LncRNA GAS6-AS1 contributes to 5-fluorouracil resistance in colorectal cancer by facilitating the binding of PCBP1 with MCM3.

5-Fluorouracil (5-FU) resistance has always been a formidable obstacle in the adjuvant treatment of advanced colorectal cancer (CRC). In recent years, long non-coding RNAs have emerged as key regulators in various pathophysiological processes including 5-FU resistance. TRG is a postoperative pathological score of the chemotherapy effectiveness for CRC, of which TRG 0-1 is classified as chemotherapy sensitivity and TRG 3 as chemotherapy resistance. Here, RNA-seq combined with weighted gene correlation network analysis confirmed the close association of GAS6-AS1 with TRG. GAS6-AS1 expression was positively correlated with advanced clinicopathological features and poor prognosis in CRC. GAS6-AS1 increased the 50% inhibiting concentration of 5-FU, enhanced cell proliferation and accelerated G1/S transition, both with and without 5-FU, both in vitro and in vivo. Mechanistically, GAS6-AS1 enhanced the stability of MCM3 mRNA by recruiting PCBP1, consequently increasing MCM3 expression. Furthermore, PCBP1 and MCM3 counteracted the effects of GAS6-AS1 on 5-FU resistance. Notably, the PDX model indicated that combining chemotherapeutic drugs with GAS6-AS1 knockdown yielded superior outcomes in vivo. Together, our findings elucidate that GAS6-AS1 directly binds to PCBP1, enhancing MCM3 expression and thereby promoting 5-FU resistance. GAS6-AS1 may serve as a robust biomarker and potential therapeutic target for combination therapy in CRC.

MCM complexes are barriers that restrict cohesin-mediated loop extrusion.

Eukaryotic genomes are compacted into loops and topologically associating domains (TADs)1-3, which contribute to transcription, recombination and genomic stability4,5. Cohesin extrudes DNA into loops that are thought to lengthen until CTCF boundaries are encountered6-12. Little is known about whether loop extrusion is impeded by DNA-bound machines. Here we show that the minichromosome maintenance (MCM) complex is a barrier that restricts loop extrusion in G1 phase. Single-nucleus Hi-C (high-resolution chromosome conformation capture) of mouse zygotes reveals that MCM loading reduces CTCF-anchored loops and decreases TAD boundary insulation, which suggests that loop extrusion is impeded before reaching CTCF. This effect extends to HCT116 cells, in which MCMs affect the number of CTCF-anchored loops and gene expression. Simulations suggest that MCMs are abundant, randomly positioned and partially permeable barriers. Single-molecule imaging shows that MCMs are physical barriers that frequently constrain cohesin translocation in vitro. Notably, chimeric yeast MCMs that contain a cohesin-interaction motif from human MCM3 induce cohesin pausing, indicating that MCMs are 'active' barriers with binding sites. These findings raise the possibility that cohesin can arrive by loop extrusion at MCMs, which determine the genomic sites at which sister chromatid cohesion is established. On the basis of in vivo, in silico and in vitro data, we conclude that distinct loop extrusion barriers shape the three-dimensional genome.

Identification of mungbean yellow mosaic India virus (MYMIV) Rep interacting partners using phage display and influence of Arabidopsis thaliana MCM3 on geminivirus DNA replication.

Geminiviruses consist of a single-stranded DNA genome that replicates by a rolling circle (RCR) and recombination-dependent (RDR) modes of replication. The AC1 or Rep is the indispensable viral protein required for the RCR mode of replication. Since these viruses encode only a few proteins, they depend on several host factors for replication, transcription, and other physiological processes. To get insights into the repertoire of host factors influencing the replication of geminiviruses, we performed phage display experiments which led to the identification of putative mungbean yellow mosaic India virus (MYMIV) Rep interacting host proteins. These proteins might directly or indirectly participate in geminivirus biology. MCM3 was one of the Rep-interacting partners obtained in the phage display results. Using bimolecular fluorescence complementation (BiFC), the interaction of the MYMIV Rep with Arabidopsis thaliana MCM3 (AtMCM3) was confirmed. We report the involvement of AtMCM3 in the replication of MYMIV DNA through an ex vivo system. The physiological relevance of the interaction between AtMCM3 and MYMIV Rep is reflected by yeast replication assay.Communicated by Ramaswamy H. Sarma.

Bioinformatics Analysis Reveals MCM3 as an Important Prognostic Marker in Cervical Cancer.

The minichromosome maintenance complex 3 (MCM3) is essential for the regulation of DNA replication and cell cycle progression. However, the expression and prognostic values of MCM3 in cervical cancer (CC) have not been well-studied. Herein, we investigated the expression patterns and survival data of MCM3 in cervical cancer patients from the ONCOMINE, GEPIA, Human Protein Atlas, UALCAN, Kaplan-Meier Plotter, and LinkedOmics databases. The expression level of MCM3 is negatively correlated with advanced tumor stage and metastatic status. Specifically, MCM3 is significantly differentially expressed between patients in stage 1 and stage 3 cervical cancer with p value 0.0138. Similarly, the p values between stage 1 and stage 4 cervical cancer, between stage 2 and stage 3, and between stage 2 and stage 4 are 0.00089, 0.0244, and 0.00197, respectively. Not only that, cervical cancer patients with high mRNA expression of MCM3 may indicate longer overall survival but indicate shorter relapse-free survival. PRIM2 and MCM6 are positively correlated genes of MCM3. Bioinformatics analysis revealed that MCM3 might be considered a biological indicator for prognostic evaluation of cervical cancer. However, it is currently limited to bioinformatics analysis, and more clinical tissue specimens and cell experiments are needed to further explore the role of MCM3 in the occurrence and progression of cervical cancer.

The Immunohistochemical Expression of MCM-3, -5, and -7 Proteins in the Uterine Fibroids.

Uterine fibroids are the most common mesenchymal uterine neoplasms; their prevalence is estimated in 40%-60% of women under 35 and in 70%-80% of women over 50 years of age. The current research aims to focus on the etiopathogenesis of uterine fibroids, the factors that affect their growth, and markers with diagnostic and prognostic properties. The MCM (minichromosome maintenance) protein family consists of peptides whose primary function is participation in the molecular mechanism of creating replication forks while regulating DNA synthesis. The aim of this work was to determine the proliferative potential of uterine fibroid cells based on the expression of the Ki-67 antigen and the MCMs-i.e., MCM-3, MCM-5, and MCM-7. In addition, the expression of estrogen (ER) and progesterone (PgR) receptors was evaluated and correlated with the expression of the abovementioned observations. Ultimately, received results were analyzed in terms of clinical and pathological data. MATERIALS AND METHODS: In forty-four cases of uterine fibroids, immunohistochemical reactions were performed. A tissue microarray (TMA) technique was utilized and analyzed cases were assessed in triplicate. Immunohistochemistry was performed using antibodies against Ki-67 antigen, ER, PgR, MCM-3, MCM-5, and MCM-8 on an automated staining platform. Reactions were digitalized by a histologic scanner and quantified utilizing dedicated software for nuclear analysis. Assessment was based on quantification expression of the three histiospots, each representing one case in TMA. RESULTS: In the study group (uterine fibroids), statistically significant stronger expression of all the investigated MCMs was observed, as compared to the control group. In addition, moderate and strong positive correlations were found between all tested proliferative markers. The expression of the MCM-7 protein also correlated positively with ER and PgR. With regard to clinical and pathological data, there was a negative correlation between the expression of MCMs and the number of both pregnancies and births. Significant reductions in MCM-5 and MCM-7 expression were observed in the group of women receiving oral hormonal contraceptives, while smoking women showed an increase in MCM-7, ER, and PgR. CONCLUSIONS: Uterine fibroid cells have greater proliferative potential, as evaluated by expression of the Ki-67 antigen and MCMs, than unaltered myometrial cells of the uterine corpus. The expression of MCM-7 was found to have strong or moderate correlations in all assessed relations. In the context of the clinical data, as well evident proliferative potential of MCMs, further studies are strongly recommended.

Downregulation of circular RNA circDOCK7 identified from diabetic rats after sleeve gastrectomy contributes to hepatocyte apoptosis through regulating miR-139-3p and MCM3.

Sleeve gastrectomy (SG) is the most widely used bariatric procedures globally, which could improve glucose and lipid metabolism dramatically. Circular RNAs (circRNAs) are being increasingly implicated in numerous pathophysiological processes. However, for diabetes mellitus (DM), the expression and function of circRNAs remain largely undetermined, in particular, whether circRNAs mediate the amelioration of DM observed after SG. Using a diabetic rat model, we subjected liver tissue from SG and sham-operated rats to RNA sequencing. Amongst the 103 differentially regulated circRNAs identified in diabetic rats after SG, we focused on circDOCK7, a highly expressed circRNA derived from the back-splicing of the DOCK7 gene. Silencing of circDOCK7 significantly inhibited cellular proliferation and induction of apoptosis in insulin-resistant rat hepatocytes. Further analysis indicated circDOCK7 harbored binding sites for miR-139-3p and regulated the expression of minichromosome maintenance 3 (MCM3) through sequestration of miR-139-3p. Our findings therefore demonstrate a novel regulatory pathway involving circDOCK7 that regulates cellular proliferation and apoptosis through increasing the expression of MCM3. Overall, our study establishes a list of specific circRNAs expressed in diabetic rat liver after SG including circDOCK7 which serve as potential biomarkers and treatment targets for DM patients.

Elevated expression of minichromosome maintenance 3 indicates poor outcomes and promotes G1/S cell cycle progression, proliferation, migration and invasion in colorectal cancer.

BACKGROUND: The minichromosome maintenance (MCM) family, a core component of DNA replication, is involved in cell cycle process. Abnormal proliferation has been identified as a crucial process in the evolution of colorectal cancer (CRC). However, the roles of the MCM family in CRC remain largely unknown. METHODS: Here, the expression, prognostic significance and functions of the MCM family in CRC were systematically analyzed through a series of online databases including CCLE, Oncomine, HPA, cBioPortal and cancerSEA. RESULTS: We found all MCM family members were highly expressed in CRC, but only elevation of MCM3 expression was associated with poor prognosis of patients with CRC. Further in vitro and in vivo experiments were performed to examine the role of MCM3 in CRC. Analysis of CCLE database and qRT-PCR assay confirmed that MCM3 was overexpressed in CRC cell lines. Moreover, knockdown of MCM3 significantly suppressed transition of G1 to S phase in CRC cells. Furthermore, down-regulation of MCM3 inhibited CRC cell proliferation, migration, invasion and promoted apoptosis. CONCLUSION: These findings reveal that MCM3 may function as an oncogene and a potential prognosis biomarker. Thus, the association between abnormal expression of MCM3 and the initiation of CRC deserves further exploration.

DNA unwinding mechanism of a eukaryotic replicative CMG helicase.

High-resolution structures have not been reported for replicative helicases at a replication fork at atomic resolution, a prerequisite to understanding the unwinding mechanism. The eukaryotic replicative CMG (Cdc45, Mcm2-7, GINS) helicase contains a Mcm2-7 motor ring, with the N-tier ring in front and the C-tier motor ring behind. The N-tier ring is structurally divided into a zinc finger (ZF) sub-ring followed by the oligosaccharide/oligonucleotide-binding (OB) fold ring. Here we report the cryo-EM structure of CMG on forked DNA at 3.9 Å, revealing that parental DNA enters the ZF sub-ring and strand separation occurs at the bottom of the ZF sub-ring, where the lagging strand is blocked and diverted sideways by OB hairpin-loops of Mcm3, Mcm4, Mcm6, and Mcm7. Thus, instead of employing a specific steric exclusion process, or even a separation pin, unwinding is achieved via a "dam-and-diversion tunnel" mechanism that does not require specific protein-DNA interaction. The C-tier motor ring contains spirally configured PS1 and H2I loops of Mcms 2, 3, 5, 6 that translocate on the spirally-configured leading strand, and thereby pull the preceding DNA segment through the diversion tunnel for strand separation.

PLK1 promotes proliferation and suppresses apoptosis of renal cell carcinoma cells by phosphorylating MCM3.

Minichromosome maintenance 3 (MCM3) protein has been widely studied due to its essential role in DNA replication. In addition, it is overexpressed in several human tumor types. However, the role of this protein in renal cell carcinoma (RCC) is not widely known. In this study, we demonstrated that polo-like kinase 1 (PLK1)-mediated MCM3 phosphorylation regulates proliferation and apoptosis in RCC. Our results confirm that PLK1 and phospho-MCM3 (p-MCM3) are highly expressed in renal cell carcinoma. The expression of PLK1 is closely related to the clinical characteristics of renal cell carcinoma. They play important roles in the proliferation and apoptosis of RCC. In vitro, after overexpression of PLK1 or MCM3, the proliferation of RCC cells was significantly enhanced and cell apoptosis was inhibited, while after knockout, the proliferation of RCC cells was weakened and cell apoptosis was promoted. In addition, Mn2+-Phos-tag SDS-PAGE, western blotting, and immunofluorescence were utilized to determine that MCM3 is a physiological substrate of PLK1, which is phosphorylated on serine 112 (Ser112) in a PLK1-dependent manner. PLK1-mediated MCM3 phosphorylation promotes RCC cell cycle proliferation and suppresses apoptosis in vitro. Moreover, we found that PLK1-mediated MCM3 phosphorylation induced cellular proliferation and decreased apoptosis, as well as tumor growth in mice. Overall, we conclude that PLK1-mediated MCM3 phosphorylation is a novel mechanism to regulate RCC proliferation and apoptosis.

Comparison of bleaching protocols utilizing hematoxylin and eosin stain and immunohistochemical proliferation marker MCM3 in pigmented melanomas.

Melanomas represent the malignant transformation of melanocytes, cells found primarily in the skin to protect epithelium and underlying connective tissues from harmful effects of ultraviolet light. Melanomas vary greatly in morphology and may continue to produce melanin markedly, mildly, or not at all. Performing and evaluating hematoxylin and eosin stains as well as immunohistochemical stains on pigmented melanomas has been a long-standing challenge due to the obscuring pigment. Protocols for removing melanin to reveal cellular morphology have been used successfully for years, but coupling these protocols with stains for immunohistochemistry represents an added challenge. In this study, the investigators evaluated results of various melanin bleaching protocols on tissue morphology, completeness of melanin removal, and immunohistochemistry staining quality. It was found that 1% formamide in 3% H2O2 under bright light without heating outperformed other tested protocols.

Positive expression of basic transcription factor 3 predicts poor survival of colorectal cancer patients: possible mechanisms involved.

Basic transcription factor 3 (BTF3) is associated with the development of several cancers. The aim of our study was to elucidate the role of BTF3 in colorectal cancer (CRC) tissues. CRC tissues or their paired adjacent noncancerous (ANCT) tissues were obtained from 90 patients who underwent operations in our hospital from November 2011 to December 2016, and then we implemented a gene microarray assay for detecting significant changes in gene expression and confirmed expression in tissues using immunohistochemistry and real-time PCR. We transfected or injected the silencing BTF3 (BTF3-siRNA) plasmid into cells and nude mice, and measured the tumorigenicity of CRC cells with flow cytometry and studied the expression level of BTF3 downstream genes (MAD2L2, MCM3 and PLK1) in CRC cells. BTF3 expression level was not only significantly higher in CRC tissue than in ANCT tissue (2.61 ± 0.07 vs 1.90 ± 0.03, P < 0.001) but BTF3-siRNA decreased tumor formation in a nude mice model. Furthermore, based on the data of gene microarray analysis, MAD2L2, MCM3 and PLK1 were detected as the downstream target genes of BTF3 and their expressions were positive related with BTF3 expression. Also, through transfecting BTF3-siRNA into HCT116 cells, we found that BTF3-siRNA could decrease cell viability and induced cell apoptosis and blocking the cell cycle. In conclusion, BTF3 is positively related to CRC and BTF3-siRNA attenuated the tumorigenicity of colorectal cancer cells via MAD2L2, MCM3 and PLK1 activity reduction.

Minichromosome maintenance 3 promotes hepatocellular carcinoma radioresistance by activating the NF-κB pathway.

BACKGROUND: Hepatocellular carcinoma (HCC) is the most common tumors in the worldwide, it develops resistance to radiotherapy during treatment, understanding the regulatory mechanisms of radioresistance generation is the urgent need for HCC therapy. METHODS: qRT-PCR, western blot and immunohistochemistry were used to examine MCM3 expression. MTT assay, colony formation assay, terminal deoxynucleotidyl transferase nick end labeling assay and In vivo xenograft assay were used to determine the effect of MCM3 on radioresistance. Gene set enrichment analysis, luciferase reporter assay, western blot and qRT-PCR were used to examine the effect of MCM3 on NF-κB pathway. RESULTS: We found DNA replication initiation protein Minichromosome Maintenance 3 (MCM3) was upregulated in HCC tissues and cells, patients with high MCM3 expression had poor outcome, it was an independent prognostic factor for HCC. Cells with high MCM3 expression or MCM3 overexpression increased the radioresistance determined by MTT assay, colony formation assay, TUNEL assay and orthotopic transplantation mouse model, while cells with low MCM3 expression or MCM3 knockdown reduced the radioresistance. Mechanism analysis showed MCM3 activated NF-κB pathway, characterized by increasing the nuclear translocation of p65, the expression of the downstream genes NF-κB pathway and the phosphorylation of IKK-ß and IκBα. Inhibition of NF-κB in MCM3 overexpressing cells using small molecular inhibitor reduced the radioresistance, suggesting MCM3 increased radioresistance through activating NF-κB pathway. Moreover, we found MCM3 expression positively correlated with NF-κB pathway in clinic. CONCLUSIONS: Our findings revealed that MCM3 promoted radioresistance through activating NF-κB pathway, strengthening the role of MCM subunits in the tumor progression and providing a new target for HCC therapy.

Long Noncoding RNA CPR (Cardiomyocyte Proliferation Regulator) Regulates Cardiomyocyte Proliferation and Cardiac Repair.

BACKGROUND: The adult mammalian cardiomyocytes lose their proliferative capacity, which is responsible for cardiac dysfunction and heart failure following injury. The molecular mechanisms underlying the attenuation of adult cardiomyocyte proliferation remain largely unknown. Because long noncoding RNAs (lncRNAs) have a critical role in the development of cardiovascular problems, we investigated whether lncRNAs have any role in the regulation of cardiomyocyte proliferation and cardiac repair. METHODS: Using bioinformatics and initial analysis, we identified an lncRNA, named CPR (cardiomyocyte proliferation regulator), that has a potential regulatory role in cardiomyocyte proliferation. For in vivo experiments, we generated CPR knockout and cardiac-specific CPR-overexpressing mice. In isolated cardiomyocytes, we used adenovirus for silencing (CPR-small interfering RNA) or overexpressing CPR. To investigate the mechanisms of CPR function in cardiomyocyte proliferation, we performed various analyses including quantitative reverse transcription-polymerase chain reaction, Western blot, histology, cardiac function (by echocardiography), transcriptome analyses (microarray assay), RNA pull-down assay, and chromatin immunoprecipitation assay. RESULTS: CPR level is comparatively higher in the adult heart than in the fetal stage. The silencing of CPR significantly increased cardiomyocyte proliferation in postnatal and adult hearts. Moreover, CPR deletion restored the heart function after myocardial injury, which was evident from increased cardiomyocyte proliferation, improvement of myocardial function, and reduced scar formation. In contrast, the neonatal cardiomyocyte proliferation and cardiac regeneration were remarkably suppressed in CPR-overexpressing mice or adeno-associated virus serotype 9-CPR-overexpressing heart. These results indicate that CPR acts as a negative regulator of cardiomyocyte proliferation and regeneration. Next, we found that CPR targets minichromosome maintenance 3, an initiator of DNA replication and cell cycle progression, to suppress cardiomyocyte proliferation. CPR silenced minichromosome maintenance 3 expression through directly interacting and recruiting DNMT3A to its promoter cysteine-phosphate-guanine sites, as evident from decreased minichromosome maintenance 3 promoter methylation and increased minichromosome maintenance 3 expression in CPR knocked-down cardiomyocytes and CPR knockout mouse heart. These results were confirmed in CPR-overexpressing cardiomyocytes and CPR-overexpressing mouse heart. CONCLUSIONS: Together, our findings identified that CPR is a suppressor of cardiomyocyte proliferation and indicated that lncRNAs take part in the regulation of cardiomyocyte proliferation and cardiac repair. Our study provides an lncRNA-based therapeutic strategy for effective cardiac repair and regeneration.

Minichromosome Maintenance Proteins MCM-3, MCM-5, MCM-7, and Ki-67 as Proliferative Markers in Adrenocortical Tumors.

BACKGROUND/AIM: Morphological features, combined with Ki-67 proliferative index, remain the standard for discriminating benign and malignant adrenocortical tumors. The aim of this study was to evaluate the role of minichromosome maintenance proteins MCM-3, MCM-5, MCM-7, and Ki-67 as proliferative markers in adrenocortical tumors. MATERIALS AND METHODS: Specimens of 81 adrenocortical adenomas and 3 adrenocortical carcinomas were stained with antibodies against MCM-3, 5, 7 and Ki-67. RESULTS: Malignant tumors were characterized by a greater size (p=0.017), volume (p=0.017), and higher levels of Ki-67 (p=0.005), MCM-3 (p=0.005), MCM-7 (p=0.008), but not MCM-5 (p=0.069). The markers' levels were independent from the tumors' size and volume, the patient's age and hormonal status. ROC curves showed Ki-67 (AUC 0.984), MCM-3 (AUC 0.984), and MCM-7 (AUC 0.950), but not MCM-5 (AUC 0.820) to be reliable markers. CONCLUSION: Ki-67, MCM-3, and MCM-7, but not MCM-5 are reliable proliferative and diagnostic markers in discerning benign and malignant adrenocortical tumors.

Destabilization of the MiniChromosome Maintenance (MCM) complex modulates the cellular response to DNA double strand breaks.

DNA replication during S phase involves thousands of replication forks that must be coordinated to ensure that every DNA section is replicated only once. The minichromosome maintenance proteins, MCM2 to MCM7, form a heteromeric DNA helicase required for both the initiation and elongation of DNA replication. Although only two DNA helicase activities are necessary to establish a bidirectional replication fork from each replication origin, a large excess of MCM complexes is amassed and distributed along the chromatin. The function of the additional MCM complexes is not well understood, as most are displaced from the DNA during the S-phase, apparently without playing an active role in DNA replication. DNA damage response (DDR) kinases activated by stalled forks prevent the replication machinery from being activated, indicating a tight relationship between DDR and DNA replication. To investigate the role of MCM proteins in the cellular response to DNA damage, we used shRNA targeting MCM2 or MCM3 to determine the impact of a reduction in MCM complex. The alteration of MCM proteins induced a change in the activation of key factors of the DDR in response to Etoposide treatment. Etoposide-induced DNA damage affected the phosphorylation of γ-H2AX, CHK1 and CHK2 without affecting cell viability. Using assays measuring homologous recombination (HR) and non-homologous end-joining (NHEJ), we identified a decrease in both HR and NHEJ associated with a decrease in MCM complex.

CDK phosphorylation regulates Mcm3 degradation in budding yeast.

Accurate regulation of activity and level of the MCM complex is critical for precise DNA replication and genome transmission. Cyclin-dependent kinase (CDK) negatively regulates nuclear localization of the MCM complex via phosphorylation of the Mcm3 subunit. More recently, we found that Mcm3 is degraded via the Skp1-Cullin-F-box (SCF)-proteasome axis in budding yeast. However, how Mcm3 degradation is regulated is largely unknown. Here, we show that CDK represses Mcm3 degradation. Phosphorylated Mcm3 was excluded from the nucleus, where SCF is predominantly located, although CDK-mediated phosphorylation itself generated a phosphodegron of Mcm3, stimulating the degradation of Mcm3 resident in the nucleus. Thus, CDK negatively regulated nuclear MCM levels by exclusion from the nucleus and degradation in the nucleus via Mcm3 phosphorylation. We will discuss the physiological importance of Mcm3 degradation.

Regulation of the Minichromosome Maintenance Protein 3 (MCM3) Chromatin Binding by the Prolyl Isomerase Pin1.

Human Pin1 is a peptidyl prolyl cis/trans isomerase with a unique preference for phosphorylated Ser/Thr-Pro substrate motifs. Here we report that MCM3 (minichromosome maintenance complex component 3) is a novel target of Pin1. MCM3 interacts directly with the WW domain of Pin1. Proline-directed phosphorylation of MCM3 at S112 and T722 are crucial for the interaction with Pin1. MCM3 as a subunit of the minichromosome maintenance heterocomplex MCM2-7 is part of the pre-replication complex responsible for replication licensing and is implicated in the formation of the replicative helicase during progression of replication. Our data suggest that Pin1 coordinates phosphorylation-dependently MCM3 loading onto chromatin and its unloading from chromatin, thereby mediating S phase control.

TORC1 signaling regulates DNA replication via DNA replication protein levels.

Accurate DNA replication is at the heart of faithful genome transmission in dividing cells. DNA replication is strictly controlled by various factors. However, how environmental stresses such as nutrient starvation impact on these factors and DNA replication is largely unknown. Here we show that DNA replication is regulated by target of rapamycin complex 1 (TORC1) protein kinase, which is a central regulator of cell growth and proliferation in response to nutrients. TORC1 inactivation reduced the levels of various proteins critical for DNA replication initiation, such as Mcm3, Orc3, Cdt1, and Sld2, and retarded DNA replication. TORC1 inactivation promoted proteasome-mediated Mcm3 degradation. Skp1-Cullin-F-box (SCF)-Grr1 and PEST motif mediated Mcm3 degradation. TORC1-downstream factors PP2A-Cdc55 protein phosphatase and protein kinase A regulated Mcm3 degradation. This study showed that TORC1 signaling modulates DNA replication to coordinate cell growth and genome replication in response to nutrient availability.

Keap1-MCM3 interaction is a potential coordinator of molecular machineries of antioxidant response and genomic DNA replication in metazoa.

Coordination of DNA replication and cellular redox homeostasis mechanisms is essential for the sustained genome stability due to the sensitivity of replicating DNA to oxidation. However, substantial gaps remain in our knowledge of underlying molecular pathways. In this study, we characterise the interaction of Keap1, a central antioxidant response regulator in Metazoa, with the replicative helicase subunit protein MCM3. Our analysis suggests that structural determinants of the interaction of Keap1 with its critical downstream target - Nrf2 master transactivator of oxidative stress response genes - may have evolved in evolution to mimic the conserved helix-2-insert motif of MCM3. We show that this has led to a competition between MCM3 and Nrf2 proteins for Keap1 binding, and likely recruited MCM3 for the competitive binding dependent modulation of Keap1 controlled Nrf2 activities. We hypothesise that such mechanism could help to adjust the Keap1-Nrf2 antioxidant response pathway according to the proliferative and replicative status of the cell, with possible reciprocal implications also for the regulation of cellular functions of MCM3. Altogether this suggests about important role of Keap1-MCM3 interaction in the cross-talk between replisome and redox homeostasis machineries in metazoan cells.

Identification of candidate biomarkers and pathways associated with SCLC by bioinformatics analysis.

Small cell lung cancer (SCLC) is one of the highly malignant tumors and a serious threat to human health. The aim of the present study was to explore the underlying molecular mechanisms of SCLC. mRNA microarray datasets GSE6044 and GSE11969 were downloaded from Gene Expression Omnibus database, and the differentially expressed genes (DEGs) between normal lung and SCLC samples were screened using GEO2R tool. Functional and pathway enrichment analyses were performed for common DEGs using the DAVID database, and the protein­protein interaction (PPI) network of common DEGs was constructed by the STRING database and visualized with Cytoscape software. In addition, the hub genes in the network and module analysis of the PPI network were performed using CentiScaPe and plugin Molecular Complex Detection. Finally, the mRNA expression levels of hub genes were validated in the Oncomine database. A total of 150 common DEGs with absolute fold­change >0.5, including 66 significantly downregulated DEGs and 84 upregulated DEGs were obtained. The Gene Ontology term enrichment analysis suggested that common upregulated DEGs were primarily enriched in biological processes (BPs), including 'cell cycle', 'cell cycle phase', 'M phase', 'cell cycle process' and 'DNA metabolic process'. The common downregulated genes were significantly enriched in BPs, including 'response to wounding', 'positive regulation of immune system process', 'immune response', 'acute inflammatory response' and 'inflammatory response'. Kyoto Encyclopedia of Genes and Genomes pathway analysis identified that the common downregulated DEGs were primarily enriched in the 'complement and coagulation cascades' signaling pathway; the common upregulated DEGs were mainly enriched in 'cell cycle', 'DNA replication', 'oocyte meiosis' and the 'mismatch repair' signaling pathways. From the PPI network, the top 10 hub genes in SCLC were selected, including topoisomerase IIα, proliferating cell nuclear antigen, replication factor C subunit 4, checkpoint kinase 1, thymidylate synthase, minichromosome maintenance protein (MCM) 2, cell division cycle (CDC) 20, cyclin dependent kinase inhibitor 3, MCM3 and CDC6, the mRNA levels of which are upregulated in Oncomine SCLC datasets with the exception of MCM2. Furthermore, the genes in the significant module were enriched in 'cell cycle', 'DNA replication' and 'oocyte meiosis' signaling pathways. Therefore, the present study can shed new light on the understanding of molecular mechanisms of SCLC and may provide molecular targets and diagnostic biomarkers for the treatment and early diagnosis of SCLC.