MiR-23a-3p promoted G1/S cell cycle transition by targeting protocadherin17 in hepatocellular carcinoma

MiR-23a-3p has been shown to promote liver cancer cell growth and metastasis and regulate that of chemosensitivity. Protocadherin17 (PCDH17) is a tumor suppressor gene and plays an essential part in cell cycle of hepatocellular carcinoma (HCC). This study aimed at evaluating the effects of miR-23a-3p and PCDH17 on HCC cell cycle and underlining the mechanism. The level of miR-23a-3p was up-regulated, while PCDH17 level was down-regulated in HCC tissues compared to adjacent tissues. For the in vitro studies, high expression of miR-23a-3p down-regulated PCDH17 level; increased cell viability; promoted G1/S cell cycle transition; up-regulated cyclin D1, cyclin E, CDK2, CDK4, p-p27, and p-RB levels; and down-regulated the expression of p27. Dual luciferase reporter assay suggested PCDH17 was a target gene of miR-23a-3p. MiR-23a-3p inhibitor and PCDH17 siRNA led to an increase in cell viability and the number of cells in the S phase and up-regulated cyclin D1 and cyclin E levels, compared with miR-23a-3p inhibitor and NC siRNA group. For the in vivo experiments, high expression of miR-23a-3p promoted tumor growth and reduced PCDH17 level in the cytoplasm. These results indicated that high expression of miR-23a-3p might promote G1/S cell cycle transition by targeting PCDH17 in HCC cells. The miR-23a-3p could be considered as a molecular target for HCC detection.

     

Boolean network model predicts cell cycle sequence of fission yeast

A Boolean network model of the cell-cycle regulatory network of fission yeast (Schizosaccharomyces Pombe) is constructed solely on the basis of the known biochemical interaction topology. Simulating the model in the computer faithfully reproduces the known activity sequence of regulatory proteins along the cell cycle of the living cell. Contrary to existing differential equation models, no parameters enter the model except the structure of the regulatory circuitry. The dynamical properties of the model indicate that the biological dynamical sequence is robustly implemented in the regulatory network, with the biological stationary state G1 corresponding to the dominant attractor in state space, and with the biological regulatory sequence being a strongly attractive trajectory. Comparing the fission yeast cell-cycle model to a similar model of the corresponding network in S. cerevisiae, a remarkable difference in circuitry, as well as dynamics is observed. While the latter operates in a strongly damped mode, driven by external excitation, the S. pombe network represents an auto-excited system with external damping.     

miR-18a Inhibits CDC42 and Plays a Tumour Suppressor Role in Colorectal Cancer Cells

The miR-17-92 cluster of microRNAs is elevated in colorectal cancer, and has a causative role in cancer development. Of the six miR-17-92 cluster members, miR-19a and b in particular are key promoters of cancer development and cell proliferation, while preliminary evidence suggests that miR-18a may act in opposition to other cluster members to decrease cell proliferation. It was hypothesised that miR-18a may have a homeostatic function in helping to contain the oncogenic effect of the entire miR-17-92 cluster, and that elevated miR-17-92 cluster activity without a corresponding increase in miR-18a may promote colorectal tumour progression. In colorectal cancer samples and corresponding normal colorectal mucosa, miR-18a displayed lower overall expression than other miR-17-92 cluster members. miR-18a was shown to have an opposing role to other miR-17-92 cluster members, in particular the key oncogenic miRNAs, miR-19a and b. Transfection of HCT116 and LIM1215 colorectal cancer cell lines with miR-18a mimics decreased proliferation, while a miR-18a inhibitor increased proliferation. miR-18a was also responsible for decreasing cell migration, altering cell morphology, inducing G1/S phase cell cycle arrest, increasing apoptosis, and enhancing the action of a pro-apoptotic agent. CDC42, a mediator of the PI3K pathway, was identified as a novel miR-18a target. Overexpression of miR-18a reduced CDC42 expression, and a luciferase assay confirmed that miR-18a directly targets the 3′UTR of CDC42. miR-18a mimics had a similar effect on proliferation as a small molecule inhibitor of CDC42. Inhibition of CDC42 expression is likely to be a key mechanism by which miR-18a impairs cancer cell growth, with a target protector experiment revealing miR-18a influences proliferation via direct inhibition of CDC42. Inhibition of CCND1 by miR-18a may also assist in this growth-suppression effect. The homeostatic function of miR-18a within the miR-17-92 cluster in colorectal cancer cells may be achieved through suppression of CDC42 and the PI3K pathway.     

The microRNA-302-367 cluster suppresses the proliferation of cervical carcinoma cells through the novel target AKT1

The miR-302-367 cluster is specifically expressed in human embryonic stem cells and has been shown to convert human somatic cells into induced pluripotent stem cells. Here, we investigated the role of the miR-302-367 cluster in cervical carcinoma. The cluster was not endogenously expressed in cervical cancer cells, and its ectopic expression did not reprogram the cervical cancer cells to an embryonic stem cell-like state. However, ectopic expression of the miR-302-367 cluster in HeLa and SiHa cervical cancer cells inhibited cell proliferation and tumor formation by blocking the G1/S cell cycle transition. We identified a new cell cycle regulatory pathway in which the miR-302-367 cluster directly down-regulated both cyclin D1 and AKT1 and indirectly up-regulated p27^Kip1 and p21^Cip1, leading to the suppression of cervical cancer cell proliferation. Our findings suggest that the miR-302-367 cluster may be used as a therapeutic reagent for the treatment of cervical carcinoma.     

A miR-206 regulated gene landscape enhances mammary epithelial differentiation

miR-206 is known to suppress breast cancer. However, while it is expressed in mammary stem cells, its function in such nontumor cells is not well understood. Here, we explore the role of miR-206 in undifferentiated, stem-like mammary cells using the murine mammary differentiation model HC11, genome-wide gene expression analysis, and functional assays. We describe the miR-206-regulated gene landscape and propose a network whereby miR-206 suppresses tumor development. We functionally demonstrate that miR-206 in nontumor stem-like cells induces a G1–S cell cycle arrest, and reduces colony formation and epithelial-to-mesenchymal transition markers. Finally, we show that addition of miR-206 accelerates the mammary differentiation process along with related accumulation of lipids. We conclude that miR-206 impacts a network of signaling pathways, and acts as a regulator of proliferation, stemness, and mammary cell differentiation in nontumor stem-like mammary cells. Our study provides a broad insight into the breast cancer suppressive functions of miR-206.     

MicroRNA-7 arrests cell cycle in G1 phase by directly targeting CCNE1 in human hepatocellular carcinoma cells

Growing evidence has demonstrated that the aberrant expression of miRNA is a hallmark of malignancies, indicating the important roles of miRNA in the development and progression of cancer. MiR-7 is considered as a tumor suppressor miRNA in multiple types of cancer. However, the role of miR-7 in human hepatocellular carcinoma (HCC) and its underlying mechanism remain elusive. In this study, we found that overexpression of miR-7 arrested cell cycle at G1 to S transition in HCC. By combinational use of bioinformatic prediction, reporter assay, quantitative real-time PCR (qRT-PCR) and Western blot, we confirmed that CCNE1, an important mediator in G1/S transition is one of new direct target genes of miR-7. Further studies revealed that silencing of CCNE1 recapitulated the effects of miR-7 overexpression, whereas enforced expression of CCNE1 reversed the suppressive effects of miR-7 in cell cycle regulation. Finally, analysis of qRT-PCR showed a reciprocal relationship between miR-7 and CCNE1 in clinical cancer tissues and multiple types of tumor cell lines. These findings indicate that miR-7 exerts tumor-suppressive effects in hepatocarcinogenesis through the suppression of oncogene CCNE1 expression and suggest a therapeutic application of miR-7 in HCC.     

A cyclin D1/microRNA 17/20 regulatory feedback loop in control of breast cancer cell proliferation

Decreased expression of specific microRNAs (miRNAs) occurs in human tumors, which suggests a function for miRNAs in tumor suppression. Herein, levels of the miR-17-5p/miR-20a miRNA cluster were inversely correlated to cyclin D1 abundance in human breast tumors and cell lines. MiR-17/20 suppressed breast cancer cell proliferation and tumor colony formation by negatively regulating cyclin D1 translation via a conserved 3' untranslated region miRNA-binding site, thereby inhibiting serum-induced S phase entry. The cell cycle effect of miR-17/20 was abrogated by cyclin D1 siRNA and in cyclin D1-deficient breast cancer cells. Mammary epithelial cell-targeted cyclin D1 expression induced miR-17-5p and miR-20a expression in vivo, and cyclin D1 bound the miR-17/20 cluster promoter regulatory region. In summary, these studies identify a novel cyclin D1/miR-17/20 regulatory feedback loop through which cyclin D1 induces miR-17-5p/miR-20a. In turn, miR-17/20 limits the proliferative function of cyclin D1, thus linking expression of a specific miRNA cluster to the regulation of oncogenesis.     

The miR-17-5p microRNA is a key regulator of the G1/S phase cell cycle transition

Background

MicroRNAs are modifiers of gene expression, acting to reduce translation through either translational repression or mRNA cleavage. Recently, it has been shown that some microRNAs can act to promote or suppress cell transformation, with miR-17-92 described as the first oncogenic microRNA. The association of miR-17-92 encoded microRNAs with a surprisingly broad range of cancers not only underlines the clinical significance of this locus, but also suggests that miR-17-92 may regulate fundamental biological processes, and for these reasons miR-17-92 has been considered as a therapeutic target.

Results

In this study, we show that miR-17-92 is a cell cycle regulated locus, and ectopic expression of a single microRNA (miR-17-5p) is sufficient to drive a proliferative signal in HEK293T cells. For the first time, we reveal the mechanism behind this response - miR-17-5p acts specifically at the G1/S-phase cell cycle boundary, by targeting more than 20 genes involved in the transition between these phases. While both pro- and anti-proliferative genes are targeted by miR-17-5p, pro-proliferative mRNAs are specifically up-regulated by secondary and/or tertiary effects in HEK293T cells.

Conclusion

The miR-17-5p microRNA is able to act as both an oncogene and a tumor suppressor in different cellular contexts; our model of competing positive and negative signals can explain both of these activities. The coordinated suppression of proliferation-inhibitors allows miR-17-5p to efficiently de-couple negative regulators of the MAPK (mitogen activated protein kinase) signaling cascade, promoting growth in HEK293T cells. Additionally, we have demonstrated the utility of a systems biology approach as a unique and rapid approach to uncover microRNA function.     

Live Imaging-Based Model Selection Reveals Periodic Regulation of the Stochastic G1/S Phase Transition in Vertebrate Axial Development

In multicellular organism development, a stochastic cellular response is observed, even when a population of cells is exposed to the same environmental conditions. Retrieving the spatiotemporal regulatory mode hidden in the heterogeneous cellular behavior is a challenging task. The G1/S transition observed in cell cycle progression is a highly stochastic process. By taking advantage of a fluorescence cell cycle indicator, Fucci technology, we aimed to unveil a hidden regulatory mode of cell cycle progression in developing zebrafish. Fluorescence live imaging of Cecyil, a zebrafish line genetically expressing Fucci, demonstrated that newly formed notochordal cells from the posterior tip of the embryonic mesoderm exhibited the red (G1) fluorescence signal in the developing notochord. Prior to their initial vacuolation, these cells showed a fluorescence color switch from red to green, indicating G1/S transitions. This G1/S transition did not occur in a synchronous manner, but rather exhibited a stochastic process, since a mixed population of red and green cells was always inserted between newly formed red (G1) notochordal cells and vacuolating green cells. We termed this mixed population of notochordal cells, the G1/S transition window. We first performed quantitative analyses of live imaging data and a numerical estimation of the probability of the G1/S transition, which demonstrated the existence of a posteriorly traveling regulatory wave of the G1/S transition window. To obtain a better understanding of this regulatory mode, we constructed a mathematical model and performed a model selection by comparing the results obtained from the models with those from the experimental data. Our analyses demonstrated that the stochastic G1/S transition window in the notochord travels posteriorly in a periodic fashion, with doubled the periodicity of the neighboring paraxial mesoderm segmentation. This approach may have implications for the characterization of the pathophysiological tissue growth mode.     

MicroRNA-26a/b and their host genes cooperate to inhibit the G1/S transition by activating the pRb protein

The functional association between intronic miRNAs and their host genes is still largely unknown. We found that three gene loci, which produced miR-26a and miR-26b, were embedded within introns of genes coding for the proteins of carboxy-terminal domain RNA polymerase II polypeptide A small phosphatase (CTDSP) family, including CTDSPL, CTDSP2 and CTDSP1. We conducted serum starvation-stimulation assays in primary fibroblasts and two-thirds partial-hepatectomies in mice, which revealed that miR-26a/b and CTDSP1/2/L were expressed concomitantly during the cell cycle process. Specifically, they were increased in quiescent cells and decreased during cell proliferation. Furthermore, both miR-26 and CTDSP family members were frequently downregulated in hepatocellular carcinoma (HCC) tissues. Gain- and loss-of-function studies showed that miR-26a/b and CTDSP1/2/L synergistically decreased the phosphorylated form of pRb (ppRb), and blocked G1/S-phase progression. Further investigation disclosed that miR-26a/b directly suppressed the expression of CDK6 and cyclin E1, which resulted in reduced phosphorylation of pRb. Moreover, c-Myc, which is often upregulated in cancer cells, diminished the expression of both miR-26 and CTDSP family members, enhanced the ppRb level and promoted the G1/S-phase transition. Our findings highlight the functional association of miR-26a/b and their host genes and provide new insight into the regulatory network of the G1/S-phase transition.     

Overexpressed MicroRNA-182 Promotes Proliferation and Invasion in Prostate Cancer PC-3 Cells by Down-Regulating N-myc Downstream Regulated Gene 1 (NDRG1)

MicroRNAs, non-coding 20–22 nucleotide single-stranded RNAs, result in translational repression or degradation and gene silencing of their target genes, and significantly contribute to the regulation of gene expression. In the current study, we report that miR-182 expression was significantly upregulated in prostate cancer tissues and four cell lines, compared to benign prostatic hyperplasia tissues and normal prostatic epithelial (RWPE-1) cells. Ectopic overexpression of miR-182 significantly promotes the proliferation, increases the invasion, promotes the G1/S cell cycle transition and reduces early apotosis of PC-3 cells, while suppression of miR-182 decreased the proliferation and invasion, inhibits the G1/S cell cycle transition and increase early apotosis of PC-3 cells. Additionally, we demonstrated that miR-182 could downregulate expression of NDRG1 by directly targeting the NDRG1 3′-untranslated region. In conclusion, our results suggest that miR-182 plays an important role in the proliferation of human prostate cancer cells by directly suppressing the tumor supressor gene NDRG1. We uncovered a new epigenetic regulation of NDRG1.     

The Gcn2 Kinase as a Cell Cycle Regulator

Cell cycle progression through G1 phase is of particular importance because this is the phase where the decision to embark on another cell cycle is made. An aberrant G1/S transition often leads to cell cycle deregulation and cancer development. Therefore, there is a complex regulatory network to ensure timely entry into S phase, coordinating initiation of DNA replication with growth and stress signals. We have studied the response of fission yeast cells to ultraviolet (UV) irradiation in G1 phase and identified a Gcn2-dependent checkpoint that delays entry into S phase. UV irradiation activates Gcn2 which, in turn, phosphorylates the translation initiation factor eIF2α and depresses translation. Phosphorylation of eIF2α is a well-known response to various forms of stress, but whether or how this response is causing the specific cell cycle effects is not known. Here we discuss the relationships between Gcn2 activity, eIF2α phosphorylation, translation downregulation and cell cycle delay.     

Bifurcation analysis of the regulatory modules of the mammalian G1/S transition

MOTIVATION: Mathematical models of the cell cycle can contribute to an understanding of its basic mechanisms. Modern simulation tools make the analysis of key components and their interactions very effective. This paper focuses on the role of small modules and feedbacks in the gene-protein network governing the G1/S transition in mammalian cells. Mutations in this network may lead to uncontrolled cell proliferation. Bifurcation analysis helps to identify the key components of this extremely complex interaction network. RESULTS: We identify various positive and negative feedback loops in the network controlling the G1/S transition. It is shown that the positive feedback regulation of E2F1 and a double activator-inhibitor module can lead to bistability. Extensions of the core module preserve the essential features such as bistability. The complete model exhibits a transcritical bifurcation in addition to bistability. We relate these bifurcations to the cell cycle checkpoint and the G1/S phase transition point. Thus, core modules can explain major features of the complex G1/S network and have a robust decision taking function.     

LncRNA SNHG17 interacts with LRPPRC to stabilize c-Myc protein and promote G1/S transition and cell proliferation

Oncogenic c-Myc is a master regulator of G1/S transition. Long non-coding RNAs (lncRNAs) emerge as new regulators of various cell activities. Here, we found that lncRNA SnoRNA Host Gene 17 (SNHG17) was elevated at the early G1-phase of cell cycle. Both gain- and loss-of function studies disclosed that SNHG17 increased c-Myc protein level, accelerated G1/S transition and cell proliferation, and consequently promoted tumor cell growth in vitro and in vivo. Mechanistically, the 1-150-nt of SNHG17 physically interacted with the 1035-1369-aa of leucine rich pentatricopeptide repeat containing (LRPPRC) protein, and disrupting this interaction abrogated the promoting role of SNHG17 in c-Myc expression, G1/S transition, and cell proliferation. The effect of SNHG17 in stimulating cell proliferation was attenuated by silencing c-Myc or LRPPRC. Furthermore, silencing SNHG17 or LRPPRC increased the level of ubiquitylated c-Myc and reduced the stability of c-Myc protein. Analysis of human hepatocellular carcinoma (HCC) tissues revealed that SNHG17, LRPPRC, and c-Myc were significantly upregulated in HCC, and they showed a positive correlation with each other. High level of SNHG17 or LRPPRC was associated with worse survival of HCC patients. These data suggest that SNHG17 may inhibit c-Myc ubiquitination and thus enhance c-Myc level and facilitate proliferation by interacting with LRPPRC. Our findings identify a novel SNHG17-LRPPRC-c-Myc regulatory axis and elucidate its roles in G1/S transition and tumor growth, which may provide potential targets for cancer therapy.Subject terms: Oncogenes, Cell growth, Long non-coding RNAs     

Mechanisms of signalling-memory governing progression through the eukaryotic cell cycle

As cells pass through each replication–division cycle, they must be able to postpone further progression if they detect any threats to genome integrity, such as DNA damage or misaligned chromosomes. Once a ‘decision’ is made to proceed, the cell unequivocally enters into a qualitatively different biochemical state, which makes the transitions from one cell cycle phase to the next switch-like and irreversible. Each transition is governed by a unique signalling network; nonetheless, they share a common characteristic of bistable behaviour, a hallmark of molecular memory devices. Comparing the cell cycle signalling mechanisms acting at the restriction point, G1/S, G2/M and meta-to-anaphase transitions, we deduce a generic network motif of coupled positive and negative feedback loops underlying each transition.