miR‐134 inhibits non‐small cell lung cancer growth by targeting the epidermal growth factor receptor

The epidermal growth factor receptor (EGFR) is frequently activated in a wide range of solid tumours and represents an important therapeutic target. MicroRNAs (miRNAs) have recently been recognized as a rational and potential modality for anti‐EGFR therapies. However, more EGFR‐targeting miRNAs need to be explored. In this study, we identified a novel EGFR‐targeting miRNA, miRNA‐134 (miR‐134), in non‐small‐cell lung cancer (NSCLC) cell lines. Luciferase assays confirmed that EGFR is a direct target of miR‐134. In addition, the overexpression of miR‐134 inhibited EGFR‐related signaling and suppressed NSCLC cells proliferation by inducing cell cycle arrest and/or apoptosis, suggesting that miR‐134 functions as a tumour suppressor in NSCLC. Further mechanistic investigation including RNAi and rescue experiments suggested that the down‐regulation of EGFR by miR‐134 partially contributes to the antiproliferative role of miR‐134. Last, in vivo experiments demonstrated that miR‐134 suppressed tumour growth of A549 xenograft in nude mice. Taken together, our findings suggest that miR‐134 inhibits non‐small cell lung cancer growth by targeting the EGFR.     

The p53/miRNAs/Ccna2 pathway serves as a novel regulator of cellular senescence: Complement of the canonical p53/p21 pathway

Aging is a multifactorial process characterized by the progressive deterioration of physiological functions. Among the multiple molecular mechanisms, microRNAs (miRNAs) have increasingly been implicated in the regulation of Aging process. However, the contribution of miRNAs to physiological Aging and the underlying mechanisms remain elusive. We herein performed high‐throughput analysis using miRNA and mRNA microarray in the physiological Aging mouse, attempted to deepen into the understanding of the effects of miRNAs on Aging process at the “network” level. The data showed that various p53 responsive miRNAs, including miR‐124, miR‐34a and miR‐29a/b/c, were up‐regulated in Aging mouse compared with that in Young mouse. Further investigation unraveled that similar as miR‐34a and miR‐29, miR‐124 significantly promoted cellular senescence. As expected, mRNA microarray and gene co‐expression network analysis unveiled that the most down‐regulated mRNAs were enriched in the regulatory pathways of cell proliferation. Fascinatingly, among these down‐regulated mRNAs, Ccna2 stood out as a common target of several p53 responsive miRNAs (miR‐124 and miR‐29), which functioned as the antagonist of p21 in cell cycle regulation. Silencing of Ccna2 remarkably triggered the cellular senescence, while Ccna2 overexpression delayed cellular senescence and significantly reversed the senescence‐induction effect of miR‐124 and miR‐29. Moreover, these p53 responsive miRNAs were significantly up‐regulated during the senescence process of p21‐deficient cells; overexpression of p53 responsive miRNAs or knockdown of Ccna2 evidently accelerated the cellular senescence in the absence of p21. Taken together, our data suggested that the p53/miRNAs/Ccna2 pathway might serve as a novel senescence modulator independent of p53/p21 pathway.     

microRNA‐128‐3p overexpression inhibits breast cancer stem cell characteristics through suppression of Wnt signalling pathway by down‐regulating NEK2

Emerging evidence has reported that dysregulation of microRNAs (miRNAs) participated in the development of diverse types of cancers. Our initial microarray‐based analysis identified differentially expressed NEK2 related to breast cancer and predicted the regulatory microRNA‐128‐3p (miR‐128‐3p). Herein, this study aimed to characterize the tumour‐suppressive role of miR‐128‐3p in regulating the biological characteristics of breast cancer stem cells (BCSCs). CD44^＋CD24^?/low cells were selected for subsequent experiments. After verification of the target relationship between miR‐128‐3p and NEK2, the relationship among miR‐128‐3p, NEK2 and BCSCs was further investigated with the involvement of the Wnt signalling pathway. The regulatory effects of miR‐128‐3p on proliferation, migration, invasion and self‐renewal in vitro as well as tumorigenicity in vivo of BCSCs were examined via gain‐ and loss‐of‐function approaches. Highly expressed NEK2 was found in breast cancer based on GSE61304 expression profile. Breast cancer stem cells and breast cancer cells showed a down‐regulation of miR‐128‐3p. Overexpression of miR‐128‐3p was found to inhibit proliferation, migration, invasion, self‐renewal in vitro and tumorigenicity in vivo of BCSCs, which was further validated to be achieved through inhibition of Wnt signalling pathway by down‐regulating NEK2. In summary, this study indicates that miR‐128‐3p inhibits the stem‐like cell features of BCSCs via inhibition of the Wnt signalling pathway by down‐regulating NEK2, which provides a new target for breast cancer treatment.     

Integrated analysis of co‐expression and ceRNA network identifies five lncRNAs as prognostic markers for breast cancer

Long non‐coding RNAs (lncRNAs), which competitively bind miRNAs to regulate target mRNA expression in the competing endogenous RNAs (ceRNAs) network, have attracted increasing attention in breast cancer research. We aim to find more effective therapeutic targets and prognostic markers for breast cancer. LncRNA, mRNA and miRNA expression profiles of breast cancer were downloaded from TCGA database. We screened the top 5000 lncRNAs, top 5000 mRNAs and all miRNAs to perform weighted gene co‐expression network analysis. The correlation between modules and clinical information of breast cancer was identified by Pearson's correlation coefficient. Based on the most relevant modules, we constructed a ceRNA network of breast cancer. Additionally, the standard Kaplan‐Meier univariate curve analysis was adopted to identify the prognosis of lncRNAs. Ultimately, a total of 23 and 5 modules were generated in the lncRNAs/mRNAs and miRNAs co‐expression network, respectively. According to the Green module of lncRNAs/mRNAs and Blue module of miRNAs, our constructed ceRNA network consisted of 52 lncRNAs, 17miRNAs and 79 mRNAs. Through survival analysis, 5 lncRNAs (AL117190.1, COL4A2‐AS1, LINC00184, MEG3 and MIR22HG) were identified as crucial prognostic factors for patients with breast cancer. Taken together, we have identified five novel lncRNAs related to prognosis of breast cancer. Our study has contributed to the deeper understanding of the molecular mechanism of breast cancer and provided novel insights into the use of breast cancer drugs and prognosis.     

Identification of micro‐RNA networks in end‐stage heart failure because of dilated cardiomyopathy

Micro‐RNAs regulate gene expression by directly binding to the target mRNAs. The goal of the study was to examine the expression profiling of miRNAs in human failing hearts and identify the key miRNAs that regulate molecular signalling networks and thus contribute to this pathological process. The levels of miRNAs and expressed genes were analysed in myocardial biopsy samples from patients with end‐stage heart failure (n = 14) and those from normal heart samples (n = 8). Four networks were built including the Gene regulatory network, Signal‐Network, miRNA‐GO‐Network and miRNA‐Gene‐Network. According to the fold change in the network and probability values in the microarray cohort, RT‐PCR was performed to measure the expression of five of the 72 differentially regulated miRNAs. miR‐340 achieved statistically significant. miR‐340 was identified for the first time in cardiac pathophysiological condition. We overexpressed miR‐340 in cultured neonatal rat cardiomyocytes to identify whether miR‐340 plays a determining role in the progression of heart failure. ANP, BNP and caspase‐3 were significantly elevated in the miR‐340 transfected cells compared with controls (P < 0.05). The cross‐sectional area of overexpressing miR‐340 cardiomyocytes (1952.22 ± 106.59) was greater (P < 0.0001) than controls (1059.99 ± 45.59) documented by Laser Confocal Microscopy. The changes of cellular structure and the volume were statistical significance. Our study provided a comprehensive miRNA expression profiling in the end‐stage heart failure and identified miR‐340 as a key miRNA contributing to the occurrence and progression of heart failure. Our discoveries provide novel therapeutic targets for patients with heart failure.     

Epigenetic regulation of miR‐129‐2 and its effects on the proliferation and invasion in lung cancer cells

MicroRNAs (miRNAs) play a pivotal role in carcinogenesis. Dysregulation of miRNAs, both oncogenic miRNAs and tumour‐suppressive miRNAs, is closely associated with cancer development and progression. The levels of miRNAs could be changed epigenetically by DNA methylation in the 5′ untranslated region (UTR) of pre‐mature miRNAs. To investigate whether DNA methylation alters the expression of miR‐129 in lung cancer, we did DNA methylation assays and found that 5′ UTR region of miR‐129‐2 gene was absolutely methylated in both A549 and SPCA‐1 lung cancer cells, but totally un‐methylated in 95‐D cells. The expression of miR‐129 was restored by 5‐Aza‐2'‐deoxycytidine (DAC), a de‐methylation agent, in both A549 and SPCA‐1 cells, resulting in attenuated cell migration and invasion ability, and decreased protein level of NF‐κB, which indicates the involvement of NF‐κB pathway. To further illustrate the roles of miR‐129 in lung tumourigenesis, we overexpressed miR‐129 in lung cancer cells by transfection of miR‐129 mimics, and found arrested cell proliferation at G2/M phase of cell cycle and inhibited cell invasion. These findings strongly suggest that miR‐129 is a tumour suppressive miRNA, playing important roles in the development and progression of human lung cancer.     

A functional high‐content miRNA screen identifies miR‐30 family to boost recombinant protein production in CHO cells

The steady improvement of mammalian cell factories for the production of biopharmaceuticals is a key challenge for the biotechnology community. Recently, small regulatory microRNAs (miRNAs) were identified as novel targets for optimizing Chinese hamster ovary (CHO) production cells as they do not add any translational burden to the cell while being capable of regulating entire physiological pathways. The aim of the present study was to elucidate miRNA function in a recombinant CHO‐SEAP cell line by means of a genome‐wide high‐content miRNA screen. This screen revealed that out of the 1, 139 miRNAs examined, 21% of the miRNAs enhanced cell‐specific SEAP productivity mainly resulting in elevated volumetric yields, while cell proliferation was accelerated by 5% of the miRNAs. Conversely, cell death was diminished by 13% (apoptosis) or 4% (necrosis) of all transfected miRNAs. Besides these large number of identified target miRNAs, the outcome of our studies suggest that the entire miR‐30 family substantially improves bioprocess performance of CHO cells. Stable miR‐30 over expressing cells outperformed parental cells by increasing SEAP productivity or maximum cell density of approximately twofold. Our results highlight the application of miRNAs as powerful tools for CHO cell engineering, identified the miR‐30 family as a critical component of cell proliferation, and support the notion that miRNAs are powerful determinants of cell viability.     

MiR‐34b‐3p represses cell proliferation,cell cycle progression and cell apoptosis in non‐small‐cell lung cancer (NSCLC) by targeting CDK4

Lung cancer is the most common incident cancer, with a high mortality worldwide, and non‐small‐cell lung cancer (NSCLC) accounts for approximately 85% of cases. Numerous studies have shown that the aberrant expression of microRNAs (miRNAs) is associated with the development and progression of cancers. However, the clinical significance and biological roles of most miRNAs in NSCLC remain elusive. In this study, we identified a novel miRNA, miR‐34b‐3p, that suppressed NSCLC cell growth and investigated the underlying mechanism. miR‐34b‐3p was down‐regulated in both NSCLC tumour tissues and lung cancer cell lines (H1299 and A549). The overexpression of miR‐34b‐3p suppressed lung cancer cell (H1299 and A549) growth, including proliferation inhibition, cell cycle arrest and increased apoptosis. Furthermore, luciferase reporter assays confirmed that miR‐34b‐3p could bind to the cyclin‐dependent kinase 4 (CDK4) mRNA 3′‐untranslated region (3′‐UTR) to suppress the expression of CDK4 in NSCLC cells. H1299 and A549 cell proliferation inhibition is mediated by cell cycle arrest and apoptosis with CDK4 interference. Moreover, CDK4 overexpression effectively reversed miR‐34‐3p‐repressed NSCLC cell growth. In conclusion, our findings reveal that miR‐34b‐3p might function as a tumour suppressor in NSCLC by targeting CDK4 and that miR‐34b‐3p may, therefore, serve as a biomarker for the diagnosis and treatment of NSCLC.     

Identification and development of long non‐coding RNA‐associated regulatory network in colorectal cancer

Colorectal cancer (CRC) is one of the leading causes of cancer‐associated death globally. Long non‐coding RNAs (lncRNAs) have been identified as micro RNA (miRNA) sponges in a competing endogenous RNA (ceRNA) network and are involved in the regulation of mRNA expression. This study aims to construct a lncRNA‐associated ceRNA network and investigate the prognostic biomarkers in CRC. A total of 38 differentially expressed (DE) lncRNAs, 23 DEmiRNAs and 27 DEmRNAs were identified by analysing the expression profiles of CRC obtained from The Cancer Genome Atlas (TCGA). These RNAs were chosen to develop a ceRNA regulatory network of CRC, which comprised 125 edges. Survival analysis showed that four lncRNAs, six miRNAs and five mRNAs were significantly associated with overall survival. A potential regulatory axis of ADAMTS9‐AS2/miR‐32/PHLPP2 was identified from the network. Experimental validation was performed using clinical samples by quantitative real‐time PCR (qRT‐PCR), which showed that expression of the genes in the axis was associated with clinicopathological features and the correlation among them perfectly conformed to the ‘ceRNA theory’. Overexpression of ADAMTS9‐AS2 in colon cancer cell lines significantly inhibited the miR‐32 expression and promoted PHLPP2 expression, while ADAMTS9‐AS2 knockdown had the opposite effects. The constructed novel ceRNA network may provide a comprehensive understanding of the mechanisms of CRC carcinogenesis. The ADAMTS9‐AS2/miR‐32/PHLPP2 regulatory axis may serve as a potential therapeutic target for CRC.     

miR379–410 cluster miRNAs regulate neurogenesis and neuronal migration by fine‐tuning N‐cadherin

N‐cadherin‐mediated adhesion is essential for maintaining the tissue architecture and stem cell niche in the developing neocortex. N‐cadherin expression level is precisely and dynamically controlled throughout development; however, the underlying regulatory mechanisms remain largely unknown. MicroRNAs (miRNAs) play an important role in the regulation of protein expression and subcellular localisation. In this study, we show that three miRNAs belonging to the miR379–410 cluster regulate N‐cadherin expression levels in neural stem cells and migrating neurons. The overexpression of these three miRNAs in radial glial cells repressed N‐cadherin expression and increased neural stem cell differentiation and neuronal migration. This phenotype was rescued when N‐cadherin was expressed from a miRNA‐insensitive construct. Transient abrogation of the miRNAs reduced stem cell differentiation and increased cell proliferation. The overexpression of these miRNAs specifically in newborn neurons delayed migration into the cortical plate, whereas the knockdown increased migration. Collectively, our results indicate a novel role for miRNAs of the miR379–410 cluster in the fine‐tuning of N‐cadherin expression level and in the regulation of neurogenesis and neuronal migration in the developing neocortex.     

Hormone replacement therapy enhances IGF‐1 signaling in skeletal muscle by diminishing miR‐182 and miR‐223 expressions: a study on postmenopausal monozygotic twin pairs

MiRNAs are fine‐tuning modifiers of skeletal muscle regulation, but knowledge of their hormonal control is lacking. We used a co‐twin case–control study design, that is, monozygotic postmenopausal twin pairs discordant for estrogen‐based hormone replacement therapy (HRT) to explore estrogen‐dependent skeletal muscle regulation via miRNAs. MiRNA profiles were determined from vastus lateralis muscle of nine healthy 54–62‐years‐old monozygotic female twin pairs discordant for HRT (median 7 years). MCF‐7 cells, human myoblast cultures and mouse muscle experiments were used to confirm estrogen's causal role on the expression of specific miRNAs, their target mRNAs and proteins and finally the activation of related signaling pathway. Of the 230 miRNAs expressed at detectable levels in muscle samples, qPCR confirmed significantly lower miR‐182, miR‐223 and miR‐142‐3p expressions in HRT using than in their nonusing co‐twins. Insulin/IGF‐1 signaling emerged one common pathway targeted by these miRNAs. IGF‐1R and FOXO3A mRNA and protein were more abundantly expressed in muscle samples of HRT users than nonusers. In vitro assays confirmed effective targeting of miR‐182 and miR‐223 on IGF‐1R and FOXO3A mRNA as well as a dose‐dependent miR‐182 and miR‐223 down‐regulations concomitantly with up‐regulation of FOXO3A and IGF‐1R expression. Novel finding is the postmenopausal HRT‐reduced miRs‐182, miR‐223 and miR‐142‐3p expression in female skeletal muscle. The observed miRNA‐mediated enhancement of the target genes' IGF‐1R and FOXO3A expression as well as the activation of insulin/IGF‐1 pathway signaling via phosphorylation of AKT and mTOR is an important mechanism for positive estrogen impact on skeletal muscle of postmenopausal women.     

miRNA engineering of CHO cells facilitates production of difficult‐to‐express proteins and increases success in cell line development

In recent years, coherent with growing biologics portfolios also the number of complex and thus difficult‐to‐express (DTE) therapeutic proteins has increased considerably. DTE proteins challenge bioprocess development and can include various therapeutic protein formats such as monoclonal antibodies (mAbs), multi‐specific affinity scaffolds (e.g., bispecific antibodies), cytokines, or fusion proteins. Hence, the availability of robust and versatile Chinese hamster ovary (CHO) host cell factories is fundamental for high‐yielding bioprocesses. MicroRNAs (miRNAs) have emerged as potent cell engineering tools to improve process performance of CHO manufacturing cell lines. However, there has not been any report demonstrating the impact of beneficial miRNAs on industrial cell line development (CLD) yet. To address this question, we established novel CHO host cells constitutively expressing a pro‐productive miRNA: miR‐557. Novel host cells were tested in two independent CLD campaigns using two different mAb candidates including a normal as well as a DTE antibody. Presence of miR‐557 significantly enhanced each process step during CLD in a product independent manner. Stable expression of miR‐557 increased the probability to identify high‐producing cell clones. Furthermore, production cell lines derived from miR‐557 expressing host cells exhibited significantly increased final product yields in fed‐batch cultivation processes without compromising product quality. Strikingly, cells co‐expressing miR‐557 and a DTE antibody achieved a twofold increase in product titer compared to clones co‐expressing a negative control miRNA. Thus, host cell engineering using miRNAs represents a promising tool to overcome limitations in industrial CLD especially with regard to DTE proteins. Biotechnol. Bioeng. 2017;114: 1495–1510. © 2017 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.     

miR‐193b represses influenza A virus infection by inhibiting Wnt/β‐catenin signalling

Due to an increasing emergence of new and drug‐resistant strains of the influenza A virus (IAV), developing novel measures to combat influenza is necessary. We have previously shown that inhibiting Wnt/β‐catenin pathway reduces IAV infection. In this study, we aimed to identify antiviral human microRNAs (miRNAs) that target the Wnt/β‐catenin signalling pathway. Using a miRNA expression library, we identified 85 miRNAs that up‐regulated and 20 miRNAs that down‐regulated the Wnt/β‐catenin signalling pathway. Fifteen miRNAs were validated to up‐regulate and five miRNAs to down‐regulate the pathway. Overexpression of four selected miRNAs (miR‐193b, miR‐548f‐1, miR‐1‐1, and miR‐509‐1) that down‐regulated the Wnt/β‐catenin signalling pathway reduced viral mRNA, protein levels in A/PR/8/34‐infected HEK293 cells, and progeny virus production. Overexpression of miR‐193b in lung epithelial A549 cells also resulted in decreases of A/PR/8/34 infection. Furthermore, miR‐193b inhibited the replication of various strains, including H1N1 (A/PR/8/34, A/WSN/33, A/Oklahoma/3052/09) and H3N2 (A/Oklahoma/309/2006), as determined by a viral reporter luciferase assay. Further studies revealed that β‐catenin was a target of miR‐193b, and β‐catenin rescued miR‐193b‐mediated suppression of IAV infection. miR‐193b induced G0/G1 cell cycle arrest and delayed vRNP nuclear import. Finally, adenovirus‐mediated gene transfer of miR‐193b to the lung reduced viral load in mice challenged by a sublethal dose of A/PR/8/34. Collectively, our findings suggest that miR‐193b represses IAV infection by inhibiting Wnt/β‐catenin signalling.     

Induced miR‐99a expression represses Mtor cooperatively with miR‐150 to promote regulatory T‐cell differentiation

Peripheral induction of regulatory T (Treg) cells provides essential protection from inappropriate immune responses. CD4⁺ T cells that lack endogenous miRNAs are impaired to differentiate into Treg cells, but the relevant miRNAs are unknown. We performed an overexpression screen with T‐cell‐expressed miRNAs in naive mouse CD4⁺ T cells undergoing Treg differentiation. Among 130 candidates, the screen identified 29 miRNAs with a negative and 10 miRNAs with a positive effect. Testing reciprocal Th17 differentiation revealed specific functions for miR‐100, miR‐99a and miR‐10b, since all of these promoted the Treg and inhibited the Th17 program without impacting on viability, proliferation and activation. miR‐99a cooperated with miR‐150 to repress the expression of the Th17‐promoting factor mTOR. The comparably low expression of miR‐99a was strongly increased by the Treg cell inducer “retinoic acid”, and the abundantly expressed miR‐150 could only repress Mtor in the presence of miR‐99a. Our data suggest that induction of Treg cell differentiation is regulated by a miRNA network, which involves cooperation of constitutively expressed as well as inducible miRNAs.