Comparative metabolomic and transcriptomic analysis of Saccharomyces cerevisiae W303a and CEN.PK2-1C.

Saccharomyces cerevisiae is a health microorganism closely related to human life, especially in food and pharmaceutical industries. S. cerevisiae W303a and CEN.PK2-1C are two commonly used strains for synthetic biology-based natural product production. Yet, the metabolomic and transcriptomic differences between these two strains have not been compared. In this study, metabolomics and transcriptomics were applied to analyze the differential metabolites and differential expression genes (DEGs) between W303a and CEN.PK2-1C cultured in YPD and SD media. The growth rate of W303a in YPD medium was the lowest compared with other groups. When cultured in YPD medium, CEN.PK2-1C produced more phenylalanine than W303a; when cultured in SD medium, W303a produced more phospholipids than CEN.PK2-1C. Transcriptomic analysis revealed that 19 out of 22 genes in glycolysis pathway were expressed at higher levels in CEN.PK2-1C than that in W303a no matter which media were used, and three key genes related to phenylalanine biosynthesis including ARO9, ARO7 and PHA2 were up-regulated in CEN.PK2-1C compared with W303a when cultured in YPD medium, whereas seven DEGs associated with phospholipid biosynthesis were up-regulated in W303a compared with CEN.PK2-1C when cultured in SD medium. The high phenylalanine produced by CEN.PK2-1C and high phospholipids produced by W303a indicated that CEN.PK2-1C may be more suitable for synthesis of natural products with phenylalanine as precursor, whereas W303a may be more appropriate for synthesis of phospholipid metabolites. This finding provides primary information for strain selection between W303a and CEN.PK2-1C for synthetic biology-based natural product production.

The inhibition of centromere protein K causes anticancer effects in breast carcinoma via effects on the FAK/PI3K/AKT/mTOR pathway.

The overexpression of centromere protein K (CENPK) is a major contributor to the malignant progression of numerous cancers. To date, the detailed functions and mechanisms of CENPK in breast carcinoma are not fully elucidated. The goals of this project were to comprehensively address the relevance of CENPK in breast carcinoma. The initial investigation by TCGA analysis revealed a high expression level of CENPK in breast carcinoma. Subsequently, an immunoblotting assay confirmed that CENPK is highly expressed in the clinical samples of breast carcinoma. In vitro experiments elucidated that the inhibition of CENPK produced substantial anticancer effects, including a reduction of proliferation, the inhibition of epithelial-mesenchymal transition, the induction of cell cycle arrest and chemosensitivity. Mechanism research unveiled a role for CENPK in mediating the focal adhesion kinase (FAK1)/PI3K/AKT/mTOR pathway. Inhibiting the FAK/PI3K/AKT/mTOR pathway was able to reverse CENPK-elicited cancer-promoting effects. Additionally, CENPK-silenced breast carcinoma cells exhibited low tumorigenicity in vivo. In summary, our data demonstrated that CENPK inhibition provided an excellent anticancer effect for breast carcinoma by regulating FAK/PI3K/AKT/mTOR pathway. This work illustrates a novel molecular mechanism for CENPK in breast carcinoma and suggests CENPK inhibition as a promising targeted therapy for breast carcinoma.

Centromeric protein K (CENPK) promotes gastric cancer proliferation and migration via interacting with XRCC5.

BACKGROUND: CENPK is a novel oncogene which is aberrantly expression in some malignant tumors. However, the role and mechanisms of CENPK in gastric cancer have not been explored. METHODS: In this study, we use RT-PCR and IHC to study CENPK expression in gastric cancer cells and tissues. In addition, we constructed the two kinds of CENPK siRNA lentivirus to knock down CENPK. Then, we use High content living cell imaging System, Cell Counting Kit-8, colony formation, wound healing and Transwell assays to demonstrate the function of CENPK on gastric cancer cells AGS and MKN45. Meanwhile, we use flow cytometry assay to study CENPK function on gastric cancer cell apoptosis and cell cycle arrest. Subcutaneous tumorigenesis in nude mice was also performed to confirm CENPK function on gastric cancer. Finally, we use Co-IP, LC-MS and function rescue assay to study the downstream interaction molecular of CENPK. RESULTS: We demonstrated that CENPK expression were up-regulated in GC cell lines. Poor differentiation and III-IV stage had more percentages of high CENPK expression. Knocking down CENPK could significantly suppress GC cells proliferation, migration and invasion, and induce GC cells apoptosis and G1/S phase transition arrest. Subcutaneous tumorigenesis confirmed the tumor-promoting effects of CENPK in vivo. Remarkably, we found for the first time that XRCC5 might be interacted with CENPK through Co-IP, LC-MS and rescue study. CONCLUSION: CENPK promotes GC cell proliferation and migration via interacting with XRCC5 and may be a novel prognostic factor or therapeutic target for CENPK.

Knockdown of CENPK inhibits cell growth and facilitates apoptosis via PTEN-PI3K-AKT signalling pathway in gastric cancer.

Previous studies have indicated that centromere protein K (CENPK) is upregulated in several cancers and related to tumorigenesis. Nevertheless, the potential function of CENPK in gastric cancer (GC) remains unknown. Here, we investigated the function of CENPK on oncogenicity and explored its underlying mechanisms in GC. Our results showed that CENPK was dramatically overexpressed in GC and was associated with poor prognosis through bioinformatics analysis. We demonstrated that CENPK is upregulated in GC tissues and cell lines. Moreover, knockdown of CENPK significantly inhibited proliferation in vitro and attenuated the growth of implanted GCs in vivo. In addition, CENPK silencing induced G1 phase cell cycle arrest and facilitated apoptosis of GC cells. KEGG pathway analysis indicated that the PI3K-AKT signalling pathway was considerably enriched. Knockdown of CENPK decreased the expression of PI3K, p-Akt (Ser437) and p-GSK3ß (Ser9) in GC cells, and increased the expression of PTEN. In conclusion, this study indicated that CENPK was overexpressed in GC and may promote gastric carcinogenesis through the PTEN-PI3K-AKT signalling pathway. Thus, CENPK may be a potential target for cancer therapeutics in GC.

Bioinformatics analysis combined with experiments predicts CENPK as a potential prognostic factor for lung adenocarcinoma.

BACKGROUND: Lung cancer is the most common malignant tumor. Identification of novel diagnostic and prognostic biomarkers for lung cancer is a key research imperative. The role of centromere protein K (CENPK) in cancer is an emerging research hotspot. However, the role of CENPK in the progression of lung adenocarcinoma (LAC) is not well characterized. METHODS: In this study, we identified CENPK as a potential new gene for lung cancer based on bioinformatics analysis. In addition, in vitro experiments were performed to verify the function of this gene. We investigated the expression of CENPK in LAC by analyses of datasets from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases. Differential expression analyses, gene ontology (GO) enrichment, Kyoto encyclopedia of genes and genomes (KEGG) analysis, and gene set enrichment analysis (GSEA) were conducted to evaluate the diagnostic and prognostic relevance of CENPK. Then, for evaluating the biological behavior and role of CENPK in lung cancer cells, we did a series of vitro experiments, such as immunohistochemistry analysis, Western blot analysis, CCK8 assay, transwell assay, flow cytometry, and wound healing assay. RESULTS: We demonstrated overexpression of CENPK in LAC; in addition, increased expression of CENPK was associated with clinical progression. Moreover, CENPK was found to be an independent risk factor in patients with LAC. Furthermore, we observed activation of CENPK-related signaling pathways in patients with LAC. CONCLUSIONS: Our findings indicate a potential role of CENPK in promoting tumor proliferation, invasion, and metastasis. It may serve as a novel diagnostic and prognostic biomarker in patients with LAC.

LINC01158 works as an oncogene in glioma via sponging miR-6734-3p to boost CENPK expression.

BACKGROUND: Long non-coding RNAs (lncRNAs) have been certified to play vital biological functions in glioma and have received considerable attention in the recent literature. Nonetheless, the role of LINC01158 in glioma remains to be elucidated. METHODS: qRT-PCR, western blot and GEPIA database were applied for reporting the expression of CENPK and LINC01158 in glioma and the correlation between LINC01158 and CENPK expression. EdU, colony formation, CCK-8, caspase-3 activity and TUNEL assays probed the impacts of LINC01158 on glioma cell growth. Subcellular fractionation and FISH assays revealed the cellular distribution of LINC01158. Luciferase reporter and RIP assays examined ceRNA network of LINC01158, CENPK and miR-6734-3p. RESULTS: LINC01158 and CENPK were both overexpressed in glioma and a positive regulation of LINC01158 on CENPK was corroborated. LINC01158 served a pro-proliferative and anti-apoptotic part in glioma by sponging miR-6734-3p to augment CENPK. CONCLUSION: LINC01158 enhances CENPK by serving as sponge for miR-6734-3p to facilitate glioma development, proposing LINC01158 as a new player in glioma.

LINC00958 promotes the proliferation of TSCC via miR-211-5p/CENPK axis and activating the JAK/STAT3 signaling pathway.

BACKGROUND: Tongue squamous cell carcinoma (TSCC) is one of the most common oral tumors. Recently, long intergenic noncoding RNA 00958 (LINC00958) has been identified as an oncogene in human cancers. Nevertheless, the role of LINC00958 and its downstream mechanisms in TSCC is still unknown. METHODS: The effect of LINC00958 on TSCC cells proliferation and growth were assessed by CCK-8, colony formation, 5-Ethynyl-2'-deoxyuridline (EdU) assay and flow cytometry assays in vitro and tumor xenograft model in vivo. Bioinformatics analysis was used to predict the target of LINC00958 in TSCC, which was verified by RNA immunoprecipitation and luciferase reporter assays. RESULTS: LINC00958 was increased in TSCC tissues, and patients with high LINC00958 expression had a shorter overall survival. LINC00958 knockdown significantly decreased the growth rate of TSCC cells both in vitro and in vivo. In mechanism, LINC00958 acted as a ceRNA by competitively sponging miR-211-5p. In addition, we identified CENPK as a direct target gene of miR-211-5p, which was higher in TSCC tissues than that in adjacent normal tissues. Up-regulated miR-211-5p or down-regulated CENPK could abolish LINC00958-induced proliferation promotion in TSCC cells. Furthermore, The overexpression of CENPK promoted the expression of oncogenic cell cycle regulators and activated the JAK/STAT3 signaling. CONCLUSIONS: Our findings suggested that LINC00958 is a potential prognostic biomarker in TSCC.

Regulation of trehalose, a typical stress protectant, on central metabolisms, cell growth and division of Saccharomyces cerevisiae CEN.PK113-7D.

Trehalose could protect the typical food microorganism Saccharomyces cerevisiae cell against environmental stresses; however, the other regulation effects of trehalose on yeast cells during the fermentation are still poorly understood. In this manuscript, different concentrations (i.e., 0, 2 and 5% g/v) of trehalose were respectively added into the medium to evaluate the effect of trehalose on growth, central metabolisms and division of S. cerevisiae CEN.PK113-7D strain that could uptake exogenous trehalose. Results indicated that addition of trehalose could inhibit yeast cell growth in the presence or absence of 8% v/v ethanol stress. Exogenous trehalose inhibited the glucose transporting efficiency and reduced intracellular glucose content. Simultaneously, increased intracellular trehalose content destroyed the steady state of trehalose cycle and caused the imbalance between the upper glycolysis part and the lower part, thereby leading to the dysfunction of glycolysis and further inhibiting the normal yeast cell growth. Moreover, energy metabolisms were impaired and the ATP production was reduced by addition of trehalose. Finally, exogenous trehalose-associated inhibition on yeast cell growth and metabolisms delayed cell cycle. These results also highlighted our knowledge about relationship between trehalose and growth, metabolisms and division of S. cerevisiae cells during fermentation.

CENPK orchestrates ovarian cancer progression via GOLPH3-Mediated activation of mTOR signaling.

Ovarian cancer stands as a formidable clinical challenge, with limited therapeutic options. This investigation delves into the intricate molecular mechanisms governing ovarian cancer progression and uncovers Centromere Protein K (CENPK) as a central figure in disease pathogenesis. Elevated CENPK levels within ovarian cancer tissues conspicuously align with adverse clinical outcomes, positioning CENPK as a promising prognostic biomarker. Deeper exploration reveals a direct transcriptional connection between CENPK and the E2F1 transcription factor and clearly establishes E2F1's role as the master regulator of CENPK expression in ovarian cancer. Our inquiry revealing a suppression of tumor-promoting signaling pathways, most notably the mTOR pathway, upon CENPK silencing. Intriguingly, CENPK renders ovarian cancer cells more responsive to the mTOR inhibitor rapamycin, introducing a promising avenue for therapeutic intervention. In summation, our study unravels the multifaceted role of CENPK in ovarian cancer progression. It emerges as a prognostic indicator, a pivotal mediator of cell proliferation and tumorigenicity, and a regulator of the mTOR pathway, shedding light on potential therapeutic avenues for this formidable disease.

Re-evaluation of the impact of BUD21 deletion on xylose utilization by Saccharomyces cerevisiae.

Various rational metabolic engineering and random approaches have been applied to introduce and improve xylose utilization and ethanol productivity by Saccharomyces cerevisiae. Among them, the BUD21 gene was identified as an interesting candidate for enhancing xylose consumption as its deletion appeared to be sufficient to improve growth, substrate utilization and ethanol productivity on xylose, even in a laboratory strain lacking a heterologous xylose pathway. The present study aimed at studying the influence of BUD21 deletion in recombinant strains carrying heterologous oxido-reductive xylose utilization pathway. The positive effect of BUD21 gene deletion on aerobic growth and xylose utilization could not be confirmed in two non-engineered laboratory strains (BY4741 and CEN.PK 113-7D) that were grown in YP rich medium with 20 g/L xylose as sole carbon source, despite the fact that effective deletion of BUD21 gene was confirmed using both genotypic (colony PCR) and phenotypic (heat sensitive phenotype of the BUD21 deletion mutant) control experiments. Therefore, the effect of BUD21 deletion on xylose fermentation might be strain- or medium-dependent.

The cell cycle gene centromere protein K (CENPK) contributes to the malignant progression and prognosis of prostate cancer.

Background: The cell cycle gene centromere protein K (CENPK) is upregulated in various cancers; however, the clinical value and mechanism of CENPK in prostate cancer (PCa) and castration-resistant prostate cancer (CRPC) remain unclear. Methods: The expression of CENPK in PCa was analyzed in both patients with PCa and cell lines using immunohistochemistry (IHC), real-time quantitative reverse transcription PCR (qRT-PCR), Western blot and bioinformatics analyses. Knockdown of CENPK in PCa cells was achieved by transfecting siRNAs and assessed using qRT-PCR and Western blotting. MTT and colony formation assays were used to assess the growth of PCa cells. The cell cycle was analyzed using propidium iodide (PI) staining and flow cytometry. To study the possible biological function of CENPK, pathway enrichment analysis was performed by dividing these groups into a high CENPK expression group and a low CENPK expression group based on the median CENPK expression level. Finally, the correlation between CENPK expression in PCa and clinical parameters was evaluated. Results: Our study revealed that CENPK was expressed at high levels in CRPC tissues and cell lines compared to primary PCa. The downregulation of CENPK significantly inhibited cell viability and reduced the number of colonies formed by LNCaP-AI and DU145 cells (two CRPC cell lines). Gene enrichment and flow cytometry analyses showed that high CENPK expression was linked to mitotic spindles and the cell cycle and may be involved in mitosis in the cell cycle of cancer cells to modulate cell proliferation and promote the development of CRPC. Moreover, patients exhibiting higher expression of the CENPK mRNA experienced shorter disease-free survival (DFS) and overall survival (OS) than the lower expression group. Conclusions: This study provides novel molecular insights into the role of CENPK in castration-resistant PCa cells and reveals that an increase in CENPK expression may indicate shorter DFS and a poor prognosis for patients with PCa. Targeting CENPK may be a novel strategy for the treatment of PCa.

Upregulation of centromere protein K is crucial for lung adenocarcinoma cell viability and invasion.

BACKGROUND: Identification of functional genes or biomarkers may be helpful for developing new treatment strategies in lung adenocarcinoma (LUAD). The centromere protein K (CENPK) gene has been discovered to be overexpressed and could influence tumor progression in several tumor types. However, its role in LUAD has never been revealed. OBJECTIVES: The purpose of the current study was to detect the effects of CENPK and its mechanisms in the progression of LUAD. MATERIAL AND METHODS: Data from The Cancer Genome Atlas (TCGA) and Oncomine databases was used to analyze the expression of CENPK. The relationship between CENPK expression and the prognosis of LUAD was investigated using Kaplan-Meier and Cox regression analyses. The cell viability was monitored with Cell Counting Kit-8 (CCK-8) and colony forming assays, while migration and invasion were analyzed with a transwell assay. The effect of CENPK on the expression of epithelial-mesenchymal transition (EMT) markers were estimated using western blotting. RESULTS: CENPK was significantly overexpressed in LUAD tissues and cells (p < 0.01). The overall survival rate in the low CENPK expression group was significantly higher than in the high CENPK expression group (p = 0.003). Furthermore, the overexpression of CENPK facilitated cell viability, migration and invasion of tumor cells, while knockdown of CENPK prevented these behaviors (p < 0.01). Moreover, upregulation of CENPK decreased the expression of E-cadherin and enhanced the expression of N-cadherin, vimentin and Snail in LUAD cells (p < 0.01). Conversely, knockdown of CENPK resulted in the opposite trend (p < 0.01). CONCLUSIONS: CENPK was upregulated in LUAD tissues and cells, and the enhancement of CENPK promoted the viability, migration, invasion, and EMT of LUAD cells.

Pan-cancer investigation of CENPK gene: clinical significance and oncogenic immunology.

Many studies have confirmed that the CENPK gene regulates the progression of cancers, but its specific molecular mechanism remains unidentified, as does its significance in the analysis of human cancers. We specify a comprehensive genomic architecture of the CENPK gene associated with the tumor immune microenvironment and its clinical relevance across a broad spectrum of solid tumors. Statistics from The Cancer Genome Atlas (TCGA) and Cancer Cell Line Encyclopedia (CCLE) of over 30 solid tumors were examined. CENPK was expressed differentially in several cancers and is significantly associated in survival outcomes, with higher CENPK signifying a worse prognosis for ACC, KICH, KIRC, KIRP, LGG, LIHC, LUAD, MESO, and SARC. We further examined its clinical relevance with tumor immunogenic features. The expression level of CENPK was not only strongly linked to the tumor infiltration, such as tumor-infiltrating immune cells and immune scores but also linked to microsatellite instability and tumor mutation burden in diverse cancers (P<0.05). I mmune markers such as TNFRSF14 and VSIR were highly expressed on over 20 kinds of human cancer and mismatch repair genes like MLH1, MSH2, MSH6, and PMS2 were positively related with CENPK expression. Moreover, the methyltransferases and functional pathways also seem to have a relationship with the CENPK. CENPK is expected to be a guiding marker gene for clinical prognosis and tumor personalized immunotherapy.

DNA Double-Strand Break-Induced Gene Amplification in Yeast.

Precise control of the gene copy number in the model yeast Saccharomyces cerevisiae may facilitate elucidation of enzyme functions or, in cell factory design, can be used to optimize production of proteins and metabolites. Currently, available methods can provide high gene-expression levels but fail to achieve accurate gene dosage. Moreover, strains generated using these methods often suffer from genetic instability resulting in loss of gene copies during prolonged cultivation. Here we present a method, CASCADE, which enables construction of strains with defined gene copy number. With our present system, gene(s) of interest can be amplified up to nine copies, but the upper copy limit of the system can be expanded. Importantly, the resulting strains can be stably propagated in selection-free media.

Downregulation of CENPK suppresses hepatocellular carcinoma malignant progression through regulating YAP1.

BACKGROUND: Several studies have found that centromere protein K (CENPK) is overexpressed in several tumour types and promotes tumor progression. However, there has been little research on the role of CENPK in the progression of hepatocellular carcinoma (HCC). MATERIALS AND METHODS: The expression of CENPK in HCC tissues was quantified by Western blot and quantitative real-time PCR. Cells were transfected with lentiviral plasmids containing shRNA sequences targeting CENPK and YAP1 to silence the expression of CENPK and YAP1. Cell Counting Kit-8 assay, colony formation assay, wound healing assay, and transwell invasion assay were performed to evaluate cell growth, migration, and invasion of HCC cells. Tumorigenicity assay was used to detect the effect of CENPK on the growth of HCC cells. Western blot assay was performed to investigate the expression of epithelial-mesenchymal transition (EMT) markers and YAP1. RESULTS: Compared to that in adjacent non-tumor tissues, CENPK was aberrantly upregulated in HCC tumor tissues. Furthermore, CENPK knockdown significantly inhibited proliferation, migration, invasion, and EMT progression in HCC cells. Mechanistically, we identified that YAP1 was responsible for the tumor-suppressive effects of CENPK knockdown in the HCC cells. The inhibitory effects of CENPK silencing on cell proliferation, migration, invasion, and EMT were partially reversed by the restoration of YAP1 expression. CONCLUSION: Our results suggested that the CENPK-YAP1-EMT axis plays a critical role in regulating HCC malignant progression, indicating the role of this axis as a potential therapeutic target for HCC.

The sole introduction of two single-point mutations establishes glycerol utilization in Saccharomyces cerevisiae CEN.PK derivatives.

BACKGROUND: Glycerol is an abundant by-product of biodiesel production and has several advantages as a substrate in biotechnological applications. Unfortunately, the popular production host Saccharomyces cerevisiae can barely metabolize glycerol by nature. RESULTS: In this study, two evolved derivatives of the strain CEN.PK113-1A were created that were able to grow in synthetic glycerol medium (strains PW-1 and PW-2). Their growth performances on glycerol were compared with that of the previously published evolved CEN.PK113-7D derivative JL1. As JL1 showed a higher maximum specific growth rate on glycerol (0.164 h-1 compared to 0.119 h-1 for PW-1 and 0.127 h-1 for PW-2), its genomic DNA was subjected to whole-genome resequencing. Two point mutations in the coding sequences of the genes UBR2 and GUT1 were identified to be crucial for growth in synthetic glycerol medium and subsequently verified by reverse engineering of the wild-type strain CEN.PK113-7D. The growth rate of the resulting reverse-engineered strain was 0.130 h-1. Sanger sequencing of the GUT1 and UBR2 alleles of the above-mentioned evolved strains PW-1 and PW-2 also revealed one single-point mutation in these two genes, and both mutations were demonstrated to be also crucial and sufficient for obtaining a maximum specific growth rate on glycerol of ~0.120 h-1. CONCLUSIONS: The current work confirmed the importance of UBR2 and GUT1 as targets for establishing glycerol utilization in strains of the CEN.PK family. In addition, it shows that a growth rate on glycerol of 0.130 h-1 can be established in reverse-engineered CEN.PK strains by solely replacing a single amino acid in the coding sequences of both Ubr2 and Gut1.

Overexpression of centromere protein K (CENPK) in ovarian cancer is correlated with poor patient survival and associated with predictive and prognostic relevance.

Ovarian cancer has a poor prognosis. Most patients are diagnosed with ovarian cancer when the disease has reached an advanced stage and cure rates are generally under 30%. Hence, early diagnosis of ovarian cancer is the best means to control the disease in the long term and abate mortality. So far, cancer antigen 125 (CA125) and human epididymis protein 4 (HE4) are the gold-standard tumor markers for ovarian cancer; however, these two markers can be elevated in a number of conditions unrelated to ovarian cancer, resulting in decreased specifically and positive predictive value. Therefore, it is urgent to identify novel biomarkers with high reliability and sensitivity for ovarian cancer. In this study for the first time, we identified a member of the centromere protein (CENP) family, CENPK, which was specifically upregulated in ovarian cancer tissues and cell lines and the overexpression of which was associated with poor prognoses in patients with ovarian cancer. In addition, the presence of CENPK significantly improved the sensitivity of CA125 or HE4 for predicting clinical outcomes of ovarian cancer patients. In conclusion, we identified that CENPK was specifically upregulated in ovarian cancer cells and can be used as a novel tumor marker of ovarian cancer.