Minichromosome Maintenance Complex Component Research Articles

Prognostic and Clinical Significance of the Proliferation Marker MCM7 in Breast Cancer.

Minichromosome maintenance complex component 7 (MCM7) plays an essential role in proliferation and DNA replication of cancer cells. However, the expression and prognostic significance of MCM7 in breast cancer (BC) remain to be defined. In this study, we aimed to evaluate the role of MCM7 in BC. We conducted immunohistochemistry staining of MCM7 in 1,156 operable early-stage BC samples and assessed MCM7 at the transcriptomic levels using publicly available cohorts (n = 13,430). MCM7 expression was evaluated and correlated with clinicopathological parameters including Ki67 labelling index and patient outcome. At the transcriptomic level, there was a significant association between high MCM7 mRNA levels and shorter patient survival in the whole cohort and in luminal BC class but not in the basal-like molecular subtype. High MCM7 protein expression was detected in 43% of patients and was significantly associated with parameters characteristic of aggressive tumour behaviour. MCM7 was independently associated with shorter survival, particularly in oestrogen receptor-positive (luminal) BC. MCM7 stratified luminal tumours with aggressive clinicopathological features into distinct prognostic groups. In endocrine therapy-treated BC patients, high MCM7 was associated with poor outcome, but such association disappeared with administration of adjuvant chemotherapy. Patients with high expression of Ki67 and MCM7 showed worst survival, while patients with double low expression BC showed the best outcome compared with single expression groups. The current findings indicate that MCM7 expression has a prognostic value in BC and can be used to identify luminal BC patients who can benefit from adjuvant chemotherapy.

Identification of biallelic mutations in MCM3AP and comprehensive literature analysis.

Minichromosome maintenance complex component 3 associated protein (MCM3AP) is a gene in which mutations can result in autosomal recessive peripheral neuropathy with or without impaired intellectual development. The MCM3AP genotype-phenotype correlation and prognosis remain unclear. The aim of this study was to explore the genotype-phenotype correlations pertaining to MCM3AP. Whole-exome sequencing (WES) combined with copy number variation sequencing (CNV-seq) were performed on the genomic DNA isolated from a Chinese family, and Sanger sequencing, quantitative PCR and cDNA analyses were performed to examine the mutations. The retrospective study was conducted on 28 individuals with biallelic MCM3AP mutation-related diseases, including features such as mutations, motor development impairment, intellectual disability, weakness/atrophy, and cerebral magnetic resonance imaging abnormalities. Sequencing identified novel compound heterozygous mutations in MCM3AP, namely, a paternal variant c.1_5426del (loss of exons 1-25) and a maternal splicing variant c.1858 + 3A>G. Functional studies revealed that the variant c.1858 + 3A>G resulted in the heterozygous deletion of exon 5, thereby affecting splicing functionality. Furthermore, the compound heterozygous mutation may affect the functionality of the protein domain. Retrospective analysis revealed different genotype-phenotype correlations for the pathogenic variants in biallelic MCM3AP: all individuals (100%) with mutations outside the Sac3 domain exhibited early-onset symptoms, motor developmental delays, and cognitive abnormalities, conversely, the proportions of individuals carrying mutations within the domain were 26.7% (motor delays) and 46.7% (cognitive abnormalities). Our findings further expand the genetic mutation spectrum of biallelic MCM3AP and highlight the genotype-phenotype associations. Additionally, we elaborate on the importance of rehabilitation intervention.

Dynamics of Replication-Associated Protein Levels through the Cell Cycle.

The measurement of dynamic changes in protein level and localization throughout the cell cycle is of major relevance to studies of cellular processes tightly coordinated with the cycle, such as replication, transcription, DNA repair, and checkpoint control. Currently available methods include biochemical assays of cells in bulk following synchronization, which determine protein levels with poor temporal and no spatial resolution. Taking advantage of genetic engineering and live-cell microscopy, we performed time-lapse imaging of cells expressing fluorescently tagged proteins under the control of their endogenous regulatory elements in order to follow their levels throughout the cell cycle. We effectively discern between cell cycle phases and S subphases based on fluorescence intensity and distribution of co-expressed proliferating cell nuclear antigen (PCNA)-mCherry. This allowed us to precisely determine and compare the levels and distribution of multiple replication-associated factors, including Rap1-interacting factor 1 (RIF1), minichromosome maintenance complex component 6 (MCM6), origin recognition complex subunit 1 (ORC1, and Claspin, with high spatiotemporal resolution in HeLa Kyoto cells. Combining these data with available mass spectrometry-based measurements of protein concentrations reveals the changes in the concentration of these proteins throughout the cell cycle. Our approach provides a practical basis for a detailed interrogation of protein dynamics in the context of the cell cycle.

Targeting histone deacetylase 6 (HDAC6) to enhance radiation therapy in meningiomas in a 2D and 3D in vitro study

External radiation therapy (RT) is often a primary treatment for inoperable meningiomas in the absence of established chemotherapy. Histone deacetylase 6 (HDAC6) overexpression, commonly found in cancer, is acknowledged as a driver of cellular growth, and inhibiting HDACs holds promise in improving radiotherapeutic efficacy. Downregulation of HDAC6 facilitates the degradation of β-catenin. This protein is a key element in the Wnt/β-catenin signalling pathway, contributing to the progression of meningiomas. In order to elucidate the associations and therapeutic potential of HDAC6 inhibitors (HDAC6i) in conjunction with RT, we administered Cay10603, HDAC6i, to both immortalised and patient-derived meningioma cells prior to RT in this study. Our findings reveal an increase in HDAC6 expression following exposure to RT, which is effectively mitigated with pre-treated Cay10603. The combination of Cay10603 with RT resulted in a synergistic augmentation of cytotoxic effects, as demonstrated through a range of functional assays conducted in both 2D as well as 3D settings; the latter containing syngeneic tumour microenvironment (TME). Radiation-induced DNA damage was augmented by pre-treatment with Cay10603, concomitant with the inhibition of β-catenin and minichromosome maintenance complex component 2 (MCM2) accumulation within the nucleus. This subsequently inhibited c-myc oncogene expression. Our findings demonstrate the therapeutic potential of Cay10603 to improve the radiosensitisation and provide rationale for combining HDAC6i with RT for the treatment of meningioma. This work was funded by Brain Tumour Research Centre of Excellence award to C Oliver Hanemann.

Beyond ribosomal function: RPS6 deficiency suppresses cholangiocarcinoma cell growth by disrupting alternative splicing

Cholangiocarcinoma (CCA) is a bile duct malignancy with a dismal prognosis. This study systematically investigated the role of the ribosomal protein S6 (RPS6) gene, which is dependent in CCA. We found that RPS6 upregulation in CCA tissues was correlated with a poor prognosis. Functional investigations have shown that alterations in RPS6 expression, both gain- and loss-of function could affect the proliferation of CCA cells. In xenograft tumor models, RPS6 overexpression enhances tumorigenicity, whereas RPS6 silencing reduces it. Integration analysis using RNA-seq and proteomics elucidated downstream signaling pathways of RPS6 depletion by affecting the cell cycle, especially DNA replication. Immunoprecipitation followed by mass spectrometry has identified numerous spliceosome complex proteins associated with RPS6. Transcriptomic profiling revealed that RPS6 affects numerous alternative splicing (AS) events, and combined with RNA immunoprecipitation sequencing, revealed that minichromosome maintenance complex component 7 (MCM7) binds to RPS6, which regulates its AS and increases oncogenic activity in CCA. Targeting RPS6 with vivo phosphorodiamidate morpholino oligomer (V-PMO) significantly inhibited the growth of CCA cells, patient-derived organoids, and subcutaneous xenograft tumor. Taken together, the data demonstrate that RPS6 is an oncogenic regulator in CCA and that RPS6-V-PMO could be repositioned as a promising strategy for treating CCA.

In silico functional, structural and pathogenicity analysis of missense single nucleotide polymorphisms in human MCM6 gene

Single nucleotide polymorphisms (SNPs) are one of the most common determinants and potential biomarkers of human disease pathogenesis. SNPs could alter amino acid residues, leading to the loss of structural and functional integrity of the encoded protein. In humans, members of the minichromosome maintenance (MCM) family play a vital role in cell proliferation and have a significant impact on tumorigenesis. Among the MCM members, the molecular mechanism of how missense SNPs of minichromosome maintenance complex component 6 (MCM6) contribute to DNA replication and tumor pathogenesis is underexplored and needs to be elucidated. Hence, a series of sequence and structure-based computational tools were utilized to determine how mutations affect the corresponding MCM6 protein. From the dbSNP database, among 15,009 SNPs in the MCM6 gene, 642 missense SNPs (4.28%), 291 synonymous SNPs (1.94%), and 12,500 intron SNPs (83.28%) were observed. Out of the 642 missense SNPs, 33 were found to be deleterious during the SIFT analysis. Among these, 11 missense SNPs (I123S, R207C, R222C, L449F, V456M, D463G, H556Y, R602H, R633W, R658C, and P815T) were found as deleterious, probably damaging, affective and disease-associated. Then, I123S, R207C, R222C, V456M, D463G, R602H, R633W, and R658C missense SNPs were found to be highly harmful. Six missense SNPs (I123S, R207C, V456M, D463G, R602H, and R633W) had the potential to destabilize the corresponding protein as predicted by DynaMut2. Interestingly, five high-risk mutations (I123S, V456M, D463G, R602H, and R633W) were distributed in two domains (PF00493 and PF14551). During molecular dynamics simulations analysis, consistent fluctuation in RMSD and RMSF values, high Rg and hydrogen bonds in mutant proteins compared to wild-type revealed that these mutations might alter the protein structure and stability of the corresponding protein. Hence, the results from the analyses guide the exploration of the mechanism by which these missense SNPs of the MCM6 gene alter the structural integrity and functional properties of the protein, which could guide the identification of ways to minimize the harmful effects of these mutations in humans.

Minichromosome Maintenance Complex Component 5 Modified by Mettl3 Inhibits the Proliferation of Liver Cancer by Regulating PI3K/AKT/mTOR Axis

Hepatocellular carcinoma (HCC) is a major contributor to global cancer-related deaths. The chromatin binding protein MCM5, part of the MCM family, plays a crucial role in regulating DNA replication, a key driver of cancer. Database analysis revealed elevated MCM5 levels in HCC, associated with shorter patient survival. Silencing MCM5 impedes liver cancer cell proliferation by halting the cell cycle at G1 phase. In vivo experiments confirm this effect, demonstrating that MCM5 knockdown suppresses HCC growth. Mechanistic studies unveil MCM5′s impact on HCC development via the PI3K/AKT/mTOR signaling pathway. Reversing liver cancer growth is possible by adding AKT agonist SC79. Additionally, inhibiting mettl3 with stm2457 downregulates MCM5, further suppressing liver cancer growth. In summary, high MCM5 expression in liver cancer correlates with poor prognosis and drives disease progression. Targeting MCM5 with mettl3 inhibitors presents a promising therapeutic strategy for HCC.

Abstract 4530: Nuclear export inhibitor cooperates with PARP inhibitor to suppress the growth of metastatic castration resistant prostate cancer in vivo

Abstract Background: Aberrant nuclear protein transport, often observed in cancer, causes mislocalization-dependent inactivation of critical cellular proteins. Earlier we showed that overexpression of nuclear export protein exportin 1 (XPO1) is linked to higher grade and Gleason score in metastatic castration resistant prostate cancer (mCRPC). The network topology computational approach (NTCP) determined a higher synthetic lethal score between XPO1 and poly (ADP-ribose) polymerase (PARP1). We showed that selective inhibitor of nuclear export (SINE) could synergized with PARP inhibitors in mCRPC cell lines. Here we evaluated the efficacy of SINE and PARP inhibitor (PARPi) combination in cell line derived as well as patient derived xenograft (CDX and PDX) models and deciphered the mechanism of synergy. Methods: For CDX model, the 22rv1 mCRPC cells were grown as subcutaneous xenografts in ICR-SCID male mice. For PDX model, tissue was collected from Champions Oncology (CTG-3581) and grown subcutaneously in CEIA/NOG male mice. SINE dosed orally at 10-15 mg/kg twice a week and PARPi dosed orally at 50 mg/kg daily. For in vitro mechanistic study, 22rv1 cells were subjected to RNAseq and proteomic analysis after treatment. Data analysis was performed using iPathwayGuide (advaitabio.com). Results: The CDX and PDX showed pronounced anti-cancer efficacy by this combination compared to single agents without any significant weight loss. Survival analysis in CDX model demonstrated enhanced benefits to the mice of combination group. Immunohistochemistry (IHC) revealed apoptotic cell death in the combination group which is evident from cleaved caspase 3 staining and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL). To decipher the mechanism of synergy we performed transcriptomic (RNAseq) and proteomic analysis in vitro. We observed downregulation of DNA replication related gene minichromosome maintenance complex component 6 (MCM6) and cell division cycle 6 (CDC6) in the combination treatment. Gene set enrichment analysis (GSEA) also showed low enrichment scores for DNA replication. Proteomic analysis revealed a down regulation of DNA replication modulators such as HMGB2 and DNAJC9 which work on DNA coiling and histone respectively. Conclusions: Taken together, this study revealed the therapeutic potential of SINE-PARPi combination via targeting DNA damage response pathway in mCRPC. Further evaluation of molecular synergy in the xenograft models are underway through RNA interference (RNAi) technique and Digital Spatial Profiling (DSP). Citation Format: Md Hafiz Uddin, Amro Aboukameel, Husain Y. Khan, Sahar F. Bannoura, Frank Cackowski, Rafic Beydoun, Gregory Dyson, Seongho Kim, Julie Boerner, Vinod Shidham, Ramzi M. Mohammad, Boris C. Pasche, Asfar S. Azmi, Elisabeth I. Heath. Nuclear export inhibitor cooperates with PARP inhibitor to suppress the growth of metastatic castration resistant prostate cancer in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4530.

Multi-omics pan-cancer analyses identify MCM4 as a promising prognostic and diagnostic biomarker

Minichromosome Maintenance Complex Component 4 (MCM4) is a vital component of the mini-chromosome maintenance complex family, crucial for initiating the replication of eukaryotic genomes. Recently, there has been a growing interest in investigating the significance of MCM4 in different types of cancer. Despite the existing research on this topic, a comprehensive analysis of MCM4 across various cancer types has been lacking. This study aims to bridge this knowledge gap by presenting a thorough pan-cancer analysis of MCM4, shedding light on its functional implications and potential clinical applications. The study utilized multi-omics samples from various databases. Bioinformatic tools were employed to explore the expression profiles, genetic alterations, phosphorylation states, immune cell infiltration patterns, immune subtypes, functional enrichment, disease prognosis, as well as the diagnostic potential of MCM4 and its responsiveness to drugs in a range of cancers. Our research demonstrates that MCM4 is closely associated with the oncogenesis, prognosis and diagnosis of various tumors and proposes that MCM4 may function as a potential biomarker in pan-cancer, providing a deeper understanding of its potential role in cancer development and treatment.

Floralozone regulates MiR-7a-5p expression through AMPKα2 activation to improve cognitive dysfunction in vascular dementia

BackgroundThe pathogenesis of vascular dementia (VD) is complex, and currently, no effective treatments have been recommended. Floralozone is a colorless liquid first discovered in Lagotis Gaertn. Recently, its medicinal value has been increasingly recognized. Our previous study has demonstrated that Floralozone can improve cognitive dysfunction in rats with VD by regulating the transient receptor potential melastatin 2 (TRPM2) and N-methyl-D-aspartate receptor (NMDAR) signaling pathways. However, the mechanism by which Floralozone regulates TRPM2 and NMDAR to improve VD remains unclear. AMP-activated protein kinase (AMPK) is an energy regulator in vivo; however, its role of AMPK activation in stroke remains controversial. MiR-7a-5p has been identified to be closely related to neuronal function. PurposeTo explore whether Floralozone can regulate the miR-7a-5p level in vivo through AMPKα2 activation, affect the TRPM2 and NR2B expression levels, and improve VD symptoms. MethodsThe VD model was established by a modified bilateral occlusion of the common carotid arteries (2-VO) of Sprague-Dawley (SD) rats and AMPKα2 KO transgenic (AMPKα2−/−) mice. Primary hippocampal neurons were modeled using oxygen and glucose deprivation (OGD). Morris water maze (MWM) test, hematoxylin-eosin staining (HE staining), and TUNEL staining were used to investigate the effects of Floralozone on behavior and hippocampal morphology in rats. Minichromosome maintenance complex component 2(MCM2) positive cells were used to investigate the effect of Floralozone on neurogenesis. Immunofluorescence staining, qRT-PCR, and western blot analysis were used to investigate the effect of Floralozone on the expression levels of AMPKα2, miR-7a-5p, TRPM2, and NR2B. ResultsThe SD rat experiment revealed that Floralozone improved spatial learning and memory, improved the morphology and structure of hippocampal neurons, reduced apoptosis of hippocampal neurons and promoted neurogenesis in VD rats. Floralozone could increase the miR-7a-5p expression level, activate AMPKα2 and NR2B expressions, and inhibit TRPM2 expression in hippocampal neurons of VD rats. The AMPKα2 KO transgenic (AMPKα2−/−) mice experiment demonstrated that Floralozone could regulate miR-7a-5p, TRPM2, and NR2B expression levels through AMPKα2 activation. The cell experiment revealed that the TRPM2 and NR2B expression levels were regulated by miR-7a-5p, whereas the AMPKα2 expression level was not. ConclusionFloralozone could regulate miR-7a-5p expression level by activating the protein expression of AMPKα2, control the protein expression of TRPM2 and NR2B, improve the morphology and structure of hippocampus neurons, reduce the apoptosis of hippocampus neurons, promote neurogenesis and improve the cognitive dysfunction.

CUL4A Ubiquitin Ligase Is an Independent Predictor of Overall Survival in Pancreatic Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy with dismal prognosis. Genomic instability due to defects in cell-cycle regulation/mitosis or deficient DNA-damage repair is a major driver of PDAC progression with clinical relevance. Deregulation of licensing of DNA replication leads to DNA damage and genomic instability, predisposing cells to malignant transformation. While overexpression of DNA replication-licensing factors has been reported in several human cancer types, their role in PDAC remains largely unknown. We aimed here to examine the expression and prognostic significance of the DNA replication-licensing factors chromatin licensing and DNA replication factor 1 (CDT1), cell-division cycle 6 (CDC6), minichromosome maintenance complex component 7 (MCM7) and also of the ubiquitin ligase regulator of CDT1, cullin 4A (CUL4A), in PDAC. Expression levels of CUL4, CDT1, CDC6 and MCM7 were evaluated by immunohistochemistry in 76 formalin-fixed paraffin-embedded specimens of PDAC patients in relation to DNA-damage response marker H2AX, clinicopathological parameters and survival. We also conducted bioinformatics analysis of data from online available databases to corroborate our findings. CUL4A and DNA replication-licensing factors were overexpressed in patients with PDAC and expression of CDT1 positively correlated with H2AX. Expression of CUL4A and CDT1 positively correlated with lymph node metastasis. Importantly, elevated CUL4A expression was associated with reduced overall survival and was an independent indicator of poor prognosis on multivariate analysis. Our findings implicate CUL4A, CDT1, CDC6 and MCM7 in PDAC progression and identify CUL4A as an independent prognostic factor for this disease.

MCM6 Inhibits Decidualization via Cross-Talking with ERK Pathway in Human Endometrial Stromal Cells.

Decidualization plays an important role in the implantation of the embryo, but the molecular action implicated in this process is not completely known. Herein, we found that, compared with the proliferative endometrial tissues, the expression of minichromosome maintenance complex component 6 (MCM6) was markedly decreased in the secretory endometrial tissues. To verify the function of MCM6 in decidualization, in vitro decidualization model was constructed by treating human endometrial stromal cells (HESCs) with estrogen (E2) and progesterone (P4). Consistently, MCM6 level was downregulated in E2P4-treated HESCs. Administration of E2P4 accumulated HESCs in G1 cell cycle phase, leading to cell growth suppression. Ectopic expression of MCM6 promoted the transition of G1/S and restored the proliferation of HESCs that were inhibited by E2P4. MCM6 overexpression led to aberrant activation of extracellular signal-regulated kinase (ERK) and treatment with ERK agonist Ro 67-7476 restored MCM6 expression and cell proliferation inhibited by E2P4. Our data suggested that MCM6/ERK feedback loop plays a negative role in E2P4-induced decidualization and implies that MCM6 may be a promising target for meliorating uterine receptivity.

MCM2 is involved in subtyping and tamoxifen resistance of ERα-positive breast cancer by acting as the downstream factor of ERα.

Tamoxifen (TAM) resistance is finally developed in over 40% of patients with estrogen receptor α-positive breast cancer (ERα+ -BC), documenting that discovering new molecular subtype is needed to confer perception to the heterogeneity of ERα+ -BC. We obtained representative gene sets subtyping ERα+ -BC using gene set variation analysis (GSVA), non-negative matrix factorization (NMF), and COX regression methods on the basis of METABRIC, TCGA, and GEO databases. Furthermore, the risk score of ERα+ -BC subtyping was established using least absolute shrinkage and selection operator (LASSO) regression on the basis of genes in the representative gene sets, thereby generating the two subtypes of ERα+ -BC. We further found that minichromosome maintenance complex component 2 (MCM2) functioned as the hub gene subtyping ERα+ -BC using GO, KEGG, and MCODE. MCM2 expression was capable for specifically predicting 1-year overall survival (OS) of ERα+ -BC and correlated with T stage, AJCC stage, and tamoxifen (TAM) sensitivity of ERα+ -BC. The downregulation of MCM2 expression inhibited proliferation, migration, and invasion of TAM-resistant cells and promoted G0/G1 arrest. Altogether, tamoxifen resistance entails that MCM2 is a hub gene subtyping ERα+ -BC, providing a novel dimension for discovering a potential target of TAM-resistant BC.

Decreased NSD2 impairs stromal cell proliferation in human endometrium via reprogramming H3K36me2.

The proliferation of the endometrium is regulated by histone methylation. This study shows that decreased NSD2 impairs proliferative-phase endometrial stromal cell proliferation in patients with recurrent implantation failure via epigenetic reprogramming of H3K36me2 methylation on the promoter region of MCM7. Recurrent implantation failure (RIF) is a formidable challenge in assisted reproductive technology because of its unclear molecular mechanism. Impaired human endometrial stromal cell (HESC) proliferation disrupts the rhythm of the menstrual cycle, resulting in devastating disorders between the embryo and the endometrium. The molecular function of histone methylation enzymes in modulating HESC proliferation remains largely uncharacterized. Herein, we found that the levels of histone methyltransferase nuclear receptor binding SET domain protein 2 (NSD2) and the dimethylation of lysine 36 on histone H3 are decreased significantly in the proliferative-phase endometrium of patients with RIF. Knockdown of NSD2 in an HESC cell line markedly impaired cell proliferation and globally reduced H3K36me2 binding to chromatin, leading to altered expression of many genes. Transcriptomic analyses revealed that cell cycle-related gene sets were downregulated in the endometrium of patients with RIF and in NSD2‑knockdown HESCs. Furthermore, RNA-sequencing and CUT&Tag sequencing analysis suggested that NSD2 knockdown reduced the binding of H3K36me2 to the promoter region of cell cycle marker gene MCM7 (encoding minichromosome maintenance complex component 7) and downregulated its expression. The interaction of H3K36me2 with the MCM7 promoter was verified using chromatin immunoprecipitation-quantitative real-time PCR. Our results demonstrated a unifying epigenome-scale mechanism by which decreased NSD2 impairs endometrial stromal cell proliferation in the proliferative-phase endometrium of patients with RIF.

Activation of Glycolysis by MCM10 Increases Stemness and Paclitaxel Resistance in Gastric Cancer Cells.

Chemotherapy is an essential avenue for curing malignancies; however, tumor cells acquire resistance to chemotherapeutic agents, eventually leading to chemotherapy failure. At present, paclitaxel (PTX) resistance seriously hinders the therapeutic efficacy of gastric cancer (GC). Investigating the molecular mechanism of PTX resistance in GC is critical. This study attempted to delineate the impact of MCM10 on GC resistance to PTX and its mechanism in GC. The expression of minichromosome maintenance complex component 10 (MCM10) in GC tissues, its enrichment pathways, and its correlation with glycolysis marker genes and stemness index (mRNAsi) were analyzed in a bioinformatics effort. Real-time quantitative polymerase chain reaction was used to assay the expression of MCM10 in cells. Cell counting kit-8 (CCK-8) was used to analyze cell viability and calculate the 50% inhibitor concentration (IC50) value. Western blot was used to measure the expression of MCM10, Hexokinase 2 (HK2) and stemness-related factors in cells. Sphere-forming assay was performed to study cell sphere-forming ability. Seahorse XF 96 was utilized to measure cell extracellular acidification and oxygen consumption rates. The content of glycolysisrelated products was tested with corresponding kits. MCM10 was significantly upregulated in GC and enriched in the glycolysis pathway, and it was positively correlated with both glycolysis-related genes and stemness index. High expression of MCM10 increased sphere-forming ability of drug-resistant cells and GC resistance to PTX. The stimulation of PTX resistance and drug-resistant cell stemness in GC by high MCM10 expression was mediated by the glycolysis pathway. MCM10 was upregulated in GC and drove stemness and PTX resistance in GC cells by activating glycolysis. These findings generated new insights into the development of PTX resistance in GC, implicating that targeting MCM10 may be a novel approach to improve GC sensitivity to PTX chemotherapy.

Coiled-coil domain-containing 154 promotes colorectal cancer proliferation and metastasis via interacting with minichromosome maintenance complex component 2

Coiled-Coil Domain-Containing (CCDC) is a large class of structural proteins containing left-handed supercoiled structure. The clinical value and the functional implication of CCDC in colorectal cancer (CRC) remain unknown. Based on the genetic, transcriptional, and clinical data from The Cancer Genome Atlas, five of thirty-six CCDC proteins were differentially expressed in the CRC and associated with the survival of patients with CRC. A CCDC-score model was established to evaluate the prognosis of patients. The potential function of Coiled-Coil Domain-Containing 154 (CCDC154) was investigated using bioinformatical methods, which unveiled that high expression of CCDC154 indicates poor survival for patients with CRC and correlates with low infiltration of CD8+ T cells and high infiltration of neutrophils, indicating that CCDC154 enhances tumor growth and metastasis. CCDC154 interacts with Minichromosome Maintenance Complex Component 2 (MCM2) protein and promotes malignant phenotype via MCM2. We validated the expression level and survival prediction value of CCDC154 in clinical samples, and analyzed its co-expression of MCM2, Ki-67 and p53. This work discloses the role of CCDC in clinical setting and CCDC154 functions in CRC.

M6A demethylase FTO stabilizes LINK-A to exert oncogenic roles via MCM3-mediated cell-cycle progression and HIF-1α activation

RNA N6-methyladenosine (m6A) modification is implicated in cancer progression, yet its role in regulating long noncoding RNAs during cancer progression remains unclear. Here, we report that the m6A demethylase fat mass and obesity-associated protein (FTO) stabilizes long intergenic noncoding RNA for kinase activation (LINK-A) to promote cell proliferation and chemoresistance in esophageal squamous cell carcinoma (ESCC). Mechanistically, LINK-A promotes the interaction between minichromosome maintenance complex component 3 (MCM3) and cyclin-dependent kinase 1 (CDK1), increasing MCM3 phosphorylation. This phosphorylation facilitates the loading of the MCM complex onto chromatin, which promotes cell-cycle progression and subsequent cell proliferation. Moreover, LINK-A disrupts the interaction between MCM3 and hypoxia-inducible factor 1α (HIF-1α), abrogating MCM3-mediated HIF-1α transcriptional repression and promoting glycolysis and chemoresistance. These results elucidate the mechanism by which FTO-stabilized LINK-A plays oncogenic roles and identify the FTO/LINK-A/MCM3/HIF-1α axis as a promising therapeutic target for ESCC.

First Report of Peanut Black Pod Rot Caused by Berkeleyomyces rouxiae in China.

Peanut (Arachis hypogaea L.) is an important oilseed and cash crop cultivated in over 100 countries worldwide. The major producers are China, India and USA (Ding et al. 2022). In September 2022, peanut pods exhibiting black necrotic symptoms on the shell surface were observed in Puyang, Henan Province, China. These black spots often merged to form larger necrotic spots on the shell. Disease incidence was 100% in susceptible varieties. Symptomatic shell pieces were surface sterilized with 75% ethanol for 3 min, rinsed three times with sterile water, and then transferred onto PDA medium supplemented with 25 µg/ml chloramphenicol (Long et al. 2022). Isolation frequency of a fungus with similar-appearing colonies from symptomatic pods was 81.7%. A pure culture of a representative isolate, PYHB, was obtained through single-sporing and maintained on PDA plates at 25℃ in darkness. The colony initially appeared white but turned black within 2 days. The isolate produced dark brown, unicellular chlamydospores, which were arranged in club-shaped chains consisting of two to seven cells. The size of the unicellular chlamydospores varied from 3.34 to 15.27 µm (average:6.81, n = 100) in length and 8.30 to 15.51 µm (average:11.29, n = 100) in width. The endoconidia were hyaline and cylindrical, measuring 7.91-22.94 × 1.69-4.81 µm (average: 12.16 × 3.13, n = 100). Based on morphological characteristics, the isolate was tentatively identified as a Berkeleyomyces sp. (Nel et al. 2018; Long et al. 2022). The ITS region of r-DNA, the ribosomal large subunit (LSU), the minichromosome maintenance complex component 7 (MCM7), and the 60S ribosomal protein RPL10 (60S) genes were amplified using ITS1/ITS4, LR0R/LR5, rouxMCM7-F/rouxMCM7-R and roux60s-F/roux60s-R primers, respectively (White et al. 1990; Vilgalys and Hester 1990; Nakane and Usami 2020). The sequences were deposited in GenBank (ITS: OR053803; LSU: OR053818; MCM7: OR058549; 60S: OR060656). Through BLASTn analysis of the NCBI GenBank database, the generated ITS and LSU sequences showed 100% identity to Berkeleyomyces rouxiae (GenBank MF952418.1 and MF948662.1, respectively) and B. basicola (GenBank MT221585.1 and MH868639.1, respectively). Importantly, the MCM7 and 60S sequences were 100% identical to B. rouxiae (GenBank MF967114.1 and MF967077.1, respectively). Phylogenetic analysis combining ITS, LSU, MCM7, and 60S sequences showed that the isolate PYHB clustered with B. rouxiae. To evaluate pathogenicity, surface-sterilized healthy peanut pods (n = 90) were immersed in a 1×106 spore/ml conidial suspension obtained from isolate PYHB for 5 min and placed in Petri dishes containing moistened cotton at 25°C for 10 days. Pods (n = 90) inoculated with sterile water served as controls. Inoculated pods displayed black necrosis 10 days after inoculation (dai), whereas no symptoms were observed on the control pods at 21 dai. The reisolated pathogen was shown to be identical to the original inoculum through morphological and phylogenetic analysis. Black root rot is a fungal disease caused by Berkeleyomyces spp. (syn. Thielaviopsis spp.) and affects various crops and ornamentals, such as cotton, tobacco, carrot, holly, and pansy (Rahnama et al. 2022). The causal agents B. rouxiae and B. basicola have similar morphological characteristics but can be differentiated through molecular characterization (Nel et al. 2018). To our knowledge, this is the first report of black pod rot in peanut caused by B. rouxiae in China. The finding from this study will contribute to the development of monitoring and management strategies to combat this destructive disease in peanut cultivation.

MCM5 is an oncogene of colon adenocarcinoma and promotes progression through cell cycle control

Many patients with colon adenocarcinoma (COAD) are diagnosed at an advanced stage, and the molecular mechanism of COAD progression is intricate and controversial. Therefore, there is an urgent need to identify more novel prognosis biomarkers for COAD and elucidate the molecular mechanism of this disease. The present study aimed to screen out key genes correlated with COAD prognosis. In this study, a key module was identified and four hub genes (MCM5 (encoding minichromosome maintenance complex component 5), NOLC1 (encoding nucleolar and coiled-body phosphoprotein 1), MYC (encoding MYC proto-oncogene, BHLH transcription factor), and CDK4 (encoding cyclin dependent kinase 4)) were selected that correlated with COAD prognosis, based on the GSE9348 dataset in Gene Expression Omnibus database. Gene ontology enrichment and Kyoto Encyclopedia of Genes and Genomes pathway analysis indicated that MCM5 correlated with the cell cycle. Furthermore, MCM5 expression was upregulated in tumor tissues of patients with COAD compared with that in adjacent tissues, based on various databases, including The Cancer Genome Atlas, the Clinical Proteomic Tumor Analysis Consortium database, and the Human Protein Atlas database. Small interfering RNA-mediated knockdown of MCM5 inhibited the cell cycle and migration of colorectal cancer cells in vitro. And western blotting results indicated that factors correlated with cell cycle (CDK2/6, Cyclin D3, P21) were downregulated after knockdown of MCM5 in vitro. Besides, downregulation of MCM5 was demonstrated to inhibit lung metastasis of COAD in nude mice model. In conclusion, MCM5 is an oncogene of COAD that promotes COAD progression via cell cycle control.

Exploration of the potential common pathogenic mechanisms in COVID-19 and silicosis by using bioinformatics and system biology

Silicosis is an occupational lung disease that is common worldwide. In recent years, coronavirus disease 2019 (COVID-19) has provided daunting challenges to public healthcare systems globally. Although multiple studies have shown a close link between COVID-19 and other respiratory diseases, the inter-relational mechanisms between COVID-19 and silicosis remain unclear. This study aimed to explore the shared molecular mechanisms and drug targets of COVID-19 and silicosis. Gene expression profiling identified four modules that were most closely associated with both diseases. Furthermore, we performed functional analysis and constructed a protein–protein interaction network. Seven hub genes (budding uninhibited by benzimidazoles 1 [BUB1], protein regulator of cytokinesis 1 [PRC1], kinesin family member C1 [KIFC1], ribonucleotide reductase regulatory subunit M2 [RRM2], cyclin-dependent kinase inhibitor 3 [CDKN3], Cyclin B2 [CCNB2], and minichromosome maintenance complex component 6 [MCM6]) were involved in the interaction between COVID-19 and silicosis. We investigated how diverse microRNAs and transcription factors regulate these seven genes. Subsequently, the correlation between the hub genes and infiltrating immune cells was explored. Further in-depth analyses were performed based on single-cell transcriptomic data from COVID-19, and the expression of hub-shared genes was characterized and located in multiple cell clusters. Finally, molecular docking results reveal small molecular compounds that may improve COVID-19 and silicosis. The current study reveals the common pathogenesis of COVID-19 and silicosis, which may provide a novel reference for further research.