Nuclear Casein Kinase And Cyclin-dependent Kinase Substrate 1 Research Articles

Micro RNA-365-3p as a Tumor Suppressor in Gastric Cancer: Inhibition of NUCKS1-Mediated PI3K/AKT Signaling and Anti-Tumor Effects

Increasing evidence supports that microRNAs (miRNAs) are essential regulators of tumor initiation and development in a wide range of human malignancies, including gastric cancer (GC). To date, it has been reported that miR-365-3p is abnormally expressed and influences tumor progression in multiple malignancies. However, the roles of miR-365-3p are yet to be known and are worth investigating in GC. Herein, we screened differentially expressed miRNAs in 3 pairs of matched gastric cancerous and adjacent normal specimens. A series of public datasets and collected clinical GC samples were employed to determine the expression profiles of miR-365-3p and its downstream target protein. Furthermore, the effects of miR-365-3p ectopic expression and depletion on GC cell proliferation, invasion and migration were explored in vitro via cell counting kit-8 (CCK-8), 5-Ethynyl-2′-deoxyuridine (EdU), clone formation and Transwell assays, and in vivo by applying the nude mice models. Dual-luciferase reporter, western blot and a series of rescue assays were carried out to investigate the potential interactions between miR-365-3p and NUCKS1 (Nuclear, casein kinase and cyclin-dependent kinase substrate 1) and downstream signaling pathways of the miR-365-3p/NUCKS1 axis. According to the analyses of the miRNA microarray and bioinformatics, we first identified miR-365-3p dysregulation in GC. Through miRNA qRT-PCR, we revealed that miR-365-3p was downregulated in GC samples and cell lines. Lower level of miR-365-3p was significantly associated with larger tumor size and advanced lymph node metastasis in GC patients. Downregulation of miR-365-3p accelerated, while ectopic miR-365-3p significantly suppressed GC cell proliferation, migration and invasion in vitro and in vivo. Results also showed that overexpression of miR-365-3p in gastric cancer cells significantly reduced the levels of pro-inflammatory cytokines, including interleukin-1β (IL-1β) and interleukin-6 (IL-6), while miR-365-3p knockdown increased their expression. Moreover, mechanistically, knockdown of miR-365-3p promoted tumor cell aggressiveness by enhancing the expression of NUCKS1 and subsequently resulted in the activation of PI3K/AKT signaling pathway in GC. In addition, either NUCKS1 silencing or the inhibition of PI3K/AKT signaling pathway, at least partially, reversed the promotive effects of miR-365-3p knockdown on GC aggressiveness. Altogether, these findings suggest that miR-365-3p inhibits tumor growth and metastasis by attenuating the expression of NUCKS1 and inactivation of its downstream PI3K/AKT signaling, thus, highlighting the potential of miR-365-3p as a therapeutic target in GC.

NUCKS1 promotes breast cancer cell proliferation and metastasis via PI3K/ AKT pathway

Purpose: To investigate the role of nuclear casein kinase and cyclin-dependent kinase substrate 1 (NUCKS1) in breast cancer. 
 Methods: Breast cancer cells were maintained in RPMI-1640 medium containing 10 % fetal bovine serum in a incubator at 37 °C. Cell proliferation was determined by CCK8 and colony formation assays. Flow cytometry and Transwell assays were used to determine cell cycle and metastasis, respectively. 
 Results: Expression of NUCKS1 was significantly elevated in breast cancer (p < 0.01). Overexpression of NUCKS1 significantly increased cell viability (p < 0.01), and promoted proliferation of breast cancer cells. Knockdown of NUCKS1 inhibited cell proliferation, and induced cell cycle arrest at G1 phase. However, overexpression of NUCKS1 promoted cell cycle progression via down-regulation of p21 and up-regulation of cyclin D1 and CDK1. Cell migration and invasion were induced by overexpression of NUCKS1, and suppressed by silencing of NUCKS1. Overexpression of NUCKS1 enhanced p-AKT and p-PI3K expression, while knockdown of NUCKS1 reduced the expression of p-AKT and p-PI3K in breast cancer cells. 
 Conclusion: NUCKS1 promotes breast cancer cell proliferation and metastasis via activation of PI3K/AKT signaling. The silencing of NUCKS1 can be used as a strategy to develop therapies for the management of breast cancer.

Hsa_circ_0001550 facilitates colorectal cancer progression through mediating microRNA-4262/nuclear casein kinase and cyclin-dependent kinase substrate 1 cascade.

BackgroundCircular RNAs (circRNAs) play important roles in various malignancies, such as colorectal cancer (CRC). However, the function of hsa_circ_0001550 in CRC remains to be elucidated.MethodsThe expression levels of hsa_circ_0001550, microRNA (miR)‐4262, and nuclear casein kinase and cyclin‐dependent kinase substrate 1 (NUCKS1) were determined by real‐time qPCR. Cell biological behaviors were evaluated via colony formation assay, transwell assay, flow cytometry, and sphere formation assays. The target relationship was validated via dual‐luciferase reporter and RNA pull‐down assays. Protein expression was analyzed by western blot. Xenograft tumor model was adopted to evaluate hsa_circ_0001550 function in vivo.ResultsHsa_circ_0001550 enrichment was enhanced in CRC tissue specimens and cell lines. Hsa_circ_0001550 absence hindered CRC cell proliferation, metastasis, stemness, and caused apoptosis. Hsa_circ_0001550 targeted miR‐4262, and hsa_circ_0001550 absence‐caused impacts were diminished by anti‐miR‐4262. MiR‐4262 targeted NUCKS1. Hsa_circ_0001550 had positive regulation on NUCKS1 expression. NUCKS1 overexpression overturned the influences of hsa_circ_0001550 silencingon CRC cell progression. Hsa_circ_0001550 interference notably blocked in vivo xenograft tumor growth.ConclusionHsa_circ_0001550 facilitated CRC progression by binding to miR‐4262 to positively regulate NUCKS1 abundance.

MiR-641 Inhibited Cell Proliferation and Induced Apoptosis by Targeting NUCKS1/PI3K/AKT Signaling Pathway in Breast Cancer.

Objective Studies revealed an important role of microRNAs (miRNAs) in multiple cancers, including breast cancer. In the present study, we evaluated the role and function of miR-641 in breast cancer. Methods The expression level of miR-641 in breast cancer cell lines (Hs-578T, MCF7, HCC1937, and MAD-MB-231) was determined by real-time PCR. Functional analyses, including CCK-8 assay, transwell assay, wound-healing assay, and apoptosis detection, were carried out to explore the roles of miRNA-641 in malignant behaviors of breast cancer. Luciferase report assay was used to investigate the regulatory association of miRNA-641 with its potential targets. Results The expression levels of miR-641 were downregulated, while the expression levels of nuclear casein kinase and cyclin-dependent kinase substrate 1 (NUCKS1) were increased in breast cancer cell lines. The in vitro results showed that miR-641 repressed proliferation and migration/invasion and promoted apoptosis of breast cancer cells. NUCKS1, a positive regulator of phosphatidylinositol-3-kinases (PI3K)/protein-serine-threonine kinase (AKT) pathway, was confirmed as a direct target of miR-641. The of treatment of the PI3K agonist, 740Y-P, could abrogate the antioncogenic potentials of miR-641 in breast cancer cells. Conclusion miR-641 functioned as a tumor suppressor through the PI3K/AKT signaling pathway via targeting NUCKS1 in breast cancer.

Tear miRNA expression analysis reveals miR-203 as a potential regulator of corneal epithelial cells

BackgroundmicroRNAs (miRNAs) are small noncoding RNAs that negatively regulate gene expression. They are found within cells and in body fluids. Extracellular miRNAs have been shown to associate with the surrounding tissues. Therefore, we predicted that miRNAs in tears may contribute to regulate corneal epithelial cell function. However, information on the miRNA expression profile of tears is limited and the specific functions of tear miRNAs for corneal epithelial cells are still unknown. To study the role of tear miRNAs, we determined which miRNAs are highly expressed in tears and examined the involvement of miRNAs in corneal epithelial cell viability.MethodsmiRNAs extracted from monkey tears and sera were subjected to microarray analysis. miRNAs of which expression levels were higher in tears than in sera were selected, and their expression levels were quantified by quantitative polymerase chain reaction (qPCR). To examine miRNA function, mimics and inhibitors of miRNAs were transfected into human corneal epithelial (HCE-T) cells and incubated for 24 or 48 h. After transfection of miRNA mimics and inhibitors, the viability of HCE-T cells was measured using the water soluble tetrazolium salt (WST) assay, and microarray analysis and qPCR were performed using total RNA extracted from HCE-T cells. siRNAs of the candidate targets for miR-203 were transfected into HCE-T cells and the WST assay was performed. To determine a direct target gene for miR-203, a dual luciferase reporter assay was performed in HCE-T cells using a luciferase reporter plasmid containing 3′-UTR of human IGFBP5.ResultsMicroarray and qPCR analyses showed that miR-184 and miR-203 were expressed significantly more highly in tears than in sera (165,542.8- and 567.8-fold, respectively, p < 0.05). Of these two miRNAs, transfection of a miR-203 mimic significantly reduced the viability of HCE-T cells (p < 0.05), while a miR-203 inhibitor significantly increased this viability (p < 0.05). miR-203 mimic downregulated insulin-like growth factor-binding protein 5 (IGFBP5) and nuclear casein kinase and cyclin-dependent kinase substrate 1 (NUCKS1), while miR-203 inhibitor upregulated these two genes. Transfection of IGFBP5-siRNA decreased the viability of HCE-T cells. miR-203 mimic significantly diminished the luciferase reporter activity.ConclusionsIn this study, we identified miRNAs that are highly expressed in tears, and the inhibition of miR-203 increases the viability of corneal epithelial cells. Our results suggest that miR-203 contributes to regulating the homeostasis of corneal epithelial cells.

NUCKS1 Acts as a Promising Novel Biomarker for the Prognosis of Patients with Hepatocellular Carcinoma.

Objective: Nuclear casein kinase and cyclin-dependent kinase substrate 1 (NUCKS1) is highly expressed in some tumors, including hepatocellular carcinoma (HCC). However, its clinical significance in HCC prognosis is still unclear. The aim of this study was to explore the expression and prognostic value of NUCKS1 in HCC. Materials and Methods: Quantitative real-time polymerase chain reaction was used to detect relative expression of NUCKS1 mRNA in HCC tissues and corresponding adjacent normal tissues. The relationship between NUCKS1 expression and clinical characteristics of patients was analyzed by χ2 test. Kaplan-Meier method and Cox regression analysis were applied to estimate prognostic value of NUCKS1 in HCC. Results: Compared with normal ones, the expression of NUCKS1 mRNA was significantly upregulated in HCC tissues (p < 0.001). Besides, NUCKS1 expression was closely associated with tumor differentiation, tumor node metastasis stage, vascular invasion, and metastasis (p < 0.05). Kaplan-Meier analysis revealed that overall survival was obviously longer in HCC patients with low expression of NUCKS1 than those with high NUCKS1 expression (log rank test, p = 0.001). NUCKS1 might be an independent prognostic factor for HCC patients (HR = 1.905, 95% CI = 1.106-3.283, p = 0.020). Conclusions: NUCKS1 may be correlated with the progression of HCC and serve as a potential predictive factor for the prognosis of this disease.

NUCKS1 Promotes Proliferation, Invasion and Migration of Non-Small Cell Lung Cancer by Upregulating CDK1 Expression.

BackgroundNon-small cell lung cancer (NSCLC) is a predominant type of lung cancer with a high mortality rate.ObjectiveThe aim of this study is to investigate the roles of nuclear casein kinase and cyclin-dependent kinase substrate 1 (NUCKS1) in NSCLC and to identify the potential mechanisms.Materials and MethodsThe expression of NUCKS1 in several NSCLC cells was detected firstly. Then, NUCKS1 was overexpressed or silenced in both A549 and NCI-H460 cells, where cell proliferation, invasion and migration were, respectively, determined, using CCK-8, colony formation assay, transwell and wound healing assays. Cell cycle analysis was performed, and the expression-associated proteins were detected by Western blotting. Subsequently, NCI-H460 cells with NUCKS1 overexpression for the subsequent tumor-bearing experiment. And the NUCKS1 expression in tumor tissues was measured by means of immunohistochemistry and Western blotting. Additionally, the STRING database predicted that Cyclin-Dependent Kinase 1 (CDK1) would bind to NUSK1, which was verified by the co-immunoprecipitation assay. Then, CDK1 was silenced by transfection with short hairpin RNA (shRNA)-CDK-1 or by exposure to CDK1 inhibitor p2767-00. And the biological characteristics of proliferation, invasion and migration were examined.ResultsResults indicated that NUCKS1 was overly expressed in NSCLC cells, and its overexpression promoted proliferation, invasion and migration of both A549 and NCI-H460 cells while NUCKS1 knockdown displayed the opposite effects. Moreover, the results of the xenograft experiments revealed that NUCKS1-upregulation promoted the tumor growth. Furthermore, the immunoprecipitation assay verified CDK1’s interaction with NUCKS1, and CDK1 knockdown alleviates the impact of NUCKS1 overexpression on NSCLC cell proliferation, invasion and migration.ConclusionTaken together, these findings demonstrated that NUCKS1 promotes proliferation, invasion and migration of NSCLC by upregulating CDK1, providing a novel putative target for the clinical treatment of NSCLC.

A microRNA profile of pediatric glioblastoma: The role of NUCKS1 upregulation.

MicroRNAs (miRNAs/miRs) are a novel class of gene regulators that may be involved in tumor chemoresistance. Recently, specific miRNA expression profiles have been identified in adult glioblastoma (aGBM), but there are only limited data available on the role of miRNAs in pediatric GBM (pGBM). In the present study, the expression profile of miRNAs was examined in seven pGBMs and three human GBM cell lines (U87MG, A172 and T98G), compared with a non-tumoral pool of pediatric cerebral cortex samples by microarray analysis. A set of differentially expressed miRNAs was identified, including miR-490, miR-876-3p, miR-876-5p, miR-448 and miR-137 (downregulated), as well as miR-501-3p (upregulated). Through bioinformatics analysis, a series of target genes was predicted. In addition, similar gene expression patterns in pGBMs and cell lines was confirmed. Of note, drug resistant T98G cells had upregulated nuclear casein kinase and cyclin-dependent kinase substrate 1 (NUCKS1) expression, a protein overexpressed in many tumors that serves an important role in cell proliferation and progression. On the basis of the present preliminary report, it could be intriguing to further investigate the relationship between each of the identified differentially expressed miRNAs and NUCKS1, in order to clarify their involvement in the multi-drug resistance mechanism of pGBMs.

Nondysplastic Ulcerative Colitis Has High Levels of the Homologous Recombination Repair Protein NUCKS1 and Low Levels of the DNA Damage Marker Gamma-H2AX.

The colon and rectum are continuously exposed to oxidative stress that generates reactive oxygen species, which are a major cause of DNA double-strand breaks (DSB). Furthermore, chronic inflammatory diseases such as ulcerative colitis (UC) are characterized by an excess of reactive nitrogen species that can also lead to DNA double-strand breakage and genomic instability. We investigated the expression of the nuclear casein kinase and cyclin-dependent kinase substrate 1 (NUCKS1) protein in UC and sporadic colorectal cancer (CRC) due to its involvement in both DNA double-strand break repair and inflammatory signaling. NUCKS1 expression and expression of the DNA double-strand break marker gamma-H2AX (γH2AX) were assessed in formalin-fixed, paraffin-embedded UC and CRC patient biopsies using peroxidase immunohistochemistry. Expression levels for both proteins were evaluated together with previously published expression-level data for hTERT and TP53 proteins in the same material. Nondysplastic UC lesions had 10-fold lower γH2AX expression and approximately 4-fold higher NUCKS1 expression compared with sporadic CRC, indicating minimal DNA DSB damage and heightened DNA DSB repair in these lesions, respectively. NUCKS1 expression in UC tended to decrease with increasing grades of dysplasia, whereas γH2AX, hTERT, and TP53 expression tended to increase with increasing grades of dysplasia. The highest γH2AX expression was seen in sporadic CRC, indicating considerable DNA DSB damage, whereas the highest NUCKS1 expression and hTERT expression were seen in nondysplastic UC. Overall, our data suggest that NUCKS1 may be involved in DNA DSB repair and/or inflammatory signaling in UC, but a more thorough investigation of both pathways in UC is warranted.

Roles of NUCKS1 in Diseases: Susceptibility, Potential Biomarker, and Regulatory Mechanisms.

Nuclear casein kinase and cyclin-dependent kinase substrate 1 (NUCKS1) is a 27 kD chromosomal, highly conserved, and vertebrate-specific protein. NUCKS1 gene encodes a nuclear protein and the conserved regions of NUCKS1 contain several consensus phosphorylation sites for casein kinase II (CK2) and cyclin-dependent kinases (Cdk) and a basic DNA-binding domain. NUCKS1 is similar to the high mobility group (HMG) family which dominates chromatin remodeling and regulates gene transcription. Meanwhile, NUCKS1 is a RAD51 associated protein 1 (RAD51AP1) paralog that is significant for homologous recombination (HR) and genome stability and also a transcriptional regulator of the insulin signaling components. NUCKS1 plays an important role in DNA damage response and metabolism, participates in inflammatory immune response, and correlates with microRNA. Although there is still not enough functional information on NUCKS1, evidences suggest that NUCKS1 can be used as the biomarker of several cancers. This review summarizes the latest research on NUCKS1 about its susceptibility in diseases, expression levels, and regulatory mechanisms as well as the possible functions in reference to diseases.

Identification of NUCKS1 as a putative oncogene and immunodiagnostic marker of hepatocellular carcinoma

Although the molecular mechanisms underpinning hepatocellular carcinoma (HCC) are unknown, gene copy number and associated mRNA expression changes are frequently reported. Comparative genomic hybridization arrays spotted with 4041 bacterial artificial chromosome clones were used to assess copy number changes in 45 HCC tissues. Seventy more HCC tissues were used to validate candidate genes by using western blots and immunohistochemistry. A total of 259 clones were associated with copy number changes that significantly differed between normal liver and HCC samples. The chromosomal region 1q32.1 containing the nuclear casein kinase and cyclin-dependent kinase substrate 1 (NUCKS1) gene was associated with tumor vascular invasion. Western blot analysis demonstrated that NUCKS1 was up-regulated in 37 of 70 (52.8%) HCC tissues compared with adjacent non-tumor tissues, and over-expressed in a vast majority of HCCs (44/52, 84.6%) as determined by immunohistochemical staining. Furthermore, immunostaining of both NUCKS1 and glypican-3 improved the diagnostic prediction of HCC. Knock-down of NUCKS1 by siRNA implied the decrease in cell viability of the Hep3B cell line and reduced tumor formation in a xenograft mouse model. NUCKS1 was identified as a potential oncogene at chromosomal 1q32.1 in patients with HCC, and it might be a valuable immunodiagnostic marker for HCC.

MicroRNA-137 inhibits tumor growth and sensitizes chemosensitivity to paclitaxel and cisplatin in lung cancer.

Chemotherapy resistance frequently drives tumour progression. However, the underlying molecular mechanisms are poorly characterized. In this study, we explored miR-137's role in the chemosensitivity of lung cancer. We found that the expression level of miR-137 is down-regulated in the human lung cancer tissues and the resistant cells strains: A549/paclitaxel(A549/PTX) and A549/cisplatin (A549/CDDP) when compared with lung cancer A549 cells. Moreover, we found that overe-expression of miR-137 inhibited cell proliferation, migration, cell survival and arrest the cell cycle in G1 phase in A549/PTX and A549/CDDP. Furthermore, Repression of miR-137 significantly promoted cell growth, migration, cell survival and cell cycle G1/S transition in A549 cells. We further demonstrated that the tumor suppressive role of miR-137 was mediated by negatively regulating Nuclear casein kinase and cyclin-dependent kinase substrate1(NUCKS1) protein expression. Importantly, miR-137 inhibits A549/PTX, A549/CDDP growth and angiogenesis in vivo. Our study is the first to identify the tumor suppressive role of over-expressed miR-137 in chemosensitivity. Identification of a novel miRNA-mediated pathway that regulates chemosensitivity in lung cancer will facilitate the development of novel therapeutic strategies in the future.

NUCKS1 is a novel RAD51AP1 paralog important for homologous recombination and genome stability.

NUCKS1 (nuclear casein kinase and cyclin-dependent kinase substrate 1) is a 27 kD chromosomal, vertebrate-specific protein, for which limited functional data exist. Here, we demonstrate that NUCKS1 shares extensive sequence homology with RAD51AP1 (RAD51 associated protein 1), suggesting that these two proteins are paralogs. Similar to the phenotypic effects of RAD51AP1 knockdown, we find that depletion of NUCKS1 in human cells impairs DNA repair by homologous recombination (HR) and chromosome stability. Depletion of NUCKS1 also results in greatly increased cellular sensitivity to mitomycin C (MMC), and in increased levels of spontaneous and MMC-induced chromatid breaks. NUCKS1 is critical to maintaining wild type HR capacity, and, as observed for a number of proteins involved in the HR pathway, functional loss of NUCKS1 leads to a slow down in DNA replication fork progression with a concomitant increase in the utilization of new replication origins. Interestingly, recombinant NUCKS1 shares the same DNA binding preference as RAD51AP1, but binds to DNA with reduced affinity when compared to RAD51AP1. Our results show that NUCKS1 is a chromatin-associated protein with a role in the DNA damage response and in HR, a DNA repair pathway critical for tumor suppression.

NUCKS1 overexpression is a novel biomarker for recurrence-free survival in cervical squamous cell carcinoma.

Nuclear casein kinase and cyclin-dependent kinase substrate 1 (NUCKS1) is overexpressed in various cancer tissues and may therefore contribute to oncogenesis. However, the status of NUCKS1 expression in cervical squamous cell carcinoma (CSCC) remains unknown. Immunohistochemistry was used to determine the expression of NUCKS1 protein in 30 cervical intraepithelial neoplasias (CINs) and 125 CSCCs compared with 20 normal cervical specimens. The correlationships of NUCKS1 protein overexpression with the clinicopathologic characteristics and clinical outcomes in patients with CSCC were analysed. The status of NUCKS1 expression was negative or weak in normal tissues, but high in 21 (70.0 %) CINs and in 44 (35.2 %) CSCCs. NUCKS1 overexpression was associated with advanced International Federation of Gynaecology and Obstetrics stage (P = 0.016), poor histologic grade (P = 0.040), large tumour size (P = 0.016), parametrial involvement (P = 0.025), deep stromal infiltration (P = 0.043), lymph node metastasis (P = 0.034) and recurrence (P < 0.001). Multivariate analysis suggested that NUCKS1 overexpression was an independent factor for recurrence-free survival (RFS) (hazard ratio, 2.193; 95 % confidence interval, 1.060 to 4.535; P = 0.034). In conclusion, NUCKS1 overexpression may be associated with tumour progression and recurrence in CSCCs and may thus serve as a new molecular marker for the prediction of RFS in these patients.

Effect of NUCKS-1 Overexpression on Cytokine Profiling in Obese Women with Breast Cancer

Overweight and obesity are recognized as major drivers of cancers including breast cancer. Several cytokines, including interleukin-6 (IL-6), IL-10 and lipocalin 2 (LCN2), as well as dysregulated cell cycle proteins are implicated in breast carcinogenesis. The nuclear, casein kinase and cyclin-dependent kinase substrate-1 (NUCKS-1), is a nuclear DNA-binding protein that has been implicated in several human cancers, including breast cancer. The present study was conducted to evaluate NUCKS-1 mRNA expression in breast tissue from obese patients with and without breast cancer and lean controls. NUCKS-1 expression was correlated to cytokine profiles as prognostic and monitoring tools for breast cancer, providing a molecular basis for a causal link between obesity and risk. This study included 39 females with breast cancer (G III) that was furtherly subdivided into two subgroups according to cancer grading (G IIIa and G IIIb) and 10 control obese females (G II) in addition to 10 age-matched healthy lean controls (G I). NUCKS-1 expression was studied in breast tissue biopsies by means of real-time PCR (RT-PCR). Serum cytokine profiles were determined by immunoassay. Lipid profiles and glycemic status as well as anthropometric measures were also recorded for all participants. IL-6, IL-12 and LCN2 were significantly higher in control obese and breast cancer group than their relevant lean controls (p<0.05), while NUCKS-1 mRNA expression was significantly higher in the breast cancer group compared to the other groups (p<0.05). Significant higher levels of IL-6, IL-12, and LCN2 as well as NUCKS-1 mRNA levels were reported in G IIIb than G IIIa, and positively correlated with obesity markers in all obese patients. Evaluation of cytokine levels as well as related gene expression may provide a new tool for understanding interactions for three axes of carcinogenesis, innate immunity, inflammation and cell cycling, and hope for new strategies of management.