Pim1 supports human colorectal cancer growth during glucose deprivation by enhancing the Warburg effect

Cancer cells metabolize glucose mainly by glycolysis and are well adapted to metabolic stress. Pim1 is an oncogene that promotes colorectal cancer (CRC) growth and metastasis, and its expression is positively correlated with CRC progression. However, the mechanism underlying Pim1 overexpression during CRC progression and the role of Pim1 in CRC metabolism remains unclear. In the present study, we discovered that Pim1 expression was significantly upregulated in response to glucose deprivation‐induced metabolic stress by AMP‐activated protein kinase signaling. Pim1 promoted CRC cell proliferation in vitro and tumorigenicity in vivo. Clinical observations showed that Pim1 expression was higher in CRC tissues than in adjacent normal tissues. Pim1 overexpression in CRC tissues not only predicted CRC prognosis in patients but also showed a positive relationship with ¹⁸F‐fluorodeoxyglucose uptake. Further in vitro experiments showed that Pim1 promoted the Warburg effect and that Pim1 expression was positively correlated with hexokinase 2 and lactate dehydrogenase A expression. Pim1‐silenced cells were more vulnerable to glucose starvation, and Pim1‐induced tumor proliferation or tolerance to glucose starvation was attenuated by blocking the Warburg effect. In conclusion, glucose deprivation is one of the mechanisms that leads to elevated Pim1 expression in CRC, and Pim1 upregulation ensures CRC growth in response to glucose deprivation by facilitating the Warburg effect in a compensatory way.     

miR-340与远端胃癌淋巴结转移的相关性及其对胃癌细胞迁移、侵袭的影响北大核心

目的:分析miR-340与远端胃癌患者淋巴结转移的相关性及其对胃癌细胞迁移、侵袭的影响。方法:采用实时荧光定量聚合酶链反应(RT-qPCR)检测远端胃癌组织及胃癌细胞系中miR-340的水平,分析miR-340与胃癌患者临床病理特征的相关性,应用受试者工作特征(ROC)曲线及Logistic回归分析探讨miR-340诊断淋巴结转移的性能;通过转染miR-340模拟物(mimic)上调胃癌细胞HGC-27和MGC-803中miR-340的表达,划痕实验和Transwell实验检测miR-340对胃癌细胞迁移、侵袭的影响。结果:miR-340在远端胃癌组织中低表达,且与淋巴结转移、浸润深度、分化程度及TNM分期相关(P<0.05)。miR-340诊断胃癌淋巴结转移的曲线下面积(AUC)为0.737,敏感性为76.32%,特异性为79.41%。低水平miR-340是胃癌淋巴结转移的独立危险因素。与正常胃黏膜上皮细胞相比,胃癌细胞中miR-340的表达水平下调。上调miR-340的表达能够抑制胃癌细胞的迁移、侵袭。结论:miR-340在胃癌组织和细胞中表达下调,能够调控胃癌细胞的迁移、侵袭,与淋巴结转移密切相关。     

Methylation of miR-124a-1, miR-124a-2, and miR-124a-3 genes correlates with aggressive and advanced breast cancer disease

Aberrant DNA methylation on CpG islands is one of the most consistent epigenetic changes in human cancers, and the process of methylation is catalyzed by the DNA methyltransferases DNMT1, DNMT3a, and DNMT3b. Recent reports demonstrate that deregulation of miR-124a, one of the frequently methylated microRNAs in human cancers, is related to carcinogenesis. The aim of this study was to evaluate the frequencies of methylation of the three genomic loci encoding the miR-124a in primary breast cancers and to investigate their relationships with the clinicopathological characteristics of the tumors and with the expression levels of DNMT1, DNMT3a, and DNMT3b. The methylation status of the three genomic loci encoding the miR-124a (miR-124a-1, miR-124a-2, and miR-124a-3) was analyzed in fresh-frozen tumor samples using methylation-specific PCR in a large series of invasive breast ductal carcinomas (n?=?60). Results were correlated to several clinicopathological characteristics of the tumors and to the expression levels of DNMT1, DNMT3a, and DNMT3b, determined by immunohistochemistry. Promoter hypermethylation of miR-124a-1, miR-124a-2, and miR-124a-3 was detected in 53.3, 70, and 36.7 % of cases, respectively. Methylation of miR-124a-2 correlated to patients with age higher than 45 years (P?=?0.008) and to postmenopausal patients (P?=?0.03), whereas methylation of miR-124a-3 correlated significantly to tumor size >20 mm (P?=?0.03). Interestingly, simultaneous methylation of the three genes encoding miR-124a correlated significantly with the presence of lymph node metastasis (P?=?0.01) and high mitotic score (P?=?0.03). No significant correlation was found between promoter hypermethylation of miR-124a and expression of hormone receptors or HER2/neu. With regard to DNMT expression, no correlation was found between DNMT1 or DNMT3a expression and promoter methylation of any tested microRNA. However, DNMT3b overexpression correlates significantly with the hypermethylation of miR-124a-3 (P?=?0.03). Our data indicates that miR-124a-1, miR-124a-2, and miR-124a-3 genes are frequently methylated in breast cancer and play a role in tumor growth and aggressivity.     

miR-124 and miR-506 inhibit colorectal cancer progression by targeting DNMT3B and DNMT1

miR-124 and miR-506 are reportedly down-regulated and associated with tumor progression in many cancers, but little is known about their intrinsic regulatory mechanisms in colorectal cancer (CRC). In this study, we found that the miR-124 and miR-506 levels were significantly lower in human CRC tissues than in controls, as indicated by qRT-PCR and in situ hybridization histochemistry. We also found that the overexpression of miR-124 or miR-506 inhibited tumor cell progression and increased sensitivity to chemotherapy in vitro. Increased miR-124 or miR-506 expression also inhibited tumor cell proliferation and invasion in vivo. Luciferase reporter assays and western blotting were used to determine the association between miR-124, miR-506 and their target genes, DNMTs. We further identified that miR-124 and miR-506 directly targeted DNMT3B and indirectly targeted DNMT1. The overexpression of miR-124 and miR-506 reduced global DNA methylation and restored the expression of E-cadherin, MGMT and P16. In conclusion, our data showed that miR-124 and miR-506 inhibit progression and increase sensitivity to chemotherapy by targeting DNMT3B and DNMT1 in CRC. These findings may provide novel avenues for the development of targeted therapies.     

miR-340通过下调 CCND1 表达增加结直肠癌细胞对5-Fu的耐药

目的:探讨细胞周期蛋白D1(cyclin D1)的编码基因CCND1 miR-340介导的逆转结直肠癌细胞对5-氟尿嘧啶(5-Fu)耐药的机制.方法:采用瞬时转染技术将结直肠癌细胞HCT116、SW480株分别转染si-CCND1和miR340-mimic.应用MTT法检测转染后的结直肠癌细胞对5-Fu敏感性的变化,应用双荧光素酶试验验证CCND1对miR340参与的影响结直肠癌细胞对5-Fu敏感性的影响.结果:瞬时转染siCCND1和过表达miR-340后,结直肠癌HCT116和SW480细胞的IC50值均显著低于对照组(10,10 vs 20 μmol/L和20,20 vs 40 μmol/L,均P＜0.05).共转染CCND1 3'UTR野生质粒和miR-340 inhibitor的结直肠癌HCT116和SW48细胞荧光素酶的活性显著高于共转染空载体和mimic细胞(P＜0.01).结论:CCND1作为不良因子通过抑制miR340的表达进而发挥增加结直肠癌细胞对5-Fu耐药的作用.     

miR-340 inhibition of breast cancer cell migration and invasion through targeting of oncoprotein c-Met

BACKGROUND:

Different microRNAs have been shown to have oncogenic and tumor‐suppressive functions in human cancers. Detection of their expression may lead to identifying novel markers for breast cancer.

METHODS:

The authors detected miR‐340 expression in 4 human breast cell lines and then focused on its role in regulation of tumor cell growth, migration, and invasion and target gene expression. They then analyzed miR‐340 expression in benign and cancerous breast tissue specimens.

RESULTS:

Endogenous miR‐340 expression was down‐regulated in the more aggressive breast cancer cell lines, which was confirmed in breast cancer tissue specimens by using quantitative real‐time polymerase chain reaction. Further studies showed that induction of miR‐340 expression was able to suppress tumor cell migration and invasion, whereas knockdown of miR‐340 expression induced breast cancer cell migration and invasion. At the gene level, the authors identified c‐Met as a direct miR‐340 target to mediate cell migration and invasion through regulation of MMP‐2 and MMP‐9 expression. Ex vivo, loss of miR‐340 expression was associated with lymph node metastasis, high tumor histological grade, clinical stage, and shorter overall survival of breast cancer as well as increased c‐Met expression in breast cancer tissue specimens.

CONCLUSIONS:

miR‐340 may play an important role in breast cancer progression, suggesting that miR‐340 should be further evaluated as a novel biomarker for breast cancer metastasis and prognosis, and potentially a therapeutic target. Cancer 2011. © 2011 American Cancer Society.     

miR-340对乳腺癌MDA-MB231细胞增殖和凋亡的影响

背景与目的：既往研究表明,微小RNA-340(miR-340)能负性调控多种肿瘤的进展,但其在乳腺癌细胞增殖和凋亡中的研究较少,本研究旨在探讨miR-340对乳腺癌MDA-MB231细胞增殖和凋亡的作用。方法：利用脂质体LipofectamineTM2000将pre-miR-340或anti-miR-340瞬时转染至乳腺癌MDA-MB231细胞,通过RT-PCR检测miR-340 mRNA的水平,蛋白质印迹法(Western blot)检测cleaved-caspase-3蛋白的表达,MTT比色法检测细胞增殖的抑制情况,流式细胞仪检测细胞凋亡。结果：Pre-miR-340增加了MDA-MB231细胞中miR-340表达,同时增加cleaved-caspase-3蛋白表达、抑制MDA-MB231细胞增殖并促进其凋亡。而anti-miR-340抑制了MDAMB231细胞中miR-340表达,并且抑制cleaved-caspase-3蛋白表达,促进了MDA-MB231细胞增殖,抑制了MDAMB231细胞的凋亡。结论：miR-340转染后能上调MDA-MB231细胞中cleaved-caspase-3蛋白的表达从而抑制细胞增殖,促进其凋亡。     

miR-618抑制结直肠癌生长转移的机制探讨

目的:探讨miR-618在结直肠癌发生发展中的作用。方法:Real-time PCR法检测结直肠癌组织及Caco-2细胞中miR-618表达情况,改变结直肠癌细胞中miR-618表达,应用平板克隆实验检测转染后的结直肠癌细胞克隆能力,创伤愈合实验检测转染后的结直肠癌细胞迁移能力,Transwell小室实验检测转染后的结直肠癌细胞侵袭能力,Western Blotting法检测结直肠癌组织、细胞中SMAD4蛋白的表达变化情况及改变结直肠癌细胞中miR-618表达对SMAD4蛋白的影响。结果:结直肠癌组织及Caco-2细胞中miR-618表达较正常癌旁组织及FHC细胞明显下降（P＜0.05）,但SMAD4蛋白的表达增高（P＜0.05）。上调Caco-2细胞中miR-618表达后,Caco-2细胞的克隆、迁移及侵袭能力均减弱（P＜0.05）,但SMAD4蛋白表达下降（P＜0.05）。结论:miR-618通过下调SMAD4蛋白表达抑制结直肠癌生长转移。     

miR-124在宫颈癌中的表达及对宫颈癌细胞增殖、迁移的影响

目的:探讨miR-124在宫颈癌中的表达,及其对宫颈癌细胞增殖、迁移的影响。方法:收集宫颈癌组织和癌旁组织各13例,qRT-PCR法检测miR-124的表达,免疫组化检测STAT3的表达。生物信息学技术预测miR-124的靶基因,双荧光素酶报告基因、qRT-PCR、Western blot实验验证。每种宫颈细胞随机分为三组,一组转染miR-124 mimics,一组不转染,一组转染miR-124 inhibitor。CCK-8实验检测细胞增殖能力,细胞划痕实验检测细胞迁移能力。结果:miR-124在宫颈癌组织和细胞系中表达均降低(P均＜0.05),STAT3在宫颈癌组织中的表达明显增高(P<0.05)。STAT3是miR-124的直接靶基因,miR-124可靶向调节STAT3的mRNA和蛋白表达水平(P均＜0.05)。在Hela及Siha细胞中,miR-124 mimics转染组细胞增殖及迁移能力均降低(P均＜0.05),miR-124 inhibitor转染组细胞增殖及迁移能力均增加（P均＜0.05)。结论:miR-124在宫颈癌中低表达,可能通过靶向调节STAT3表达抑制宫颈癌细胞的增殖及迁移能力。     

Expression of miR-124 inhibits growth of medulloblastoma cells

Medulloblastoma is the most common malignant brain tumor in children, and a substantial number of patients die as a result of tumor progression. Overexpression of CDK6 is present in approximately one-third of medulloblastomas and is an independent poor prognostic marker for this disease. MicroRNA (miR)-124 inhibits expression of CDK6 and prevents proliferation of glioblastoma and medulloblastoma cells in vitro. We examined the effects of miR-124 overexpression on medulloblastoma cells both in vitro and in vivo and compared cell lines that have low and high CDK6 expression. MiR-124 overexpression inhibits the proliferation of medulloblastoma cells, and this effect is mediated mostly through the action of miR-124 upon CDK6. We further show that induced expression of miR-124 potently inhibits growth of medulloblastoma xenograft tumors in rodents. Further testing of miR-124 will help define the ultimate therapeutic potential of preclinical models of medulloblastoma in conjunction with various delivery strategies for treatment.     

miR-335 directly,while miR-34a indirectly modulate survivin expression and regulate growth,apoptosis, and invasion of gastric cancer cells

Tumor Biology - miR-335 and miR-34a are two microRNAs (miRNAs) usually downregulated in gastric cancer (GC). But, their exact regulative roles were not fully elucidated. In this study, we studied...     

Promotion of the Warburg effect is associated with poor benefit from adjuvant chemotherapy in colorectal cancer

Metabolic reprogramming, including the Warburg effect, is a hallmark of cancer. Indeed, the diversity of cancer metabolism leads to cancer heterogeneity, but accurate assessment of metabolic properties in tumors has not yet been undertaken. Here, we performed absolute quantification of the expression levels of 113 proteins related to carbohydrate metabolism and antioxidant pathways, in stage III colorectal cancer surgical specimens from 70 patients. The Warburg effect appeared in absolute protein levels between tumor and normal mucosa specimens demonstrated. Notably, the levels of proteins associated with the tricarboxylic citric acid cycle were remarkably reduced in the malignant tumors which had relapsed after surgery and treatment with 5‐fluorouracil‐based adjuvant therapy. In addition, the efficacy of 5‐fluorouracil also decreased in the cultured cancer cell lines with promotion of the Warburg effect. We further identified nine and eight important proteins, which are closely related to the Warburg effect, for relapse risk and 5‐fluorouracil benefit, respectively, using a biomarker exploration procedure. These results provide us a clue for bridging between metabolic protein expression profiles and benefit from 5‐fluorouracil adjuvant chemotherapy.     

MicroRNAs miR-7 and miR-340 predict response to neoadjuvant chemotherapy in breast cancer

Purpose

miRNAs have been linked to chemosensitivity of breast cancer cells in vitro. In patients, however, there is no clinically validated method for predicting chemotherapy response. The aim of this study was to assess whether (I) a specific pattern of miRNA expression in pretherapeutic biopsies can predict response to neoadjuvant chemotherapy, and (II) differential miRNA expression in residual tumor after completion of chemotherapy allows further prognostic stratification of non-responding patients.

Methods

Sixty-four patients with newly diagnosed large (≥3 cm) or locally advanced primary breast cancers who underwent neoadjuvant anthracycline/taxane-based chemotherapy were included. Relative expression of 10 miRNAs likely to be associated with chemotherapy response (miR-7,-21,-29a,-29b,-34a,-125b,-155,-200c,-340,-451) was determined by quantitative RT-PCR from pretherapeutic biopsies (n = 64) and residual invasive tumor after chemotherapy (n = 42). Pathologic complete response (pCR) defined by absence of invasive tumor served as reference standard. In addition, miRNA expression was compared with disease-free and overall survival.

Results

Nine (14%) of 64 patients achieved pCR. High expression of miR-7 and low expression of miR-340 in pretherapeutic biopsies predicted pCR with a negative predictive value of 96 and 97%, respectively (specificity 54 and 57%). The combined profile of miR-7^high/miR-340^low demonstrated improved specificity of 86% while maintaining a high negative predictive value (96%) to identify non-responders. Pretherapeutic expression of miR-200c and miR-155 showed prognostic information, and low expression was associated with increased overall survival (115 vs. 90 months, p ≤ 0.03). After chemotherapy, the overall survival of patients with residual invasive tumor was better for those demonstrating low miR-7 or high miR-125b (p = 0.01).

Conclusions

Intratumoral expression of miR-7 and miR-340 prior to neoadjuvant chemotherapy could be used to predict pCR and a profile of miR-7^low or miR-340^high identified patients unlikely to achieve pCR who might benefit from alternative treatment options including earlier surgery. Our study identifies miRNAs as promising predictive biomarkers, which could aid in optimization of breast cancer management and treatment stratification.

     

Expression of CD137 and CD137 ligand in colorectal cancer patients

The cytokine CD137, a member of the TNF receptor family, is expressed by T cells and regulates activation and proliferation of these cells. The CD137 ligand (CD137L) is expressed by antigen-presenting cells including macrophages, but also on various carcinoma cells. CD137/CD137L interaction plays a central role in sustaining T cell and macrophage activation, i.e. in antitumour immunity. The present study was designed to investigate whether CD137 and CD137L protein levels are altered in colorectal tumours compared with paired normal tissues. The CD137 and CD137L plasma levels from patients with colorectal cancer were also examined. Collectively, we noted a significantly lower CD137L level in cancerous tissue compared with paired normal tissue, and the difference in CD137L protein level was significantly lower in the colon cancer subgroup compared with paired normal colon tissue. On the other hand, we found an elevated CD137 protein level in the rectal cancer subgroup compared with paired normal rectal tissue. Patients with a tumour localised in the colon revealed significantly higher soluble CD137 protein concentration in the plasma than patients with a tumour localised in the rectum, and there was a tendency toward a higher concentration of CD137L protein in the plasma from patients with tumour localised in the colon. Moreover, the plasma concentrations of CD137 and CD137L proteins were strongly and significantly correlated. The different expression levels of CD137 and CD137L in the colon and rectum may reflect divergent mechanisms involved in the pathogenesis of colorectal cancer and lead to dissimilar protective immunity.     

miR-124在胃癌中的表达及临床意义

背景与目的：miR-124在多种肿瘤中发挥抑癌基因样功能,如肺癌、前列腺癌、膀胱癌和乳腺癌,但是其在胃癌中的表达及临床意义尚不清楚。该研究旨在研究miR-124在正常胃黏膜上皮细胞与不同胃癌细胞及胃癌组织及正常胃黏膜组织中的表达,分析其表达与胃癌患者性别、年龄、组织学分级、T分期、TNM分期、淋巴结转移及预后之间的相关性。方法：采用实时荧光定量聚合酶链反应(real-time fluorescent quantitative polymerase chain-reaction,RTFQ-PCR)检测miR-124在人胃黏膜上皮细胞及胃癌细胞中的表达,采用原位杂交法检测miR-124在胃癌及癌旁正常组织中的表达。结果：RTFQ-PCR结果显示,miR-124在胃癌MKN-74、MKN-28、MKN-45、MGC-803、SGC-7901及AGS细胞中的表达均低于GES-1细胞;原位杂交实验结果显示,miR-124在正常胃黏膜中呈强阳性表达,在胃腺癌组织中表达下降、局灶阳性或缺失;统计分析显示,miR-124与胃腺癌患者组织学分级、TNM分期及淋巴结转移密切相关,与患者的年龄、性别及肿瘤大小无关;Kaplan-Meier生存曲线统计分析显示,miR-124低表达患者的总生存时间和无病生存时间显明低于miR-124高表达患者;多因素分析结果提示,miR-124表达下调是影响患者生存的独立预后因素。结论：miR-124在胃癌细胞及组织中表达下调,并且与患者的组织学分级、TNM分期、淋巴结转移及预后密切相关。     

miR-124 在乳腺癌中的表达及其作用机制研究

目的：探讨m iR- 124 表达与乳腺癌发生、发展的相关性及机制。方法：运用实时定量聚合酶链反应（qRT-PCR）检测乳腺癌细胞系以及52例患者乳腺癌癌组织和对应的癌旁正常组织样本中miR-124 的表达水平。在乳腺癌细胞株MDA-MB-231和T-47D 中过表达miR-124 后,测定细胞增殖活性以及侵袭转移能力。构建荧光素酶报告载体pMIR- 特异性蛋白1（specificityprotein 1,SP1）的3'UTR,利用荧光素酶活性检测鉴定miR-124 的预测靶基因SP1。qRT-PCR和Westernblot法分别检测SP1 的mRNA 和蛋白质的表达水平。结果：miR-124 在乳腺癌细胞系和癌组织中表达量下调,差异具有统计学意义（P < 0.01）,并与肿瘤的转移、分期、分级和预后相关。在乳腺癌细胞株MDA-MB-231 和T-47D 中过表达miR-124 后抑制乳腺癌细胞系的增殖、侵袭以及迁移（P < 0.01）。 转染miR-124 模拟物显著抑制荧光素酶的活性（P < 0.05）。 转染miR-124 模拟物显著下调MDA-MB-231 和T-47D 细胞中SP1 的mRNA（P < 0.05）和蛋白质的表达水平。结论：miR-124 在乳腺癌癌组织中低表达,miR-124 低表达与乳腺癌不良预后有关,且miR-124 可通过调控转录因子SP1 抑制乳腺癌癌细胞的增殖、侵袭和转移。miR-124 表达异常减少可能是乳腺癌发生、发展的重要因素。     

miR-27b通过c-MET抑制胃癌细胞的生长、增殖及侵袭转移

目的:找出调控胃癌细胞c-MET的miRNA,研究该miRNA能否通过c-MET抑制胃癌细胞的生长、增殖及侵袭转移。方法:预测软件筛选出可能调节胃癌c-MET的miRNA。荧光定量PCR检测胃癌细胞株(N87、MKN45、AGS)及正常胃黏膜细胞(GES1)中该miRNA的表达水平。过表达miRNA后检测胃癌细胞株AGS中c-MET的表达水平,选择对c-MET抑制最强的miRNA行双荧光素酶实验。平板克隆及Transwell实验检测过表达该miRNA后胃癌细胞的生长、增殖及侵袭转移能力,过表达c-MET后再次检测胃癌细胞的生长、增殖及侵袭转移能力。结果:预测软件显示miR-34a、miR-27b及miR-31可能调节胃癌cMET表达。miR-34a、miR-27b及miR-31在胃癌细胞株中表达较正常胃黏膜明显降低(P<0.05)。Western bolt显示miR-27b对c-MET的抑制能力最强。双荧光素酶实验同样证实了c-MET是miR-27b的直接作用靶点。平板克隆及Transwell实验显示过表达miR-27b能抑制胃癌细胞的生长、增殖及侵袭转移能力,而过表达c-MET后能恢复胃癌细胞的生长、增殖及侵袭转移能力。结论:miR-27b能作用于c-MET 3’UTR端从而抑制c-MET的表达,并能通过c-MET抑制胃癌细胞的生长、增殖及侵袭转移。