量与miR-129-5p表达呈负相关（r= -0.420,P＜0.05）。血清外泌体lncRNA PCGEM1、miR-129-5p表达与患者TNM分期、分化程度、淋巴结转移有关（P＜0.05）。Kplan-Meier生存曲线显示,lncRNA PCGEM1低表达组5年生存率69.05%高于lncRNA PCGEM1高表达组35.53%,miR-129-5p高表达组5年生存率68.09%高于miR-129-5p低表达组33.80%。多因素Cox比例风险回归显示,TNM分期III期、有淋巴结转移、lncRNA PCGEM1高表达、miR-129-5p低表达为NSCLC患者预后不良的独立危险因素（P＜0.05）。ROC曲线显示,lncRNA PCGEM1、miR-129-5p联合检测对NSCLC预后的预测曲线下面积（AUC）为0.865,预测价值高于两者单独预测。结论：NSCLC患者血清外泌体lncRNA PCGEM1表达上调、miR-129-5p表达下调,二者表达与NSCLC患者TNM分期、分化程度、淋巴结转移有关,且与患者预后密切相关,对NSCLC预后不良具有较好预测价值。     

miR-20a and miR-290, multi-faceted players with a role in tumourigenesis and senescence

Expression of microRNAs changes markedly in tumours and evidence indicates that they are causatively related to tumourigenesis, behaving as tumour suppressor microRNAs or onco microRNAs; in some cases they can behave as both depending on the type of cancer. Some tumour suppressor microRNAs appear to be an integral part of the p53 and Retinoblastoma (RB) network, the main regulatory pathways controlling senescence, a major tumour suppressor mechanism. The INK4a/ARF locus which codifies for two proteins, p19ARF and p16INK4a, plays a central role in senescence by controlling both p53 and RB. Recent evidence shows that the proto-oncogene leukaemia/lymphoma related factor, a p19ARF specific repressor, is controlled by miRNAs and that miRNAs, in particular miR-20a and miR-290, are causatively involved in mouse embryo fibroblasts (MEF) senescence in culture. Intriguingly, both miR-20a, member of the oncogenic miR-17-92 cluster, and miR-290, belonging to the miR-290-295 cluster, are highly expressed in embryonic stem (ES) cells. The pro-senescence role of miR-20a and miR-290 in MEF is apparently in contrast with their proliferative role in tumour and ES cells. We propose that miRNAs may exert opposing functions depending on the miRNAs repertoire as well as target/s level/s present in different cellular contexts, suggesting the importance of evaluating miRNAs activity in diverse genetic settings before their therapeutic use as tumour suppressors.