Depletion of key protein components of the RISC pathway impairs pre-ribosomal RNA processing

Little is known about whether components of the RNA-induced silencing complex (RISC) mediate the biogenesis of RNAs other than miRNA. Here, we show that depletion of key proteins of the RISC pathway by antisense oligonucleotides significantly impairs pre-rRNA processing in human cells. In cells depleted of Drosha or Dicer, different precursors to 5.8S rRNA strongly accumulated, without affecting normal endonucleolytic cleavages. Moderate yet distinct processing defects were also observed in Ago2-depleted cells. Physical links between pre-rRNA and these proteins were identified by co-immunoprecipitation analyses. Interestingly, simultaneous depletion of Dicer and Drosha led to a different processing defect, causing slower production of 28S rRNA and its precursor. Both Dicer and Ago2 were detected in the nuclear fraction, and reduction of Dicer altered the structure of the nucleolus, where pre-rRNA processing occurs. Together, these results suggest that Drosha and Dicer are implicated in rRNA biogenesis.     

沉默PRR14基因抑制结肠癌HCT116细胞增殖

已有报道显示,富脯氨酸蛋白 14（proline-rich protein 14,PRR14）促进肿瘤的发生发展,但具体作用机制仍不清楚。本文以结肠癌细胞为模型,探索其对细胞增殖和细胞周期进程的影响。qPCR和Western 印迹检测发现,PRR14在4个结肠癌细胞系中呈现高水平表达。合成特异靶向PRR14基因的siRNA,转染结肠癌HCT116细胞。检测发现,PRR14基因表达下调约70%。CCK8结果显示,沉默PRR14后各时间点细胞增殖能力均显著降低,克隆形成实验细胞克隆数减少约40%;流式细胞仪结果显示,沉默PRR14后,G1期细胞比例升高约10%,S期细胞比例降低约14%;BrdU标记免疫荧光检测结果显示,BrdU阳性细胞比例减少约50%,表明细胞DNA合成速率显著降低。机制分析表明：促G1/S期转换基因周期蛋白依赖性激酶2（cyclin dependent kinase 2, CDK2）mRNA水平降低约85%,对应的蛋白质水平也明显降低,G1/S期转换抑制因子周期蛋白依赖性激酶抑制因子1A（cyclin dependent kinase inhibitor 1A,CDKN1A/P21）和周期蛋白依赖性激酶抑制因子1B（cyclin dependent kinase inhibitor 1B,CDKN1B/P27）mRNA水平分别升高约1.8倍和5倍,对应的蛋白质水平也明显升高。沉默PRR14表达,G1/S期相关基因表达紊乱,导致细胞G1期阻滞并抑制细胞增殖。结肠癌细胞中PRR14高表达可促进癌细胞恶性增殖。     

沉默PRR14基因抑制结肠癌HCT116细胞增殖

已有报道显示,富脯氨酸蛋白 14（proline-rich protein 14,PRR14）促进肿瘤的发生发展,但具体作用机制仍不清楚。本文以结肠癌细胞为模型,探索其对细胞增殖和细胞周期进程的影响。qPCR和Western 印迹检测发现,PRR14在4个结肠癌细胞系中呈现高水平表达。合成特异靶向PRR14基因的siRNA,转染结肠癌HCT116细胞。检测发现,PRR14基因表达下调约70%。CCK8结果显示,沉默PRR14后各时间点细胞增殖能力均显著降低,克隆形成实验细胞克隆数减少约40%;流式细胞仪结果显示,沉默PRR14后,G1期细胞比例升高约10%,S期细胞比例降低约14%;BrdU标记免疫荧光检测结果显示,BrdU阳性细胞比例减少约50%,表明细胞DNA合成速率显著降低。机制分析表明：促G1/S期转换基因周期蛋白依赖性激酶2（cyclin dependent kinase 2, CDK2）mRNA水平降低约85%,对应的蛋白质水平也明显降低,G1/S期转换抑制因子周期蛋白依赖性激酶抑制因子1A（cyclin dependent kinase inhibitor 1A,CDKN1A/P21）和周期蛋白依赖性激酶抑制因子1B（cyclin dependent kinase inhibitor 1B,CDKN1B/P27）mRNA水平分别升高约1.8倍和5倍,对应的蛋白质水平也明显升高。沉默PRR14表达,G1/S期相关基因表达紊乱,导致细胞G1期阻滞并抑制细胞增殖。结肠癌细胞中PRR14高表达可促进癌细胞恶性增殖。     

Inducible deletion of epidermal Dicer and Drosha reveals multiple functions for miRNAs in postnatal skin

MicroRNAs (miRNAs) regulate the expression of many mammalian genes and play key roles in embryonic hair follicle development; however, little is known of their functions in postnatal hair growth. We compared the effects of deleting the essential miRNA biogenesis enzymes Drosha and Dicer in mouse skin epithelial cells at successive postnatal time points. Deletion of either Drosha or Dicer during an established growth phase (anagen) caused failure of hair follicles to enter a normal catagen regression phase, eventual follicular degradation and stem cell loss. Deletion of Drosha or Dicer in resting phase follicles did not affect follicular structure or epithelial stem cell maintenance, and stimulation of anagen by hair plucking caused follicular proliferation and formation of a primitive transient amplifying matrix population. However, mutant matrix cells exhibited apoptosis and DNA damage and hair follicles rapidly degraded. Hair follicle defects at early time points post-deletion occurred in the absence of inflammation, but a dermal inflammatory response and hyperproliferation of interfollicular epidermis accompanied subsequent hair follicle degradation. These data reveal multiple functions for Drosha and Dicer in suppressing DNA damage in rapidly proliferating follicular matrix cells, facilitating catagen and maintaining follicular structures and their associated stem cells. Although Drosha and Dicer each possess independent non-miRNA-related functions, the similarity in phenotypes of the inducible epidermal Drosha and Dicer mutants indicates that these defects result primarily from failure of miRNA processing. Consistent with this, Dicer deletion resulted in the upregulation of multiple direct targets of the highly expressed epithelial miRNA miR-205.     

miRNA的重要调节者Dicer和Drosha与子宫内膜异位症的相关性研究

利用荧光定量PCR和Western blot检测证实,在异位的子宫内膜组织中Dicer和Drosha的表达低于在位子宫内膜组织,随后体外培养在位子宫内膜组织,采用siRNA干扰Dicer和Drosha,发现与干扰对照组相比,子宫内膜细胞的增殖加快而凋亡减少;同时ELISA检测显示转化生长因子-β1(transforming growth factor-beta 1, TGF-β1)的表达上调;Western blot检测显示凋亡抑制蛋白Bcl2表达增加而促凋亡蛋白Bax的表达减少．结果表明,miRNA的重要调节者Dicer和Drosha可以影响TGF-β1及Bcl2/Bax的表达进而影响细胞的增殖和凋亡,从而参与了子宫内膜异位症的形成．     

The Effect of GADD45a on Furazolidone‐Induced S‐Phase Cell‐Cycle Arrest in Human Hepatoma G2 Cells

In this study, overexpression of GADD45a induced by furazolidone in HepG2 cells could arouse S‐phase cell cycle arrest, suppress cell proliferation, and increase the activities of cyclin D1, cyclin D3, and cyclin‐dependent kinase 6 (CDK6). To the opposite, GADD45a knockdown cells by RNAi could reduce furazolidone‐induced S‐phase cell cycle arrest, increase the cell viability, decrease the activities of cyclin D1, cyclin D3, and CDK6; however, cyclin‐dependent kinase 4 (CDK4) showed no change. Moreover, data from our current studies show that cyclin D1, cyclin D3, and CDK6 are target genes functioning at the downstream of the GADD45a pathway induced by furazolidone. These results demonstrate that the GADD45a pathway is partially responsible for the furazolidone‐induced S‐phase cell cycle arrest. GADD45a influences furazolidone‐induced S‐phase cell cycle arrest in human hepatoma G2 cells via cyclin D1, cyclin D3, and CDK6, but not CDK4.     

Knock-down of core proteins regulating microRNA biogenesis has no effect on sensitivity of lung cancer cells to ionizing radiation

Recent studies underline the important role of microRNAs (miRNA) in the development of lung cancer. The main regulators of miRNA biogenesis are the ribonucleases Drosha, Dicer and Ago2. Here the role of core proteins of miRNA biogenesis machinery in the response of human non-small and small cell lung carcinoma cell lines to treatment with ionizing radiation was assessed. We found that Drosha and Dicer were expressed at higher levels in radioresistant but not in sensitive cell lines. However, down-regulation of either Dicer or Drosha had no effect on the sensitivity of cells to irradiation. Elimination of components of the RNA-induced silencing complex Ago2 and Tudor staphylococcal nuclease also did not sensitize cells to the same treatment. Thus, modulation of miRNA biogenesis machinery is not sufficient to increase the radiosensitivity of lung tumors and other strategies are required to combat lung cancer.     

miRNA sensitivity to Drosha levels correlates with pre-miRNA secondary structure

microRNAs (miRNAs) are crucial for cellular development and homeostasis. In order to better understand regulation of miRNA biosynthesis, we studied cleavage of primary miRNAs by Drosha. While Drosha knockdown triggers an expected decrease of many mature miRNAs in human embryonic stem cells (hESC), a subset of miRNAs are not reduced. Statistical analysis of miRNA secondary structure and fold change of expression in response to Drosha knockdown showed that absence of mismatches in the central region of the hairpin, 5 and 9–12 nt from the Drosha cutting site conferred decreased sensitivity to Drosha knockdown. This suggests that, when limiting, Drosha processes miRNAs without mismatches more efficiently than mismatched miRNAs. This is important because Drosha expression changes over cellular development and the fold change of expression for miRNAs with mismatches in the central region correlates with Drosha levels. To examine the biochemical relationship directly, we overexpressed structural variants of miRNA-145, miRNA-137, miRNA-9, and miRNA-200b in HeLa cells with and without Drosha knockdown; for these miRNAs, elimination of mismatches in the central region increased, and addition of mismatches decreased their expression in an in vitro assay and in cells with low Drosha expression. Change in Drosha expression can be a biologically relevant mechanism by which eukaryotic cells control miRNA profiles. This phenomenon may explain the impact of point mutations outside the seed region of certain miRNAs.     

DEAD-box RNA helicase subunits of the Drosha complex are required for processing of rRNA and a subset of microRNAs

MicroRNAs (miRNAs) control cell proliferation, differentiation and fate through modulation of gene expression by partially base-pairing with target mRNA sequences. Drosha is an RNase III enzyme that is the catalytic subunit of a large complex that cleaves pri-miRNAs with distinct structures into pre-miRNAs. Here, we show that both the p68 and p72 DEAD-box RNA helicase subunits in the mouse Drosha complex are indispensable for survival in mice, and both are required for primary miRNA and rRNA processing. Gene disruption of either p68 or p72 in mice resulted in early lethality, and in both p68(-/-) and p72(-/-) embryos, expression levels of a set of, but not all, miRNAs and 5.8S rRNA were significantly lowered. In p72(-/-) MEF cells, expression of p72, but not a mutant lacking ATPase activity, restored the impaired expression of miRNAs and 5.8S rRNA. Furthermore, we purified the large complex of mouse Drosha and showed it could generate pre-miRNA and 5.8S rRNA in vitro. Thus, we suggest that DEAD-box RNA helicase subunits are required for recognition of a subset of primary miRNAs in mDrosha-mediated processing.     

Ectopic expression of cyclin D1 but not cyclin E induces anchorage-independent cell cycle progression.

Normal fibroblasts are dependent on adhesion to a substrate for cell cycle progression. Adhesion-deprived Rat1 cells arrest in the G1 phase of the cell cycle, with low cyclin E-dependent kinase activity, low levels of cyclin D1 protein, and high levels of the cyclin-dependent kinase inhibitor p27kip1. To understand the signal transduction pathway underlying adhesion-dependent growth, it is important to know whether prevention of any one of these down-regulation events under conditions of adhesion deprivation is sufficient to prevent the G1 arrest. To that end, sublines of Rat1 fibroblasts capable of expressing cyclin E, cyclin D1, or both in an inducible manner were used. Ectopic expression of cyclin D1 was sufficient to allow cells to enter S phase in an adhesion-independent manner. In contrast, cells expressing exogenous cyclin E at a level high enough to overcome the p27kip1-imposed inhibition of cyclin E-dependent kinase activity still arrested in G1 when deprived of adhesion. Moreover, expression of both cyclins D1 and E in the same cells did not confer any additional growth advantage upon adhesion deprivation compared to the expression of cyclin D1 alone. Exogenously expressed cyclin D1 was down-regulated under conditions of adhesion deprivation, despite the fact that it was expressed from a heterologous promoter. The ability of cyclin D1-induced cells to enter S phase in an adhesion-independent manner disappears as soon as cyclin D1 proteins disappear. These results suggest that adhesion-dependent cell cycle progression is mediated through cyclin D1, at least in Rat1 fibroblasts.     

Role of the F-box protein Skp2 in adhesion-dependent cell cycle progression.

Cell adhesion to the extracellular matrix (ECM) is a requirement for proliferation that is typically lost in malignant cells. In the absence of adhesion, nontransformed cells arrest in G1 with increased levels of the cyclin-dependent kinase inhibitor p27. We have reported previously that the degradation of p27 requires its phosphorylation on Thr-187 and is mediated by Skp2, an F-box protein that associates with Skp1, Cul1, and Roc1/Rbx1 to form the SCF(Skp2) ubiquitin ligase complex. Here, we show that the accumulation of Skp2 protein is dependent on both cell adhesion and growth factors but that the induction of Skp2 mRNA is exclusively dependent on cell adhesion to the ECM. Conversely, the expression of the other three subunits of the SCF(Skp2) complex is independent of cell anchorage. Phosphorylation of p27 on Thr-187 is also not affected significantly by the loss of cell adhesion, demonstrating that increased p27 stability is not dependent on p27 dephosphorylation. Significantly, ectopic expression of Skp2 in nonadherent G1 cells resulted in p27 downregulation, entry into S phase, and cell division. The ability to induce adhesion-independent cell cycle progression was potentiated by coexpressing Skp2 with cyclin D1 but not with cyclin E, indicating that Skp2 and cyclin D1 cooperate to rescue proliferation in suspension cells. Our study shows that Skp2 is a key target of ECM signaling that controls cell proliferation.     

MicroRNA 16 enhances differentiation of human bone marrow mesenchymal stem cells in a cardiac niche toward myogenic phenotypes in vitro

AimUpregulation of microRNA 16 (miR-16) contributed to the differentiation of human bone marrow mesenchymal stem cells (hMSCs) toward myogenic phenotypes in a cardiac niche, the present study aimed to determine the role of miR-16 in this process.Main methodshMSCs and neonatal rat ventricular myocytes were co-cultured indirectly in two chambers to set up a cardiac microenvironment (niche). miRNA expression profile in cardiac-niche‐induced hMSCs was detected by miRNA microarray. Cardiac marker expression and cell cycle analysis were determined in different treatment hMSCs. Quantitative real-time PCR and Western blot were used to identify the expression of mRNA, mature miRNA and protein of interest.Key findingsmiRNA dysregulation was shown in hMSCs after cardiac niche induction. miR-16 was upregulated in cardiac-niche‐induced hMSCs. Overexpression of miR-16 significantly increased G1-phase arrest of the cell cycle in hMSCs and enhanced the expression of cardiac marker genes, including GATA4, NK2-5, MEF2C and TNNI3. Differentiation-inducing factor 3 (DIF-3), a G0/G1 cell cycle arrest compound, was used to induce G1 phase arrest in cardiac-niche‐induced hMSCs, and the expression of cardiac marker genes was up-regulated in DIF-3-treated hMSCs. The expression of CCND1, CCND2 and CDK6 was suppressed by miR-16 in hMSCs. CDK6, CCND1 or CCND2 knockdown resulted in G1 phase arrest in hMSCs and upregulation of cardiac marker gene expression in hMSCs in a cardiac niche.SignificancemiR-16 enhances G1 phase arrest in hMSCs, contributing to the differentiation of hMSCs toward myogenic phenotypes when in a cardiac niche. This mechanism provides a novel strategy for pre-modification of hMSCs before hMSC-based transplantation therapy for severe heart diseases.     

Characterization of the effects of butyric acid on cell proliferation, cell cycle distribution and apoptosis

We examined concentration-dependent changes in cell cycle distribution and cell cycle-related proteins induced by butyric acid. Butyric acid enhanced or suppressed the proliferation of Jurkat human T lymphocytes depending on concentration. A low concentration of butyric acid induced a massive increase in the number of cells in S and G2/M phases, whereas a high concentration significantly increased the accumulation of cells in G2/M phase, suppressed the accumulation of cells in G0/G1 and S phases, and induced apoptosis that cell cycle-related protein expression in Jurkat cells treated with high levels of butyric acid caused a marked decrease in cyclin A, cyclin E, cyclin-dependent kinase 2 (CDK2), CDK4 and CDK6 protein levels in G0/G1 and S phases, with apoptosis induction, and a decrease in cyclin B, Cdc25c and p27KIP1 protein levels, as well as an increase in p21CIP1/WAF1 protein level, in the G2/M phase. Taken together, our results indicate that butyric acid has bimodal effects on cell proliferation and survival. The inhibition of cell growth followed by the increase in apoptosis induced by high levels of butyric acid were related to an increase in cell death in G0/G1 and S phases, as well as G2/M arrest of cells. Finally, these results were further substantiated by the expression profile of butyric acid-treated Jurkat cells obtained by means of cDNA array.     

miRNA Biogenesis Enzyme Drosha Is Required for Vascular Smooth Muscle Cell Survival

miRNA biogenesis enzyme Drosha cleaves double-stranded primary miRNA by interacting with double-stranded RNA binding protein DGCR8 and processes primary miRNA into precursor miRNA to participate in the miRNA biogenesis pathway. The role of Drosha in vascular smooth muscle cells (VSMCs) has not been well addressed. We generated Drosha conditional knockout (cKO) mice by crossing VSMC-specific Cre mice, SM22-Cre, with Drosha ^loxp/loxp mice. Disruption of Drosha in VSMCs resulted in embryonic lethality at E14.5 with severe liver hemorrhage in mutant embryos. No obvious developmental delay was observed in Drosha cKO embryos. The vascular structure was absent in the yolk sac of Drosha homozygotes at E14.5. Loss of Drosha reduced VSMC proliferation in vitro and in vivo. The VSMC differentiation marker genes, including αSMA, SM22, and CNN1, and endothelial cell marker CD31 were significantly downregulated in Drosha cKO mice compared to controls. ERK1/2 mitogen-activated protein kinase and the phosphatidylinositol 3-kinase/AKT were attenuated in VSMCs in vitro and in vivo. Disruption of Drosha in VSMCs of mice leads to the dysregulation of miRNA expression. Using bioinformatics approach, the interactions between dysregulated miRNAs and their target genes were analyzed. Our data demonstrated that Drosha is required for VSMC survival by targeting multiple signaling pathways.     

Inhibition of G1 phase cyclin dependent kinases by transforming growth factor β1

Transforming growth factor β1 (TGFβ1) inhibits epithelial cell proliferation late in the G1 phase of the cell cycle. We examined the effect of TGFβ1 on known late G1 cell cycle regulators in an attempt to determine the molecular mechanism of growth inhibition by this physiological inhibitor. The results demonstrate the TGFβ1 inhibits the late G1 and S phase specific histone H1 kinase activity of p33^cdk2. This inhibitiion is not dur to TGFβ1's effect on p33^cdk2 synthesis, but rather due to its negative effect on the late G1 phosphorylation of p33^cdk2. It is also shown that TGFβ1 inhibits both late G1 cyclin A and cyclin E associated histon H1 kinase activities. The inhibitor has no effects on the synthesis of cyclin E but to inhibit the synthesis of cyclin A protein in a cell cycle dependent manner. If TGFβ1 is added to cells which have progressed futher than 8 hours into G1, then it is without inhibitory effect on cyclin A synthesis. These effect on TGFβ1 on late G1 cell cycle regulators correlate well with its inhibitory effects on cellular growth and suggest that these G1 cyclin dependent kinases might serve as targets for TGFβ1-mediated growth arrest.     

Novel control of S phase of the cell cycle by ubiquitin-conjugating enzyme H7

Timely degradation of regulatory proteins by the ubiquitin proteolytic pathway (UPP) is an established paradigm of cell cycle regulation during the G2/M and G1/S transitions. Less is known about roles for the UPP during S phase. Here we present evidence that dynamic cell cycle–dependent changes in levels of UbcH7 regulate entrance into and progression through S phase. In diverse cell lines, UbcH7 protein levels are dramatically reduced in S phase but are fully restored by G2. Knockdown of UbcH7 increases the proportion of cells in S phase and doubles the time to traverse S phase, whereas UbcH7 overexpression reduces the proportion of cells in S phase. These data suggest a role for UbcH7 targets in the completion of S phase and entry into G2. Notably, UbcH7 knockdown was coincident with elevated levels of the checkpoint kinase Chk1 but not Chk2. These results argue that UbcH7 promotes S phase progression to G2 by modulating the intra-S phase checkpoint mediated by Chk1. Furthermore, UbcH7 levels appear to be regulated by a UPP. Together the data identify novel roles for the UPP, specifically UbcH7 in the regulation of S phase transit time as well as in cell proliferation.