MicroRNA expression signature of castration-resistant prostate cancer: the microRNA-221/222 cluster functions as a tumour suppressor and disease progression marker

Background:

Our present study of the microRNA (miRNA) expression signature in castration-resistant prostate cancer (CRPC) revealed that the clustered miRNAs microRNA-221 (miR-221) and microRNA-222 (miR-222) are significantly downregulated in cancer tissues. The aim of this study was to investigate the functional roles of miR-221 and miR-222 in prostate cancer (PCa) cells.

Methods:

A CRPC miRNA signature was constructed by PCR-based array methods. Functional studies of differentially expressed miRNAs were analysed using PCa cells. The association between miRNA expression and overall survival was estimated by the Kaplan–Meier method. In silico database and genome-wide gene expression analyses were performed to identify molecular targets regulated by the miR-221/222 cluster.

Results:

miR-221 and miR-222 were significantly downregulated in PCa and CRPC specimens. Kaplan–Meier survival curves showed that low expression of miR-222 predicted a short duration of progression to CRPC. Restoration of miR-221 or miR-222 in cancer cells revealed that both miRNAs significantly inhibited cancer cell migration and invasion. Ecm29 was directly regulated by the miR-221/222 cluster in PCa cells.

Conclusions:

Loss of the tumour-suppressive miR-221/222 cluster enhanced migration and invasion in PCa cells. Our data describing targets regulated by the tumour-suppressive miR-221/222 cluster provide insights into the mechanisms of PCa and CRPC progression.     

Circulating microRNAs predict biochemical recurrence in prostate cancer patients

Background:

Circulating microRNAs (miRNAs) are emerging as promising biomarkers for prostate cancer. Here, we investigated the potential of these molecules to assist in prognosis and treatment decision-making.

Methods:

MicroRNAs in the serum of patients who had experienced rapid biochemical recurrence (BCR) (n=8) or no recurrence (n=8) following radical prostatectomy (RP) were profiled using high-throughput qRT-PCR. Recurrence-associated miRNAs were subsequently quantitated by qRT-PCR in a validation cohort comprised of 70 patients with Gleason 7 cancers treated by RP, 31 of whom had undergone disease progression following surgery. The expression of recurrence-associated miRNAs was also examined in tumour tissue cohorts.

Results:

Three miRNAs – miR-141, miR-146b-3p and miR-194 – were elevated in patients who subsequently experienced BCR in the screening study. MiR-146b-3p and miR-194 were also associated with disease progression in the validation cohort, as determined by log-rank tests and Cox proportional hazards regression. Multivariate analysis revealed that miR-146b-3p possessed prognostic information beyond standard clinicopathological parameters. Analysis of tissue cohorts revealed that miR-194 was robustly expressed in the prostate, elevated in metastases, and its expression in primary tumours was associated with a poor prognosis.

Conclusion:

Our study suggests that circulating miRNAs, measured at the time of RP, could be combined with current prognostic tools to predict future disease progression in men with intermediate risk prostate cancers.     

The microRNA-23b/27b/24-1 cluster is a disease progression marker and tumor suppressor in prostate cancer

Our recent study of microRNA (miRNA) expression signatures in prostate cancer (PCa) has revealed that all members of the miR-23b/27b/24-1 cluster are significantly downregulated in PCa tissues. The aim of this study was to investigate the effectiveness of these clustered miRNAs as a disease progression marker and to determine the functional significance of these clustered miRNAs in PCa. Expression of the miR-23b/27b/24-1 cluster was significantly reduced in PCa tissues. Kaplan-Meier survival curves showed that low expression of miR-27b predicted a short duration of progression to castration-resistant PCa. Gain-of-function studies using mature miR-23b, miR-27b, and miR-24-1 significantly inhibited cell proliferation, migration and invasion in PCa cells (PC3 and DU145). To identify the molecular targets of these miRNAs, we carried out gene expression and in silico database analyses. GOLM1 was directly regulated by miR-27b in PCa cells. Elucidation of the molecular targets and pathways regulated by the tumor-suppressive microRNAs should shed light on the oncogenic and metastatic processes in PCa.     

Methylation-associated silencing of microRNA-34b/c in gastric cancer and its involvement in an epigenetic field defect

Altered expression of microRNA (miRNA) is strongly implicated in cancer, and recent studies have shown that the silencing of some miRNAs is associated with CpG island hypermethylation. To identify epigenetically silenced miRNAs in gastric cancer (GC), we screened for miRNAs induced by treatment with 5-aza-2'-deoxycytidine and 4-phenylbutyrate. We found that miR-34b and miR-34c are epigenetically silenced in GC and that their downregulation is associated with hypermethylation of the neighboring CpG island. Methylation of the miR-34b/c CpG island was frequently observed in GC cell lines (13/13, 100%) but not in normal gastric mucosa from Helicobacter pylori-negative healthy individuals. Transfection of a precursor of miR-34b and miR-34c into GC cells induced growth suppression and dramatically changed the gene expression profile. Methylation of miR-34b/c was found in a majority of primary GC specimens (83/118, 70%). Notably, analysis of non-cancerous gastric mucosae from GC patients (n = 109) and healthy individuals (n = 85) revealed that methylation levels are higher in gastric mucosae from patients with multiple GC than in mucosae from patients with single GC (27.3 versus 20.8%; P < 0.001) or mucosae from H. pylori-positive healthy individuals (27.3 versus 20.7%; P < 0.001). These results suggest that miR-34b and miR-34c are novel tumor suppressors frequently silenced by DNA methylation in GC, that methylation of miR-34b/c is involved in an epigenetic field defect and that the methylation might be a predictive marker of GC risk.     

Analysis of miR-205 and miR-155 expression in the blood of breast cancer patients

The purpose of this study was to identify and validate circulating microRNAs (miRNAs) in human plasma for use as breast cancer (BC) biomarkers and to analyze their relationship to clinicopathologic features and its preliminary biological function. Genome-wide expression profiling of miRNAs in BC was investigated by microarray analysis. miR-155 was up-regulated greater than two-fold in BC compared with Normal Adjacent Tissue (NAT), whereas let-7b, miR-381, miR-10b, miR-125a-5p, miR-335, miR-205 and miR-145 were down- regulated greater than two-fold. Our hypothesis was that circulating miRNAs are also present and differentially expressed in the serum of BC patients compared to controls. Using real-time PCR (RT-PCR), we analyzed miR-205 and miR-155 in archived serum from 30 participants, 20 with breast cancer and 10 healthy people. miR-205 was down-regulated in BC patient serum while miR-155 was up-regulated. Furthermore, we analyzed the relationship between the expression levels of these two miRNAs and the clinicopathologic parameters of BC patients. High expression of miR155 was associated with clinical stage, molecular type, Ki-67 and p53 in BC patients (P<0.05). By contrast, we found no significant correlation between miR-205 and BC patient clinicopathologic parameters. Functional analysis showed that ectopic expression of miR-205 significantly inhibits cell proliferation and promotes apoptosis. miR-205 was down-regulated and miR-155 was up-regulated in BC patient serum. miR-155 was positive correlated with clinical stage and ki-67 and negatively correlated with p53 status.     

Exosome-delivered miR-221/222 exacerbates tumor liver metastasis by targeting SPINT1 in colorectal cancer

MicroRNAs (miRNAs) are involved in the progression of many cancers through largely unelucidated mechanisms. The results of our present study identified a gene cluster, miR-221/222, that is constitutively upregulated in serum exosome samples of patients with colorectal carcinoma (CRC) with liver metastasis (LM); this upregulation predicts a poor overall survival rate. Using an in vitro cell coculture model, we demonstrated that CRC exosomes harboring miR-221/222 activate liver hepatocyte growth factor (HGF) by suppressing SPINT1 expression. Importantly, miR-221/222 plays a key role in forming a favorable premetastatic niche (PMN) that leads to the aggressive nature of CRC, which was further shown through in vivo studies. Overall, our results show that exosomal miR-221/222 promotes CRC progression and may serve as a novel prognostic marker and therapeutic target for CRC with LM.     

miRNA在肺腺癌恶性胸腔积液中的表达及临床意义

目的 检测肺腺癌恶性胸腔积液中相关的miRNA表达,探讨miRNA表达与预后的关系。方法 采用miRNA芯片技术筛选10例肺腺癌患者恶性胸腔积液miRNA表达谱,结合临床分析可能与预后有关的miRNA。采用实时荧光定量PCR（QPCR）技术检测84例肺腺癌恶性胸腔积液标本中miRNA表达,结合临床预后分析确认表达差异的miRNA。结果 在5例无进展生存期（PFS）≥5个月和5例PFS≤2个月患者中,芯片筛查显示共13种miRNAs 表达变化倍数＞2倍。在84例不同预后肺腺癌胸腔积液患者中选取miR-146a、miR-16、miR-155、miR-484、miR-134、miR-141、miR-106b和miR-93共8个miRNA进行QPCR验证,发现仅miR-93和miR-146a表达存在差异（P＜0.05）,miR-93在预后好的患者中表达升高,变化倍数为2.41倍,miR-146a在预后好的患者中表达下降,变化倍数为0.46倍。结论 miR-93在预后好的患者恶性胸腔积液中高表达,miR-146a则相反,其可能成为判断肺癌恶性胸腔积液患者预后新的分子标志物。     

Expression of 6 MicroRNAs in Prostate Cancer and Its Significance

OBJECTIVE Numerous microRNAs (miRNAs) are deregulated in human cancers. The experimental evidence supports that miRNAs plays a role in the initiation and progression of human malignancies.The present study was undertaken to evaluate the differential expression of 6 miRNAs as biomarker for early detection of prostate cancer, and then to determine whether the expression profiling of these miRNAs could predict the prognosis of prostate cancer.METHODS The expression profilings of these 6 miRNAs were investigated using the method of locked nucleic acid (LNA)-modified oligonucleotide in situ hybridization (ISH). And the technology of tissue microarray (TMA) was employed using the formalin-fixed, paraffin-embedd (FFPE) specimens taken from 52 patients with prostate carcinoma (PCa) and 38 patients with benign prostatic hyperplasia (BPH).RESULTS The rates of positive expression for 6 miRNAs (miR-15b, miR-16, let-7g, miR- 96,miR-182 and miR-183) were 26.92%,15.38%o, 15.38%, 67.31%, 61.54% and 71.15% in the specimens of prostate cancer, and 57.89%, 76.32%, 68.42%, 44.74%, 31.58%,47.37% in the tissues of benign prostatic hyperplasia, respectively.The expressions of all 6 miRNAs between the prostate cancer and benign prostatic hyperplasia tissues were significantly different (P < 0.05). The positive rate of these 6 miRNAs was significantly related to the Gleason Grading of prostate cancer (P < 0.01). There was no significant correlation between the expression of these miRNAs and age and the concentration of serum PSA of the patient (P > 0.05). We also found that the expression of miR-15b, miR-96 and miR-182 correlated with clinical stages of tumor (P <0.05). The expression of miR-96 correlated with lobus prostatae of tumor invasion (P < 0.01), but the expressions of the remaining five miRNAs were not correlated with that (P > 0.05). In addition, the expression of miR-15b was negatively related to that of miR-96,miR-182 and miR-183, respectively (P < 0.01, r < 0.00).There was a positive correlation among the expressions of miR-96, miR-182 and miR-183 in prostate cancer (P < 0.01, r > 0.00). The expression of miR-16 was positively related to that of miR-let-7g (P < 0.01, r >0.00).CONCLUSION The results suggest that miRNA expression profiling could have relevance to the biological and clinical behavior of prostate cancer, and they might be important biomarkers for early detection and prognostic assessment of prostate cancer.     

卵巢癌血清相关miRNAs的筛选及其临床意义

背景与目的：卵巢癌预后较差,发现时通常是晚期,需找寻与卵巢癌发生、发展相关的诊治方法。该研究检测miRNA在上皮性卵巢癌患者术前外周血清及良性卵巢肿瘤患者外周血清中表达情况的差异,筛选差异有统计学意义的miRNA并分析其与上皮性卵巢癌患者临床病理特征、预后等的关系。方法：定制研究相关的48种miRNA表达谱芯片,通过TaqMan低密度微阵列芯片,筛选出有统计学意义的miRNA。采用实时荧光定量聚合酶链反应(real-time fluorescent quantitative polymerase chain reaction,RTFQ-PCR)法验证筛选的miRNA在卵巢良、恶性肿瘤患者血清中的表达情况,选择具有统计学意义的miRNA行大样本验证并分析其与肿瘤分期、组织病理及预后等的关系。结果：通过TaqMan低密度微阵列芯片筛选和RTFQ-PCR验证,发现miR-125b在上皮性卵巢癌患者血清中的表达高于良性肿瘤患者(P=0.039),miR-125b在早期患者中的表达量高于晚期患者(P=0.003),术后无残余肿瘤患者表达量高于术后有残余肿瘤患者(P=0.013)。血清miR-125b高表达有利于卵巢癌患者无进展生存期(progression-free survival,PFS)延长(P=0.003),但对总生存期(overall survival,OS)无明显影响(P=0.069)。结论：miR-125b在上皮性卵巢癌的发生、发展中起着关键作用,与患者预后相关,是预测卵巢癌复发的潜在基因,但在肿瘤不同期别的表达情况发生变化,在早期作用比较明显,在晚期或肿瘤残余较多的患者表达较不明显,其作用机制有待进一步研究。     

CpG island methylation status of miRNAs in esophageal squamous cell carcinoma

Previous studies on esophageal squamous cell carcinoma (ESCC) indicated that it contains much dysregulation of microRNAs (miRNAs). DNA hypermethylation in the miRNA 5' regulatory region is a mechanism that can account for the downregulation of miRNA in tumors (Esteller, N Engl J Med 2008;358:1148-59). Among those dysregulated miRNAs, miR-203, miR-34b/c, miR-424 and miR-129-2 are embedded in CpG islands, as is the promoter of miR-34a. We investigated their methylation status in ESCC by bisulfite sequencing PCR (BSP) and methylation specific PCR (MSP). The methylation frequency of miR-203 and miR-424 is the same in carcinoma and in the corresponding non-tumor tissues. The methylation ratio of miR-34a, miR-34b/c and miR-129-2 is 66.7% (36/54), 40.7% (22/54) and 96.3% (52/54), respectively in ESCC, which are significantly higher than that in the corresponding non-tumor tissues(p < 0.01). Quantitative RT-PCR analysis in clinical samples suggested that CpG island methylation is significantly correlated with their low expression in ESCC, 5-aza-2'-deoxycytidine (DAC) treatment partly recovered their expression in EC9706 cell line. We conclude that CpG island methylation of miR-34a, miR-34b/c and miR-129-2 are frequent events and important mechanism for their low expression in ESCC. DNA methylation changes have been reported to occur early in carcinogenesis and are potentially good early indicators of carcinoma (Laird, Nat Rev Cancer 2003;3:253-66). The high methylation ratio of miR-129-2 indicated its potential as a methylation biomarker in early diagnosis of ESCC.     

Association of miR-193b Down-regulation and miR-196a up-Regulation with Clinicopathological Features and Prognosis in Gastric Cancer

Dysregulated expression of microRNAs (miRNAs) has been shown to be closely associated with tumordevelopment, progression, and carcinogenesis. However, their clinical implications for gastric cancer remainelusive. To investigate the hypothesis that genome-wide alternations of miRNAs differentiate gastric cancer tissuesfrom those matched adjacent non-tumor tissues (ANTTs), miRNA arrays were employed to examine miRNAexpression profiles for the 5-pair discovery stage, and the quantitative real-time polymerase chain reaction (qRTPCR)was applied to validate candidate miRNAs for 48-pair validation stage. Furthermore, the relationshipbetween altered miRNA and clinicopathological features and prognosis of gastric cancer was explored. Amonga total of 1,146 miRNAs analyzed, 16 miRNAs were found to be significantly different expressed in tissues fromgastric cancer compared to ANTTs (p<0.05). qRT-PCR further confirmed the variation in expression of miR-193band miR-196a in the validation stage. Down-expression of miR-193b was significantly correlated with Laurentype, differentiation, UICC stage, invasion, and metastasis of gastric cancer (p<0.05), while over-expression ofmiR-196a was significantly associated with poor differentiation (p=0.022). Moreover, binary logistic regressionanalysis demonstrated that the UICC stage was a significant risk factor for down-expression of miR-193b (adjustedOR=8.69; 95%CI=1.06-56.91; p=0.043). Additionally, Kaplan-Meier survival curves indicated that patientswith a high fold-change of down-regulated miR-193b had a significantly shorter survival time (n=19; mediansurvival=29 months) compared to patients with a low fold-change of down-regulated miR-193b (n=29; mediansurvival=54 months) (p=0.001). Overall survival time of patients with a low fold-change of up-regulated miR-196a (n=27; median survival=52 months) was significantly longer than that of patients with a high fold-changeof up-regulated miR-196a (n=21; median survival=46 months) (p=0.003). Hence, miR-193b and miR-196a maybe applied as novel and promising prognostic markers in gastric cancer.     

组织miR-193b表达及甲基化状态对前列腺癌患者术后骨转移的预测价值北大核心

目的:探究组织miR-193b表达及甲基化状态对前列腺癌(prostate cancer,PCa)患者术后发生骨转移的预测价值。方法:回顾性分析2016年09月至2018年07月期间在我院首次接受根治性前列腺切除术的154例患者,将骨转移作为本研究的主要研究终点,比较组织miR-193b表达及其甲基化状态与PCa患者发生骨转移的相关性。结果:骨转移组肿瘤组织miR-193b相对表达量低于非骨转移组,同时miR-193b基因启动子甲基化率高于非骨转移组(P<0.05)。miR-193b基因启动子甲基化组织中miR-193b相对表达量较非甲基化组织降低(P<0.05)。经Pearson法分析,肿瘤组织中miR-193b表达和基因启动子甲基化呈负相关性(r=-0.45,P<0.05)。经单因素和多因素Logistic回归分析,bGS>7分、cTx:T_(3)-T_(4)、tPSA水平升高、组织miR-193b基因启动子甲基化都是影响PCa患者发生骨转移的独立危险因素。经受试者工作特征曲线分析,组织miR-193b表达预测PCa患者发生骨转移的AUC为0.912(95%CI:0.863~0.960)。结论:miR-193b基因启动子高甲基化是PCa患者术后发生骨转移的独立危险因素,且肿瘤组织中miR-193b表达与基因启动子甲基化状态呈负相关性,因此检测组织miR-193b表达可用于预测PCa患者术后骨转移风险。     

Changes in microRNA (miRNA) expression during pancreatic cancer development and progression in a genetically engineered KrasG12D;Pdx1-Cre mouse (KC) model

Differential expression of microRNAs (miRNAs) has been demonstrated in various cancers, including pancreatic cancer (PC). Due to the lack of tissue samples from early-stages of PC, the stage-specific alteration of miRNAs during PC initiation and progression is largely unknown. In this study, we investigated the global miRNA expression profile and their processing machinery during PC progression using the Kras^G12D;Pdx1-Cre (KC) mouse model. At 25 weeks, the miRNA microarray analysis revealed significant downregulation of miR-150, miR-494, miR-138, miR-148a, miR-216a, and miR-217 and upregulation of miR-146b, miR-205, miR-31, miR-192, and miR-21 in KC mice compared to controls. Further, expression of miRNA biosynthetic machinery including Dicer, Exportin-5, TRKRA, and TARBP2 were downregulated, while DGCR8 and Ago2 were upregulated in KC mice. In addition, from 10 to 50 weeks of age, stage-specific expression profiling of miRNA in KC mice revealed downregulation of miR-216, miR-217, miR-100, miR-345, miR-141, miR-483-3p, miR-26b, miR-150, miR-195, Let-7b and Let-96 and upregulation of miR-21, miR-205, miR-146b, miR-34c, miR-1273, miR-223 and miR-195 compared to control mice. Interestingly, the differential expression of miRNA in mice also corroborated with the miRNA expression in human PC cell lines and tissue samples; ectopic expression of Let-7b in CD18/HPAF and Capan1 cells resulted in downregulation of KRAS and MSST1 expression. Overall, the present study aids an understanding of miRNA expression patterns during PC pathogenesis and helps to facilitate the identification of promising and novel early diagnostic/prognostic markers and therapeutic targets.     

MicroRNA与DNA甲基化相互调节机制在肝细胞癌中的研究进展

肝细胞癌是原发性肝癌的主要类型,也是人类恶性程度较高的肿瘤之一,其发病机制至今尚未完全阐明.表观遗传学机制在肿瘤的发生、发展中起重要作用,DNA甲基化和微小RNA (microRNA,miRNA)的调控机制属于表观遗传学的研究范畴.研究表明,DNA甲基化及miRNA在肝细胞癌的形成中分别或协同发挥着重要作用,miRNA是一类在转录后水平调节基因表达的非编码短链RNA.研究表明,DNA甲基化和组蛋白修饰不仅可以调节蛋白编码基因的表达,而且可以调节miRNA的表达.在肝细胞癌中,一些异常表达的miRNAs(如miR-125b、miR-1-1、miR-124、miR-203和miR-191)是通过表观遗传学机制调控的.另外,在肝细胞癌中还发现了一类miRNAs通过调控表观遗传学通路中关键分子来改变整个基因组的表观遗传学状态.本文就DNA甲基化和miRNA之间复杂的相互调节机制在肝细胞癌发生和发展中的研究进展进行综述.     

miR-125a-5p is a prognostic biomarker that targets HDAC4 to suppress breast tumorigenesis

Identifying stably expressed tumor markers that can be used easily to detect cancer is currently an important area of cancer research. By using miRNA microarray, we identified 20 differentially expressed miRNAs in serum samples of breast cancer patients. Expression of miR-125a-5p was relatively lower in patients with shorter survival compared to long-term survivors. In a cohort of breast cancer patients (N = 300), serum expression of miR-125a-5p was negatively and significantly correlated with tumor grade (P = 0.004), lymph-node status (P = 0.004), and tumor size (P < 0.001). Low miR-125a-5p expression was an independent prognostic marker (OR = 0.421; 95% CI = 0.184 to 0.961; P = 0.04) associated with poor survival rates (P = 0.0062). We show that miR-125a-5p directly inhibits expression of the HDAC4 gene, resulting in tumor suppression in vitro and in vivo. Together these results demonstrate that serum miR-125a-5p level in breast cancer may be a useful prognostic biomarker and offer a novel therapeutic avenue by targeting HDAC4 in breast cancer.