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Long noncoding (Lnc) RNAs are novel regulators in melanoma. Lnc nuclear enriched autosomal transcript 1 (NEAT1) was reportedly upregulated in melanoma; however, the functional roles and mechanisms of Lnc NEAT1 need further investigation. Therefore, we used quantitative real-time PCR to determine the mRNA levels of Lnc NEAT1, miR-152-3p, and cyclin-dependent protein kinase 6 (CDK6). The protein level of CDK6 was determined by Western blot. Cell counting kit 8 and colony formation assays were used to assess cell proliferation. Cell migration was measured by wound healing and Transwell assays. Direct binding of the indicated molecules was verified by an RNA-binding protein immunoprecipitation assay and a dual luciferase reporter assay. The results revealed that Lnc NEAT1 and CDK6 were elevated, while miR-152-3p was downregulated in melanoma. Furthermore, Lnc NEAT1 was positively correlated with CDK6 expression and negatively correlated with miR-152-3p level. Furthermore, Lnc NEAT1 facilitated proliferation, migration, and invasion of melanoma cells. The underlying mechanism is that Lnc NEAT1 serves as a sponge for miR-152-3p to suppress the inhibitory effect of miR-152-3p on CDK6. Furthermore, the miR-152-3p/ CDK6 axis was implicated in the progression of melanoma accelerated by Lnc NEAT1. Taken together, Lnc NEAT1 may promote melanoma development by serving as an endogenous sponge of miR-152-3p, increasing CDK6 expression, and identifying a new target for the treatment of melanoma., Competing Interests: The authors have no conflicts of interest to disclose., (Copyright © 2024 the Author(s). Published by Wolters Kluwer Health, Inc.)

Colorectal cancer (CRC) represents a substantial challenge to public health. Despite extensive research, the pathogenesis of CRC is not yet fully elucidated, hindering the development of effective therapeutic strategies. Recent advancements have underscored the importance of Non-coding RNAs in tumor biology. Our research identified a significant upregulation of Linc00475 in CRC, which correlated with reduced survival rates among CRC patients. Consequently, this study aimed to elucidate the mechanisms by which Linc00475 contributed to CRC progression. Employing a comprehensive array of experimental techniques-including CCK-8 assays, colony formation assays, flow cytometry, quantitative PCR (qPCR), western blot analysis, and in vivo tumorigenesis assays-we have demonstrated that Linc00475 enhances CRC cell proliferation. Further analysis revealed that Linc00475 directly interacted with miR-107, leading to its downregulation. Moreover, our findings confirmed that miR-107 directly targeted CDK6, which was markedly downregulated following Linc00475 silencing. In vivo experiments further indicated that the silencing of Linc00475 markedly inhibited the proliferation of CRC cells. Collectively, our findings suggested that Linc00475 facilitated CRC cell proliferation through the regulation of the miR-107/CDK6 axis, thereby providing a novel perspective for understanding the molecular mechanisms underlying CRC development., (© 2024 Wiley Periodicals LLC.)

Purpose: Treatment paradigms for isocitrate dehydrogenase (IDH)-mutant gliomas are rapidly evolving. Although typically indolent and responsive to initial treatment, these tumors invariably recur at a higher grade and require salvage treatment. Homozygous deletion of the tumor suppressor gene CDKN2A/B frequently emerges at recurrence in these tumors, driving poor patient outcomes. We investigated the effect of CDK-Rb pathway blockade on IDH-mutant glioma growth in vitro and in vivo using CDK4/6 inhibitors (CDKi)., Experimental Design: Cell viability, proliferation assays, and flow cytometry were used to examine the pharmacologic effect of two distinct CDKi, palbociclib and abemaciclib, in multiple patient-derived IDH-mutant glioma lines. Isogenic models were used to directly investigate the influence of CDKN2A/B status on CDKi sensitivity. Orthotopic xenograft tumor models were used to examine the efficacy and tolerability of CDKi in vivo., Results: CDKi treatment leads to decreased cell viability and proliferative capacity in patient-derived IDH-mutant glioma lines, coupled with enrichment of cells in the G1 phase. CDKN2A inactivation sensitizes IDH-mutant glioma to CDKi in both endogenous and isogenic models with engineered CDKN2A deletion. CDK4/6 inhibitor administration improves survival in orthotopically implanted IDH-mutant glioma models., Conclusions: IDH-mutant gliomas with deletion of CDKN2A/B are sensitized to CDK4/6 inhibitors. These results support the investigation of the use of these agents in a clinical setting., (©2024 American Association for Cancer Research.)

Pharyngeal arch artery (PAA) progenitors undergo proliferative expansion and angioblast differentiation to build vessels connecting the heart with the dorsal aortae. However, it remains unclear whether and how these two processes are orchestrated. Here we demonstrate that Tmem88 is required to fine-tune PAA progenitor proliferation and differentiation. Loss of zebrafish tmem88a/b leads to an excessive expansion and a failure of differentiation of PAA progenitors. Moreover, tmem88a/b deficiency enhances cyclin D1 expression in PAA progenitors via aberrant Wnt signal activation. Mechanistically, cyclin D1-CDK4/6 promotes progenitor proliferation through accelerating the G1/S transition while suppressing angioblast differentiation by phosphorylating Nkx2.5/Smad3. Ectodermal Wnt2bb signaling is confined by Tmem88 in PAA progenitors to ensure a balance between proliferation and differentiation. Therefore, the proliferation and angioblast differentiation of PAA progenitors manifest an inverse relationship and are delicately regulated by cell cycle machinery downstream of the Tmem88-Wnt pathway., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

The present study aimed to investigate the effect of miR-186 on proliferation, migration, invasion, and epithelialmesenchymal transition (EMT) of hepatocellular carcinoma (HCC). In this work, miR-186 was downregulated in HCC tissues and cells, and low miR-186 level helped predict the occurrence of vascular invasion and poor prognosis in patients with HCC. miR-186 overexpression inhibited cell proliferation and tumor growth in nude mice, repressed migration and invasion abilities, and enhanced apoptosis in HCC cells. miR-186 also retarded progression of EMT. miR-186 directly bound to the 3-untranslated regions of cyclin-dependent kinase 6 (CDK6) to inhibit its expression. Overexpression of CDK6 markedly reversed inhibitory effects of miR-186 on proliferation, apoptosis, migration, and invasion of HCC cells. Conversely, inhibition of CDK6 exerted synergic effect on the biological functions of miR-186. In conclusion, miR-186 represses proliferation, migration, invasion, and EMT, and induces apoptosis through targeting CDK6 in HCC, which may provide a new therapeutic target for HCC.

Non-small-cell lung cancer (NSCLC) is the main pathological type of lung cancer and has a low overall five-year survival rate. miR-187 has been reported to play major roles in various tumor types. In this study, we explored the impact of miR-187 on NSCLC. qRT-PCR results demonstrated that miR-187 expression is lower in NSCLC and cancer cells than normal tissues and normal lung cells. miR-187 expression levels are associated with tumor size, TNM stage and overall survival rate. MTS and colony formation assays showed that high miR-187 expression inhibits NSCLC cell proliferation and colony formation ability, and flow cytometry showed that miR-187 overexpression induces cell cycle arrest at the G0/G1 phase. A luciferase reporter assay showed that FGF9 is a target of miR-187. miR-187 overexpression reduces the expression of FGF9, cyclin D1 CDK4 and CDK6. Therefore, miR-187 may present a new NSCLC treatment target by regulates cyclins-related protein expression.

The present study aimed to investigate the expression of cyclin-dependent kinase 6 (CDK6) and microRNA-126-5p (miR-126-5p) in esophageal cancer tissues and cells, and their effect on esophageal cancer cell proliferation and invasion, and to explore the potential molecular mechanisms. The relative expression levels of CDK6 and miR-126-5p in esophageal cancer tissue, paracancerous tissue, and HEEC and EC109 cells were determined and compared using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). A miR-126-5p overexpression vector was constructed and a stable EC109 cell line expressing miR-126-5p was established. The EC109 cell line was transfected with a CDK6 small interfering RNA sequence. The rate of cell proliferation was determined using the WST-8 method, and cell invasion was determined using a Transwell assay. In addition, the relative expression levels of genes were determined using RT-qPCR; the relative expression levels of proteins were determined by western blot analysis; the binding sites of CDK6 and miR-126-5p were analyzed using TargetScan software; and the interaction of CDK6 and miR-126-5p was verified using dual-fluorescence reporter gene expression. Esophageal tissues and EC109 cells expressed higher levels of CDK6 but significantly lower levels of miR-126-5p compared with adjacent tissues and HEEC cells, and their correlation coefficient between esophageal tissues and matched adjacent tissues was -7.526. The overexpression of miR-126-5p and CDK6 knockdown in the EC109 cell line inhibited cell proliferation and invasion compared with the control and NC (negative control) groups. miR-126-5p overexpression reduced the relative expression level of CDK6, and CDK6 knockdown by siRNA increased the expression of miR-126-5p. miR-126-5p regulated CDK6 expression by binding to the 3'-untranslated region of its mRNA. Overexpression miR-126-5p inhibited the proliferation and migration of esophageal cancer cells by targeting CDK6 and negatively regulating its expression. These findings contribute to the understanding of the underlying molecular mechanism of esophageal cancer., (© 2020 American Association for Anatomy.)

This study aimed to investigate the function of long non-coding RNA FOXD2 adjacent opposite strand RNA 1 (lncRNA FOXD2-AS1) during the progression of esophagus cancer (EC) and explore its underlying molecular mechanisms. The level of FOXD2-AS1 in EC tissues and paracancerous tissues was detected by using RT-qPCR; ROC curve was used to evaluate the diagnostic value of FOXD2-AS1 for EC. In addition, CCK8 assay and immunofluorescence staining assay were used to detect the proliferation of Eca-109 and TE-1 cells. To investigate the function of FOXD2-AS1 on cell apoptosis and cell cycle, flow cytometry was performed. To detect the invasion ability of EC cells, transwell invasion assay was performed. Starbase3.0 and Targetscan were used to predict the target genes of FOXD2-AS1 and miR-145-5p, and protein expressions were detected with western blot. We found FOXD2-AS1 was significantly upregulated in EC tissues compared with adjacent normal tissues, which was positively correlated with clinicopathological parameters of patients with EC. Downregulation of FOXD2-AS1 inhibited the proliferation and invasion by inducing apoptosis of EC cells. Moreover, FOXD2-AS1 may regulate the expression of CDK6 by targeting miR-145-3p. Meanwhile, silencing of FOXD2-AS1 caused G1 phase arrest of EC cells by reducing the expression of CDK6. In conclusion, silening FOXD2-AS1 significantly inhibited the proliferation and invasion of EC cells by regulating the miR-145-5p/CDK6 axis. Therefore, FOXD2-AS1 might be used as diagnostic biomarker and therapeutic target for EC.

Upregulation of histone methyltransferase SET domain bifurcated 1 (SETDB1) is associated with poor prognosis in cancer patients. However, the mechanism of oncogenicity of SETDB1 in cancer is hitherto unknown. Here, we show that SETDB1 is upregulated in human colorectal cancer (CRC) where its level correlates with poor clinical outcome. Ectopic SETDB1 promotes CRC cell proliferation, whereas SETDB1 attenuation inhibits this process. Flow cytometry reveals that SETDB1 promotes proliferation by driving the CRC cell cycle from G0/G1 phase to S phase. Mechanistically, SETDB1 binds directly to the STAT1 promoter region resulting in increased STAT1 expression. Functional characterization reveals that STAT1-CCND1/CDK6 axis is a downstream effector of SETDB1-mediated CRC cell proliferation. Furthermore, SETDB1 upregulation is sufficient to accelerate in vivo proliferation in xenograft animal model. Taken together, our results provide insight into the upregulation of SETDB1 within CRC and can lead to novel treatment strategies targeting this cell proliferation-promoting gene., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Increasing evidence indicates that miRNAs are involved in tumorigenesis of human renal cell carcinoma (RCC). However, the role of miR-206 is still unknown. This study aimed to investigate the possible mechanism of miR-206 in progression of RCC. Here, compared with adjacent normal renal tissues and HK-2 cells, miR-206 level was markedly decreased, whereas CDK6 level was obviously increased in RCC tissues and cell lines. MiR-206 was inversely associated with lymph node metastasis and TNM stage, and acted as an independent prognostic factor in RCC. MiR-206 effectively caused apoptosis and cell cycle arrest at G0/G1 phase, and affected the growth of xenograft tumor of nude mice. MiR-206 also inhibited migration and invasion of RCC cells by modulating the expressions of EMT-related genes. Dual-luciferase assay demonstrated CDK6 was a direct target of miR-206. CDK6 silencing aggravated the inhibition effects of miR-206. In conclusion, miR-206 suppresses proliferation and EMT of RCC by inhibiting CDK6 expression. The miR-206/CDK6 axis may provide a novel insight into tumorigenesis of RCC.

The aim of this study was to investigate the effects and mechanisms of baicalin against prostate cancer cell growth and cell cycle progression. A human prostate cancer cell line LNCaP was engrafted into nude mice, and the oncogenicity of LNCaP cells was analyzed. The 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay was used to measure LNCaP and PC3 proliferation capability. CDK6 mRNA level was detected using qRT-PCR. Western blotting was performed to assess the levels of cell cycle-associated proteins. In addition, cell cycle and apoptosis were analyzed using flow cytometry. Baicalin significantly decreased the expression of cell cycle-associated proteins. Furthermore, baicalin showed an observable effect on proliferation, cell cycle progression and apoptosis in LNCaP and PC3 cells. Upregulation of CDK6 and FOXM1 reversed the effect of baicalin. CDK6- and FOXM1-mediated cell growth attenuated the protective effect of baicalin in prostate cancer. This study presented an understanding of the role and mechanism of baicalin in prostate cancer cells, providing a new target and therapies for the prevention and treatment of prostate cancer.

Because bladder cancer (BC) is one of the most common malignant cancers of the urinary system, identification of BC cell growth-associated effectors is of great significance. Cyclin-dependent kinase (CDK)6 is a member of the CDK family of cell cycle-related proteins and plays an important role in cancer cell growth. This is borne out by the fact that a CDK6 inhibitor had been approved to treat several types of cancers. Nevertheless, underlying molecular mechanisms concerning how to regulate CDK6 expression in BC remains unclear. In the present study, it was observed that miR-934 was much higher in human BCs and human BC cell lines as well. The results also revealed that miR-934 inhibition dramatically decreased human BC cell monolayer growth in vitro and xenograft tumor growth in vivo ; the outcomes were accompanied by CDK6 protein down-regulation and G 0 -G 1 cell cycle arrest. Moreover, overexpression of CDK6 reversed the inhibition of BC cell growth induced by miR-934. Further studies showed that miR-934 binds to a 3'-UTR of ubiquitin-conjugating enzyme 2N ( ube2n ) mRNA, down-regulated UBE2N protein expression; this, in turn, attenuated CDK6 protein degradation and led to CDK6 protein accumulation as well as the promotion of BC tumor growth. Collectively, this study not only establishes a novel regulatory axis of miR-934/UBE2N of CDK6 but also provides data suggesting that miR-934 and UBE2N may be potentially promising targets for therapeutic strategies against BC.-Yan, H., Ren, S., Lin, Q., Yu, Y., Chen, C., Hua, X., Jin, H., Lu, Y., Zhang, H., Xie, Q., Huang, C., Huang, H. Inhibition of UBE2N-dependent CDK6 protein degradation by miR-934 promotes human bladder cancer cell growth.

Background: Nicotine, an important component of tobacco, is a major risk factor of lung cancer, but the mechanism through which nicotine promotes lung cancer development remains unclear., Methods: Eighty patients with lung cancer were enrolled in this study, 34 of whom did not smoke and the others did. The expression of miR-218 and CDK6 messenger RNA (mRNA) was measured using quantitative reverse transcription polymerase chain reaction (qRT-PCR). A luciferase reporter system was used to identify the direct target of miR-218. The protein expression of CDK6 was analyzed by using Western blotting. Cell proliferation was analyzed using an approach of calculation of cell number under a microscope., Results: Nicotine decreased miR-218 expression in non-small cell lung cancer (NSCLC) cells and promoted proliferation of NSCLC cells. Smoking patients with NSCLC had lower expression of miR-218 in tumor compared with NSCLC patients who did not smoke. We found that miR-218 directly targeted the CDK6 mRNA 3'untranslated region and inhibited its expression in NSCLC cells and also observed a negative correlation between the expression of miR-218 and CDK6 mRNA in lung cancer tissues. Furthermore, miR-218- or nicotine-induced proliferative effects of NSCLC cells were rescued by the recovery of the expression level of CDK6., Conclusion: Nicotine promotes proliferation of NSCLC cells through regulating the miR-218/CDK6 axis, which may be a potential therapeutic target for lung cancer., (© 2019 Wiley Periodicals, Inc.)

Growth differentiation factor 11 (GDF11) has been characterized as a key regulator of differentiation in cells that retain stemness features, despite some controversies in age-related studies. GDF11 has been poorly investigated in cancer, particularly in those with stemness capacity, such as hepatocellular carcinoma (HCC), one of the most aggressive cancers worldwide. Here, we focused on investigating the effects of GDF11 in liver cancer cells. GDF11 treatment significantly reduced proliferation, colony and spheroid formation in HCC cell lines. Consistently, down-regulation of CDK6, cyclin D1, cyclin A, and concomitant upregulation of p27 was observed after 24 h of treatment. Interestingly, cell viability was unchanged, but cell functionality was compromised. These effects were potentially induced by the expression of E-cadherin and occludin, as well as Snail and N-cadherin repression, in a time-dependent manner. Furthermore, GDF11 treatment for 72 h induced that cells were incapable of sustaining colony and sphere capacity in the absent of GDF11, up to 5 days, indicating that the effect of GDF11 on self-renewal capacity is not transient. Finally, in vivo invasion studies revealed a significant decrease in cell migration of hepatocellular carcinoma cells treated with GDF11 associated to a decreased proliferation judged by Ki67 staining. Data show that exogenous GDF11 displays tumor suppressor properties in HCC cells., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The cyclin-dependent kinases Cdk4 and Cdk6 form complexes with D-type cyclins to drive cell proliferation. A well-known target of cyclin D-Cdk4,6 is the retinoblastoma protein Rb, which inhibits cell-cycle progression until its inactivation by phosphorylation. However, the role of Rb phosphorylation by cyclin D-Cdk4,6 in cell-cycle progression is unclear because Rb can be phosphorylated by other cyclin-Cdks, and cyclin D-Cdk4,6 has other targets involved in cell division. Here, we show that cyclin D-Cdk4,6 docks one side of an alpha-helix in the Rb C terminus, which is not recognized by cyclins E, A, and B. This helix-based docking mechanism is shared by the p107 and p130 Rb-family members across metazoans. Mutation of the Rb C-terminal helix prevents its phosphorylation, promotes G1 arrest, and enhances Rb's tumor suppressive function. Our work conclusively demonstrates that the cyclin D-Rb interaction drives cell division and expands the diversity of known cyclin-based protein docking mechanisms., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Aims: We aimed to explore the impact of long noncoding RNA (lncRNA) nuclear enriched abundant transcript 1 (NEAT1) on cell proliferation, invasion, and migration of glioma., Methods: Differentially expressed genes were screened out from Gene Expression Omnibus data set based on the microarray analysis. The expression levels of lncRNA NEAT1, miR-139-5p, and CDK6 in glioma cells and tissues were examined by quantitative reverse transcription polymerase chain reaction, and the protein level of CDK6 in glioma cells was determined by western blot and immunohistochemistry. Glioma cell viability, cell cycle, and apoptosis were detected by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) and flow cytometry, respectively, whereas cell invasion and migration were analyzed by transwell assay. The target relationships among NEAT1, miR-139-5p, and CDK6 were confirmed by dual-luciferase reporter gene assay. The effects of lncRNA NEAT1 on tumor growth were further testified through glioma xenografts in nude mice., Results: LncRNA NEAT1 and CDK6 were highly expressed in glioma tissues and cells, whereas miR-139-5p was lowly expressed. There were target relationships and correlations on expressions between miR-139-5p and NEAT1/ CDK6. NEAT1 and CDK6 could promote cell proliferation and metastasis of glioma cells and impeded cell apoptosis, whereas miR-139-5p exerted suppressive effects on the biological functions of glioma cells. NEAT1 regulated CDK6 to affect glioma growth through sponging miR-139-5p., Conclusions: LncRNA NEAT1 promotes cell proliferation, invasion, and migration of glioma through regulating miR-139-5p/CDK6 pathway., (© 2018 Wiley Periodicals, Inc.)

Purpose: Melanoma is a malignant skin tumor that can easily metastasize, while no effective treatment exists for this disease. This study explored the mechanism of microRNA-29c in inhibiting melanoma cell growth., Methods: Bioinformatics analysis and polymerase chain reaction (PCR) experiments were performed to analyze the expression of microRNA-29c in various samples. The Cell Counting Kit-8 (CCK-8) experiment was used to detect cell viability. The mimic and inhibitor of microRNA-29c were transfected into melanoma cells to achieve microRNA-29c overexpression or knockdown so as to observe the biological effect on the melanoma cells. Flow cytometry was used to detect cell cycle, while the luciferase reporter gene assay was used for predicting microRNA-29c target genes. Western blot was performed to determine the cellular protein expression., Results: microRNA-29c was highly expressed in melanoma cells. Overexpression of microRNA-29c inhibited cell viability and induced G1 cell cycle arrest. Conversely, cell proliferation and cycle progression were promoted by transfection of microRNA-29c inhibitor in melanoma cells. In addition, CDK6 served as a microRNA-29c target gene. G1 phase of melanoma cells was blocked by knockdown of CDK6., Conclusions: microRNA-29c can inhibit the growth of melanoma cells by targeting CDK6, which could trigger G1 arrest of melanoma cells.

Neural stem cells give rise to granule dentate neurons throughout life in the hippocampus. Upon activation, these stem cells generate fast proliferating progenitors that complete several rounds of divisions before differentiating into neurons. Although the mechanisms regulating the activation of stem cells have been intensively studied, little attention has been given so far to the intrinsic machinery allowing the expansion of the progenitor pool. The cell cycle protein Cdk6 positively regulates the proliferation of hippocampal progenitors, but the mechanism involved remains elusive. Whereas Cdk6 functions primarily as a cell cycle kinase, it can also act as transcriptional regulator in cancer cells and hematopoietic stem cells. Using mouse genetics, we show here that the function of Cdk6 in hippocampal neurogenesis relies specifically on its kinase activity. The present study also reveals a specific regulatory mechanism for Cdk6 in hippocampal progenitors. In contrast to the classical model of the cell cycle, we observe that the Cip/Kip family member p27, rather than the Ink4 family, negatively regulates Cdk6 in the adult hippocampus. Altogether, our data uncover a unique, cell type-specific regulatory mechanism controlling the expansion of hippocampal progenitors, where Cdk6 kinase activity is modulated by p27.

Long noncoding RNA (lncRNA) is yet to be linked to cancer metabolism. Here, we report that upregulation of the lncRNA LINC00538 ( YIYA ) promotes glycolysis, cell proliferation, and tumor growth in breast cancer. YIYA is associated with the cytosolic cyclin-dependent kinase CDK6 and regulated CDK6-dependent phosphorylation of the fructose bisphosphatase PFK2 (PFKFB3) in a cell-cycle-independent manner. In breast cancer cells, these events promoted catalysis of glucose 6-phosphate to fructose-2,6-bisphosphate/fructose-1,6-bisphosphate. CRISPR/Cas9-mediated deletion of YIYA or CDK6 silencing impaired glycolysis and tumor growth in vivo In clinical specimens of breast cancer, YIYA was expressed in approximately 40% of cases where it correlated with CDK6 expression and unfavorable survival outcomes. Our results define a functional role for lncRNA in metabolic reprogramming in cancer, with potential clinical implications for its therapeutic targeting. Significance: These findings offer a first glimpse into how a long-coding RNA influences cancer metabolism to drive tumor growth. Cancer Res; 78(16); 4524-32. ©2018 AACR ., (©2018 American Association for Cancer Research.)

THIS ARTICLE WAS WITHDRAWN BY THE PUBLISHERS IN OCTOBER 2020.

Little is known about the molecular players driving proliferation of neural progenitor cells (NPCs) during embryonic mouse development. Here, we demonstrate that proliferation of NPCs in the developing forebrain depends on a particular combination of cell cycle regulators. We have analyzed the requirements for members of the cyclin-dependent kinase (cdk) family using cdk-deficient mice. In the absence of either cdk4 or cdk6, which are both regulators of the G1 phase of the cell cycle, we found no significant effects on the proliferation rate of cortical progenitor cells. However, concomitant loss of cdk4 and cdk6 led to a drastic decrease in the proliferation rate of NPCs, specifically the basal progenitor cells of both the dorsal and ventral forebrain at embryonic day 13.5 (E13.5). Moreover, basal progenitors in the forebrain of Cdk4;Cdk6 double mutant mice exhibited altered cell cycle characteristics. Cdk4;cdk6 deficiency led to an increase in cell cycle length and cell cycle exit of mutant basal progenitor cells in comparison to controls. In contrast, concomitant ablation of cdk2 and cdk6 had no effect on the proliferation of NCPs. Together, our data demonstrate that the expansion of the basal progenitor pool in the developing telencephalon is dependent on the presence of distinct combinations of cdk molecules. Our results provide further evidence for differences in the regulation of proliferation between apical and basal progenitors during cortical development. © 2018 Wiley Periodicals, Inc. Develop Neurobiol 78: 660-670, 2018., (© 2018 Wiley Periodicals, Inc.)

Background: Angiogenesis is an important process in the development of tumor. PD 0332991, a cell cycle inhibitor, can specifically inhibit CD4/6 phosphorylation and cell cycle progression. In xeongraft mice models, PD 0332991 treated mice had significantly decreased angiogenesis and vascular density compared with the control group, but the mechanism remains unknown. The purpose of this study is to investigate the role and molecular mechanism of PD 0332991 on vascular endothelial cells., Methods: EA.hy926 cells, a kind of vascular endothelial cell, were used as the research model. The effects of PD 0332991 on the activity and proliferation of EA.hy926 cells were detected by the MTT, EdU assays. Wound-healing assays and transwell assays were used to determine the effects of PD 0332991 on the mobility of EA.hy926. The influence of PD 0332991 on cell cycle and apoptosis of endothelial cells was tested by flow cytometry, and the Western blot was applied to observe the expression of cell cycle related proteins in EA.hy926 cells treated by PD 0332991., Results: PD 0332991 significantly inhibited the proliferation and mobility of EA.hy926 cells, caused cell cycle arrest and apoptosis. At the same time, PD 0332991 inhibited the expression of CDK4/6 and phosphorylation of Rb, and thus inhibited the cell cycle progression of EA.hy926 cells., Conclusions: PD 0332991 can inhibit the proliferation and activity of endothelial cells and induces apoptosis.

Medulloblastoma, an aggressive cancer of the cerebellum, is among the most common pediatric brain tumors. Approximately one-third of medulloblastomas are associated with misactivation of the Hedgehog (Hh) pathway. GLI family zinc finger 2 (GLI2) coordinates the Hh transcriptional program; however, the GLI2 targets that promote cancer cell proliferation are unknown. Here, we incorporated a Gli2-EGFP allele into 2 different genetic mouse models of Hh-associated medulloblastoma. Hh signaling induced GLI2 binding to the Cdk6 promoter and activated Cdk6 expression, thereby promoting uncontrolled cell proliferation. Genetic or pharmacological inhibition of CDK6 in mice repressed the growth of Hh-associated medulloblastoma and prolonged survival through inhibition of cell proliferation. In human medulloblastoma, misactivation of Hh signaling was associated with high levels of CDK6, pointing to CDK6 as a direct transcriptional target of the Hh pathway. These results suggest that CDK6 antagonists may be a promising therapeutic approach for Hh-associated medulloblastoma in humans.

p27 plays critical roles in cell proliferation, differentiation, and apoptosis, which have been well studied in mammals and Drosophila. However, the mechanisms underlying p27 regulation of the cell cycle have not been thoroughly researched. In this study, Genevestigator, Kaplan-Meier Plotter, and the Human Protein Atlas databases were used to analyze the expression of p27, cell division protein kinase 6 (CDK6), and cyclin D1 (CCND1), as well as its prognostic value in different tumor tissues and corresponding normal tissues. Quantitative PCR and immunohistochemistry were used to detect the expression of p27, CDK6, and CCND1 in the tissues of cancer patients. The effects of p27, CDK6, and CCND1 on the proliferation of lung cancer cells were examined by the MTT assay, and flow cytometry was used to investigate the mechanism by which p27 affected cell proliferation. Immunofluorescence, co-immunoprecipitation, and Western blotting were used to determine if p27 interacted with CDK and CCND1 to regulate the cell cycle. The results showed that p27, CDK6, and CCND1 played different roles in tumorigenesis and development, which are in accordance with CDK6 and CCND1 in affecting the cell cycle and cell proliferation. p27 regulated the cell cycle and inhibited cell proliferation by affecting formation of the cell cycle-dependent complex CDK6/CCND1, but did not directly affect the expression of CDK6 and CCND1. Moreover, CCND1 did not regulate the cell cycle alone, but rather, functioned together with CDK6. This study provides insights into the effects of p27 on tumor formation and development, and the underlying regulatory mechanisms.

Circular RNAs (circRNAs), a novel class of widespread and diverse endogenous RNAs, can regulate gene expression in mammals. CircRNAs have recently been identified as microRNA sponges and involved in the development of some human diseases. However, the role of circRNAs in the process of tumorigenesis and development of lung cancer remains vague. The purpose of this study is to investigate the role of circRNAs in the lung cancer. In this study, we chose hsa_circ_0000064 as a targeted circRNA to investigate its clinical significances in lung cancer patients. The result indicated that hsa_circ_0000064 was up-regulated in lung cancer tissues and lung cancer cell lines (A549 and H1229). Moreover, its aberrant expression was correlated with several clinical characteristics, including T stage, lymphatic metastasis, and TNM stage. Fluorescence in situ hybridization detected that hsa_circ_0000064 was mostly located in the cytoplasm in A549 and H1229 cells. In addition, knockdown of hsa_circ_0000064 with siRNA dramatically attenuated the proliferation, blocked cell cycle progression, and promoted cell apoptosis. Western blot analysis showed that the protein levels of caspase-3, caspase-9, bax, p21, CDK6 and cyclin D1 significantly restrained by si-hsa_circ_0000064, while the expression of bcl-2 notably increased in A549 and H1229 cells. Further, si-hsa_circ_0000064 also abated migration and invasion activities of A549 and H1229 cells, which may be associated with reduced expressions of MMP-2 and MMP-9. In general, our data suggest that hsa_circ_0000064 represents a novel potential biomarker and therapeutic target of lung cancer., (Copyright © 2017 Elsevier Masson SAS. All rights reserved.)

Studies in several cancers have suggested that miR-218 has anti-tumor activities, but its function is yet to be elucidated. In this study, we investigated the regulation and function of miR-218 (miR-218-5p) in the cell cycle progression of gastric cancer (GC). We found that miR-218 could suppress proliferation of gastric cancer cells, induce cell cycle arrest at the G1 phase and inhibit tumor growth and metastasis in vivo. We also demonstrated that miR-218 specifically targeted the 3'-UTR regions of CDK6 and cyclin D1 and inhibited the expression of these molecules, which in turn repressed the pRb/E2F1 signaling pathway. Overexpression of CDK6 and Cyclin D1 reversed miR-218-mediated inhibition of pRB/E2F1 signaling and attenuated the miR-218-induced cell cycle arrest. More importantly, miR-218 expression was significantly reduced and inversely correlated with the levels of CDK6 and Cyclin D1 in gastric cancer tissues. Decreased miR-218 expression was also correlated with advanced clinical stage, lymph node metastasis, and poor prognosis in gastric cancer patients. Furthermore, we showed that miR-218 expression was directly activated by E2F1 through the transactivation of miR-218 host genes, SLIT2 and SLIT3, revealing a negative feedback regulation of miR-218 expression. Taken together, our results describe a regulatory loop miR-218-CDK6/CyclinD1-E2F1 whose disruption may contribute to cell cycle progression in gastric cancer and indicate the potential application of miR-218 in cancer therapy., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Heterochromatin protein 1γ (CBX3) links histone methylation marks to transcriptional silence, DNA repair and RNA splicing, but a role for CBX3 in cancer remains largely unknown. In this study, we show that CBX3 in colon cancer cells promotes the progression of the cell cycle and proliferation in vitro and in vivo. Cell cycle (G1 phase to S phase) related gene CDK6 and p21 were further identified as targets of CBX3. In addition, we found that enhancing CDK6 suppresses cell proliferation by upregulating inhibitor p21 in the absence of CBX3, and this function is independent of the kinase activity of CDK6. Our results demonstrate a key role of CBX3 in colon carcinogenesis via suppressing the expression of CDK6/p21, which may disrupt the role of CDK6 in transcriptionally regulating p21, as part of a negative feedback loop to limit CDK6 excessive activation.

The transcription factor, v-maf avian musculoaponeurotic fibrosarcoma oncogene homolog B (MAFB), promotes tumorigenesis in some cancers. In this study, we found that MAFB levels were increased in clinical colorectal cancer (CRC) samples, and higher expression correlated with more advanced TNM stage. We identified MAFB amplifications in a majority of tumor types in an assessment of The Cancer Genome Atlas database. Altered MAFB levels due to gene amplification, deletion, mutation, or transcription upregulation occurred in 9% of CRC cases within the database. shRNA knockdown experiments demonstrated that MAFB deficiency blocked CRC cell proliferation by arresting the cell cycle at G0/G1 phase in vitro. We found that MAFB could be SUMOylated by SUMO1 at lysine 32, and this modification was critical for cell cycle regulation by MAFB in CRC cells. SUMOylated MAFB directly regulated cyclin-dependent kinase 6 transcription by binding to its promoter. MAFB knockdown CRC cell xenograft tumors in mice grew more slowly than controls, and wild-type MAFB-overexpressing tumors grew more quickly than tumors overexpressing MAFB mutated at lysine 32. These data suggest that SUMOylated MAFB promotes CRC tumorigenesis through cell cycle regulation. MAFB and its SUMOylation process may serve as novel therapeutic targets for CRC treatment.

Background and Aims: The role of transforming growth factor beta 1 (TGFβ1) in cholangiocarcinoma (CCA) initiation and growth requires further definition., Methods: We employed pharmacological and genetic approaches to inhibit or enhance TGFβ1 signaling, respectively, and determine the cellular mechanisms involved., Results: It was observed that inhibiting TGFβ1 activity with short hairpin RNA (shRNA) or pharmaceutical agents suppressed CCA development and growth, whereas overexpression of TGFβ1 enhanced CCA tumor size and promoted intrahepatic metastasis in a rat model. Suppression of TGFβ1 activity inhibits downstream target gene expression mediated by miR-34a that includes cyclin D1, CDK6, and c-Met. In addition, "knockdown" of TGFβ1 expression revealed a miR-34a positive feedback mechanism for enhanced p21 expression in CCAs. A miR-34a inhibitor reversed the effects of "knocking down" TGFβ1 on cell growth, migration, cyclin D1, CDK6 and c-Met expression, suggesting that TGFβ1 mediated effects occur, in part, through this miR-34a signaling pathway. Overexpression of TGFβ1 was associated with CCA tumor progression., Conclusions: This study suggests that TGFβ1 is involved in CCA tumor progression and participates through miR-34a mediated downstream cascades, and is a target to inhibit CCA development and growth., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Posttranscriptional control of gene expression is important for defining both normal and pathological cellular phenotypes. In vitro, RNA-binding proteins (RBPs) have recently been shown to play important roles in posttranscriptional regulation; however, the contribution of RBPs to cell specification is not well understood. Here, we determined that the RBP insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3) is specifically overexpressed in mixed lineage leukemia-rearranged (MLL-rearranged) B-acute lymphoblastic leukemia (B-ALL), which constitutes a subtype of this malignancy associated with poor prognosis and high risk of relapse. IGF2BP3 was required for the survival of B-ALL cell lines, as knockdown led to decreased proliferation and increased apoptosis. Enforced expression of IGF2BP3 provided murine BM cells with a strong survival advantage, led to proliferation of hematopoietic stem and progenitor cells, and skewed hematopoietic development to the B cell/myeloid lineage. Cross-link immunoprecipitation and high throughput sequencing uncovered the IGF2BP3-regulated transcriptome, which includes oncogenes MYC and CDK6 as direct targets. IGF2BP3 regulated transcripts via targeting elements within 3' untranslated regions (3'UTR), and enforced IGF2BP3 expression in mice resulted in enhanced expression of Myc and Cdk6 in BM. Together, our data suggest that IGF2BP3-mediated targeting of oncogenic transcripts may represent a critical pathogenetic mechanism in MLL-rearranged B-ALL and support IGF2BP3 and its cognate RNA-binding partners as potential therapeutic targets in this disease.

The elucidation of the molecular mechanisms underlying the differentiation and proliferation of human adipose tissue-derived stromal cells (hADSCs) represents a critical step in the development of hADSCs-based cellular therapies. To examine the role of the microRNA-103a-3p (miR-103a-3p) in hADSCs functions, miR-103a-3p mimics were transfected into hADSCs in order to overexpress miR-103a-3p. Osteogenic differentiation was induced for 14 days in an osetogenic differentiation medium and assessed by using an Alizarin Red S stain. The regulation of the expression of CDK6 (cyclin-dependent kinase 6), a predicted target of miR-103a-3p, was determined by western blot, real-time PCR and luciferase reporter assays. Overexpression of miR-103a-3p inhibited the proliferation and osteogenic differentiation of hADSCs. In addition, it downregulated protein and mRNA levels of predicted target of miR-103a-3p (CDK6 and DICER1). In contrast, inhibition of miR-103a-3p with 2'O methyl antisense RNA increased the proliferation and osteogenic differentiation of hADSCs. The luciferase reporter activity of the construct containing the miR-103a-3p target site within the CDK6 and DICER1 3'-untranslated regions was lower in miR-103a-3p-transfected hADSCs than in control miRNA-transfected hADSCs. RNA interference-mediated downregulation of CDK6 and DICER1 in hADSCs inhibited their proliferation and osteogenic differentiation. The results of the current study indicate that miR-103a-3p regulates the osteogenic differentiation of hADSCs and proliferation of hADSCs by direct targeting of CDK6 and DICER1 partly. These findings further elucidate the molecular mechanisms governing the differentiation and proliferation of hADSCs.

Glioblastoma development is often associated with alteration in the activity and expression of cell cycle regulators, such as cyclin-dependent kinases (CKDs) and cyclins, resulting in aberrant cell proliferation. Recent studies have highlighted the pivotal roles of miRNAs in controlling the development and growth of glioblastoma. Here, we provide evidence for a function of miR-340 in the inhibition of glioblastoma cell proliferation. We found that miR-340 is downregulated in human glioblastoma tissue samples and several established glioblastoma cell lines. Proliferation and neurosphere formation assays revealed that miR-340 plays an oncosuppressive role in glioblastoma, and that its ectopic expression causes significant defect in glioblastoma cell growth. Further, using bioinformatics, luciferase assay and western blot, we found that miR-340 specifically targets the 3'UTRs of CDK6, cyclin-D1 and cyclin-D2, leading to the arrest of glioblastoma cells in the G0/G1 cell cycle phase. Confirming these results, we found that re-introducing CDK6, cyclin-D1 or cyclin-D2 expression partially, but significantly, rescues cells from the suppression of cell proliferation and cell cycle arrest mediated by miR-340. Collectively, our results demonstrate that miR-340 plays a tumor-suppressive role in glioblastoma and may be useful as a diagnostic biomarker and/or a therapeutic avenue for glioblastoma., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Purposes: Uncontrolled proliferation is a key characteristic of gastric carcinogenesis and the precise mechanisms underlying the altered proliferation behaviors of GC cells have not been clearly elucidated. miRNAs has been suggested to play a crucial role in the pathogenesis and development of various cancers. In the present study, we employed an impedance-based real-time cell electronic sensing (RT-CES) system to detect the effects of ectopically expressed miRNAs on GC cell proliferation., Methods: miRNA mimics were transfected into gastric cancer cell line SGC7901 and the effect of individual miRNA on the proliferation rate of the cells was measured by the RT-CES system. The screening results were validated with qRT-PCR and miR-137 was selected for further research. The effects of ectopically expressed miR-137 on GC cell growth and cell cycle progress were measured using MTT assay and flow cytometry. The target gene of miR-137 was predicted using different bioinformatics tools and the direct interaction between miR-137 and the 3'-UTR was confirmed with a luciferase reporter assay. The in vivo effect of miR-137 on GC cell proliferation was examined with a tumor-bearing nude mouse model. The correlation between miR-137 expression and patients' prognosis was explored in a cohort of 38 patients. Prognosis was explored in a cohort of 38 patients., Results: Ectopic expression of miR-137 was sufficient to inhibit GC cell proliferation both in vitro and in vivo. Bioinformatics prediction and luciferase reporter assay revealed CDK6 as a target gene through which miR-137 exerted an inhibitory function. Moreover, miR-137 expression positively correlated with better prognosis., Conclusion: Our data indicated an important regulatory role of miR-137 in GC cell proliferation and that it may be explored as a prognostic marker for GC.

Backgrounds/aims: MicroRNAs (MiRNAs) control many biological events and play critical roles in the development of tumor. Among all miRNAs, miR203 has been recently shown to have an inhibitory effect on prostate cancer. However, its involvement in the carcinogenesis of breast cancer has not been reported., Methods: We examined the levels of miR203 in the breast cancer from the patients compared to the paired normal breast tissue. We also examined the levels of miR203 in several commonly used breast cancer cell lines. The effects of overexpression or depletion of miR203 on breast cancer cell growth were analyzed by a MTT assay, and on breast cancer cell invasion were examined by a scratch wound healing assay and a transwell cell migration assay. MiR203-targeted genes were analyzed by Western blot., Results: We detected significantly lower levels of miR203 in the breast cancer from the patients compared to the paired normal breast tissue. Moreover, the levels of miR203 were significantly lower in breast cancer tissue from the patients with cancer metastasis. Decreased miR203 levels were detected in all examined breast cancer lines. Overexpression of miR203 inhibited breast cancer cell growth and invasion, while antisense-mediated inhibition of miR203 enhanced cancer cell growth and invasion. Further analyses show that miR203 may inhibit cell growth through decreasing cell-cycle activator cyclinD2 and CDK6, increasing cell-cycle suppressor p21 and p27, and increasing apoptosis-associated protein Bcl-2. MiR203 may also inhibit cell metastasis through suppressing matrix metalloproteinase 2 (MMP2), MMP7 and MMP9., Conclusion: Our data thus highlight miR203 as a novel therapeutic target for breast cancer., (© 2015 S. Karger AG, Basel.)

Background: Increasing evidence has suggested that dysregulation of microRNAs (miRNAs) could contribute to human disease including cancer. Previous miRNA microarray analysis illustrated that miR-320c is down-regulated in various cancers. However, the roles of miR-320c in human bladder cancer have not been well elucidated. Therefore, this study was performed to investigate the biological functions and molecular mechanisms of miR-320c in human bladder cancer cell lines, discussing whether it could be a therapeutic biomarker of bladder cancer in the future., Methods: Two human bladder cancer cell lines and samples from thirteen patients with bladder cancer were analyzed for the expression of miR-320c by quantitative RT-PCR. Over-expression of miR-320c was established by transfecting mimics into T24 and UM-UC-3. Cell proliferation and cell cycle were assessed by cell viability assay, flow cytometry and colony formation assay. Cell motility ability was evaluated by transwell assay. The target gene of miR-320c was determined by luciferase assay, quantitative RT-PCR and western blot. The regulation of cell cycle and mobility by miR-320c was analyzed by western blot., Results: We observed that miR-320c was down-regulated in human bladder cancer tissues and bladder cancer cell lines T24 and UM-UC-3. Over-expression of miR-320c could induce G1 phase arrest in UM-UC-3 and T24 cells, and subsequently inhibited cell growth. We also indentified miR-320c could impair UM-UC-3 and T24 cell motility. In addition, we identified CDK6, a cell cycle regulator, as a novel target of miR-320c. Moreover, we demonstrated miR-320c could induce bladder cancer cell cycle arrest and mobility via regulating CDK6. We also observed that inhibition of miR-320c or restoration of CDK6 in miR-320c-over-expressed bladder cancer cells partly reversed the suppressive effects of miR-320c., Conclusions: miR-320c could inhibit the proliferation, migration and invasion of bladder cancer cells via regulating CDK6. Our study revealed that miR-320c could be a therapeutic biomarker of bladder cancer in the future.

Ovarian carcinoma is the most lethal gynaecological malignancy. Better understanding of the molecular pathogenesis of this disease and effective targeted therapies are needed to improve patient outcomes. MicroRNAs play important roles in cancer progression and have the potential for use as either therapeutic agents or targets. Studies in other cancers have suggested that miR-506 has anti-tumour activity, but its function has yet to be elucidated. We found that deregulation of miR-506 in ovarian carcinoma promotes an aggressive phenotype. Ectopic over-expression of miR-506 in ovarian cancer cells was sufficient to inhibit proliferation and to promote senescence. We also demonstrated that CDK4 and CDK6 are direct targets of miR-506, and that miR-506 can inhibit CDK4/6-FOXM1 signalling, which is activated in the majority of serous ovarian carcinomas. This newly recognized miR-506-CDK4/6-FOXM1 axis provides further insight into the pathogenesis of ovarian carcinoma and identifies a potential novel therapeutic agent., (Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

A significant role for micro (mi)RNA in the regulation of gene expression in tumours has been recently established. In order to further understand how miRNA expression may contribute to prostate tumour growth and progression, we evaluated expression of miRNA in two invasive prostate tumour lines, PC3 and DU145, and compared it to that in normal prostate epithelial cells. Although a number of miRNAs were differentially expressed, we focused our analysis on miR-105, a novel miRNA not previously linked to prostate cancer. miR-105 levels were significantly decreased in both tumour cell lines in comparison to normal prostate epithelial cells. To determine its potential role in prostate cancer pathogenesis, we overexpressed miR-105 in both PC3 and DU145 cells and determined its effect on various tumourigenic properties. miR-105 overexpression inhibited tumour cell proliferation, tumour growth in anchorage-independent three-dimensional conditions and tumour invasion in vitro, properties of highly aggressive tumour cells. Of potential clinical significance, miR-105 overexpression inhibited tumour growth in vivo in xenograft models using these cell lines. We further identified CDK6 as a putative target of miR-105 which is likely a main contributor to the inhibition of tumour cell growth observed in our assays. Our results suggest that miR-105 inhibits tumour cell proliferation and hence may represent a novel therapeutically relevant cellular target to inhibit tumour growth or a marker of aggressive tumours in prostate cancer patients.

Recent literature suggests that cyclin-dependent kinases (CDKs) mediate cell migration. However, the mechanisms were not known. Therefore, the objective of this study is to test whether cyclin/CDKs activate Pak1, an effector of Rac1, whose involvement in the modulation of cell migration and proliferation is well established. Monocyte chemotactic protein 1 (MCP1) induced Pak1 phosphorylation/activation in human aortic smooth muscle cells (HASMCs) in a delayed time-dependent manner. MCP1 also stimulated F-actin stress fiber formation in a delayed manner in HASMCs, as well as the migration and proliferation of these cells. Inhibition of Pak1 suppressed MCP1-induced HASMC F-actin stress fiber formation, migration, and proliferation. MCP1 induced cyclin D1 expression as well as CDK6 and CDK4 activities, and these effects were dependent on activation of NFATc1. Depletion of NFATc1, cyclin D1, CDK6, or CDK4 levels attenuated MCP1-induced Pak1 phosphorylation/activation and resulted in decreased HASMC F-actin stress fiber formation, migration, and proliferation. CDK4, which appeared to be activated downstream of CDK6, formed a complex with Pak1 in response to MCP1. MCP1 also activated Rac1 in a time-dependent manner, and depletion/inhibition of its levels/activation abrogated MCP1-induced NFATc1-cyclin D1-CDK6-CDK4-Pak1 signaling and, thereby, decreased HASMC F-actin stress fiber formation, migration, and proliferation. In addition, smooth muscle-specific deletion of NFATc1 led to decreased cyclin D1 expression and CDK6, CDK4, and Pak1 activities, resulting in reduced neointima formation in response to injury. Thus, these observations reveal that Pak1 is a downstream effector of CDK4 and Rac1-dependent, NFATc1-mediated cyclin D1 expression and CDK6 activity mediate this effect. In addition, smooth muscle-specific deletion of NFATc1 prevented the capacity of vascular smooth muscle cells for MCP-1-induced activation of the cyclin D1-CDK6-CDK4-Pak1 signaling axis, affecting their migration and proliferation in vitro and injury-induced neointima formation in vivo.

Background: Glioblastoma multiforme (GBM) is the most aggressive type of glioma and carries the poorest chances of survival. There is therefore an urgent need to understand the mechanisms of glioma tumorigenesis and develop or improve therapeutics. The aim of this study was to assess the possible prognostic value of cyclin-dependent kinase 6 (CDK6) and the effects of microRNA-495 (miR-495) manipulation on CDK6 expression and cell survival in glioma cells., Methods: Analyses of clinical specimens from GBM patients were used. Expression of CDK6 was analyzed by real-time polymerase chain reaction (RT-PCR), Western blotting, and immunohistochemistry. Expression of CDK6 was also analyzed after over-expression of miR-495 in T98 cells; both cell proliferation and RB phosphorylation were examined. Cell proliferation, cell cycle distribution, and RB phosphorylation were also examined after knockdown of CDK6 in U87-MG and T98 cells., Results: Analyses of clinical specimens from GBM patients identified that CDK6 is significantly expressed in gliomas. CDK6 antigen expression was higher in tumor cores and margins than in adjacent normal brain tissues, and higher levels of CDK6 expression in the tumor margin correlated with decreased survival. Over-expression of miR-495 in T98 cells downregulated the expression of CDK6 and inhibited retinoblastoma phosphorylation, and knockdown of CDK6 in U87-MG and T98 cells by siRNAs resulted in cell cycle arrest at the G1/S transition and inhibition of cell proliferation., Conclusions: This study revealed miR-495 is down-regulated in glioma tissues. Furthermore, miR-495 regulated CDK6 expression and involved in glioma cell growth inhibition, which indicated the possible role of miR-495 in tumor progression.

Achyranthes bidentata polysaccharides (ABPS) are the major bioactive constituents of Radix Achyranthes bidentata (AB), which has been widely used in traditional Chinese medicine for the treatment of osteoarthritis. However, the molecular mechanisms behind the therapeutic effect of ABPS remain unclear. In the present study, chondrocytes were isolated from Sprague-Dawley rats. The effects of ABPS on the G1/S cell cycle transition in primary chondrocytes were investigated. The chondrocytes treated with and without ABPS were analyzed and it was observed that ABPS treatment was able to enhance chondrocyte proliferation in a dose- and time-dependent manner and promote the progression of chondrocyte cell cycle proliferation via the promotion of the G1 to S phase transition. Furthermore, using RT-PCR and western blot analysis, ABPS were observed as significantly upregulating the expression of cyclin D1 and the cyclin-dependent kinases (CDKs) CDK4 and CDK6. These results suggest that ABPS are able to promote chondrocyte proliferation via the promotion of the G1/S cell cycle transition.

Cells derived from the amniotic foetal membrane of human term placenta have drawn particular attention mainly for their plasticity and immunological properties, which render them interesting for stem-cell research and cell-based therapeutic applications. In particular, we have previously demonstrated that amniotic mesenchymal tissue cells (AMTC) inhibit lymphocyte proliferation in vitro and suppress the generation and maturation of monocyte-derived dendritic cells. Here, we show that AMTC also significantly reduce the proliferation of cancer cell lines of haematopoietic and non-haematopoietic origin, in both cell-cell contact and transwell co-cultures, therefore suggesting the involvement of yet-unknown inhibitory soluble factor(s) in this 'cell growth restraint'. Importantly, we provide evidence that the anti-proliferative effect of AMTC is associated with induction of cell cycle arrest in G0/G1 phase. Gene expression analyses demonstrate that AMTC can down-regulate cancer cells' mRNA expression of genes associated with cell cycle progression, such as cyclins (cyclin D2, cyclin E1, cyclin H) and cyclin-dependent kinase (CDK4, CDK6 and CDK2), whilst they up-regulate cell cycle negative regulator such as p15 and p21, consistent with a block in G0/G1 phase with no progression to S phase. Taken together, these findings warrant further studies to investigate the applicability of these cells for controlling cancer cell proliferation in vivo., (© 2012 The Authors Journal of Cellular and Molecular Medicine © 2012 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd.)

Millimeter waves, high-frequency electromagnetic waves, can effectively alleviate the clinical symptoms in osteoarthritis patients, as a non-pharmaceutical and non-invasive physical therapy regimen. However, the molecular mechanisms of the therapeutic effects of millimeter wave treatment are not well understood. In the present study, the effect of millimeter waves on the G1/S cell cycle progression in chondrocytes and the underlying mechanism was investigated. Chondrocytes isolated from the knee of SD rats were cultured and identified using toluidine blue staining. The second generation chondrocytes were collected and stimulated with or without millimeter waves for 48 h. Chondrocyte viability was analyzed using the MTT assay. The cell cycle distribution of chondrocytes was analyzed by flow cytometry. mRNA and protein expression levels of cyclin D1, cyclin-dependent kinases 4 and 6 (CDK4 and CDK6) and p21 were detected using real-time PCR and western blotting, respectively. Millimeter wave stimulation was found to significantly enhance chondrocyte viability. Moreover, the percentage of chondrocytes in the G0/G1 phase was significantly decreased, whereas that in the S phase was significantly increased. In addition, following millimeter wave treatment, cyclin D1, CDK4 and CDK6 expression was significantly upregulated, whereas p21 expression was significantly downregulated. The results indicate that millimeter wave treatment promotes chondrocyte proliferation via cell cycle progression.

RGS19 is a regulator of G protein signaling which is upregulated in ovarian cancers and its overexpression promotes cell proliferation in several mammalian cell types. Here we showed that cyclin D1/3 and Cdk6 were upregulated in HEK293 cells overexpressing RGS19, while INK4A and INK4B were reduced. Moreover, RGS19 augmented serum-stimulated PTEN/PDK/Akt and Rb phosphorylations in 293/RGS19 and Caco2/RGS19 cells. These changes were reversed upon the knockdown of RGS19. Consistent with an elevated Akt activity, increased levels of phosphorylated Bad and c-Raf and a diminished expression of TSC2 were detected, thus demonstrating that RGS19 can deregulate cell proliferation via multiple pathways., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Fbxo7 is an unusual F-box protein because most of its interacting proteins are not substrates for ubiquitin-mediated degradation. Fbxo7 directly binds p27 and Cdk6, enhances the level of cyclin D-Cdk6 complexes, and its overexpression causes Cdk6-dependent transformation of immortalised fibroblasts. Here, we test the ability of Fbxo7 to transform haematopoietic pro-B (Ba/F3) cells which, unexpectedly, it was unable to do despite high levels of Cdk6. Instead, reduction of Fbxo7 expression increased proliferation, decreased cell size and shortened G1 phase. Analysis of cell cycle regulators showed that cells had decreased levels of p27, and increased levels of S phase cyclins and Cdk2 activity. Also, Fbxo7 protein levels correlated inversely with those of CD43, suggesting direct regulation of its expression and, therefore, of B cell maturation. Alterations to Cdk6 protein levels did not affect the cell cycle, indicating that Cdk6 is neither rate-limiting nor essential in Ba/F3 cells; however, decreased expression of Cdk6 also enhanced levels of CD43, indicating that expression of CD43 is independent of cell cycle regulation. The physiological effect of reduced levels of Fbxo7 was assessed by creating a transgenic mouse with a LacZ insertion into the Fbxo7 locus. Homozygous Fbxo7(LacZ) mice showed significantly increased pro-B cell and pro-erythroblast populations, consistent with Fbxo7 having an anti-proliferative function and/or a role in promoting maturation of precursor cells.

Cyclin-dependent kinase-6 (CDK6) is required for early thymocyte development and tumorigenesis. To mechanistically dissect the role of CDK6 in thymocyte development, we generated and analyzed mutant knock-in mice and found that mice expressing a kinase-dead Cdk6 allele (Cdk6(K43M)) had a pronounced reduction in thymocytes and hematopoietic stem cells and progenitor cells (Lin⁻Sca-1⁺c-Kit⁺ [LSK]). In contrast, mice expressing the INK4-insensitive, hyperactive Cdk6(R31C) allele displayed excess proliferation in LSK and thymocytes. However, this is countered at least in part by increased apoptosis, which may limit progenitor and thymocyte expansion in the absence of other genetic events. Our mechanistic studies demonstrate that CDK6 kinase activity contributes to Notch signaling because inactive CDK6 kinase disrupts Notch-dependent survival, proliferation, and differentiation of LSK, with concomitant alteration of Notch target gene expression, such as massive up-regulation of CD25. Further, knockout of CD25 in Cdk6(K43M) mice rescued most defects observed in young mice. These results illustrate an important role for CDK6 kinase activity in thymocyte development that operates partially through modulating Notch target gene expression. This role of CDK6 as a downstream mediator of Notch identifies CDK6 kinase activity as a potential therapeutic target in human lymphoid malignancies.

Maintenance of oral epithelial homeostasis requires a fine balance between cell proliferation and differentiation. However, the molecular mechanisms that couple these processes, and its deregulation in tumorigenesis are not fully understood. Cyclin D1 and its kinase partners CDK4 and CDK6 play an important role in regulating the G1-S phase of the cell cycle. Deregulation of cyclin D1 is a frequent event in oral squamous cell carcinoma. Here, we examined whether overexpression of cyclin D1, CDK4 and CDK6 can deregulate the link between oral keratinocyte proliferation and differentiation. Our results show that cyclin D1 and its kinase partners CDK4 and CDK6 enhance keratinocyte proliferation, but are not sufficient to block calcium-induced keratinocyte differentiation and suggest that deregulation of these G1-regulatory kinases alone is insufficient to uncouple the link between proliferation and differentiation.

E2F1 is a positive regulator of cell cycle progression and also a potent inducer of apoptosis, especially when activated by DNA damage. We identified E2F1-inducible microRNAs (miRNAs) by microarray hybridization and found that the levels of miRNAs 449a and 449b, as well as their host gene CDC20B, are strongly upregulated by E2F1. High miR-449 levels were found in testes, lung, and trachea, but not in testicular and other cancer cells. MiR-449a/b structurally resemble the p53-inducible miRNA 34 family. In agreement with a putative tumor-suppressive role, miR-449a as well as miR-34a reduced proliferation and strongly promoted apoptosis by at least partially p53-independent mechanisms. Both miRNAs reduced the levels of CDK6, implying miR-449 in a negative feedback mechanism for E2F1. Moreover, miR-449a and miR-34a diminished the deacetylase Sirt1 and augmented p53 acetylation. We propose that both miRNAs provide a twofold safety mechanism to avoid excessive E2F1-induced proliferation by cell cycle arrest and by apoptosis. While responding to different transactivators, miRNAs 449 and 34 each repress E2F1, but promote p53 activity, allowing efficient cross-talk between two major DNA damage-responsive gene regulators.

Cyclin-dependent kinase 6 (CDK6) is the key element of the D-type cyclin holoenzymes which has been found to function in the regulation of G1-phase of the cell cycle and is presumed to play important roles in T cell function. In this study, Znhit1, a member of a new zinc finger protein family defined by a conserved Zf-HIT domain, induced arrest in the G1-phase of the cell cycle in NIH/3T3 cells. Of the G1 cell cycle factors examined, the expression of CDK6 was found to be strongly down-regulated by Znhit1 via transcriptional repression. This effect may have correlations with the decreased acetylation level of histone H4 in the CDK6 promoter region. In addition, considering that CDK6 expression predominates in T cells, the negative regulatory role of Znhit1 in TCR-induced T cell proliferation was validated using transgenic mice. These findings identified Znhit1 as a CDK6 regulator that plays an important role in cell proliferation.