MicroRNA‐mediated non‐cell‐autonomous regulation of cortical radial glial transformation revealed by a Dicer1 knockout mouse model

Radial glia (RG), as neurogenic progenitors and neuronal migration scaffolds, play critical roles during cortical neurogenesis. RG transformation into astrocytes, marking the transition from developmental to physiological function of these cells, is an important step during cortical development. In this study, we aim to determine the roles of microRNAs (miRNAs) during this biological process. In a conditional Dicer1‐null mouse where Dicer1 is deleted in both RG and their neuronal progeny, we observe delayed RG transformation as revealed by the persistence of their radial processes, and reduced number and complexity of translocated RG cell bodies in the postnatal cerebral cortex. Downregulation of Notch1 signaling is crucial to RG transformation, and consistently we find that Notch1 signaling is enhanced in the Dicer1‐null cerebral cortex. In addition, we show that, among the Notch1 ligands, Jagged2 (Jag2) is preferentially upregulated in the postnatal Dicer1‐null cerebral cortex as well as primary embryonic cortical cultures with instant Dicer1 deletion. Functionally, Dicer1‐deleted postnatal cerebellar cells with elevated Jag2 expression stimulate a stronger Notch1 signaling in a RG clone L2.3 when co‐cultured than control cells. Therefore, we unravel a novel non‐cell‐autonomous mechanism that regulates RG transformation by modulating Notch1 signaling via miRNA‐mediated suppression of the Nocth1 ligand Jag2. Furthermore, we validate Jag2 as a miR‐124 target gene and demonstrate in vitro that Jag2 expression is highly sensitive to Dicer1 deletion. Finally, we propose a new concept of MiRNA‐Sensitive target genes, identification of which may unravel a unique mode of miRNA‐mediated gene expression regulation. GLIA 2015;63:860–876     

K14‐EGFP‐miR‐31 transgenic mice have high susceptibility to chemical‐induced squamous cell tumorigenesis that is associating with Ku80 repression

Squamous cell carcinoma (SCC) occurring in the head and neck region and the esophagus causes tremendous cancer mortality around the world. miR‐31 is among the most eminently upregulated MicroRNAs in SCC, when it occurs in the head and neck region and the esophagus. We established miR‐31 transgenic mouse lines, in which miR‐31 is under the control of the K14 promoter. 4‐nitroquinoline 1‐oxide (4NQO) is a mutagen that causes double strand breaks. The transgenic mice exhibited a higher potential for tumor induction than wild‐type (Wt) mice of the tongue and esophagus after 4NQO treatment. After 4NQO treatment or irradiation, p‐γH2AX expression in squamous epithelium of transgenic mice was increased more than in Wt mice. Exogenous expression of miR‐31 was also found to be associated with the higher p‐γH2AX expression induced by 4NQO in human oral SCC (OSCC) cell lines. The repair genes PARP1 and Ku80 were validated as new targets of miR‐31 in human OSCC cell lines, and were found to be downregulated in the squamous epithelium of the tongue in transgenic mice. However, only the downregulation of Ku80 was essential for maintaining the high level of p‐γH2AX induced by 4NQO in OSCC cells. Inverse expression profiles for miR‐31 and Ku80 were noted in human OSCC tissue. Our study identifies the high sensitivity of K14‐EGFP‐miR‐31 transgenic mice to chemical carcinogen‐induced squamous cell tumorigenesis and shows that this seems to be associated with the downregulation of Ku80 and an impairment of repair activity in squamous cells, which are mediated by miR‐31.     

Tumor‐suppressive microRNA‐145 induces growth arrest by targeting SENP1 in human prostate cancer cells

Prostate cancer (PCa) prevails as the most commonly diagnosed malignancy in men and the third leading cause of cancer‐related deaths in developed countries. One of the distinct characteristics of prostate cancer is overexpression of the small ubiquitin‐like modifier (SUMO)‐specific protease 1 (SENP1), and the upregulation of SENP1 contributes to the malignant progression and cell proliferation of PCa. Previous studies have shown that the expression of microRNA‐145 (miRNA‐145) was extensively deregulated in PCa cell lines and primary clinical prostate cancer samples. Independent target prediction methods have indicated that the 3′‐untranslated region of SENP1 mRNA is a potential target of miR‐145. Here we found that low expression of miR‐145 was correlated with high expression of SENP1 in PCa cell line PC‐3. The transient introduction of miR‐145 caused cell cycle arrest in PC‐3 cells, and the opposite effect was observed when miR‐145 inhibitor was transfected. Further studies revealed that the SENP1 3′‐untranslated region was a regulative target of miR‐145 in vitro. MicroRNA‐145 also suppressed tumor formation in vivo in nude mice. Taken together, miR‐145 plays an important role in tumorigenesis of PCa through interfering SENP1.     

Combining TGF-β1 knockdown and miR200c administration to optimize antitumor efficacy of B16F10/GPI-IL-21 vaccine

TGF-β1 secreted abundantly by tumors cells as well as present in the local microenvironment promotes neoplasm invasion and metastasis by triggering the epithelial to mesenchymal transition (EMT). MiR200c has been shown to suppress EMT and to regulate the cellular epithelial and interstitial state conversion, whereas the tumor vaccines are intended to specifically initiate or amplify a host response against evolving tumor cells. Our study aimed at optimizing the antitumor effects of the B16F10/glycosylphosphatidylinositol-interleukin 21 (B16F10/GPI-IL-21) tumor vaccine on melanoma bearing mice by combining the TGF-β1 knockdown and the administration of miR200c agomir. The mice were subcutaneously vaccinated with inactivated B16F10/GPI-IL-21 vaccine and challenged by B16F10 cells transfected with shTGF-β1 (B16F10/shTGF-β1 cells) or B16F10/shTGF-β1 cells with the administration of miR200c agomir. The later combination showed that, when compared with the mice in the control group that received no vaccination, vaccinated mice significantly increased NK and CTL activities, enhanced levels of IFN-γ, and reduced expression of TGF-β1, N-cadherin, Vimentin, Gli1/2, P-Smad2/3 and others involved in promoting expression of EMT-related molecules in tumor areas, and inhibited the melanoma metastasis in lungs and lymph nodes. Altogether, our findings demonstrate that this synergistic anti-cancer regimen effectively induces strong immune response and diminishes the melanoma progression.     

MiR‐34a deficiency accelerates medulloblastoma formation in vivo

Previous studies have evaluated the role of miRNAs in cancer initiation and progression. MiR‐34a was found to be downregulated in several tumors, including medulloblastomas. Here we employed targeted transgenesis to analyze the function of miR‐34a in vivo. We generated mice with a constitutive deletion of the miR‐34a gene. These mice were devoid of mir‐34a expression in all analyzed tissues, but were viable and fertile. A comprehensive standardized phenotypic analysis including more than 300 single parameters revealed no apparent phenotype. Analysis of miR‐34a expression in human medulloblastomas and medulloblastoma cell lines revealed significantly lower levels than in normal human cerebellum. Re‐expression of miR‐34a in human medulloblastoma cells reduced cell viability and proliferation, induced apoptosis and downregulated the miR‐34a target genes, MYCN and SIRT1. Activation of the Shh pathway by targeting SmoA1 transgene overexpression causes medulloblastoma in mice, which is dependent on the presence and upregulation of Mycn. Analysis of miR‐34a in medulloblastomas derived from ND2:SmoA1(tg) mice revealed significant suppression of miR‐34a compared to normal cerebellum. Tumor incidence was significantly increased and tumor formation was significantly accelerated in mice transgenic for SmoA1 and lacking miR‐34a. Interestingly, Mycn and Sirt1 were strongly expressed in medulloblastomas derived from these mice. We here demonstrate that miR‐34a is dispensable for normal development, but that its loss accelerates medulloblastomagenesis. Strategies aiming to re‐express miR‐34a in tumors could, therefore, represent an efficient therapeutic option.     

Novel functions and targets of miR‐944 in human cervical cancer cells

Altered expression of specific microRNAs (miRNAs) has been observed in human cervical cancer. However, the biological functions of many of these miRNAs are yet to be discovered. We previously showed that miR‐944 is significantly more abundant in cervical cancer tissues than their normal counterparts. In this study, we investigated the functions and targets of miR‐944 in human cervical cancer cells. MiR‐944 is located in the intron of the tumor protein p63 (TP63) gene, which is frequently overexpressed in cervical carcinomas. Using gain‐ and loss‐of‐function experiments in vitro, we demonstrate that miR‐944 promotes cell proliferation, migration and invasion, but has no effect on apoptosis, in human cervical cancer cells. To identify the targets of miR‐944, we performed photoactivatable‐ribonucleoside‐enhanced crosslinking and immunoprecipitation followed by deep sequencing. Among the candidate targets, we validated HECW2 (HECT domain ligase W2) and S100PBP (S100P binding protein) as direct targets of miR‐944 using luciferase reporter assays and western blot analysis. Our findings reveal novel functions and targets of miR‐944 in human cervical cancer cells, which may provide new insights of its role in cervical carcinogenesis.     

Expression of tumor suppressive microRNA‐34a is associated with a reduced risk of bladder cancer recurrence

Bladder cancer is the fourth most common cancer among men in the United States and more than half of patients experience recurrences within 5 years after initial diagnosis. Additional clinically informative and actionable biomarkers of the recurrent bladder cancer phenotypes are needed to improve screening and molecular therapeutic approaches for recurrence prevention. MicroRNA‐34a (miR‐34a) is a short noncoding regulatory RNA with tumor suppressive attributes. We leveraged our unique, large, population‐based prognostic study of bladder cancer in New Hampshire, United States to evaluate miR‐34a expression levels in individual tumor cells to assess prognostic value. We collected detailed exposure and medical history data, as well as tumor tissue specimens from bladder patients and followed them long‐term for recurrence, progression and survival. Fluorescence‐based in situ hybridization assays were performed on urothelial carcinoma tissue specimens (n = 229). A larger proportion of the nonmuscle invasive tumors had high levels of miR‐34a within the carcinoma cells compared to those tumors that were muscle invasive. Patients with high miR‐34a levels in their baseline nonmuscle invasive tumors experienced lower risks of recurrence (adjusted hazard ratio 0.57, 95% confidence interval 0.34–0.93). Consistent with these observations, we demonstrated a functional tumor suppressive role for miR‐34a in cultured urothelial cells, including reduced matrigel invasion and growth in soft agar. Our results highlight the need for further clinical studies of miR‐34a as a guide for recurrence screening and as a possible candidate therapeutic target in the bladder.     

Association of Fusobacterium nucleatum with clinical and molecular features in colorectal serrated pathway

Human gut microbiota is being increasingly recognized as a player in colorectal cancers (CRCs). Evidence suggests that Fusobacterium nucleatum (F. nucleatum) may contribute to disease progression and is associated with CpG island methylator phenotype (CIMP) and microsatellite instability (MSI) in CRCs; however, to date, there are no reports about the relationship between F. nucleatum and molecular features in the early stage of colorectal tumorigenesis. Therefore, we investigated the presence of F. nucleatum in premalignant colorectal lesions. In total, 465 premalignant lesions (343 serrated lesions and 122 non‐serrated adenomas) and 511 CRCs were studied. We determined the presence of F. nucleatum and analyzed its association with molecular features including CIMP, MSI and microRNA‐31 status. F. nucleatum was detected in 24% of hyperplastic polyps, 35% of sessile serrated adenomas (SSAs), 30% of traditional serrated adenomas (TSAs) and 33% of non‐serrated adenomas. F. nucleatum was more frequently detected in CIMP‐high premalignant lesions than in CIMP‐low/zero lesions (p = 0.0023). In SSAs, F. nucleatum positivity increased gradually from sigmoid colon to cecum (p = 0.042). F. nucleatum positivity was significantly higher in CRCs (56%) than in premalignant lesions of any histological type (p < 0.0001). In conclusion, F. nucleatum was identified in premalignant colorectal lesions regardless of histopathology but was more frequently associated with CIMP‐high lesions. Moreover, F. nucleatum positivity increased according to histological grade, suggesting that it may contribute to the progression of colorectal neoplasia. Our data also indicate that F. nucleatum positivity in SSAs may support the “colorectal continuum” concept.