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.

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.

MCM3 is a novel proliferation marker associated with longer survival for patients with tubo-ovarian high-grade serous carcinoma.

Tubo-ovarian high-grade serous carcinomas (HGSC) are highly proliferative neoplasms that generally respond well to platinum/taxane chemotherapy. We recently identified minichromosome maintenance complex component 3 (MCM3), which is involved in the initiation of DNA replication and proliferation, as a favorable prognostic marker in HGSC. Our objective was to further validate whether MCM3 mRNA expression and possibly MCM3 protein levels are associated with survival in patients with HGSC. MCM3 mRNA expression was measured using NanoString expression profiling on formalin-fixed and paraffin-embedded tissue (N = 2355 HGSC) and MCM3 protein expression was assessed by immunohistochemistry (N = 522 HGSC) and compared with Ki-67. Kaplan-Meier curves and the Cox proportional hazards model were used to estimate associations with survival. Among chemotherapy-naïve HGSC, higher MCM3 mRNA expression (one standard deviation increase in the score) was associated with longer overall survival (HR = 0.87, 95% CI 0.81-0.92, p < 0.0001, N = 1840) in multivariable analysis. MCM3 mRNA expression was highest in the HGSC C5.PRO molecular subtype, although no interaction was observed between MCM3, survival and molecular subtypes. MCM3 and Ki-67 protein levels were significantly lower after exposure to neoadjuvant chemotherapy compared to chemotherapy-naïve tumors: 37.0% versus 46.4% and 22.9% versus 34.2%, respectively. Among chemotherapy-naïve HGSC, high MCM3 protein levels were also associated with significantly longer disease-specific survival (HR = 0.52, 95% CI 0.36-0.74, p = 0.0003, N = 392) compared to cases with low MCM3 protein levels in multivariable analysis. MCM3 immunohistochemistry is a promising surrogate marker of proliferation in HGSC.

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.

Single-cell chromatin accessibility landscape of human umbilical cord blood in trisomy 18 syndrome.

BACKGROUND: Trisomy 18 syndrome (Edwards syndrome, ES) is a type of aneuploidy caused by the presence of an extra chromosome 18. Aneuploidy is the leading cause of early pregnancy loss, intellectual disability, and multiple congenital anomalies. The research of trisomy 18 is progressing slowly, and the molecular characteristics of the disease mechanism and phenotype are still largely unclear. RESULTS: In this study, we used the commercial Chromium platform (10× Genomics) to perform sc-ATAC-seq to measure chromatin accessibility in 11,611 single umbilical cord blood cells derived from one trisomy 18 syndrome patient and one healthy donor. We obtained 13 distinct major clusters of cells and identified them as 6 human umbilical cord blood mononuclear cell types using analysis tool. Compared with the NC group, the ES group had a lower ratio of T cells to NK cells, the ratio of monocytes/DC cell population did not change significantly, and the ratio of B cell nuclear progenitor and megakaryocyte erythroid cells was higher. The differential genes of ME-0 are enriched in Human T cell leukemia virus 1 infection pathway, and the differential peak genes of ME-1 are enriched in apopotosis pathway. We found that CCNB2 and MCM3 may be vital to the development of trisomy 18. CCNB2 and MCM3, which have been reported to be essential components of the cell cycle and chromatin. CONCLUSIONS: We have identified 6 cell populations in cord blood. Disorder in megakaryocyte erythroid cells implicates trisomy 18 in perturbing fetal hematopoiesis. We identified a pathway in which the master differential regulatory pathway in the ME-0 cell population involves human T cell leukemia virus 1 infection, a pathway that is dysregulated in patients with trisomy 18 and which may increase the risk of leukemia in patients with trisomy 18. CCNB2 and MCM3 in progenitor may be vital to the development of trisomy 18. CCNB2 and MCM3, which have been reported to be essential components of the cell cycle and chromatin, may be related to chromosomal abnormalities in trisomy 18.

MCM complex members MCM3 and MCM7 are associated with a phenotypic spectrum from Meier-Gorlin syndrome to lipodystrophy and adrenal insufficiency.

The MCM2-7 helicase is a heterohexameric complex with essential roles as part of both the pre-replication and pre-initiation complexes in the early stages of DNA replication. Meier-Gorlin syndrome, a rare primordial dwarfism, is strongly associated with disruption to the pre-replication complex, including a single case described with variants in MCM5. Conversely, a biallelic pathogenic variant in MCM4 underlies immune deficiency with growth retardation, features also seen in individuals with pathogenic variants in other pre-initiation complex encoding genes such as GINS1, MCM10, and POLE. Through exome and chromium genome sequencing, supported by functional studies, we identify biallelic pathogenic variants in MCM7 and a strong candidate biallelic pathogenic variant in MCM3. We confirm variants in MCM7 are deleterious and through interfering with MCM complex formation, impact efficiency of S phase progression. The associated phenotypes are striking; one patient has typical Meier-Gorlin syndrome, whereas the second case has a multi-system disorder with neonatal progeroid appearance, lipodystrophy and adrenal insufficiency. We provide further insight into the developmental complexity of disrupted MCM function, highlighted by two patients with a similar variant profile in MCM7 but disparate clinical features. Our results build on other genetic findings linked to disruption of the pre-replication and pre-initiation complexes, and the replisome, and expand the complex clinical genetics landscape emerging due to disruption of DNA replication.

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.

MCM3 proliferative index is worthier over Ki-67 in the characterization of salivary gland tumors.

BACKGROUND: Salivary gland tumors bear uncanny characteristics of being different based on their morphological aspects rather than the presence of clear demarcation. This ambiguity in the spectrum from benign to malignant salivary gland neoplasms while categorizing the neoplasm is having inherent pitfalls. The present study was, therefore, designed to characterize benign and malignant salivary gland tumors based on their proliferative indices. MATERIALS AND METHOD: Study samples comprised of 97 cases of histopathologically confirmed benign and malignant salivary gland tumors. The cases were immunohistochemically assessed for MCM3 and Ki-67 expressions and the molecular characterization was performed based on the findings. RESULTS: The majority of benign and malignant salivary gland tumors were from the parotid gland, (51.2%) and (42.4%), respectively. Overall mean labeling index of MCM3 was higher i.e., (5.60 ± 3.99) in comparison to Ki-67 i.e., (2.82 ± 3.14) with P = 0.05 using paired t-test. Besides, malignant salivary gland neoplasms represented a higher mean score of MCM3 and Ki-67 than benign neoplasms. CONCLUSION: The requirement of a novel marker has led to the use of MCM3 which has a characteristic role in the entire spectrum of the cell cycle. The present study highlighted the extrapolation of MCM3 over Ki-67 for diagnosis and for true characterization of biologic behavior of salivary gland pathologies which may, in turn, influence the treatment modality employed for such lesions.

Biological features, gene expression profile, and mechanisms of drug resistance of two- and three-dimensional hepatocellular carcinoma cell cultures.

Hepatocellular carcinoma (HCC) is a common malignant tumor with insidious onset and rapid progression. Its treatment is often difficult owing to tumor resistance. In this study, we aimed to understand the different biological characteristics, gene expression profiles, and drug resistance mechanisms of HCC cells cultured under different conditions. A conventional adherence method and a liquid overlay technique were used to prepare two- and three-dimensional cultures of Bel-7402 and 5-fluorouracil (5-Fu)-resistant Bel-7402 (Bel-7402/5-Fu) cells. Morphological characteristics were assessed via microscopy, and cell cycle distribution and apoptotic rate were obtained using flow cytometry. Cell sensitivity to different concentrations of drugs was detected with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays. Gene expression profiles and signal transduction pathways of Bel-7402 and Bel-7402/5-Fu cells under different culture conditions were determined using gene chips. Cells in three-dimensional culture were suspended and they grew into dense multicellular spheroid (MCS) structures, aggregating with each other. In contrast to cells in the two-dimensional culture, cell cycle arrest was observed in MCSs. The sensitivity of Bel-7402 cells in the two-dimensional culture to drugs at high concentrations was significantly higher than that of cells in the three-dimensional culture (p < .05). The apoptotic rate of Bel-7402 and Bel-7402/5-Fu cells was also higher in the two-dimensional culture (p < .05). Signal transduction pathway analysis showed that after Bel-7402 cells acquired resistance to 5-Fu, CCND1, MCM2, and MCM3 gene expression was upregulated in the G1 to S cell cycle control signal transduction pathway, CDKN1C and CCNG2 gene expression was downregulated, and MCM2 and MCM3 gene expression was upregulated in the DNA replication signal transduction pathway. Therefore, the liquid overlay technique is a simple, low-cost procedure to successfully construct three-dimensional culture models of HCC. This study provides new information and methods for exploring the molecular mechanisms of liver cancer resistance, clinical treatment, development of molecular information, and interventional prevention.

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.

Gene expression profiling revealed MCM3 to be a better marker than Ki67 in prognosis of invasive ductal breast carcinoma patients.

Invasive ductal carcinoma (IDC) is the most common breast cancer. Our study used gene microarray data to select differentially expressed genes between normal and IDC mammary tissues. From these, we selected genes related to the proliferation of tumor cells and compared their prognostic value with known biomarker Ki67 for IDC. Analysis of publicly available Gene Expression Omnibus (GEO) data revealed 24 differentially expressed genes (DEGs) in normal and 31 DEGS in IDC tissues that were used for further analyses. Gene chip analysis software was used to identify DEGs. DEG profiles were confirmed using quantitative PCR (qPCR). DEG functions where shown to be related to cell proliferation. We confirmed MCM3 expression using immunohistochemical staining in 45 IDC patients. The relationship between MCM3 expression and survival was investigated using Kaplan-Meier survival curves and Cox proportional hazard regression models. A total of 1307 differentially expressed genes were identified between IDC and normal tissues, which were enriched in 32 Gene Ontology (GO) terms and 9 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. qPCR demonstrated that both COL1A1 and MCM3 were significantly up-regulated in IDC tissues, of which only MCM3 was related to cell proliferation. Ki67 is closely associated with the tumor grade, ER status, PR status and HER2 status, while MCM3 was shown to relate to tumor size, lymph node, and PR status. There was significant association between survival and MCM3, but not for Ki67. High MCM3 expression demonstrated statistically significant associations with poor prognosis in IDC patients. Findings from the gene microarray data analysis confirmed that MCM3 is associated with the response to cell proliferation. MCM3 represents a better proliferation marker than Ki67 making it a valuable prognostic tool that is independent of ER and HER2 status.

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.

Changes in MCM2-7 proteins at senescence.

Cellular aging is characterized by the loss of DNA replication capability and is mainly brought about by various changes in chromatin structure. Here, we examined changes in MCM2-7 proteins, which act as a replicative DNA helicase, during aging of human WI38 fibroblasts at the single-cell level. We used nuclear accumulation of p21 as a marker of senescent cells, and examined changes in MCM2-7 by western blot analysis. First, we found that senescent cells are enriched for cells with a DNA content higher than 4N. Second, the levels of MCM2, MCM3, MCM4 and MCM6 proteins decreased in senescent cells. Third, cytoplasmic localization of MCM2 and MCM7 was observed in senescent cells, from an analysis of MCM2-7 except for MCM5. Consistent with this finding, fragmented MCM2 was predominant in these cells. These age-dependent changes in MCM2-7, a protein complex that directly affects cellular DNA replication, may play a critical role in cellular senescence.

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.

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.

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.

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.