m6A modification of CDC5L promotes lung adenocarcinoma progression through transcriptionally regulating WNT7B expression.

Cell division cycle 5-like (CDC5L) protein is implicated in the development of various cancers. However, its role in the progression of lung adenocarcinoma (LUAD) remains uncertain. Our findings revealed frequent upregulation of CDC5L in LUAD, which correlated with poorer overall survival rates and advanced clinical stages. In vitro experiments demonstrated that CDC5L overexpression stimulated the proliferation, migration, and invasion of LUAD cells, whereas CDC5L knockdown exerted suppressive effects on these cellular processes. Furthermore, silencing CDC5L significantly inhibited tumor growth and metastasis in a xenograft mouse model. Mechanistically, CDC5L activates the Wnt/ß-catenin signaling pathway by transcriptionally regulating WNT7B, thereby promoting LUAD progression. Besides, METTL14-mediated m6A modification contributed to CDC5L upregulation in an IGF2BP2-dependent manner. Collectively, our study uncovers a novel molecular mechanism by which the m6A-induced CDC5L functions as an oncogene in LUAD by activating the Wnt/ß-catenin pathway through transcriptional regulation of WNT7B, suggesting that CDC5L may serve as a promising prognostic marker and therapeutic target for LUAD.

FOXA1 prolongs S phase and promotes cancer progression in non-small cell lung cancer through upregulation of CDC5L and activation of the ERK1/2 and JAK2 pathways.

Non-small cell lung cancer (NSCLC) causes high mortality worldwide; however, its molecular pathways have not been fully investigated. The relationship between FOXA1 and CDC5L as well as their roles in NSCLC have not been comprehensively studied. Clinical tissues were collected from 78 NSCLC patients for clinical studies. The BEAS-2B human normal lung epithelial cell line and the A549, Calu-3, H526 and H2170 human NSCLC cell lines were used for in vitro studies. sh-FOXA1 and oe-CDC5L constructs were used to generate knockdown and overexpression models, respectively. The CCK-8 assay was used to analyze cell viability. The cell cycle and apoptosis were evaluated by flow cytometry analysis. The relationship between FOXA1 and CDC5L was demonstrated using dual-luciferase and ChIP assays. Gene levels were examined via immunohistochemistry, qRT-PCR and western blot analysis. FOXA1 levels were increased in NSCLC clinical tissues and cell lines. Depletion of FOXA1 increased the apoptosis rate and increased the proportion of cells in G2/M phase. In addition, we demonstrated that FOXA1 was directly bound to the promoter of CDC5L and that depletion of FOXA1 inhibited CDC5L expression. Overexpression of CDC5L induced ERK1/2 phosphorylation, induced JAK2 phosphorylation, inhibited cell apoptosis, prolonged S phase, and significantly reversed the effects of FOXA1 knockdown on the progression of NSCLC. The present study demonstrated that FOXA1 prolongs S phase and promotes NSCLC progression through upregulation of CDC5L and activation of the ERK1/2 and JAK2 pathways.

Knockdown of LncRNA DICER1-AS1 arrests the cell cycle, inhibits cell proliferation, and induces cell apoptosis by regulating CDC5L nuclear transfer in osteosarcoma.

BACKGROUND: DICER1-AS1 is reported to promote the progression and disturb the cell cycle in osteosarcoma; however, its mechanism has rarely been studied. MATERIALS AND METHODS: DICER1-AS1 expression levels were evaluated by qPCR and fluorescence in situ hybridization (FISH). The total, nuclear, and cytosolic levels of CDC5L were measured by western blotting and immunofluorescence (IF). Cell proliferation, apoptosis, and cell cycle analyses were conducted using the colony formation, CCK-8 assay, terminal transferase-mediated UTP nick end-labeling kit (TUNEL) assay, and flow cytometry. Levels of cell proliferation-, cell cycle-, and cell apoptosis-related proteins were determined by western blotting. RNA immunoprecipitation (RIP) and RNA pull-down assays were conducted to evaluate the relationship between DICER1-AS1 and CDC5L. RESULTS: LncRNA DICER1-AS1 was highly expressed in samples of osteosarcoma tissue and in osteosarcoma cell lines. DICER1-AS1 knockdown inhibited cell proliferation, promoted cell apoptosis, and disturbed the cell cycle. Moreover, DICER1-AS1 was found to bind with CDC5L, and knockdown of DICER-AS1 inhibited the nuclear transfer of CDC5L. DICER1-AS1 knockdown also reversed the effects of CDC5L overexpression on cell proliferation, apoptosis, and the cell cycle. Moreover, CDC5L inhibition suppressed cell proliferation, promoted cell apoptosis, and disturbed the cell cycle, and those effects were further enhanced by DICER1-AS1 knockdown. Finally, DICER1-AS knockdown inhibited tumor growth and proliferation, and promoted cell apoptosis in vivo. CONCLUSION: LncRNA DICER1-AS1 knockdown inhibits the nuclear transfer of CDC5L protein, arrests the cell cycle, and induces apoptosis to suppress the development of osteosarcoma. Our results suggest a novel target (DICER1-AS1) for treatment of osteosarcoma.

[Bioinformatics-based identification of key genes CDC5L and related pathways in osteosarcoma and Ewing's sarcoma].

OBJECTIVE: Osteosarcoma(OS) and Ewing's sarcoma (EWS) are the two most common primary malignant bone tumors in children. The aim of the study was to identify key genes in OS and EWS and investigate their potential pathways. METHODS: Expression profiling (GSE16088 and GSE45544) were obtained from GEO DataSets. Differentially expressed genes were identified using GEO2R and key genes involved in the occurrence of both OS and EWS were selected using venn diagram. Gene ontology and pathway enrichment analyses were performed for the ensembl. Protein-protein interaction (PPI) networks were established by STRING. Further, UCSC was used to predict the transcription factors of the cell division cycke 5-like(CDC5L) gene, and GEPIA was used to analyze the correlation between the transcription factors and the CDC5L gene. RESULTS: The results showed that CDC5L gene was the key gene involved in the pathogenesis of OS and EWS. The gene is mainly involved in mitosis, and is related to RNA metabolism, processing of capped intron-containing pre-mRNA, mRNA and pre-mRNA splicing. CONCLUSION: CDC5L, as a key gene, plays a role in development of OS and EWS, which may be reliable targets for diagnosis and treatment of these primary malignant tumors.

Protein network analysis to prioritize key genes in amyotrophic lateral sclerosis.

Amyotrophic Lateral Sclerosis (ALS) is a fatal disease, progressive nature characterizes by loss of both upper and lower motor neuron functions. One of the major challenge is to understand the mechanism of ALS multifactorial nature. We aimed to explore some key genes related to ALS through bioinformatics methods for its therapeutic intervention. Here, we applied a systems biology approach involving experimentally validated 148 ALS-associated proteins and construct ALS protein-protein interaction network (ALS-PPIN). The network was further statistically analysed and identified bottleneck-hubs. The network is also subjected to identify modules which could have similar functions. The interaction between the modules and bottleneck-hubs provides the functional regulatory role of the ALS mechanism. The ALS-PPIN demonstrated a hierarchical scale-free nature. We identified 17 bottleneck-hubs, in which CDC5L, SNW1, TP53, SOD1, and VCP were the high degree nodes (hubs) in ALS-PPIN. CDC5L was found to control highly cluster modules and play a vital role in the stability of the overall network followed by SNW1, TP53, SOD1, and VCP. HSPA5 and HSPA8 acting as a common connector for CDC5L and TP53 bottleneck-hubs. The functional and disease association analysis showed ALS has a strong correlation with mRNA processing, protein deubiquitination, and neoplasms, nervous system, immune system disease classes. In the future, biochemical investigation of the observed bottleneck-hubs and their interacting partners could provide a further understanding of their role in the pathophysiology of ALS.

Downregulation of DEAD-box helicase 21 (DDX21) inhibits proliferation, cell cycle, and tumor growth in colorectal cancer via targeting cell division cycle 5-like (CDC5L).

Identification of novel anti-tumor target is crucial for cancer diagnosis, prognosis, and therapeutic strategy. The study aimed to explore the roles and interaction of DEAD-box helicase 21 (DDX21) and cell division cycle 5-like (CDC5L) in colorectal cancer (CRC) progression. Levels of DDX21 and CDC5L were detected in colorectal cancer cell lines by RT-qPCR and Western blot assay. The role of DDX21 and CDC5L on the cell proliferation, cell cycle and tumor growth were evaluated both in vitro and in vivo. The interaction of DDX21 and CDC5L was predicted by The STRING publicly available data and verified by immunoprecipitation. The results showed that DDX21 was dramatically upregulated in colorectal cancer cells. In vivo and in vitro experiments revealed that downregulation of DDX21 suppressed colorectal cancer cell proliferation, colony formation, cell cycle development, and tumor growth, while overexpression of CDC5L reversed the suppressive effects of DDX21 silencing. Furthermore, DDX21 interacted with CDC5L to exert the tumor-promoting effects in CRC. In summary, the data indicate a novel role for DDX21/CDC5L in the development of CRC, which enrich the therapeutic strategy for CRC.

Cell Division Cycle 5-Like Regulates Metaphase-to-Anaphase Transition in Meiotic Oocyte.

The quality of oocytes is a vital factor for embryo development. Meiotic progression through metaphase I usually takes a relatively long time to ensure correct chromosome separation, a process that is critical for determining oocyte quality. Here, we report that cell division cycle 5-like (Cdc5L) plays a critical role in regulating metaphase-to-anaphase I transition during mouse oocyte meiotic maturation. Knockdown of Cdc5L by small interfering RNA injection did not affect spindle assembly but caused metaphase I arrest and subsequent reduced first polar body extrusion due to insufficient anaphase-promoting complex/cyclosome activity. We further showed that Cdc5L could also directly interact with securin, and Cdc5L knockdown led to a continuous high expression level of securin, causing severely compromised meiotic progression. The metaphase-to-anaphase I arrest caused by Cdc5L knockdown could be rescued by knockdown of endogenous securin. In summary, we reveal a novel role for Cdc5L in regulating mouse oocyte meiotic progression by interacting with securin.

CDC5L promotes early chondrocyte differentiation and proliferation by modulating pre-mRNA splicing of SOX9, COL2A1, and WEE1.

Ossification of the posterior longitudinal ligament (OPLL) of the spine is a common pathological condition that causes intractable myelopathy and radiculopathy, mainly the result of an endochondral ossification-like process. Our previous genome-wide association study identified six susceptibility loci for OPLL, including the cell division cycle 5-like (CDC5L) gene region. Here, we found CDC5L to be expressed in type II collagen-producing chondrocyte-like fibroblasts in human OPLL specimens, as well as in differentiating ATDC5 chondrocytes. Cdc5l siRNA transfection in murine chondrocytes decreased the expression of the early chondrogenic genes Sox9 and Col2a1, diminished the cartilage matrix production, and enhanced the expression of parathyroid-hormone-related protein (a resting chondrocyte marker). We also showed that Cdc5l shRNA suppressed the growth of cultured murine embryonal metatarsal cartilage rudiments and that Cdc5l knockdown suppressed the growth of ATDC5 cells. Fluorescence-activated cell sorting analysis revealed that the G2/M cell cycle transition was blocked; our data showed that Cdc5l siRNA transfection enhanced expression of Wee1, an inhibitor of the G2/M transition. Cdc5l siRNA also decreased the pre-mRNA splicing efficiency of Sox9 and Col2a1 genes in both ATDC5 cells and primary chondrocytes; conversely, loss of Cdc5l resulted in enhanced splicing of Wee1 pre-mRNA. Finally, an RNA-binding protein immunoprecipitation assay revealed that Cdc5l bound directly to these target gene transcripts. Overall, we conclude that Cdc5l promotes both early chondrogenesis and cartilage growth and may play a role in the etiology of OPLL, at least in part by fine-tuning the pre-mRNA splicing of chondrogenic genes and Wee1, thus initiating the endochondral ossification process.

Long Noncoding RNA LINC00963 Promotes CDC5L-Mediated Malignant Progression in Gastric Cancer.

BACKGROUND: Gastric cancer (GC) is a common cancer with high incidence and mortality worldwide. In recent years, accumulating evidence has shown that long noncoding RNAs (lncRNAs) exert critical roles in the development and progression of cancer by acting as a tumor initiator or suppressor. LINC00963 is a newly reported lncRNA related to cancer, and its role in GC remains unclear. MATERIALS AND METHODS: The expression levels of LINC00963, miR-612, and cell division cycle 5-like protein (CDC5L) were measured using quantitative real-time PCR or Western blot. The biological functions of LINC00963, miR-612, and CDC5L in GC cells were analyzed by transwell and proliferation experiments. The expression of CDC5L in patients with GC was evaluated using the Oncomine database. Bone marrow-derived dendritic cells (DCs) were derived from C57BL/6 mice. RESULTS: LINC00963 expression was higher in GC tissues than in adjacent normal tissues. Similar results were found in GC cell lines and normal human gastric epithelial cells. Upregulation of LINC00963 was related to the poor prognosis of patients with GC. Knockdown of LINC00963 inhibited the proliferation, invasion, and metastasis but promoted the apoptosis of GC cells. Furthermore, silencing of LINC00963 in GC cells significantly suppressed the tumor growth of GC. Bioinformatics analysis indicated that LINC00963 could target miR-612 by functioning as a competing endogenous RNA. The expression of miR-612 decreased in GC tissues and cell lines. Meanwhile, LINC00963 expression was negatively associated with miR-612. CDC5L was a direct target of miR-612. miR-612 suppressed the expression of CDC5L in GC tissues and cells. Moreover, LINC00963 inhibited the differentiation and maturation of DCs by regulating miR-612 expression in DCs. CONCLUSION: LINC00963 promoted the progression of GC by competitively binding to miR-612 to regulate the expression of CDC5L and mediated DC-related anti-tumor immune response. Thus, targeting LINC00963 may be a promising therapeutic strategy for GC.

Prognostic value of aberrantly expressed methylation genes in human hepatocellular carcinoma.

OBJECTIVES: To identify the prognostic value of aberrantly methylated differentially expressed genes (DEGs) in hepatocellular carcinoma (HCC) and to explore the underlying mechanisms of tumorigenesis. METHODS: Gene expression profiles (GSE65372 and GSE37988) were analyzed using GEO2R to obtain aberrantly methylated DEGs. Functional enrichment analysis of screened genes was performed by the Database for Annotation, Visualization, and Integrated Discovery (DAVID). Cytoscape software was used to analyze the PPI network and to select hub genes. Transcriptional and proteinic expression data of hub genes were obtained through UALCAN and the Human Protein Reference Database. Finally, we analyzed the prognostic value of hub genes with the Kaplan-Meier Plotter and MethSurv database. RESULTS: In total, 24 up-hypomethylated oncogenes and 37 down-hypermethylated tumor suppressor genes (TSGs) were identified, and 8 hub genes, including 4 up-hypomethylated oncogenes (CDC5L, MERTK, RHOA and YBX1) and 4 down-hypermethylated TSGs (BCR, DFFA, SCUBE2 and TP63), were selected by PPI. Higher expression of methylated CDC5L-cg05671347, MERTK-cg08279316, RHOA-cg05657651 and YBX1-cg16306148, and lower expression of methylated BCR-cg25410636, DFFA-cg20696875, SCUBE2-cg19000089 and TP63-cg06520450, were associated with better overall survival (OS) in HCC patients. Multivariate analysis also showed they were independent prognostic factors for OS of HCC patients. CONCLUSIONS: In summary, different expression of methylated genes above mentioned were associated with better prognosis in HCC patients. Altering the methylation status of these genes may be a therapeutic target for HCC, but it should be further evaluated in clinical studies.

ANXA7 promotes the cell cycle, proliferation and cell adhesion-mediated drug resistance of multiple myeloma cells by up-regulating CDC5L.

This study aimed to investigate whether annexin A7 (ANXA7) could promote the cell cycle, proliferation and cell adhesion-mediated drug resistance (CAM-DR) of multiple myeloma (MM) cells by up-regulating cell division cycle 5-like (CDC5L). As a result, ANXA7 expression was increased in the serum of MM patients and the expression of ANXA7 and CDC5L was also increased in MM cell lines. ANXA7 overexpression promoted the proliferation and cycle of U266 and RPMI8226 cells. The expression of proliferation cell nuclear antigen (PCNA), KI67, cyclin dependent kinase 1 (CDK1) and cyclinB1 in transfected cells was consistent with the changes of proliferation and cell cycle. In co-culture system of BMSC cells and MM cells, expression of CD44, ICAM1 and VCAM1 in MM cells was increased, which was further increased by ANXA7 overexpression. Bortezomib could increase the apoptosis of U266 and RPMI8226 cells. In co-culture system of BMSC cells and MM cells, the promotion effects of bortezomib on apoptosis of MM cells was decreased, which was further suppressed by ANXA7 overexpression. The above effects exerted by ANXA7 overexpression could be reversed by ANXA7 interference. Moreover, ANXA7 was proved to be combined with CDC5L. CDC5L interference could inhibit the promotion effects of ANXA7 overexpression on proliferation and cell cycle and inhibition effects of ANXA7 overexpression on apoptosis of MM cells treated with bortezomib in co-culture system. In conclusion, ANXA7 could promote the cell cycle, proliferation and CAM-DR of MM cells by up-regulating CDC5L.

Identification of CDC5L as bridge gene between chronic obstructive pulmonary disease and lung adenocarcinoma.

Aim: This study aimed to explore the genetic and epigenetic similarities between chronic obstructive pulmonary disease (COPD) and lung adenocarcinoma (LUAD). Materials & methods: We mainly used Weighted correlation network analysis, protein-protein interaction network and pivot analysis to identify hub modules, bridge regulators, bridge genes and hub-driving genes in both diseases and carried out verifying using external datasets. Results: We identified eight bridge regulators, 19 key molecules in the COPD model and ten key molecules in the LUAD model. Moreover, we validated that CDC5L could be a reliable biomarker in COPD and may regulate cell proliferation and metastasis in LUAD via promoter methylation. Conclusion: Our results might form a theoretical foundation for future study at an epigenetic level.

Circ-PGAM1 promotes malignant progression of epithelial ovarian cancer through regulation of the miR-542-3p/CDC5L/PEAK1 pathway.

BACKGROUND: Epithelial ovarian cancer (EOC) is the most common ovarian malignant cancer. Circular RNA is a type of endogenous noncoding RNA and is considered as a novel regulatory molecule in the development and progression of tumors. This study investigated the expression and functions of a circular RNA, circular-phosphoglycerate mutase 1 (circ-PGAM1), in EOC tissues and cells. METHODS: The expression of circ-PGAM1 and miR-542-3p in EOC was analyzed using quantitative RT-PCR. Immunohistochemistry and western blot were performed to confirm the localization and expression of cell division cycle 5-like (CDC5L) and pseudopodium enriched atypical kinase 1 (PEAK1) in EOC tissues. Cell lines (CAOV3 and OVCAR3) overexpressing or silencingcirc-PGAM1 and miR-542-3p were established to explore the functions of circ-PGAM1 and miR-542-3p in ovarian cancer cells. Furthermore, dual-luciferase reporter assay was performed to study the interactions between circ-PGAM1 and miR-542-3p and between miR-542-3p and CDC5L. CCK-8, transwell, and flow cytometry were used to study the effect of circ-PGAM1 and miR-542-3p on cell biological behaviors including proliferation, migration, invasion, and apoptosis. The interaction between CDC5L and the PEAK1 gene promoter was confirmed using chromatin immunoprecipitation (ChIP). RESULTS: Circ-PGAM1 was upregulated in EOC tissues, whereas linear PGAM1 was not deregulated in EOC tissues. Silencing of circ-PAGM1 inhibited proliferation, migration, and invasion of ovarian cancer cells and promoted cell apoptosis. MiR-542-3p was downregulated in EOC tissues, and miR-542-3p overexpression inhibited malignant progression of ovarian cancer cells. Circ-PGAM1 directly interacted with miR-542-3p, with mutual negative feedback between them. CDC5L was a direct target of miR-542-3p and played an oncogenic role in ovarian cancer cells. Furthermore, the CDC5L protein binds directly to the PEAK1 promoter to promote its transcription. PEAK1 overexpression activated ERK1/2 and JAK2 signaling pathways and promoted malignant biological behaviors of ovarian cancer cells. Circ-PAGM1 silencing combined with miR-542-3p overexpression played the greatest anticancer role in vivo. CONCLUSION: The circ-PGAM1/miR-542-3p/CDC5L/PEAK1 pathway played an important role in the progression of ovarian cancer and might be a novel therapeutic target for ovarian cancer.

Depletion of CDC5L inhibits bladder cancer tumorigenesis.

Cell division cycle 5-like (CDC5L) protein is a cell cycle regulator of the G2/M transition and has been reported to participate in the catalytic step of pre-messenger RNA (mRNA) splicing and DNA damage repair. Recently, CDC5L was also found to act as a candidate oncogene in osteosarcoma and cervical tumours. However, the role of CDC5L expression in bladder cancer remains unclear. Here, we analysed the expression and clinical significance of CDC5L in bladder cancer tissues. The expression of CDC5L in fresh bladder cancer tissues and paraffin-embedded slices was evaluated by western blot and immunohistochemistry, respectively. We found that CDC5L was highly expressed in bladder cancer. The expression of CDC5L was significantly associated with bladder cancer pathology grade and Ki67 expression. Univariate and multivariate analyses showed that high CDC5L expression was an independent prognostic factor for the survival of bladder cancer patients. To determine whether CDC5L could regulate the proliferation of bladder cancer cells, we transfected bladder cancer cells with an interfering RNA targeting CDC5L and then investigated cell proliferation with a cell counting kit (CCK)-8, flow cytometry assays, colony formation and xenograft assay analyses. Our results indicate that knockdown of CDC5L inhibits proliferation of bladder cancer cells. In addition, reduced expression of CDC5L induced apoptosis of bladder cancer cells and inhibited their migration, invasion and EMT. These findings suggest that CDC5L might play an important role in bladder cancer and thus be a promising therapeutic target of bladder cancer.

A novel and effective inhibitor combination involving bortezomib and OTSSP167 for breast cancer cells in light of label-free proteomic analysis.

PURPOSE: The 26S proteasome plays important roles in many intracellular processes and is therefore a critical intracellular cellular target for anticancer treatments. The primary aim of the current study was to identify critical proteins that may play roles in opposing the antisurvival effect of the proteasome inhibitor bortezomib together with the calcium-chelator BAPTA-AM in cancer cells using label-free LC-MS/MS. In addition, based on the results of the proteomic technique, a novel and more effective inhibitor combination involving bortezomib as well as OTSSP167 was developed for breast cancer cells. METHODS AND RESULTS: Using label-free LC-MS/MS, it was found that expressions of 1266 proteins were significantly changed between the experimental groups. Among these proteins were cell division cycle 5-like (Cdc5L) and drebrin-like (DBNL). We then hypothesized that inhibition of the activities of these two proteins may lead to more effective anticancer inhibitor combinations in the presence of proteasomal inhibition. In fact, as presented in the current study, Cdc5L phosphorylation inhibitor CVT-313 and DBNL phosphorylation inhibitor OTSSP167 were highly cytotoxic in 4T1 breast cancer cells and their IC50 values were 20.1 and 43 nM, respectively. Under the same experimental conditions, the IC50 value of BAPTA-AM was found 19.9 µM. Using WST 1 cytotoxicity assay, it was determined that 10 nM bortezomib + 10 nM CVT-313 was more effective than the control, the single treatments, or than 5 nM bortezomib + 5 nM CVT-313. Similarly, 10 nM bortezomib + 10 nM OTSSP167 was more cytotoxic than the control, the monotherapies, 5 nM bortezomib + 5 nM OTSSP167, or than 5 nM bortezomib + 10 nM OTSSP167, indicating that bortezomib + OTSSP167 was also more effective than bortezomib + CVT-313 in a dose-dependent manner. Furthermore, the 3D spheroid model proved that bortezomib + OTSSP167 was more effective than the monotherapies as well as bortezomib + CVT-313 and bortezomib + BAPTA-AM combinations. Finally, the effect of bortezomib + OTSSP167 combination was tested on MDA-MB-231 breast cancer cells, and it similarly determined that 20 nM bortezomib +40 nM OTSSP167 combination completely blocked the formation of 3D spheroids. CONCLUSIONS: Altogether, the results presented here indicate that bortezomib + OTSSP167 is a novel and effective combination and may be tested further for cancer treatment in vivo and in clinical settings.

Single-cell transcriptome sequencing reveals that cell division cycle 5-like protein is essential for porcine oocyte maturation.

The brilliant cresyl blue (BCB) test is used in both basic biological research and assisted reproduction to identify oocytes likely to be developmentally competent. However, the underlying molecular mechanism targeted by the BCB test is still unclear. To explore this question, we first confirmed that BCB-positive porcine oocytes had higher rates of meiotic maturation, better rates of cleavage and development into blastocysts, and lower death rates. Subsequent single-cell transcriptome sequencing on porcine germinal vesicle (GV)-stage oocytes identified 155 genes that were significantly differentially expressed between BCB-negative and BCB-positive oocytes. These included genes such as cdc5l, ldha, spata22, rgs2, paip1, wee1b, and hsp27, which are enriched in functionally important signaling pathways including cell cycle regulation, oocyte meiosis, spliceosome formation, and nucleotide excision repair. In BCB-positive GV oocytes that additionally had a lower frequency of DNA double-strand breaks, the CDC5L protein was significantly more abundant. cdc5l/CDC5L inhibition by short interference (si)RNA or antibody microinjection significantly impaired porcine oocyte meiotic maturation and subsequent parthenote development. Taken together, our single-oocyte sequencing data point to a potential new role for CDC5L in porcine oocyte meiosis and early embryo development, and supports further analysis of this protein in the context of the BCB test.

CDC5L Promotes hTERT Expression and Colorectal Tumor Growth.

BACKGROUND/AIMS: Colorectal cancer (CRC) is the third leading cause of cancer-related death worldwide because the survival rate remains low. Cell division cycle 5-like (CDC5L) is highly expressed in some cancer cells, but the mechanism requires clarification. Human telomerase reverse transcriptase (hTERT) plays important roles in CRC. METHODS: This study aimed to identify a link between CDC5L and hTERT and to determine their effects on the signaling pathways, migration and prognosis of CRC cells. We first treated LoVo cells with biotin-labeled hTERT and identified CDC5L. Then, pulldown and ChIP assays were used to verify whether CDC5L was a promoter of hTERT. The roles of CDC5L and hTERT in cell growth and migration were studied using siRNA in vivo and in vitro. 130 human CRC specimens were analyzed using immunohistochemistry. Western blot and wound scratch analyses were used to determine the signaling pathway for CDC5L-mediated activation of CRC growth and migration. RESULTS: We identified CDC5L as a new hTERT promoter-binding protein. Clinically, CDC5L and hTERT expression levels were key factors in the prognosis of CRC patients. CDC5L knockdown inhibited tumor growth by down-regulating hTERT expression, and CDC5L was shown to be a transcriptional activator of hTERT in a luciferase reporter assay. CONCLUSION: Altogether, the above results demonstrated that CDC5L was positively correlated with hTERT as a key promoter of CRC cells. To some extent, our findings suggest that CDC5L may serve as a novel therapeutic target for human colorectal cancer.

CDC5L drives FAH expression to promote metabolic reprogramming in melanoma.

Metabolic reprogramming allows tumor cells to thrive in the typically hypoxic tumor microenvironment. Using immunodetection and clinical data analyses, we demonstrate here that fumarylacetoacetate hydrolase (FAH) is highly expressed in melanoma and correlates with poor survival. FAH knockdown inhibits proliferation and migration, while promoting apoptosis in melanoma cells, result in prolonged survival in tumor-bearing mice. Molecular analyses using real time RT-PCR, western blot, and 13C tracing showed that these changes are driven by strong stimulation of anaplerotic reactions through the TCA cycle and the pentose-phosphate pathway, resulting in increased fatty acid and nucleotide synthesis. Using bioinformatic, ChIP-PCR, and gene silencing analyses, we determined that cell division cycle 5-like protein (CDC5L) is an important transcription factor regulating FAH expression in melanoma cells. These findings reveal that FAH induces metabolic reprogramming in melanoma and so emerges as both a potentially useful independent prognostic indicator and an attractive therapeutic target.

ATPγS stalls splicing after B complex formation but prior to spliceosome activation.

The ATP analog ATPγS inhibits pre-mRNA splicing in vitro, but there have been conflicting reports as to which step of splicing is inhibited by this small molecule and its inhibitory mechanism remains unclear. Here we have dissected the effect of ATPγS on pre-mRNA splicing in vitro. Addition of ATPγS to splicing extracts depleted of ATP inhibited both catalytic steps of splicing. At ATPγS concentrations ≥0.5 mM, precatalytic B complexes accumulate, demonstrating a block prior to or during the spliceosome activation stage. Affinity purification of the ATPγS-stalled B complexes (B(ATPγS)) and subsequent characterization of their abundant protein components by 2D gel electrophoresis revealed that B(ATPγS) complexes are compositionally more homogeneous than B complexes previously isolated in the presence of ATP. In particular, they contain little or no Prp19/CDC5L complex proteins, indicating that these proteins are recruited after assembly of the precatalytic spliceosome. Under the electron microscope, B(ATPγS) complexes exhibit a morphology highly similar to B complexes, indicating that the ATPγS-induced block in the transformation of the B to B(act) complex is not due to a major structural defect. Likely mechanisms whereby ATPγS blocks spliceosome assembly at the activation stage, including inhibition of the RNA helicase Brr2, are discussed. Given their more homogeneous composition, B complexes stalled by ATPγS may prove highly useful for both functional and structural analyses of the precatalytic spliceosome and its conversion into an activated B(act) spliceosomal complex.

Expression of CDC5L is associated with tumor progression in gliomas.

Cell division cycle 5-like (CDC5L) protein is a cell cycle regulator of the G2/M transition and has been reported to participate in the catalytic step of pre-messenger RNA (mRNA) splicing and DNA damage repair. Recently, it was also found to act as a candidate oncogene in osteosarcoma and cervical tumors. However, the role of CDC5L expression in tumor biology was still unclear. Here, we analyzed the expression and clinical significance of CDC5L in gliomas. The expression of CDC5L in fresh glioma tissues and paraffin-embedded slices was evaluated by western blot and immunohistochemistry, respectively. We found that CDC5L was highly expressed in glioma tissues. The expression of CDC5L was significantly associated with glioma pathology grade and Ki-67 expression. Univariate and multivariate analyses showed that high CDC5L expression was an independent prognostic factor for glioma patients' survival. To determine whether CDC5L could regulate the proliferation of glioma cells, we transfected glioma cells with interfering RNA target CDC5L, then investigated cell proliferation with cell counting kit (CCK)-8, flow cytometry assays and colony formation analyses. Our results indicated that knockdown of CDC5L would inhibit proliferation of glioma cells. Besides, reduced expression of CDC5L could induce the apoptosis of glioma cells. These findings suggested that CDC5L might play an important role in glioma and thus be a promising therapeutic target of glioma.