Minichromosome Maintenance Protein Complex Research Articles

Unwinding Helicase MCM Functionality for Diagnosis and Therapeutics of Replication Abnormalities Associated with Cancer: A Review.

The minichromosome maintenance (MCM) protein is a component of an active helicase that is essential for the initiation of DNA replication. Dysregulation of MCM functions contribute to abnormal cell proliferation and genomic instability. The interactions of MCM with cellular factors, including Cdc45 and GINS, determine the formation of active helicase and functioning of helicase. The functioning of MCM determines the fate of DNA replication and, thus, genomic integrity. This complex is upregulated in precancerous cells and can act as an important tool for diagnostic applications. The MCM protein complex can be an important broad-spectrum therapeutic target in various cancers. Investigations have supported the potential and applications of MCM in cancer diagnosis and its therapeutics. In this article, we discuss the physiological roles of MCM and its associated factors in DNA replication and cancer pathogenesis.

Glutamine Regulates Gene Expression Profiles to Increase the Proliferation of Porcine Intestinal Epithelial Cells and the Expansion of Intestinal Stem Cells.

The intestinal epithelium is known for its rapid self-renewal, and glutamine is crucial in providing carbon and nitrogen for biosynthesis. However, understanding how glutamine affects gene expression in the intestinal epithelium is limited, and identifying the essential genes and signals involved in regulating intestinal epithelial cell growth is particularly challenging. In this study, glutamine supplementation exhibited a robust acceleration of intestinal epithelial cell proliferation and stem cell expansion. RNA sequencing indicated diverse transcriptome changes between the control and glutamine supplementation groups, identifying 925 up-regulated and 1152 down-regulated genes. The up-regulated DEGs were enriched in the KEGG pathway of cell cycle and GO terms of DNA replication initiation, regulation of phosphatidylinositol 3-kinase activity, DNA replication, minichromosome maintenance protein (MCM) complex, and ATP binding, whereas the down-regulated DEGs were enriched in the KEGG pathway of p53 signaling pathway, TNF signaling pathway, and JAK-STAT signaling pathway and GO terms of inflammatory response and intrinsic apoptotic signaling pathway in response to endoplasmic reticulum stress. Furthermore, GSEA analysis revealed a significant up-regulation of the cell cycle, DNA replication initiation, ATP-dependent RNA helicase activity, and down-regulation of the TNF signaling pathway. The protein-protein association network of the intersecting genes highlighted the significance of DNA replication licensing factors (MCM3, MCM6, and MCM10) in promoting intestinal epithelial growth in response to glutamine. Based on these findings, we propose that glutamine may upregulate DNA replication licensing factors, leading to increased PI3K/Akt signaling and the suppression of TNF, JAK-STAT, and p53 pathways. Consequently, this mechanism results in the proliferation of porcine intestinal epithelial cells and the expansion of intestinal stem cells.

New Resinogalea species from Araucaria araucana resin in Chile and reclassification of the genus in the Cryptocaliciomycetidae.

Araucaria araucana is an ancient conifer, native to the mountain ranges in Chile and Argentina. These trees host a large number of organisms, mainly insects, strongly or even exclusively associated with them. The recent emergence of a novel canker disease on A. araucana has emphasised the importance of fungi associated with these iconic trees and has resulted in the discovery of various new species. In this study, we considered the identity of an unknown calicioid fungus consistently found on resin on the branches of A. araucana. Preliminary phylogenetic analyses placed isolates in the recently described sub-class Cryptocaliciomycetidae, closest to Cryptocalicium blascoi. However, the morphology of the ascomata and its occurrence in a unique niche suggested that the closest relative could be Resinogalea humboldtensis (Bruceomycetaceae, incertae sedis), a fungus with similar sporing structures found on resin of Araucaria humboldtensis in New Caledonia. There are no living cultures or sequence data available for either R. humboldtensis or its supposed closest relative, Bruceomyces castoris, precluding sequence-based comparisons. Morphological comparisons of the sporing structures on A. araucana confirmed that the ascomatal morphology of our unknown calicioid fungus and R. humboldtensis are almost identical and resemble each other more so than B. castoris or Cr. blascoi. A phylogenetic analysis based on the small subunit (SSU), internal transcribed spacer (ITS) and large subunit (LSU) rDNA regions resolved our strains into two clades with Cr. blascoi as its closest relative. Further analyses applying the Genealogical Concordance Phylogenetic Species Recognition (GCPSR) based on ITS, mini chromosome maintenance protein complex (MCM7), RNA polymerase II second largest subunit (RPB2) and translation elongation factor 1-alpha (TEF) gene regions, confirmed that strains represent two new species. Based on our morphological observations and phylogenetic analyses, we introduce two new Resinogalea species, R. araucana and R. tapulicola, and reclassify the genus in the subclass Cryptocaliciomycetidae.

MCM4 is a novel prognostic biomarker and promotes cancer cell growth in glioma

Gliomas account for 75% of all primary malignant brain tumors in adults and result in high mortality. Accumulated evidence has declared the minichromosome maintenance protein complex (MCM) gene family plays a critical role in modulating the cell cycle and DNA replication stress. However, the biological function and clinic characterization of nine MCM members in low-grade glioma are not yet clarified. In this study, we utilized diverse public databases, including The Cancer Genome Atlas (TCGA), Chinese Glioma Genome Atlas (CGGA), Rembrandt, Human Protein Atlas (HPA), Linkedomics, cbioportal, Tumor and Immune System Interaction Database (TISIDB), single-sample GSEA (ssGSEA), Tumor Immune Estimation Resource (TIMER), Genomics of Drug Sensitivity in Cancer (GDSC) and Cancer Therapeutics Response Portal databases to explore the mRNA and protein expression profiles, gene mutation, clinical features, diagnosis, prognosis, signaling pathway, tumor mutational burden (TMB), immune subtype, immune cell infiltration, immune modulator and drug sensitivity of nine MCMs. Afterward, qRT-PCR was utilized to detect the expression of the MCM family in glioblastoma multiforme (GBM) cell lines. The one-, three-, or five-year survival rate was predicted by utilizing a nomogram established by cox proportional hazard regression. In this study, we found that nine MCMs were consistently up-regulated in glioma tissues and glioma cell lines. Elevated nine MCMs expressions were significantly correlated with a higher tumor stage, isocitrate dehydrogenase (IDH) mutates, 1p/19q codeletion, histological type, and primary therapy outcome. Survival analyses showed that higher expression of MCM2-MCM8 (minichromosome maintenance protein2-8) and MCM10 (minichromosome maintenance protein 10) were linked with poor overall survival (OS) and progression-free survival (PFS) in glioma patients. On the other hand, up-regulated MCM2-MCM8 and MCM10 were significantly associated with shorter disease-specific survival (DSS) in glioma patients. Univariate and multivariate analyses revealed that MCM2 (minichromosome maintenance protein2), MCM4 (minichromosome maintenance protein 4), MCM6 (minichromosome maintenance protein 6), MCM7 (minichromosome maintenance protein 7) expression and tumor grade, 1p/19q codeletion, age, and primary therapy outcome were independent factors correlated with the clinical outcome of glioma patients. More importantly, a prognostic MCMs model constructed using the above five prognostic genes could predict the overall survival of glioma patients with medium-to-high accuracy. Furthermore, functional enrichment analysis indicated that MCMs principal participated in regulating cell cycle and DNA replication. DNA copy number variation (CNV) and DNA methylation significantly affect the expression of MCMs. Finally, we uncover that MCMs expression is highly correlated with immune cell infiltration, immune modulator, TMB, and drug sensitivity. In summary, this finding confirmed that MCM4 is a potential target of precision therapy for patients with glioma.

Low-molecular-weight cyclin E deregulates DNA replication and damage repair to promote genomic instability in breast cancer.

Low-molecular-weight cyclin E (LMW-E) is an N-terminus deleted (40 amino acid) form of cyclin E detected in breast cancer, but not in normal cells or tissues. LMW-E overexpression predicts poor survival in breast cancer patients independent of tumor proliferation rate, but the oncogenic mechanism of LMW-E and its unique function(s) independent of full-length cyclin E (FL-cycE) remain unclear. In the current study, we found LMW-E was associated with genomic instability in early-stage breast tumors (n = 725) and promoted genomic instability in human mammary epithelial cells (hMECs). Mechanistically, FL-cycE overexpression inhibited the proliferation of hMECs by replication stress and DNA damage accumulation, but LMW-E facilitated replication stress tolerance by upregulating DNA replication and damage repair. Specifically, LMW-E interacted with chromatin and upregulated the loading of minichromosome maintenance complex proteins (MCMs) in a CDC6 dependent manner and promoted DNA repair in a RAD51- and C17orf53-dependent manner. Targeting the ATR-CHK1-RAD51 pathway with ATR inhibitor (ceralasertib), CHK1 inhibitor (rabusertib), or RAD51 inhibitor (B02) significantly decreased the viability of LMW-E-overexpressing hMECs and breast cancer cells. Collectively, our findings delineate a novel role for LMW-E in tumorigenesis mediated by replication stress tolerance and genomic instability, providing novel therapeutic strategies for LMW-E-overexpressing breast cancers.

OAB-020: RAD51 interacts with minichromosome maintenance protein complex (MCM2–7) providing strong dependency in myeloma

OAB-020: RAD51 interacts with minichromosome maintenance protein complex (MCM2–7) providing strong dependency in myeloma

Novel protein contact points among TP53 and minichromosome maintenance complex proteins 2, 3, and 5.

Identify protein contact points between TP53 and minichromosome maintenance (MCM) complex proteins 2, 3, and 5 with high resolution allowing for potential novel Cancer drug design. A next-generation sequencing-based protein-protein interaction method developed in our laboratory called AVA-Seq was applied to a gold-standard human protein interaction set. Proteins including TP53, MCM2, MCM3, MCM5, HSP90AA1, PCNA, NOD1, and others were sheared and ligated into the AVA-Seq system. Protein-protein interactions were then identified in both mild and stringent selective conditions. Known interactions among MCM2, MCM3, and MCM5 were identified with the AVA-Seq system. The interacting regions detected between these three proteins overlap with the structural data of the MCM complex, and novel domains were identified with high resolution determined by multiple overlapping fragments. Fragments of wild type TP53 were shown to interact with MCM2, MCM3, and MCM5, and details on the location of the interactions were provided. Finally, a mini-network of known and novel cancer protein interactions was provided, which could have implications for fundamental changes in multiple cancers. We provide a high-resolution mini-interactome that could direct novel drug targets and implicate possible effects of specific cancer mutations.

Targeted inhibition of the expression of both MCM5 and MCM7 by miRNA-214 impedes DNA replication and tumorigenesis in hepatocellular carcinoma cells

MicroRNAs are noncoding RNAs with a typical length of 22 nucleotides that post-transcriptionally suppress gene expression by inducing target mRNA degradation and/or impairing translation in eukaryotes. Thousands of miRNA genes in the human genome are involved in various physiological and pathological processes. Each miRNA targets many different mRNAs, while each mRNA may be targeted by various miRNAs. Mini-chromosome maintenance (MCM2-7) protein complex functions as essential components of the pre-replicative complex (pre-RC) and forms a helicase together with other proteins to unwind the DNA duplex in S phase. MCM proteins are overexpressed in all cancer cells, while they are strictly regulated in normal cells, with no expression in non-proliferating normal cells. Here we report that miRNA-214–3p (miR-214) targets both MCM5 and MCM7. The level of miR-214 is lower in HepG2 and Hep3B hepatocellular carcinoma cells than the L-02 normal liver cells. Introduction of miRNA-214 mimic into HepG2 and Hep3B cells reduced the mRNA and protein levels of MCM5/7 and inhibited DNA replication, cell cycle progression, cell proliferation and colony formation. Comparatively, miRNA-214 mimic had little effect in L-02 cells. Importantly, miR-214 mimic can also inhibit the growth of HepG2 xenografts in nude mice. Our data suggest that miRNA-214 regulates DNA replication by targeting MCM5/7 and has the potential to be developed into a liver cancer drug. ImplicationsThis study supports the notion that DNA replication-initiation proteins (DRIPs), including MCM2-7 proteins, are attractive anticancer targets. Furthermore, the potential of miR-214 as an anticancer agent, with activity against liver cancer cells but not normal livre cells, may be of high significance.

In Vivo Screen Identifies Liver X Receptor Alpha Agonism Potentiates Sorafenib Killing of Hepatocellular Carcinoma.

In Vivo Screen Identifies Liver X Receptor Alpha Agonism Potentiates Sorafenib Killing of Hepatocellular Carcinoma.

Significance of Irisin (FNDC5) Expression in Colorectal Cancer.

The role of irisin, the extracellular part of fibronectin type III domain containing 5 (FNDC5), in colorectal cancer (CRC) is unclear. The aim of this study was to investigate immunohistochemical (IHC) expression level of irisin and correlations with clinicopathological factors in patients with CRC. We collected 222 archived CRC samples and 26 control samples from autopsies conducted at the Department of Forensic Medicine. They were used to perform IHC reactions detecting irisin, Ki-67, minichromosome maintenance protein complex component 3 (MCM3), and urine diphosphate-galactose ceramide galactosyltransferase (UGT3) expression. The correlations with Ki-67, MCM3, and UGT3 were analyzed. Irisin expression was also evaluated in cancer cell lines by immunofluorescence reaction and western blot. Irisin expression was higher in cancer cells compared to the control tissues (p<0.0001). Irisin expression was significantly higher in stage I than in stage III (p=0.013) and IV CRC (p=0.05). The correlation between higher expression of irisin and cancer stages indicates its potential usefulness as a marker in CRC.

Scaling-up a fragment-based protein-protein interaction method using a human reference interaction set.

Protein–protein interactions (PPIs) are essential in understanding numerous aspects of protein function. Here, we significantly scaled and modified analyses of the recently developed all‐vs‐all sequencing (AVA‐Seq) approach using a gold‐standard human protein interaction set (hsPRS‐v2) containing 98 proteins. Binary interaction analyses recovered 20 of 47 (43%) binary PPIs from this positive reference set (PRS), comparing favorably with other methods. However, the increase of 20× in the interaction search space for AVA‐Seq analysis in this manuscript resulted in numerous changes to the method required for future use in genome‐wide interaction studies. We show that standard sequencing analysis methods must be modified to consider the possible recovery of thousands of positives among millions of tested interactions in a single sequencing run. The PRS data were used to optimize data scaling, auto‐activator removal, rank interaction features (such as orientation and unique fragment pairs), and statistical cutoffs. Using these modifications to the method, AVA‐Seq recovered >500 known and novel PPIs, including interactions between wild‐type fragments of tumor protein p53 and minichromosome maintenance complex proteins 2 and 5 (MCM2 and MCM5) that could be of interest in human disease.

Inhibition of DNA replication initiation by silver nanoclusters.

Silver nanoclusters (AgNCs) have outstanding physicochemical characteristics, including the ability to interact with proteins and DNA. Given the growing number of diagnostic and therapeutic applications of AgNCs, we evaluated the impact of AgNCs on DNA replication and DNA damage response in cell-free extracts prepared from unfertilized Xenopus laevis eggs. We find that, among a number of silver nanomaterials, AgNCs uniquely inhibited genomic DNA replication and abrogated the DNA replication checkpoint in cell-free extracts. AgNCs did not affect nuclear membrane or nucleosome assembly. AgNCs-supplemented extracts showed a strong defect in the loading of the mini chromosome maintenance (MCM) protein complex, the helicase that unwinds DNA ahead of replication forks. FLAG-AgNCs immunoprecipitation and mass spectrometry analysis of AgNCs associated proteins demonstrated direct interaction between MCM and AgNCs. Our studies indicate that AgNCs directly prevent the loading of MCM, blocking pre-replication complex (pre-RC) assembly and subsequent DNA replication initiation. Collectively, our findings broaden the scope of silver nanomaterials experimental applications, establishing AgNCs as a novel tool to study chromosomal DNA replication.

Transcriptome analyses of 7-day-old zebrafish larvae possessing a familial Alzheimer\u2019s disease-like mutation in psen1 indicate effects on oxidative phosphorylation, ECM and MCM functions, and iron homeostasis

BackgroundEarly-onset familial Alzheimer’s disease (EOfAD) is promoted by dominant mutations, enabling the study of Alzheimer’s disease (AD) pathogenic mechanisms through generation of EOfAD-like mutations in animal models. In a previous study, we generated an EOfAD-like mutation, psen1Q96_K97del, in zebrafish and performed transcriptome analysis comparing entire brains from 6-month-old wild type and heterozygous mutant fish. We identified predicted effects on mitochondrial function and endolysosomal acidification. Here we aimed to determine whether similar effects occur in 7 day post fertilization (dpf) zebrafish larvae that might be exploited in screening of chemical libraries to find ameliorative drugs.ResultsWe generated clutches of wild type and heterozygous psen1Q96_K97del 7 dpf larvae using a paired-mating strategy to reduce extraneous genetic variation before performing a comparative transcriptome analysis. We identified 228 differentially expressed genes and performed various bioinformatics analyses to predict cellular functions.ConclusionsOur analyses predicted a significant effect on oxidative phosphorylation, consistent with our earlier observations of predicted effects on ATP synthesis in adult heterozygous psen1Q96_K97del brains. The dysregulation of minichromosome maintenance protein complex (MCM) genes strongly contributed to predicted effects on DNA replication and the cell cycle and may explain earlier observations of genome instability due to PSEN1 mutation. The upregulation of crystallin gene expression may be a response to defective activity of mutant Psen1 protein in endolysosomal acidification. Genes related to extracellular matrix (ECM) were downregulated, consistent with previous studies of EOfAD mutant iPSC neurons and postmortem late onset AD brains. Also, changes in expression of genes controlling iron ion transport were observed without identifiable changes in the prevalence of transcripts containing iron responsive elements (IREs) in their 3′ untranslated regions (UTRs). These changes may, therefore, predispose to the apparent iron dyshomeostasis previously observed in 6-month-old heterozygous psen1Q96_K97del EOfAD-like mutant brains.

Role of Amygdalin in Blocking DNA Replication in Breast Cancer In Vitro.

Amygdalin has anticancer benefits because of its active component, hydrocyanic acid. However, the underlying molecular mechanism is unclear. This study aimed to investigate the molecular mechanism by which amygdalin exerts antiproliferative effects in the human Michigan Cancer Foundation-7 (MCF-7) breast cancer cell line. MCF-7 cells were exposed to amygdalin at a particular IC50 value for 24 and 48 hours and compared to non-treated cells. An Affymetrix whole-transcript expression array was used to analyze the expression of 32 genes related to DNA replication. Among the 32 genes, amygdalin downregulated the expression of 16 genes and 19 genes by >1.5-fold at 24 and 48 hours, respectively. At 24 hours, the downregulated genes from the DNA polymerase α-primase complex were POLA1, POLA2, PRIM1, and PRIM2; DNA polymerase δ complex: POLD3; DNA polymerase ε complex: POLE4, Minichromosome Maintenance protein (MCM) complex (helicase): MCM2, MCM3, MCM4, MCM6, and MCM7; clamp and clamp loader: PCNA; nuclease: FEN1; and DNA ligase: LIG1. At 48 hours, the downregulated genes from the DNA polymerase α-primase complex were POLA1, POLA2, and PRIM1; DNA polymerase δ complex: POLD3; DNA polymerase ε complex: POLE and POLE2; MCM complex (helicase): MCM2, MCM3, MCM4, MCM5, MCM6, and MCM7; clamp and clamp loader: PCNA, RFC2, and RFC3; RNase H: RNASEH2A; nucleases: DNA2 and FEN1; and DNA ligase: LIG1. Amygdalin treatment caused downregulation of several genes that play critical roles in DNA replication in the MCF-7 cell line. Thus, it might be useful as an anticancer agent.

Atomic Force Microscopy Investigation of the Interactions between the MCM Helicase and DNA.

The MCM (minichromosome maintenance) protein complex forms an hexameric ring and has a key role in the replication machinery of Eukaryotes and Archaea, where it functions as the replicative helicase opening up the DNA double helix ahead of the polymerases. Here, we present a study of the interaction between DNA and the archaeal MCM complex from Methanothermobacter thermautotrophicus by means of atomic force microscopy (AFM) single molecule imaging. We first optimized the protocol (surface treatment and buffer conditions) to obtain AFM images of surface-equilibrated DNA molecules before and after the interaction with the protein complex. We discriminated between two modes of interaction, one in which the protein induces a sharp bend in the DNA, and one where there is no bending. We found that the presence of the MCM complex also affects the DNA contour length. A possible interpretation of the observed behavior is that in one case the hexameric ring encircles the dsDNA, while in the other the nucleic acid wraps on the outside of the ring, undergoing a change of direction. We confirmed this topographical assignment by testing two mutants, one affecting the N-terminal β-hairpins projecting towards the central channel, and thus preventing DNA loading, the other lacking an external subdomain and thus preventing wrapping. The statistical analysis of the distribution of the protein complexes between the two modes, together with the dissection of the changes of DNA contour length and binding angle upon interaction, for the wild type and the two mutants, is consistent with the hypothesis. We discuss the results in view of the various modes of nucleic acid interactions that have been proposed for both archaeal and eukaryotic MCM complexes.

Increased expression of MCM4 is associated with poor prognosis in patients with hepatocellular carcinoma.

The minichromosome maintenance (MCM) protein complex is important for DNA replication. Moreover, the expression of specific MCM complex components has been associated with the survival of hepatocellular carcinoma (HCC) patients. However, the expression and functional roles of minichromosome maintenance complex component 4 (MCM4) in HCC development and progression have not yet been explored. We analyzed the expression and clinical significance of MCM4, including its association with liver cancer patient survival. Oncomine, UALCAN, and HCCDB (a database of HCC expression atlas) were used to characterize the expression of MCM4 in tumor and normal tissues. The expression of MCM4 at the protein level was confirmed based on immunohistochemistry (IHC) data obtained from the Human Protein Atlas (HPA) database. The level of MCM4 was measured in tumor and adjacent normal tissues by RT-qPCR, western blot and IHC staining. The copy number alterations (CNAs) and mutations in MCM4 were analyzed by cBioPortal, whereas the co-expression genes of MCM4 in HCC were obtained from Oncomine, and used for gene ontology and pathway analysis via the NetworkAnalyst 3.0 tool, to explore the predictive signaling pathway in HCC. The levels of MCM4 messenger (m)RNA and protein were found to be significantly higher in liver cancer tissues than in normal liver tissues. Kaplan-Meier analysis showed that the upregulation of MCM4 was significantly negatively correlated with the survival of HCC patients. Our data suggest that MCM4 may be used as a potential prognostic marker and therapeutic target for HCC.

A human cancer cell line initiates DNA replication normally in the absence of ORC5 and ORC2 proteins

The origin recognition complex (ORC), composed of six subunits, ORC1-6, binds to origins of replication as a ring-shaped heterohexameric ATPase that is believed to be essential to recruit and load MCM2-7, the minichromosome maintenance protein complex, around DNA and initiate DNA replication. We previously reported the creation of viable cancer cell lines that lacked detectable ORC1 or ORC2 protein without a reduction in the number of origins firing. Here, using CRISPR-Cas9-mediated mutations, we report that human HCT116 colon cancer cells also survive when ORC5 protein expression is abolished via a mutation in the initiator ATG of the ORC5 gene. Even if an internal methionine is used to produce an undetectable, N terminally deleted ORC5, the protein would lack 80% of the AAA+ ATPase domain, including the Walker A motif. The ORC5-depleted cells show normal chromatin binding of MCM2-7 and initiate replication from a similar number of origins as WT cells. In addition, we introduced a second mutation in ORC2 in the ORC5 mutant cells, rendering both ORC5 and ORC2 proteins undetectable in the same cells and destabilizing the ORC1, ORC3, and ORC4 proteins. Yet the double mutant cells grow, recruit MCM2-7 normally to chromatin, and initiate DNA replication with normal number of origins. Thus, in these selected cancer cells, either a crippled ORC lacking ORC2 and ORC5 and present at minimal levels on the chromatin can recruit and load enough MCM2-7 to initiate DNA replication, or human cell lines can sometimes recruit MCM2-7 to origins independent of ORC.

MCM10 Acts as a Potential Prognostic Biomarker and Promotes Cell Proliferation in Hepatocellular Carcinoma: Integrated Bioinformatics Analysis and Experimental Validation.

BackgroundHepatocellular carcinoma (HCC) is one of the most common human malignant tumors. The prognosis of HCC patients is still unsatisfying. Thus, it is of great importance to identify novel molecules and functional pathways associated with the pathophysiology of HCC. In this study, we performed the integrated bioinformatics analysis and experiment validation to identify novel biomarkers in the prognosis and progression of HCC.Materials and MethodsGene expression profiles were obtained from Gene Expression Omnibus database (GSE33294) for the screening of the differentially expressed genes (DEGs) between HCC tissues and matched non-tumor tissues. The DEGs were subjected to Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis and Gene Set Enrichment Analysis (GSEA). The key genes in HCC were further subjected to overall survival analysis of HCC patients. The in vitro functional studies were performed to validate the biological functions of the key gene in HCC cell progression.ResultsA total of 2,334 DEGs were screened from GSE33294 dataset, including 1,120 up-regulated and 1,214 down-regulated genes. GO, KEGG and GSEA results showed that DEGs are significantly associated with the biological process of cell cycle, cell division and DNA replication. The Kaplan–Meier survival analysis results showed that the key genes from the minichromosome maintenance protein complex (MCM) family including MCM8, MCM10, MCM2, MCM3, MCM4, MCM6 and MCM7 were significantly correlated with the overall survival of the HCC patients. Further validation studies showed that MCM10 was significantly up-regulated in the HCC cell lines, and knockdown of MCM10 significantly suppressed cell proliferation as determined by the cell counting kit-8 and BrdU incorporation assays and increased the caspase-3 activity of HCC cells.ConclusionThe comprehensive bioinformatics analysis identified several key genes that were associated with the prognosis of HCC patients. The validation study results indicated that MCM10 may be an important predictor for poorer prognosis of HCC patients and may act as an oncogene to promote HCC cell progression.

Molecular genetics of maternally-controlled cell divisions.

Forward genetic screens remain at the forefront of biology as an unbiased approach for discovering and elucidating gene function at the organismal and molecular level. Past mutagenesis screens targeting maternal-effect genes identified a broad spectrum of phenotypes ranging from defects in oocyte development to embryonic patterning. However, earlier vertebrate screens did not reach saturation, anticipated classes of phenotypes were not uncovered, and technological limitations made it difficult to pinpoint the causal gene. In this study, we performed a chemically-induced maternal-effect mutagenesis screen in zebrafish and identified eight distinct mutants specifically affecting the cleavage stage of development and one cleavage stage mutant that is also male sterile. The cleavage-stage phenotypes fell into three separate classes: developmental arrest proximal to the mid blastula transition (MBT), irregular cleavage, and cytokinesis mutants. We mapped each mutation to narrow genetic intervals and determined the molecular basis for two of the developmental arrest mutants, and a mutation causing male sterility and a maternal-effect mutant phenotype. One developmental arrest mutant gene encodes a maternal specific Stem Loop Binding Protein, which is required to maintain maternal histone levels. The other developmental arrest mutant encodes a maternal-specific subunit of the Minichromosome Maintenance Protein Complex, which is essential for maintaining normal chromosome integrity in the early blastomeres. Finally, we identify a hypomorphic allele of Polo-like kinase-1 (Plk-1), which results in a male sterile and maternal-effect phenotype. Collectively, these mutants expand our molecular-genetic understanding of the maternal regulation of early embryonic development in vertebrates.

Down-regulation of CK2\u03b1 correlates with decreased expression levels of DNA replication minichromosome maintenance protein complex (MCM) genes

Protein kinase CK2 is a serine/threonine kinase composed of two catalytic subunits (CK2α and/or CK2α’) and two regulatory subunits (CK2β). It is implicated in every stage of the cell cycle and in the regulation of various intracellular pathways associated with health and disease states. The catalytic subunits have similar biochemical activity, however, their functions may differ significantly in cells and in vivo. In this regard, homozygous deletion of CK2α leads to embryonic lethality in mid-gestation potentially due to severely impaired cell proliferation. To determine the CK2α-dependent molecular mechanisms that control cell proliferation, we established a myoblast-derived cell line with inducible silencing of CK2α and carried out a comprehensive RNA-Seq analysis of gene expression. We report evidence that CK2α depletion causes delayed cell cycle progression through the S-phase and defective response to replication stress. Differential gene expression analysis revealed that the down-regulated genes were enriched in pathways implicated in cell cycle regulation, DNA replication and DNA damage repair. Interestingly, the genes coding for the minichromosome maintenance proteins (MCMs), which constitute the core of the replication origin recognition complex, were among the most significantly down-regulated genes. These findings were validated in cells and whole mouse embryos. Taken together, our study provides new evidence for a critical role of protein kinase CK2 in controlling DNA replication initiation and the expression levels of replicative DNA helicases, which ensure maintenance of proliferative potential and genome integrity in eukaryotic cells.