Noncoding RNAs within the HOX gene network in tumor pathogenesis and progression

HOX genes are involved with normal development, cell identity, cell differentiation, cell metabolism, apoptosis, autophagy as well as with diseases such as tumor pathogenesis and progression. In particular, the genes belonging to HOX paralogous 13 seem to carry out a relevant role in both tumor development and disease progression. In recent years, several noncoding RNAs (ncRNA) sequences have been identified in HOX loci, including long noncoding RNA (lncRNA) and microRNA (miRNA), highly conserved during evolution. Many studies have shown that specific intergenic ncRNAs in HOX loci could directly modulate HOX genes expression in normal and pathological conditions. In the present review we attempt to describe the role of these ncRNAs, through the regulation of the HOX gene network, in normal cell biology, and, with particular emphasis, in diseases such as in cancer pathogenesis and progression.     

A Long Non-Coding RNA snaR Contributes to 5-Fluorouracil Resistance in Human Colon Cancer Cells

Several types of genetic and epigenetic regulation have been implicated in the development of drug resistance, one significant challenge for cancer therapy. Although changes in the expression of non-coding RNA are also responsible for drug resistance, the specific identities and roles of them remain to be elucidated. Long non-coding RNAs (lncRNAs) are a type of ncRNA (> 200 nt) that influence the regulation of gene expression in various ways. In this study, we aimed to identify differentially expressed lncRNAs in 5-fluorouracil-resistant colon cancer cells. Using two pairs of 5-FU-resistant cells derived from the human colon cancer cell lines SNU-C4 and SNU-C5, we analyzed the expression of 90 lncRNAs by qPCR-based profiling and found that 19 and 23 lncRNAs were differentially expressed in SNU-C4R and SNU-C5R cells, respectively. We confirmed that snaR and BACE1AS were downregulated in resistant cells. To further investigate the effects of snaR on cell growth, cell viability and cell cycle were analyzed after transfection of siRNAs targeting snaR. Down-regulation of snaR decreased cell death after 5-FU treatment, which indicates that snaR loss decreases in vitro sensitivity to 5-FU. Our results provide an important insight into the involvement of lncRNAs in 5-FU resistance in colon cancer cells.     

长链非编码RNA与表观遗传调控

近年来,表观遗传学(epigenetics)备受关注.表观遗传调控的方式主要包括DNA甲基化、组蛋白修饰和染色质重塑等.ENCODE计划及随后的研究发现,人类基因组中仅有很小一部分DNA序列负责编码蛋白质,而其余大部分被转录为非编码RNA(non-codingRNA,ncRNA).其中长链非编码RNA(long non-codingRNA,lncRNA)是一类长度大于200nt并且缺乏蛋白质编码能力的RNA分子.越来越多的研究表明,lncRNAs能够通过表观遗传调控、转录调控以及转录后调控等多个层面调节基因的表达,从而参与细胞增殖、分化和凋亡等多种生物学过程.本文将着重综述lncRNAs在表观遗传调控中的作用及其最新的研究进展.     

Genome-wide identification of chromatin-enriched RNA reveals that unspliced dentin matrix protein-1 mRNA regulates cell proliferation in squamous cell carcinoma

Chromatin-enriched noncoding RNAs (ncRNAs) have emerged as key molecules in epigenetic processes by interacting with chromatin-associated proteins. Recently, protein-coding mRNA genes have been reported to be chromatin-tethered, similar with ncRNA. However, very little is known about whether chromatin-enriched mRNA is involved in the chromatin modification process. Here, we comprehensively examined chromatin-enriched RNA in squamous cell carcinoma (SQCC) cells by RNA subcellular localization analysis, which was a combination of RNA fractionation and RNA-seq. We identified 11 mRNAs as highly chromatin-enriched RNAs. Among these, we focused on the dentin matrix protein-1 (DMP-1) gene because its expression in SQCC cells has not been reported. Furthermore, we clarified that DMP-1 mRNA was retained in chromatin in its unspliced form in SQCC in vitro and in vivo. As the inhibition of the unspliced DMP-1 mRNA (unspDMP-1) expression resulted in decreased cellular proliferation in SQCC cells, we performed ChIP-qPCR to identify cell cycle-related genes whose expression was epigenetically modified by unspDMP-1, and found that the CDKN1B promoter became active in SQCC cells by inhibiting unspDMP-1 expression. This result was further validated by the increased CDKN1B gene expression in the cells treated with siRNA for unspDMP-1 and by restoration of the decreased cellular proliferation rate by simultaneously inhibiting CDKN1B expression in SQCC cells. Further, to examine whether unspDMP-1 was able to associate with the CDKN1B promoter region, SQCC cells stably expressing PP7-mCherry fusion protein were transiently transfected with the unspDMP-1 fused to 24 repeats of the PP7 RNA stem loop (unspDMP-1-24xPP7) and we found that unspDMP-1-24xPP7 was efficiently precipitated with the antibody against mCherry and was significantly enriched in the CDKN1B promoter region. Thus, unspDMP-1 is a novel chromatin-enriched RNA that epigenetically regulates cellular proliferation of SQCC.     

Regulatory non‐coding RNAs in acute myocardial infarction

Acute myocardial infarction (AMI) is one of the most common cardiovascular diseases that leads to high mortality and morbidity globally. Various therapeutic targets for AMI have been investigated in recent years, including the non‐coding RNAs (ncRNAs). NcRNAs, a class of RNA molecules that typically do not code proteins, are divided into several subgroups. Among them, microRNAs (miRNAs) are widely studied for their modulation of several pathological aspects of AMI, including cardiomyocyte apoptosis, inflammation, angiogenesis and fibrosis. It has emerged that long ncRNAs (lncRNAs) and circular RNAs (circRNAs) also regulate these processes via interesting mechanisms. However, the regulatory functions of ncRNAs in AMI and their underlying functional mechanisms have not been systematically described. In this review, we summarize the recent findings involving ncRNA actions in AMI and briefly describe the novel mechanisms of these ncRNAs, highlighting their potential application as therapeutic targets in AMI.     

Rn7SK small nuclear RNA is involved in cellular senescence

Rn7SK is a conserved small nuclear noncoding RNA which its function in aging has not been studied. Recently, we have demonstrated that Rn7SK overexpression reduces cell viability and is significantly downregulated in stem cells, human tumor tissues, and cell lines. In this study, we analyzed the role of Rn7SK on senescence in adipose tissue-derived mesenchymal stem cells (AD-MSCs). For this purpose, Rn7SK expression was downregulated and upregulated via transfection and transduction, respectively, in AD-MSCs and subsequently, various distinct characteristics of senescence including cell viability, proliferation, colony formation, senescence-associated β galactosidase activity, and differentiation potency was analyzed. Our results demonstrated the transient knockdown of Rn7SK in MSCs leads to delayed senescence, while its overexpressions shows opposite effects. When osteogenic differentiation was started, however, they exhibited a greater differentiation potential than the original MSCs, suggesting a potential tool for stem cell-based regenerative medicine.     

Regulation of apoptosis by a prostate-specific and prostate cancer-associated noncoding gene, PCGEM1

PCGEM1 is a prostate tissue-specific, and prostate cancer-associated noncoding RNA (ncRNA) gene. Previous results revealed a significant association of elevated PCGEM1 expression levels in prostate cancer cells of African-American patients, whose mortality rate is the highest among prostate cancer patients. Functional study of PCGEM1 demonstrated a marked increase in colony formation in LNCaP prostate cancer cells and NIH3T3 mouse fibroblast cells. This study demonstrates that PCGEM1 overexpression in LNCaP cell culture model results in the inhibition of apoptosis induced by doxorubicin (DOX). Induction of p53 and p21(Waf1/Cip1) by DOX were delayed in LNCaP cells stably overexpressing PCGEM1 (LNCaP-PCGEM1 cells) compared to control LNCaP cells. The protein levels of cleaved caspase 7, and cleaved PARP were attenuated in DOXtreated LNCaP-PCGEM1 cells compared to control LNCaP cells. Similar results were observed in LNCaP cells transiently overexpressing PCGEM1. The inhibition of PARP cleavage by PCGEM1 overexpression was also observed in LNCaP-PCGEM1 cells incubated with etoposide and sodium selenite. Fluorescence-Activated Cell Sorter Annexin-V analysis revealed significantly lower percentage of apoptotic cells in DOX-treated LNCaP-PCGEM1 cells compared to control LNCaP cells. The attenuation of apoptic response appears to be androgen dependent in this experimental model, as androgen-independent variants of LNCaP cells did not exhibit this response. In summary, this study provides new insights into cell biologic functions and novel features of an ncRNA. Further, these data unravel biological mechanisms of cell growth/cell survival-associated functions of this ncRNA in a widely used prostate cancer cell culture model.     

Dedifferentiation derived cells exhibit phenotypic and functional characteristics of epidermal stem cells

Differentiated epidermal cells can dedifferentiate into stem cells or stem cell‐like cells in vivo. In this study, we report the isolation and characterization of dedifferentiation‐derived cells. Epidermal sheets eliminated of basal stem cells were transplanted onto the skin wounds in 47 nude athymic (BALB/c‐nu/nu) mice. After 5 days, cells negative for CK10 but positive for CK19 and β₁‐integrin emerged at the wound‐neighbouring side of the epidermal sheets. Furthermore, the percentages of CK19 and β₁‐integrin⁺ cells detected by flow cytometric analysis were increased after grafting (P < 0.01) and CK10⁺ cells in grafted sheets decreased (P < 0.01). Then we isolated these cells on the basis of rapid adhesion to type IV collagen and found that there were 4.56% adhering cells (dedifferentiation‐derived cells) in the grafting group within 10 min. The in vitro phenotypic assays showed that the expressions of CK19, β₁‐integrin, Oct4 and Nanog in dedifferentiation‐derived cells were remarkably higher than those in the control group (differentiated epidermal cells) (P < 0.01). In addition, the results of the functional investigation of dedifferentiation‐derived cells demonstrated: (1) the numbers of colonies consisting of 5–10 cells and greater than 10 cells were increased 5.9‐fold and 6.7‐fold, respectively, as compared with that in the control (P < 0.01); (2) more cells were in S phase and G2/M phase of the cell cycle (proliferation index values were 21.02% in control group, 45.08% in group of dedifferentiation); (3) the total days of culture (28 days versus 130 days), the passage number of cells (3 passages versus 20 passages) and assumptive total cell output (1 × 10⁵ cells versus 1 × 10¹² cells) were all significantly increased and (4) dedifferentiation‐derived cells, as well as epidermal stem cells, were capable of regenerating a skin equivalent, but differentiated epidermal cells could not. These results suggested that the characteristics of dedifferentiation‐derived cells cultured in vitro were similar to epidermal stem cells. This study may also offer a new approach to yield epidermal stem cells for wound repair and regeneration.     

Identification of small non-coding RNAs from mitochondria and chloroplasts

Small non-protein-coding RNAs (ncRNAs) have been identified in a wide spectrum of organisms ranging from bacteria to humans. In eukarya, systematic searches for ncRNAs have so far been restricted to the nuclear or cytosolic compartments of cells. Whether or not small stable non-coding RNA species also exist in cell organelles, in addition to tRNAs or ribosomal RNAs, is unknown. We have thus generated cDNA libraries from size-selected mammalian mitochondrial RNA and plant chloroplast RNA and searched for small ncRNA species in these two types of DNA-containing cell organelles. In total, we have identified 18 novel candidates for organellar ncRNAs in these two cellular compartments and confirmed expression of six of them by northern blot analysis or RNase A protection assays. Most candidate ncRNA genes map to intergenic regions of the organellar genomes. As found previously in bacteria, the presumptive ancestors of present-day chloroplasts and mitochondria, we also observed examples of antisense ncRNAs that potentially could target organelle-encoded mRNAs. The structural features of the identified ncRNAs as well as their possible cellular functions are discussed. The absence from our libraries of abundant small RNA species that are not encoded by the organellar genomes suggests that the import of RNAs into cell organelles is of very limited significance or does not occur at all.     

Deep sequencing of mycovirus‐derived small RNAs from Botrytis species

RNA silencing is an ancient regulatory mechanism operating in all eukaryotic cells. In fungi, it was first discovered in Neurospora crassa, although its potential as a defence mechanism against mycoviruses was first reported in Cryphonectria parasitica and, later, in several fungal species. There is little evidence of the antiviral potential of RNA silencing in the phytopathogenic species of the fungal genus Botrytis. Moreover, little is known about the RNA silencing components in these fungi, although the analysis of public genome databases identified two Dicer‐like genes in B. cinerea, as in most of the ascomycetes sequenced to date. In this work, we used deep sequencing to study the virus‐derived small RNA (vsiRNA) populations from different mycoviruses infecting field isolates of Botrytis spp. The mycoviruses under study belong to different genera and species, and have different types of genome [double‐stranded RNA (dsRNA), (+)single‐stranded RNA (ssRNA) and (–)ssRNA]. In general, vsiRNAs derived from mycoviruses are mostly of 21, 20 and 22 nucleotides in length, possess sense or antisense orientation, either in a similar ratio or with a predominance of sense polarity depending on the virus species, have predominantly U at their 5′ end, and are unevenly distributed along the viral genome, showing conspicuous hotspots of vsiRNA accumulation. These characteristics reveal striking similarities with vsiRNAs produced by plant viruses, suggesting similar pathways of viral targeting in plants and fungi. We have shown that the fungal RNA silencing machinery acts against the mycoviruses used in this work in a similar manner independent of their viral or fungal origin.     

circRNA-14723 promotes hepatocytes proliferation in rat liver regeneration by sponging rno-miR-16-5p

Circular RNA (circRNA) is a subclass of noncoding RNA (ncRNA) detected within mammalian tissues and cells. However, its regulatory role during the proliferation phase of rat liver regeneration (LR) remains unreported. This study was designed to explore their regulatory mechanisms in cell proliferation of LR. The circRNA expression profile was detected by high-throughput sequencing. It was indicated that 260 circRNAs were differentially expressed during the proliferation phase of rat LR. Among them, circ-14723 displayed a significantly differential expression. We further explored its regulatory mechanism in rat hepatocytes (BRL-3A cells). First, EdU, flow cytometry and western blot (WB) indicated that knocking down circ-14723 inhibited BRL-3A cells proliferation. Second, RNA-Pulldown and dual-luciferase report assay showed that circ-14723 could sponge rno-miR-16-5p. At last, WB showed that the reported target genes of rno-miR-16-5p, CCND1, and CCNE1 were downregulated after knocking down circ-14723. In conclusion, we found that circ-14723 exerted a critical role in G1/S arrest to promote cell proliferation via rno-miR-16-5p/CCND1 and CCNE1 axis in rat LR. This finding further revealed the regulatory mechanisms of circRNA on cell proliferation of LR, and might provide a potential target for clinical problems.     

Phorbol ester-induced G1 phase arrest selectively mediated by protein kinase Cdelta-dependent induction of p21

Although protein kinase C (PKC) has been widely implicated in the positive and negative control of proliferation, the underlying cell cycle mechanisms regulated by individual PKC isozymes are only partially understood. In this report, we show that PKCdelta mediates phorbol ester-induced G1 arrest in lung adenocarcinoma cells and establish an essential role for this novel PKC in controlling the expression of the cell cycle inhibitor p21. Activation of PKC with phorbol 12-myristate 13-acetate (PMA) in early G1 phase impaired progression of lung adenocarcinoma cells into S phase, an effect that was completely abolished by specific depletion of PKCdelta, but not PKCalpha. Although the PKC effect was unrelated to the inhibition of cyclin D1 expression, PKC activation significantly up-regulated p21 and down-regulated Rb hyperphosphorylation and cyclin A expression. Elevations in p21 mRNA and protein by PMA were mediated by PKCdelta but not PKCalpha. Studies using luciferase reporters also revealed an essential role for PKCdelta in the PMA-induced inhibition of Rb-dependent cyclin A promoter activity. Finally, we showed that the cell cycle inhibitory effect of PKCdelta is greatly attenuated by RNA interference-mediated knock-down of p21. Our results identify a novel link between PKCdelta and G1 arrest via p21 up-regulation and highlight the complexities in the downstream effectors of PKC isozymes in the context of cell cycle progression and proliferation.