miR-142-3p is a tumor suppressor that inhibits estrogen receptor expression in ER-positive breast cancer

Estrogen receptors (ERs) are involved in the development of many types of malignant tumors, in particular, breast cancer. Among others, ERs affect cell growth, proliferation, and differentiation. The microRNA (miRNA) miR-142-3p has been shown to inhibit carcinogenesis by regulating various cellular processes, including cell cycle progression, cell migration, apoptosis, and invasion. It does so via targeting molecules involved in a range of signaling pathways. We surgically collected 20 ER-positive breast cancer samples, each with matched adjacent normal breast tissue, and measured the expression of miR-142-3p via quantitative real-time polymerase chain reaction (qRT-PCR). Bioinformatics methods, luciferase reporter assay, qRT-PCR, and western blot analysis were used to assess whether miR-142-3p could target ESR1, which encodes the estrogen receptor, in ER-positive breast cancer cells and patient samples. We also restored miRNA expression and performed cell viability, cytotoxicity, and colony formation assays. Western blot analysis and qRT-PCR were used to study the expression of apoptosis and stemness markers. We found that miR-142-3p is downregulated in ER-positive breast cancers. Restoration of miR-142-3p expression in ER-positive breast cancer cells reduced cell viability, induced apoptosis via the intrinsic pathway and decreased both colony formation and the expression of stem cell markers. Bioinformatic analysis predicted miR-142-3p could bind to 3′-untranslated region ESR1 messenger RNA (mRNA). Consistently, we demonstrated that miR-142-3p reduced luciferase activity in ER-positive breast cancer cells, and decreased ESR1 expression in both mRNA and protein levels. The results revealed miR-142-3p and ESR1 expression correlated negatively in ER-positive breast cancer samples. The results suggest miR-142-3p acts as a tumor suppressor via multiple mechanisms. Thus, restoration of miR-142-3p expression, for example, via miRNA replacement therapy, may represent an effective strategy for the treatment of ER-positive breast cancer patients.     

miR-34a is associated with clinical outcome of colorectal cancer patients

Aberrant expression of microRNA-34a (miR-34a) has been reported to be involved in the tumorigenesis and progression of various classes of malignancies. However, its role in colorectal cancer (CRC) has not been completely clarified. In the current study, we have investigated the clinical significance of miR-34a. MiR-34a expression in forty-three cases of colorectal cancer tissues decreased significantly compared to that in the adjacent non-tumorous colorectal tissues (P＜0.05), as detected by real-time quantitative RT-PCR (qRT-PCR). Significantly, the expression of miR-34a was correlated with infiltration depth and clinical TNM stage (P <0.05). The miR-34a however had no correlation with other features, such as age, gender, site, tumor sizes, lymph node metastasis, serous membrane infiltration ( all P> 0.05). MiR-34a is a tumor suppressor miRNA that plays a vital role in the oncogenesis and progression of colorectal cancer. This study suggests that miR-34a may be a new tumor marker or prognostic factor in colorectal cancer. The strategies to increase miR-34a level might be a critical targeted therapy for CRC in the future.     

Global microRNA expression profiling identifies MiR-210 associated with tumor proliferation, invasion and poor clinical outcome in breast cancer

Purpose

Aberrant microRNA (miRNA) expression is associated with cancer and has potential diagnostic and prognostic value in various malignancies. In this study, we investigated miRNA profiling as a complementary tool to improve our understanding of breast cancer (BC) biology and to assess whether miRNA expression could predict clinical outcome of BC patients.

Experimental Design

Global miRNA expression profiling using microarray technology was conducted in 56 systemically untreated BC patients who had corresponding mRNA expression profiles available. Results were further confirmed using qRT-PCR in an independent dataset of 89 ER-positive BC patients homogeneously treated with tamoxifen only. MiR-210 functional analyses were performed in MCF7 and MDA-MB-231 BC cell lines using lentiviral transduction.

Results

Estrogen receptor (ER) status, tumor grade and our previously developed gene expression grade index (GGI) were associated with distinct miRNA profiles. Several miRNAs were found to be clinically relevant, including miR-210, its expression being associated with tumor proliferation and differentiation. Furthermore, miR-210 was associated with poor clinical outcome in ER-positive, tamoxifen-treated BC patients. Interestingly, the prognostic performance of miR-210 was similar to several reported multi-gene signatures, highlighting its important role in BC differentiation and tumor progression. Functional analyses in BC cell lines revealed that miR-210 is involved in cell proliferation, migration and invasion.

Conclusions

This integrated analysis combining miRNA and mRNA expression demonstrates that miRNA expression provides additional biological information beyond mRNA expression. Expression of miR-210 is linked to tumor proliferation and appears to be a strong potential biomarker of clinical outcome in BC.     

The p53 tumor suppressor gene. A preliminary clinical study in breast cancer patients.

p53 was originally considered to be a nuclear oncogene, but several convergent lines of research have indicated that the wild-type gene functions as a tumor suppressor gene negatively regulating the cell cycle. Mutations in the p53 gene have been detected in many tumor types and seem to be the most common genetic alterations in human cancer. In this preliminary study, sera of 92 patients (pts) with breast disease were analyzed for the presence of the mutant p53 protein (mp53) with a selective immunoenzyme assay employing a monoclonal antibody (PAb 240) specific for the majority of mammalian m p53 but not for the wild-type protein. Of the 10 patients with benign breast disease, only two (20%) showed detectable m p53 levels in the serum. In the breast cancer group, sera from 7 of the 30 pts (23%) without lymph node involvement were positive for m p53, as were 7 out of the 45 pts (15%) with metastatic lymph nodes and 1 out of the 7 pts (14%) with disseminated disease. The specificity of m p53 assay evaluated in 20 healthy controls was 100%. These preliminary results showed that serum positivity for m p53 is not related to breast disease extension. Further studies to assess the utility of m p53 as a possible prognosis factor in breast cancer are currently in progress.     

MicroRNA-487a-3p functions as a new tumor suppressor in prostate cancer by targeting CCND1

Prostate cancer (PCa) is one of the major health problems of the aging male. The roles of dysregulated microRNAs in PCa remain unclear. In this study, we mined the public published data and found that miR-487a-3p was significantly downregulated in 38 pairs of clinical prostate tumor tissues compared with the normal tissues. We further verified this result by in situ hybridization on tissue chip and quantitative real-time polymerase chain reaction (qRT-PCR) in PCa/normal cells. miR-487a-3p targeting of cyclin D1 (CCND1) was identified using bioinformatics, qRT-PCR and western blot analyses. The cellular proliferation, cell cycle, migration, and invasion were assessed by cell counting kit-8, flow cytometry analysis and transwell assay. We discovered that overexpression of miR-487a-3p suppressed PCa cell growth, migration, invasion by directly targeting CCND1. Knockdown of CCND1 in PCa cells showed similar results. Meanwhile, the expression level of CCND1 was significantly upregulated in the PCa tissues and cell lines, which presented negative correlation with the expression of miR-487a-3p. More important, we demonstrated significantly reduced growth of xenograft tumors of stable miR-487a-3p-overexpressed human PCa cells in nude mice. Taken together, for the first time, our results revealed that miR-487a-3p as a tumor suppressor of PCa could target CCND1. Our finding might reveal miR-487a-3p could be potentially contributed to the pathogenesis and a clinical biomarker or the new potential therapeutic target of PCa.     

MicroRNA-1 is a candidate tumor suppressor and prognostic marker in human prostate cancer

We previously reported that miR-1 is among the most consistently down-regulated miRs in primary human prostate tumors. In this follow-up study, we further corroborated this finding in an independent data set and made the novel observation that miR-1 expression is further reduced in distant metastasis and is a candidate predictor of disease recurrence. Moreover, we performed in vitro experiments to explore the tumor suppressor function of miR-1. Cell-based assays showed that miR-1 is epigenetically silenced in human prostate cancer. Overexpression of miR-1 in these cells led to growth inhibition and down-regulation of genes in pathways regulating cell cycle progression, mitosis, DNA replication/repair and actin dynamics. This observation was further corroborated with protein expression analysis and 3'-UTR-based reporter assays, indicating that genes in these pathways are either direct or indirect targets of miR-1. A gene set enrichment analysis revealed that the miR-1-mediated tumor suppressor effects are globally similar to those of histone deacetylase inhibitors. Lastly, we obtained preliminary evidence that miR-1 alters the cellular organization of F-actin and inhibits tumor cell invasion and filipodia formation. In conclusion, our findings indicate that miR-1 acts as a tumor suppressor in prostate cancer by influencing multiple cancer-related processes and by inhibiting cell proliferation and motility.     

Immunohistochemical detection of mutant p53 protein in regional lymph nodes is associated with adverse outcome in stage II colorectal cancer

Epidemiologic data identifies a cohort of Duke's B (CRC) patients whose survival more closely matches that of Duke's C. Lymph node micrometastases may account for this discrepancy. Lymph node expression of mutant p53 protein (Mp53P) has been linked to a reduction in survival in Japanese Duke's B patients. We aimed to determine the significance of nodal p53 expression in European Duke's B patients using immunohistochemistry. The study comprised 134 consecutive patients who had resections for CRC between 1984 and 1991. End points were 5 year disease free survival or CRC related death. Thirty-four subjects did not achieve end points and were excluded. We examined tumour and nodal sections for Mp53P by immunohistochemistry and correlated this with survival using a Kaplan-Meier (KM) and a Cox Proportional hazards model (CPHT). Five year survival was 73%. Fifty-eight percent of primary tumours expressed Mp53P. Tumour p53 expression did modulate survival behavior. Twenty-six percent of subjects' lymph nodes expressed Mp53P. Fifty-three per cent of those with positive and 17% of those with negative lymph nodes died of recurrence. The relative risk for nodal Mp53P expression was 3.1. There was a significant univariate relationship between lymph node p53 expression and mortality. (Log Rank p = 0.028). Multivariate analysis also showed a significant relationship with mortality. (CPHT p = .03). We conclude that lymph node expression of Mp53P is associated with increased mortality in Duke's B CRC.     

Metadherin is an apoptotic modulator in prostate cancer through miR-342-3p regulation

Prostate cancer is the second most common cancer in men worldwide. This study focused to clarify the roles of Metadherin (MTDH) and miR-342-3p in prostate cancer. We identified that MTDH was up-regulated and miR-342-3p was down-regulated in the prostate tissues, and there is an inverse correlation between MTDH and miR-342-3p. Functional studies revealed that miR-342-3p directly targets MTDH via binding to the 3′ untranslated regions (UTRs) in the prostate cancer cells. Moreover, we also found MTDH overexpression in DU145 and PC3 cells inhibited apoptosis. Subsequently, miR-342-3p has been revealed to reverse the MTDH effect on the cellular apoptosis in the further studies. Our results indicate that MTDH repress apoptosis of prostate cancer in vitro and provides a new strategy for human prostate cancer therapy in the future.     

肝癌组织中miR-338-3p的表达及与临床病理参数的关系

目的:探讨肝癌组织中微小RNA-338-3p(miR-338-3p)的表达及与临床病理参数的关系。方法:选取2015年1月至2016年6月我院手术获得的67例肝癌组织标本,同时每例标本均取癌旁正常组织标本作为配对对照,采用实时定量逆转录聚合酶链反应(RT-qPCR)对两组组织标本中的miR-338-3p进行检测,并分析其与肝癌临床病理特征的关系。结果:45例(67.16%)miR-338-3p表达下调,22例(32.85%)表达上调;RT-qPCR结果显示,肝癌组织中miR-338-3p的相对含量为(0.76±0.38),低于癌旁正常组织中的(1.23±0.45),差异有统计学意义(t=-6.259,P=0.000)。miR-338-3p在低分化、TNM分期Ⅲ+Ⅳ期、肿瘤浸润深度T3+T4期、有淋巴结转移肝癌患者肝癌组织中的表达下调率高于中高分化、Ⅰ+Ⅱ期、T1+T2期、无淋巴结转移肝癌患者,差异有统计学意义(P0.05)。不同性别、年龄、病理类型、肿瘤大小肝癌患者肝癌组织中miR-338-3p表达下调率差异无统计学意义(P0.05)。结论:miR-338-3P在肝癌组织中呈低表达水平,与分化程度、TNM分期、肿瘤浸润深度、淋巴结转移有关,可能参与了肝癌的发生发展过程,早期检测可作为评估肝癌病情的指标。     

MicroRNA‐137 is negatively associated with clinical outcome and regulates tumor development through EZH2 in cervical cancer

We intend to evaluate the expression, clinical relevance, and functional role of microRNA‐137 (miR‐137) in human cervical cancer (CC). MiR‐137 expressions were assessed by qPCR in CC cell lines and human CC tumors. The correlation between endogenous miR‐137 expression and CC patients’ postoperative overall survival was examined statistically. CC cell lines, Ca‐Ski, and SiHa cells were transduced with lentivirus to ectopically upregulate endogenous miR‐137 expressions. Possible inhibitory effects of miR‐137 upregulation on CC in vitro proliferation and migration, as well as in vivo transplantation were evaluated. Targeting of enhancer of zeste homolog 2 (EZH2) gene by miR‐137 in CC was assessed by dual‐luciferase activity assay and qPCR. In CC cells with upregulated miR‐137, EZH2 was overexpressed to assess its direct function in miR‐137 mediated CC proliferation and migration. MiR‐137 was downregulated in both CC cells and human CC tumors. Downregulation of endogenous miR‐137 was significantly correlated with CC patients’ short overall survival. In CC cells, miR‐137 upregulation is tumor‐suppressive by inhibiting proliferation and migration in vitro, and transplantation in vivo. EZH2 was a direct downstream target gene of miR‐137 in CC. Forced overexpression of EZH2 in miR‐137‐upregulated CC cells reversed the tumor‐suppression induced by miR‐137. MiR‐137 is lowly expressed in CC and possibly acting as a negative biomarker for CC patients’ clinical outcome. MiR‐137 upregulation may suppress CC, very likely by inversely regulating EZH2.     

Next generation sequencing demonstrates association between tumor suppressor gene aberrations and poor outcome in patients with cancer

Next generation sequencing is transforming patient care by allowing physicians to customize and match treatment to their patients’ tumor alterations. Our goal was to study the association between key molecular alterations and outcome parameters. We evaluated the characteristics and outcomes (overall survival (OS), time to metastasis/recurrence, and best progression-free survival (PFS)) of 392 patients for whom next generation sequencing (182 or 236 genes) had been performed. The Kaplan-Meier method and Cox regression models were used for our analysis, and results were subjected to internal validation using a resampling method (bootstrap analysis). In a multivariable analysis (Cox regression model), the parameters that were statistically associated with a poorer overall survival were the presence of metastases at diagnosis (P = 0.014), gastrointestinal histology (P < 0.0001), PTEN (P < 0.0001), and CDKN2A alterations (P = 0.0001). The variables associated with a shorter time to metastases/recurrence were gastrointestinal histology (P = 0.004), APC (P = 0.008), PTEN (P = 0.026) and TP53 (P = 0.044) alterations. TP53 (P = 0.003) and PTEN (P = 0.034) alterations were independent predictors of a shorter best PFS. A personalized treatment approach (matching the molecular aberration with a cognate targeted drug) also correlated with a longer best PFS (P = 0.046). Our study demonstrated that, across diverse cancers, anomalies in specific tumor suppressor genes (PTEN, CDKN2A, APC, and/or TP53) were independently associated with a worse outcome, as reflected by time to metastases/recurrence, best PFS on treatment, and/or overall survival. These observations suggest that molecular diagnostic tests may provide important prognostic information in patients with cancer.     

14-3-3sigma is down-regulated in human prostate cancer

The 14-3-3sigma is a negative regulator of the cell cycle, which is induced by p53 in response to DNA damage. It has been characterized as an epithelium-specific marker and down-regulation of the protein has been shown in breast cancers, suggesting its tumor-suppressive activity in epithelial cells. Here we demonstrate that 14-3-3sigma protein is down-regulated in human prostate cancer cell lines, LNCaP, PC3, and DU145 compared with normal prostate epithelial cells. Immunohistochemical analysis of primary prostate cells shows that the expression of 14-3-3sigma protein is epithelial cell-specific. Among prostate pathological specimens, > 95% of benign hyperplasia samples show significant and diffuse immunostaining of 14-3-3sigma in the cytoplasm whereas < 20% of carcinoma samples show positive staining. In terms of mechanisms for the down-regulation of 14-3-3sigma in prostate cancer cells, hypermethylation of the gene promoter plays a causal role in LNCaP cells as 14-3-3sigma mRNA level was elevated by 5-aza-2'-deoxycytidine demethylating treatment. Intriguingly, the proteasome-mediated proteolysis is responsible for 14-3-3sigma reduction in DU145 and PC3 cells, as 14-3-3sigma protein expression was increased by treatment with a proteasome inhibitor MG132. Furthermore, tumor necrosis factor-related apoptosis-inducing ligand enhances 14-3-3sigma gene and protein expression in DU145 and PC3 cells. These data suggest that 14-3-3sigma expression is down-regulated during the neoplastic transition of prostate epithelial cells.     

LINC00052 functions as a tumor suppressor through negatively modulating miR-330-3p in pancreatic cancer

Pancreatic cancer is a serious solid malignant tumor worldwide. Increasing evidence has pointed out that abnormal expressions of long noncoding RNAs are involved in various tumors. Meanwhile, LINC00052 is reported as a famous tumor regulator in several cancers. Nevertheless, the biological role of LINC00052 in pancreatic cancer progression is still unknown. Our study was to explore the specific mechanism of LINC00052 in pancreatic cancer. First, we observed that the LINC00052 was obviously downregulated in several pancreatic cancer cell lines. Overexpression of LINC00052 greatly repressed AsPC-1 and SW1990 cell proliferation, triggered the apoptosis and prevented cell cycle in the G1 phase. For another, AsPC-1 and SW1990 cell migration and invasion capacity were also obviously repressed by LINC00052 upregulation. Moreover, miR-330-3p was elevated in pancreatic cancer cells and can function as a target of LINC00052 confirmed by luciferase reporter and RNA Immunoprecipitation (RIP) experiments. Inhibition of miR-330-3p could depress pancreatic cancer progression while overexpressed miR-330-3p exhibited an opposite process. Finally, our data indicated that the LINC00052 also remarkably suppressed pancreatic tumor growth via modulating miR-330-3p in vivo. To conclude, our study revealed that the LINC00052 might provide a new perspective for pancreatic cancer therapy.     

miR-183-5p functions as a tumor suppressor in lung cancer through PIK3CA inhibition

Background

Lung cancer is the leading cause of cancer-related death worldwide. Previous studies revealed that miR-183-5p is frequently involved in various human cancers. However, the exact role of miR-183-5p in regulating the pathogenesis of lung cancer remains unclear.

Direct interaction and cooperative role of tumor suppressor p16 with band 3 (AE1)

By using the C-terminal 112-residue of band 3 to screen the K562 cDNA library, we find that the p16 interacts with band 3, which was confirmed both in yeast and in mammalian cells. Functional experiments show that p16 facilitates the movement of band 3 to plasma membrane with increased anion transport activity in 293t cells. Moreover, expression of endogenous p16 in 293t cells was increased at 24 and 36 h after transfection with band 3. Our findings provide a novel regulation pathway for both band 3 and p16.     

Expression of the class II tumor suppressor gene RIG1 is directly regulated by p53 tumor suppressor in cancer cell lines

Recent studies indicated that the RIG1 (RARRES3/TIG3) plays an important role in cell proliferation, differentiation, and apoptosis. However, the regulatory mechanism of RIG1 gene expression has not been clearly elucidated. In this study, we identified a functional p53 response element (p53RE) in the RIG1 gene promoter. Transfection studies revealed that the RIG1 promoter activity was greatly enhanced by wild type but not mutated p53 protein. Sequence specific mutation of the p53RE abolished p53-mediated transactivation. Specific binding of p53 protein to the rig-p53RE was demonstrated using electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) assay. Further studies confirmed that the expression of RIG1 mRNA and protein is enhanced through increased p53 protein in HepG2 or in H24-H1299 cells. In conclusion, our results indicated that RIG1 gene is a downstream target of p53 in cancer cell lines.