Expression Of Cyclin-dependent Kinase Research Articles

Role and mechanism of Circ-PDE7B in the formation of keloid.

The excessive proliferation of keloid fibroblasts is one of the important reasons leading to the formation of keloids. Circular RNA (circRNA) is an important regulator that regulates the biological functions of cells. However, the role and mechanism of circ-PDE7B in keloid formation have not been studied yet. QRT-PCR was used to detect the circ-PDE7B, miR-331-3p and cyclin-dependent kinase 6 (CDK6) expression. The biological functions of keloid fibroblasts were determined by MTT assay, flow cytometry, transwell assay and wound healing assay. Western blot analysis was used to measure the protein levels of extracellular matrix (ECM) markers and CDK6. The interaction between miR-331-3p and circ-PDE7B or CDK6 was confirmed by dual-luciferase reporter assay and RIP assay. Circ-PDE7B was found to be upregulated in keloid tissues and fibroblasts. Downregulation of circ-PDE7B could suppress the proliferation, invasion, migration, ECM accumulation and accelerate the apoptosis of keloid fibroblasts. Circ-PDE7B could serve as a sponge of miR-331-3p, and the regulation of silenced circ-PDE7B on the biological functions of keloid fibroblasts could be abolished by miR-331-3p inhibitor. Additionally, CDK6 was a target of miR-331-3p, and its overexpression could reverse the negative regulation of miR-331-3p on the biological functions of keloid fibroblasts. Circ-PDE7B sponged miR-331-3p to positively regulate CDK6 expression. Taken together, circ-PDE7B promoted the proliferation, invasion, migration and ECM accumulation of keloid fibroblasts by regulating the miR-331-3p/CDK6 axis, suggesting that circ-PDE7B might be a potential target for keloid treatment.

In vitro study of piwi interaction RNA-31106 promoting breast carcinogenesis by regulating METTL3-mediated m6A RNA methylation.

Breast cancer is the most common gynecological malignancy and the leading cause of cancer-related deaths in women. P-element induced wimpy testis (PIWI)-interacting RNAs (piRNAs) are novel non-coding RNAs whose abnormal expressions have been closely associated with multiple cancers. This study explored the roles and possible mechanisms of piRNA-31106 in breast cancer. The expression of piRNA-31106 in breast cancer tissues and cells was detected by reverse transcription polymerase chain reaction (RT-PCR). The pcDNA vector containing piRNA-31106 (pcDNA-piRNA-31106) and a short hairpin (sh)RNA containing piRNA-31106 (shRNA-piRNA-31106) were used to interfere with piRNA-31106 expression in breast cancer cells. The effects on cell proliferation, apoptosis/cell cycle, invasion, and metastasis were detected via Cell Counting Kit-8 (CCK-8), flow cytometry, transwell assays, and scratch tests, respectively. The protein expressions of murine double minute 2 (MDM2), cyclin-dependent kinase 4 (CDK4), and cyclinD1 were detected by Western blot analysis. The N6-methyladenosine (m6A) RNA methylation level and the binding relationship between piRNA-31106 and METTL3 were analyzed. The role of METTL3 in the regulation of breast cancer by piRNA-31106 was further analyzed by using small interfering (si)RNA targeting METTL3. PiRNA-31106 was highly expressed in breast cancer tissues and cell lines MDA-MB-231 and MCF-7. Overexpression of piRNA-31106 promoted the viability, invasion, and migration of breast cancer, inhibited apoptosis, and promoted the expressions of MDM2, CDK4, and cyclinD1. Inhibition of piRNA-31106 showed the opposite effect. In addition, piRNA-31106 promoted the m6A methylation levels and facilitated methyltransferase-like 3 (METTL3) expression in MDA-MB-231 and MCF-7 cells. RNA immunoprecipitation (RIP) assays confirmed the binding relationship between piRNA-31106 and METTL3. Further experiments demonstrated that si-METTL3 could inhibit the regulatory effects of piRNA-31106 on breast cancer. PiRNA-31106 was significantly highly expressed in breast cancer and could promote breast cancer progression by regulating METTL3-mediated m6A RNA methylation.

Inhibition of p90RSK Ameliorates PDGF-BB-Mediated Phenotypic Change of Vascular Smooth Muscle Cell and Subsequent Hyperplasia of Neointima.

Platelet-derived growth factor type BB (PDGF-BB) regulates vascular smooth muscle cell (VSMC) migration and proliferation, which play critical roles in the development of vascular conditions. p90 ribosomal S6 kinase (p90RSK) can regulate various cellular processes through many different target substrates in several cell types, but the regulatory function of p90RSK on PDGF-BB-mediated cell migration and proliferation and subsequent vascular neointima formation has not yet been extensively examined. In this study, we investigated whether p90RSK inhibition protects VSMCs against PDGF-BB-induced cellular phenotypic changes and the molecular mechanisms underlying the effect of p90RSK inhibition on neointimal hyperplasia in vivo. Pretreatment of cultured primary rat VSMCs with FMK or BI-D1870, which are specific inhibitors of p90RSK, suppressed PDGF-BB-induced phenotypic changes, including migration, proliferation, and extracellular matrix accumulation, in VSMCs. Additionally, FMK and BI-D1870 repressed the PDGF-BB-induced upregulation of cyclin D1 and cyclin-dependent kinase-4 expression. Furthermore, p90RSK inhibition hindered the inhibitory effect of PDGF-BB on Cdk inhibitor p27 expression, indicating that p90RSK may induce VSMC proliferation by regulating the G0/G1 phase. Notably, treatment with FMK resulted in attenuation of neointima development in ligated carotid arteries in mice. The findings imply that p90RSK inhibition mitigates the phenotypic switch and neointimal hyperplasia induced by PDGF-BB.

Abstract 5993: Dissecting the role of CDK4 in liposarcoma

Abstract Background: Liposarcoma (LPS) encompass one of most common soft tissue sarcoma (STS) histological subtypes accounting for 15% of all cases. They can be divided into four entities including atypical lipomatous tumor or well-differentiated LPS (ALT/WDLPS), dedifferentiated LPS (DDLPS), myxoid LPS (MLPS) and pleomorphic LPS (PLS). Their heterogeneity is reflected in their morphology, molecular landscape, prognosis and clinical behavior. Current treatment options for localized disease include surgery, (neo)adjuvant radiotherapy and chemotherapy; for the metastatic setting chemotherapy represents the cornerstone but its role needs to be elucidated. The unavailability of predictive biomarkers makes the management of these mesenchymal diseases even more challenging. Recent evidences have shed light on the key role of cyclin-dependent kinase 4 (CDK4) in cancer cells proliferation. Methods: This study involved 21 adult patients affected by liposarcoma (n= 5 ALT/WDLPS and n= 16 DDLPS). IHC analyses of CDK4 and MDM2 were performed on FFPE surgically resected specimens by experienced sarcoma pathologists. Moreover, a patient-derived DDLPS cell line was established and pharmacological profiling was performed. Next, standard and innovative drugs currently used for LPS management were assessed in both 2D and 3D culture systems. Finally, in silico analyses based on public repositories on STS were carried out including 260 sarcoma patients. Results: IHC results highlighted an higher expression of CDK4 and MDM2 biomarkers in DDLPS compared to ALT/WDLSP. Moreover, although CDK4 and MDM2 are codified by the same genomic region, their expression did not correlate (e.g. 100% of CDK4 and 2% of MDM2 in the same patient). Therefore we hypothesized that even exhibiting a low MDM2 expression, a patient could benefit from CDK4 inhibition. Furthermore, our data showed that in DDLPS cases CDK4 expression ranged from 90% to 100% in spermatic cord, skin and abdomen while it ranged from 5% to 50% in the retroperitoneum. The above observations prompted us to hypothesize a correlation of CDK4 expression with specific anatomical sites. Moreover, pharmacological profiling showed a synergistic effect exerted by sequential treatment with palbociclib and some chemotherapeutics including trabectedin, dacarbazine, eribulin and lenvatinib. Indeed we observed a 10% increase in cell proliferation inhibition compared to standard treatment. In addition, in silico analyses revealed the role of CDK4 as negative prognostic biomarker for PFS and OS in STS. Conclusions: Our study highlighted the promising role of CDK4 in the management of LPS patients. In particular, we pointed out encouraging results of sequential treatment combining CDK4 inhibitor palbociclib and chemotherapy. Furthermore, preliminary analyses highlighted the involvement of this biomarker as a potential prognostic tool for STS. Citation Format: Silvia Vanni, Graziana Gallo, Valentina Fausti, Giacomo Miserocchi, Chiara Liverani, Chiara Spadazzi, Claudia Cocchi, Chiara Calabrese, Giovanni De Luca, Massimo Bassi, Manlio Gessaroli, Angelo Campobassi, Federica Pieri, Giorgio Ercolani, Davide Cavaliere, Lorena Gurrieri, Nada Riva, Giovanni Martinelli, Laura Mercatali, Alessandro De Vita. Dissecting the role of CDK4 in liposarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5993.

Abstract 3759: Impact of individual and combined deregulation of miR-143 and miR-506 on cytokinetic and morphometric parameters in A549 lung cancer cells

Abstract Lung cancer (LC) is among the leading causes of death from malignancies worldwide. miRNAs (miRs), small noncoding RNAs capable of regulating gene expression through the cell’s RNAi mechanism, have emerged as promising molecules for cancer treatment. In our previous work, we identified two miRs, miR-143 and miR-506, whose transient transfection induced apoptosis, cell cycle inhibition, and cyclin-dependent kinase (CDK) 1, 4, and 6 downregulations. Here, we developed stable deregulations of the miRs’ expressions in A549 cells, individually or in combination, to evaluate their effect on LC cell behavior. To establish homogeneous populations of deregulated cells, A549 cells with stable up and downregulated miR-143 and miR-506, alone or in combination, were sorted according to marker expression using fluorescence-activated cell sorting (FACS). miR deregulations were confirmed with quantitative real-time polymerase chain reaction (qRT -PCR) analysis, and the cells were analyzed for changes in cell proliferation through cell cycle analysis using flow cytometry and cell doubling time using Quantitative Phase Imaging (QPI). With the same method, morphometric characteristics were quantified. Finally, we evaluated the motility of the cells using a scratch assay and determined the gene expression of CDKs. Taqman qPCR analysis indicated a >5-fold increase of the respective miR’s basal expression due to the stable transfections. The cell cycle analysis indicated a complex behavior for the individual miRs, while, in contrast, the combined upregulation of the two miRs indicated a G2 inhibition, representing a well-defined behavior in comparison to both the control and downregulation groups. Furthermore, the combined miR upregulation increased the cell doubling time compared to the control and respective downregulations. Similarly, using QPI microscopy, we observed that the miR combination upregulation had higher cellular sphericity, perimeter, and area compared to the control group. The wound healing assay did not indicate statistically significant differences among the different groups, with miR-506 upregulation demonstrating the slowest closure rate, followed by miR -143. In contrast, the differences between the miR-combination and control group were small, although the anti-miR-combination indicated an accelerated closure. In conclusion, the combined upregulation of both miRs inhibited cell proliferation through a G2 arrest, while the deregulation of the individual miRs demonstrated a complex behavior. Thus, the miR-combination’s consistent, promising behavior would indicate potential benefits using the two miRs against LC that merits further evaluation. Citation Format: Archana Shrestha, Behnaz Lahooti, Mahboubeh Madadi, Constantinos M. Mikelis, George Mattheolabakis. Impact of individual and combined deregulation of miR-143 and miR-506 on cytokinetic and morphometric parameters in A549 lung cancer cells. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3759.

Favorable Immunotherapy Plus Tyrosine Kinase Inhibition Outcome of Renal Cell Carcinoma Patients with Low CDK5 Expression.

Immunotherapy (IO) plus tyrosine kinase inhibitor (TKI) has become the first-line treatment for advanced renal cell carcinoma, despite the lack of prognostic biomarkers. Cyclin-dependent kinase 5 (CDK5) affects the tumor microenvironment, which may influence the efficacy of TKI+IO. Two cohorts from our center (Zhongshan Metastatic Renal Cell Carcinoma [ZS-MRCC] cohort, Zhongshan High-risk Localized Renal Cell Carcinoma [ZS-HRRCC] cohort) and one cohort from a clinical trial (JAVELIN-101) were enrolled. The expression of CDK5 of each sample was determined by RNA sequencing. Immune infiltration and T cell function were evaluated by flow cytometry and immunohistochemistry. Response and progression-free survival (PFS) were set as primary endpoints. Patients of low CDK5 expression showed higher objective response rate (60.0% vs. 23.3%) and longer PFS in both cohorts (ZS-MRCC cohort, p=0.014; JAVELIN-101 cohort, p=0.040). CDK5 expression was enhanced in non-responders (p < 0.05). In the ZS-HRRCC cohort, CDK5 was associated with decreased tumor-infiltrating CD8+ T cells, which was proved by immunohistochemistry (p < 0.05) and flow cytometry (Spearman's ρ=-0.49, p < 0.001). In the high CDK5 subgroup, CD8+ T cells revealed a dysfunction phenotype with decreased granzyme B, and more regulatory T cells were identified. A predictive score was further constructed by random forest, involving CDK5 and T cell exhaustion features. The RFscore was also validated in both cohorts. By utilizing the model, more patients might be distinguished from the overall cohort. Additionally, only in the low RFscore did TKI+IO outperform TKI monotherapy. High-CDK5 expression was associated with immunosuppression and TKI+IO resistance. RFscore based on CDK5 may be utilized as a biomarker to determine the optimal treatment strategy.

Sevoflurane-induced P300 promotes neuron apoptosis via Sp1/CDK9 pathway.

Exposure to sevoflurane leads to serious neurological side effects, including neuronal apoptosis and cognitive impairment. In the mouse model, cyclin dependent kinase 9 (CDK9) was significantly downregulated after exposure to sevoflurane, but the effect of CDK9 on neuronal apoptosis and cognitive impairment after sevoflurane exposure has not been elucidated. Here, we found that the upregulation of P300 by sevoflurane in vitro and in vivo inhibited the expression of CDK9 and induced neuron apoptosis. The effect of sevoflurane on CDK9 expression is based on inhibition of its transcription process. P300 inhibited the binding of Sp1 to DNA by affecting the level of Sp1 acetylation, thereby inhibiting the expression of CDK9, cell-cycle arrest and increasing neuron apoptosis. After the use of P300 inhibitor, the acetylation level of Sp1 decreased, thereby increasing binding in the CDK9 promoter region and exerting anti-apoptosis effects. Mice exposed to sevoflurane using P300 inhibitor also showed decreased levels of apoptosis of cortical cells and a decrease in recent cognitive impairment. In summary, sevoflurane-induced P300 inhibited activity of Sp1 by increasing Sp1 acetylation modification, down-modulates CDK9 expression and promotes the occurrence of neuronal apoptosis.

Combining bioinformatics and machine learning to identify common mechanisms and biomarkers of chronic obstructive pulmonary disease and atrial fibrillation.

Patients with chronic obstructive pulmonary disease (COPD) often present with atrial fibrillation (AF), but the common pathophysiological mechanisms between the two are unclear. This study aimed to investigate the common biological mechanisms of COPD and AF and to search for important biomarkers through bioinformatic analysis of public RNA sequencing databases. Four datasets of COPD and AF were downloaded from the Gene Expression Omnibus (GEO) database. The overlapping genes common to both diseases were screened by WGCNA analysis, followed by protein-protein interaction network construction and functional enrichment analysis to elucidate the common mechanisms of COPD and AF. Machine learning algorithms were also used to identify key biomarkers. Co-expression analysis, "transcription factor (TF)-mRNA-microRNA (miRNA)" regulatory networks and drug prediction were performed for key biomarkers. Finally, immune cell infiltration analysis was performed to evaluate further the immune cell changes in the COPD dataset and the correlation between key biomarkers and immune cells. A total of 133 overlapping genes for COPD and AF were obtained, and the enrichment was mainly focused on pathways associated with the inflammatory immune response. A key biomarker, cyclin dependent kinase 8 (CDK8), was identified through screening by machine learning algorithms and validated in the validation dataset. Twenty potential drugs capable of targeting CDK8 were obtained. Immune cell infiltration analysis revealed the presence of multiple immune cell dysregulation in COPD. Correlation analysis showed that CDK8 expression was significantly associated with CD8+ T cells, resting dendritic cell, macrophage M2, and monocytes. This study highlights the role of the inflammatory immune response in COPD combined with AF. The prominent link between CDK8 and the inflammatory immune response and its characteristic of not affecting the basal expression level of nuclear factor kappa B (NF-kB) make it a possible promising therapeutic target for COPD combined with AF.

Evaluation of CDK9 Inhibition by Dinaciclib in Combination with Apoptosis Modulating izTRAIL for the Treatment of Colorectal Cancer.

Treatment options for colorectal cancer (CRC), especially in advanced stages are still insufficient. There, the discovery of Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) was a bright spot. However, most cancers show resistance toward apoptotic signals. Cyclin-dependent kinase 9 (CDK9) plays a crucial role in cell cycle progression in most tissues. We recently demonstrated the role of CDK9 in mediating TRAIL resistance. In this work, we investigated the role of CDK9 in colorectal cancer. Immunohistochemical analysis of CDK9 expression in cancer and normal tissues of CRC specimens was performed. The effect of selective CDK9 inhibition in combination with TRAIL on CRC cells was analyzed via cell viability, colony formation, and induction of apoptosis by flow cytometry. The mechanism of action was conducted via western blotting. We now have confirmed overexpression of CDK9 in cancer tissues, with low expression associated with poorer survival in a subset of CRC patients. In-vitro, CDK9 inhibition could strongly promote TRAIL-induced cell death in TRAIL-resistant CRC cells. Mechanistically, CDK9 inhibition induced apoptosis by downregulation of antiapoptotic proteins, myeloid leukemia cell differentiation protein 1 (Mcl-1) and FLICE-inhibitory protein (c-FLIP). Overall, we identified CDK9 as a prognostic marker and combined CDK9 inhibition and TRAIL as a novel and promising therapeutic approaches for colorectal cancer.

Immunohistochemical Expression of p16 and CDK4 in Soft Tissue Tumors.

The aim of this study was to determine the immunohistochemical expression of p16 (p16INK4a) and cyclin-dependent kinase 4 (CDK4) and CDK4 markers in various lineages of soft tissue tumors and to evaluate their role in differentiating atypical lipomatous tumors/well-differentiated liposarcomas from benign lipomas. A total of 70 cases of both excisional and incisional biopsies of soft tissue tumors were included in this study. Histopathological examination was done by using formalin-fixed, paraffin-embedded tissue samples. After that, we performed IHC expressions of p16 and CDK4 markers on the unstained slides of these soft tissue tumors. Results: Immunohistochemical study showed that positive expressions of p16, CDK4, and combined (p16+CDK4) markers were 51.4%, 10.0%, and 12.9%, respectively in soft tissue tumors. Positive p16 expression was observed among a higher proportion of malignant cases (66.7%) as compared to benign (20.0%) and intermediate (50.0%) cases. This difference was found to be statistically significant (p=0.009). Negative expression of only CDK4 and combined (p16 and CDK4) were observed among a higher proportion of benign as compared to malignant and intermediate cases (90.0% vs. 78.6% & 75.0%, p=0.393 and 65.0% vs. 26.2% & 37.5%, p=0.028, respectively). This difference was not found to be statistically significant. For adipocytic tumors, the majority of malignant and intermediate tumors had positive p16 (7/7; 100%) and CDK4 (6/7; 85.7%) immunohistochemical expression. These differences were found to be statistically significant. Immunohistochemical marker p16 can be used to differentiate between malignant and benign soft tissue tumors. Amongst adipocytic tumors, combination of p16 and CDK4 immunohistochemical expression can be used to differentiate liposarcomasfrom benign ones.

Identification of prognosis-related cyclin-dependent kinases and potential response drugs in hepatocellular carcinoma

Which cyclin-dependent kinases (CDKs) involved in the progress of hepatocellular carcinoma (HCC) need to be further clarified. To identify prognostic-relevant biomarkers in HCC through a systematic investigation of the prognostic value of CDKs. We explored the relationship between CDKs expression and the prognosis of patients with HCC using multiple online databases. In addition, their biological functions and correlation with the immune system and drug response were investigated. Among the 20 CDKs (CDK1 ~20) altered in HCC, the significantly high expression of CDK1 and CDK4 in patients with HCC was significantly associated with worse prognosis. Interestingly, CDK1 had significant co-occurrence with CDK4 and CDK1-related and CDK4-related signaling pathways are closely related to hepatitis virus-related HCC. We identified multiple transcription factors of CDK1 and CDK4; of those, only four (E2F1, PTTG1, RELA, and SP1) were significantly associated with the prognosis of HCC patients. Genetic alterations in CDKs were significantly correlated with disease-free and progression-free survival, which may be associated with aberrant expression of progesterone receptor. Moreover, we found a significantly positive correlation between CDK1 and CDK4 expression and tumor-infiltrating activated CD4+ T cell and exhausted T cell-related signature. Finally, we identified drugs with good potential prognostic value predicted by CDK1 and CDK4 levels. CDK1 and CDK4 may be potential prognostic biomarkers for HCC. Moreover, targeting four transcription factors (E2F1, PTTG1, RELA, and SP1) combined with immunotherapy may be a new therapeutic strategy for treating HCC patients with high CDK1 and CDK4 expression, especially hepatitis-related HCC.

Comprehensive analysis ofCDK5 as a novel biomarker for progression in esophageal cancer.

Cyclin-dependent kinase 5 (CDK5) is a member of the cyclin-dependent kinase family, and unlike the rest of the members of the family, its kinase activity is independent of cyclins. Accumulating evidence has shown that CDK5 plays a significant role in the progress of tumorigenesis except in nervous system. In particular, the expression of CDK5 and its function in esophageal cancer (ESCA) remain unknown. With TCGA and GEO databases, CDK5 was analyzed with the expression, predicted value, clinical relationship, functional enrichment, immune cell infiltration and immune molecules in ESCA. In addition, we explored the CDK5 expression with local datasets and the influence of CDK5 on proliferation, migration and invasion behaviors of the esophageal squamous cell carcinoma (ESCC) cells in vitro and in vivo experiments. CDK5 expression was upregulated in ESCA, and this regulation has been verified in cell lines of ESCC. Further analysis has found that the expression of CDK5 was correlated with race, weight, BMI, histological type and tumor central location in ESCA. KEGG analysis revealed that CDK5 was involved in the progress of cancers, innate immune system and PI3K-Akt signaling pathway. CDK5 was closely related to immune cells and immune molecules in ESCA. Functional experiments confirmed CDK5 was an oncogene in ESCC by in vivo and in vitro models. This study shows that CDK5 is a risk factor to promote tumor progression, and Roscovitine could be one of the effective tools in the therapy of ESCA.

Identification of potential biomarkers for colorectal cancer by clinical database analysis and Kaplan-Meier curves analysis.

This study aimed to explore critical genes as potential biomarkers for the diagnosis and prognosis of colorectal cancer (CRC) for clinical utility. To identify and screen candidate genes involved in CRC carcinogenesis and disease progression, we downloaded microarray datasets GSE89076, GSE73360, and GSE32323 from the GEO database identified differentially expressed genes (DEGs), and performed a functional enrichment analysis. A protein-protein interaction network was constructed, and correlated module analysis was performed using STRING and Cytoscape. The Kaplan-Meier survival curve shows the survival of the hub genes. The expression of cyclin-dependent kinase (CDK1), cyclin B1 (CCNB1), and PCNA in tissues and changes in tumor grade were analyzed. A total of 329 DEGs were identified, including 264 upregulated and 65 downregulated genes. The functions and pathways of DEGs include the mitotic cell cycle, poly(A) RNA binding replication, ATP binding, DNA replication, ribosome biogenesis in eukaryotes, and RNA transport. Forty-seven Hub genes were identified, and biological process analysis showed that these genes were mainly enriched in cell cycle and DNA replication. Patients with mutations in CDK1, PCNA, and CCNB1 had poorer survival rates. CDK1, PCNA, and CCNB1 were significantly overexpressed in the tumor tissues. The expression of CDK1 and CCNB1 gradually decreased with increasing tumor grade. CDK1, CCNB1, and PCNA can be used as potential markers for the diagnosis and prognosis of CRC. These genes are overexpressed in colon cancer tissues and are associated with low survival rates in CRC patients.

MiR-195-5p Targets CDK1 To Regulate New DNA Synthesis and Inhibit the Proliferation of Hepatocellular Carcinoma Cells.

In cell biological functions and viability, cyclin-dependent kinase 1 (CDK1) takes an essential part. miR-195-5p is pivotal in pathogenesis and development of hepatocellular carcinoma (HCC). But in HCC, whether there is a connection between CDK1 and miR-195-5p remains an unanswered question. In view of this, this study focuses on exploring the mechanism of miR-195-5p/CDK1 in the progression of HCC. The bioinformatics method was applied to predict target mRNA and upstream miRNAs, and further analyzes the signal enrichment pathway of target mRNA. We utilized qRT-PCR and Western blot for detecting expression of genes, as well as their corresponding protein levels. Cell cycle was assayed through flow cytometry. As for the examination of DNA replication, the EDU staining was employed. Cell proliferation was determined via plate colony formation assay. The combined application of bioinformatics analysis and dual-luciferase gene assay assisted in figuring out the binding relationship between miR-195-5p and CDK1. DNA damage was marked by immunofluorescence staining. CDK1 was overexpressed in HCC cells, and enriched in cell cycle and DNA replication pathway. Silencing CDK1 modulated cell cycle of HCC cells and inhibited DNA replication and proliferation. In HCC cells, miR-195-5p targeted and reduced CDK1 expression, inhibited the G1 phase-to-S phase transition, induced DNA damage response, and inhibited DNA replication and proliferation. miR-195-5p targeted CDK1 and repressed synthesis of new DNA in HCC cells, thus restraining HCC cell proliferation.

MAST4 controls cell cycle in spermatogonial stem cells.

Spermatogonial stem cell (SSC) self-renewal is regulated by reciprocal interactions between Sertoli cells and SSCs in the testis. In a previous study, microtubule-associated serine/threonine kinase 4 (MAST4) has been studied in Sertoli cells as a regulator of SSC self-renewal. The present study focused on the mechanism by which MAST4 in Sertoli cells transmits the signal and regulates SSCs, especially cell cycle regulation. The expression of PLZF, CDK2 and PLZF target genes was examined in WT and Mast4 KO testes by Immunohistochemistry, RT-qPCR and western blot. In addition, IdU and BrdU were injected into WT and Mast4 KO mice and cell cycle of SSCs was analysed. Finally, the testis tissues were cultured in vitro to examine the regulation of cell cycle by MAST4 pathway. Mast4 KO mice showed infertility with Sertoli cell-only syndrome and reduced sperm count. Furthermore, Mast4 deletion led to decreased PLZF expression and cell cycle progression in the testes. MAST4 also induced cyclin-dependent kinase 2 (CDK2) to phosphorylate PLZF and activated PLZF suppressed the transcriptional levels of genes related to cell cycle arrest, leading SSCs to remain stem cell state. MAST4 is essential for maintaining cell cycle in SSCs via the CDK2-PLZF interaction. These results demonstrate the pivotal role of MAST4 regulating cell cycle of SSCs and the significance of spermatogenesis.

Higher Levels of Tumour-Infiltrating Lymphocytes (TILs) are Associated with a Better Prognosis, While CDK5 Plays a Different Role Between Nonmetastatic and Metastatic Colonic Carcinoma.

We investigated the prognostic value of cyclin-dependent kinase 5 (CDK5) in a true population-based cohort of patients with colon cancer. 1. Immunohistochemical (IHC) staining was used to retrospectively analyse the expression of CDK5 in colon cancer tissue samples of 296 patients. The χ2 test, Kaplan-Meier method and Cox proportional regression model were used to analyse the difference between the patients with differential expression of CDK5 and with different stages (metastatic and nonmetastatic); 2. The number of tumour-infiltrating lymphocytes (TILs) in tumour sections was determined, and its relationship with prognosis was explored. 1. Among 296 patients stained for CDK5, 18 cases (6.09%) showed negative expression, 77 cases (26.01%) showed weak expression (+1), 124 cases (41.89%) showed medium positive expression (2+), and 77 cases (26.01%) showed strong positive expression (3+). The expression of CDK5 was neither related to mismatch repair nor TILs (p > .05). In non-metastatic patients, longer progression-free survival (PFS) and cancer-specific survival (CSS) were observed in patients with high CDK5 expression (2+ or 3+) than low CDK5 expression (- or 1+), while in metastatic disease, the opposite was true (p < .001). 2. TILs in 226 patients were detected in the study. Among them, 115 cases (50.88%) showed a low number of TILs (TILs-L), and 111 cases (49.12%) showed a high number of TILs (TILs-H). Patients with a TIL ratio greater than .2 had a significantly better CSS (p < .001) or PFS (p = .008) than patients with a lower TIL ratio. By multivariate analysis, TILs-H was a protective factor for CSS, however failed to reach a significant difference (hazard ratio: .59, 95% CI: .33∼1.06, p = .079), and so was the PFS (HR: .65, 95% CI: .29∼1.43, p = .279). High expression of CDK5 indicates a good prognosis in nonmetastatic colon cancer, while it is the opposite in metastatic colon cancer, and the expression of CDK5 is unrelated to TILs. Patients with TIL-H have a better prognosis, with a proper cut-off value of 20% for colon cancer.

Nc-RNA-mediated high expression of CDK6 correlates with poor prognosis and immune infiltration in pancreatic cancer.

Emerging evidence manifests that cyclin-dependent kinase 6 (CDK6) plays an essential part in the initiation and progression of several types of human cancer, and its descending expression is correlated with an adverse prognosis. However, the precise role of CDK6 in Pancreatic cancer (PC) remains obscure. To identify the potential ceRNA regulatory axis of CDK6 in PC and explore its relationship with immune cells and immune checkpoints. Using The Cancer Genome Atlas TCGA and GTEx data analyze the expression and survival of CDK6 in patients in pan-cancer, and cellular experiments were performed to verify the effect of CDK6 on cell function. Using GEPIA and STARBASE databases to analyze prognosis, expression and survival, and identify non coding RNA (ncRNA) that mediates CDK6 overexpression. The TIMER 2.0 database was used for immune correlation analysis. We revealed CDK6 might be an oncogene in PC, and the HOXA11-AS /NR2F1-AS1- miR-454-3p axis was identified as the possible upstream ncRNA-associated pathway of CDK6 in PC. In addition, CDK6 show significant association with three immune checkpoints (PD-L1, PD-L2, and HAVCR2), the infiltration level of immune cells, and immunity biomarkers. We discussed some applications of CDK6 in breast cancer, melanoma, and hemorrhagic malignancies. The role of miR-15a-5p, HOXA11-AS and NR2F1-AS1 in tumor development was also discussed based on existing studies. The potential mechanism of CDK6 affecting immune cells in pancreatic cancer was discussed. Overall, these results established that nc-RNA-mediated high expression of CDK6 is associated with patient outcomes and immune invasion in pancreatic cancer.

Used Toothbrush as a Potential Source of Gene Expression Among Subjects With Systemic Disease and Adverse Habits.

Purpose To explore the viability of used toothbrush as a source of gene expression and comparison of the same among tobacco, alcohol, diabetic and hypertensive subjects. Materials and methods Fourteen subjects with a history of type 2 diabetes mellitus (T2DM) and hypertension were allocated to Group I, 14 subjects with tobacco and alcohol habits allocated to Group II and 14 healthy subjects allocated to Group III. Genetic materials retrieved from the used toothbrush of the study subjects were assessed for genetic expression of cyclin-dependent kinase 4 (CDK4), Bcl-2-like protein 4 (BAX), cyclin-dependent kinase inhibitor 2A (CDKN2A), B-cell lymphoma 2 (BCL2), G-protein beta-3 (GNB3), and subunit gene and transcription factor 7-like 2 (TCF7L2) by polymerase chain reaction (PCR). Results BAX gene showed reduced expression in tobacco and alcohol users (p=0.0497). BCL2, CDK4, and GNB3 showed no significant difference in expression in both the groups and with control. CDKN2A was expressed at a lower level in Group I and II participants. TCF7L2 showed higher expression in Group 2 participants (p=0.001). Conclusion The study concluded that used toothbrush is a reliable source for genetic expression. There was no difference in BCL2, CDK4, and GNB3 gene expression between subjects with systemic disease, adverse habits and healthy controls. There is a downregulation of BAX and upregulation of TCF7L2 gene in subjects with adverse habits.

Prognostic Impact of Caspase-8, CDK9 and Phospho-CDK9 (Thr 186) Expression in Patients with Uterine Cervical Cancer Treated with Definitive Chemoradiation and Brachytherapy.

Introduction: After primary platinum-based chemoradiation of locally advanced uterine cervical cancer, a substantial proportion of women present with persistent, recurrent or metastatic disease, indicating an unmet need for biomarker development. Methods: We evaluated the clinical records of 69 cervical cancer patients (Federation of Gynecology and Obstetrics, FIGO Stage &gt; IB3) who were subjected to definitive CRT. Immunohistochemical scoring of caspase-8, cyclin dependent kinase 9 (CDK9) and phosphorylated (phospho-)CDK9 (threonine (Thr) 186) was performed on pretreatment samples and correlated with the histopathological and clinical endpoints, including relapse-free survival (RFS), distant metastasis-free survival (DMFS), cancer-specific survival (CSS) and overall survival (OS). Results: Lower levels of caspase-8 were more prevalent in patients with a higher T-stage (p = 0.002) and a higher FIGO stage (p = 0.003), and were significantly correlated with CDK9 expression (p = 0.018) and inversely with pCDK9 detection (p = 0.014). Increased caspase-8 levels corresponded to improved RFS (p = 0.005), DMFS (p = 0.038) and CSS (p = 0.017) in the univariate analyses. Low CDK9 expression was associated with worse RFS (p = 0.008), CSS (p = 0.015) and OS (p = 0.007), but not DMFS (p = 0.083), and remained a significant prognosticator for RFS (p = 0.003) and CSS (p = 0.009) in the multivariate analyses. Furthermore, low pCDK9 staining was significantly associated with superior RFS (p = 0.004) and DMFS (p = 0.001), and increased CSS (p = 0.022), and remained significant for these endpoints in the multivariate analyses. Conclusion: Increased caspase-8 and CDK9 levels correlate with improved disease-related outcomes in cervical cancer patients treated with CRT, whereas elevated pCDK9 levels predict worse survival in this patient population.

MicroRNA-15 suppresses viability, migration and invasion of the human MG-63 osteosarcoma cells via inhibition of cyclin dependent kinase 6 (CDK6).

MicroRNA-15a-3p (miR-15) acts as tumor-suppressor in different human cancers including osteosarcoma. Nonetheless, the molecular function of miR-15 in osteosarcoma via suppression of cyclin dependent kinase 6 (CDK6) is yet to be studied. The results showed significant downregulation of miR-15 in osteosarcoma tissues and cell lines. Overexpression of miR-15 inhibited the proliferation and colony formation of the MG-63 osteosarcoma cells via induction of apoptosis. Moreover, miR-15 inhibited the migration and invasion of MG-63 osteosarcoma cells. The tumor-suppressive functional role of miR-15 was shown to be exerted via suppression of CDK6. The expression of CDK6 was upregulated in osteosarcoma and its silencing could exert growth inhibitory effects on human osteosarcoma cells. However, overexpression of CDK6 could nullify the tumor-suppressive effects of miR-15 on the MG-63 osteosarcoma cells. Taken together, miR-15 negatively regulates growth, migration and invasion of osteosarcoma cells by targeting CDK6 at post-transcriptional level. These findings suggest the therapeutic potential of miR-15/CDK6 in human osteosarcoma.