TG-interacting factor 1 regulates mitotic clonal expansion during adipocyte differentiation.

Obesity is one of the significant health challenges in the world and is highly associated with abnormal adipogenesis. TG-interacting factor 1 (TGIF1) is essential for differentiating murine adipocytes and human adipose tissue-derived stem cells. However, the mode of action needs to be better elucidated. To investigate the roles of TGIF1 in differentiation in-depth, CRISPR/Cas9 knockout technology was performed to generate TGIF1-silenced preadipocytes. The absence of TGIF1 in 3 T3-F442A preadipocytes abolished lipid accumulation throughout the differentiation using Oil Red O staining. Conversely, we established 3 T3-F442A preadipocytes stably expressing TGIF1 and doxycycline-inducible TGIF1 in TGIF1-silenced 3 T3-F442A preadipocytes. Remarkably, the induction of TGIF1 by doxycycline during the initial differentiation phase successfully promoted lipid accumulation in TGIF1-silenced 3 T3-F442A cells. We further explored the mechanisms of TGIF1 in early differentiation. We demonstrated that TGIF1 promoted the mitotic clonal expansion via upregulation of CCAAT/enhancer-binding proteins ß expression, interruption with peroxisome proliferators activated receptor γ downstream regulation, and inhibition of p27kip1 expression. In conclusion, we strengthen the pivotal roles of TGIF1 in early differentiation, which might contribute to resolving obesity-associated metabolic syndromes.

Phosphorylation of TG-interacting factor 1 at carboxyl-terminal sites in response to insulin regulates adipocyte differentiation.

TG-interacting factor 1 (TGIF1) contributes to the differentiation of murine white preadipocyte and human adipose tissue-derived stem cells; however, its regulation is not well elucidated. Insulin is a component of the adipogenic cocktail that induces ERK signaling. TGIF1 phosphorylation and sustained stability in response to insulin were reduced through the use of specific MEK inhibitor U0126. Mutagenesis at T235 or T239 residue of TGIF1 in preadipocytes led to dephosphorylation of TGIF1. The reduced TGIF1 stability resulted in an increase in p27kip1 expression, a decrease in phosphorylated Rb expression and cellular proliferation, and a reduced accumulation of lipids compared to the TGIF1-overexpressed cells. These findings highlight that insulin/ERK-driven phosphorylation of the T235 or T239 residue at TGIF1 is crucial for adipocyte differentiation.

Janus Kinase 3 phosphorylation and the JAK/STAT pathway are positively modulated by follicle-stimulating hormone (FSH) in bovine granulosa cells.

Janus kinase 3 (JAK3) is a member of the JAK family of tyrosine kinase proteins involved in cytokine receptor-mediated intracellular signal transduction through the JAK/STAT signaling pathway. JAK3 was previously shown as differentially expressed in granulosa cells (GC) of bovine pre-ovulatory follicles suggesting that JAK3 could modulate GC function and activation/inhibition of downstream targets. We used JANEX-1, a JAK3 inhibitor, and FSH treatments and analyzed proliferation markers, steroidogenic enzymes and phosphorylation of target proteins including STAT3, CDKN1B/p27Kip1 and MAPK8IP3/JIP3. Cultured GC were treated with or without FSH in the presence or not of JANEX-1. Expression of steroidogenic enzyme CYP11A1, but not CYP19A1, was upregulated in GC treated with FSH and both were significantly decreased when JAK3 was inhibited. Proliferation markers CCND2 and PCNA were reduced in JANEX-1-treated GC and upregulated by FSH. Western blots analyses showed that JANEX-1 treatment reduced pSTAT3 amounts while JAK3 overexpression increased pSTAT3. Similarly, FSH treatment increased pSTAT3 even in JANEX-1-treated GC. UHPLC-MS/MS analyses revealed phosphorylation of specific amino acid residues within JAK3 as well as CDKN1B and MAPK8IP3 suggesting possible activation or inhibition post-FSH or JANEX-1 treatments. We show that FSH activates JAK3 in GC, which could phosphorylate target proteins and likely modulate other signaling pathways involving CDKN1B and MAPK8IP3, therefore controlling GC proliferation and steroidogenic activity.

The involvement of let-7 in hCG-induced progesterone synthesis via regulating p27Kip1 and p21Cip1 expression.

Progesterone is essential in females to maintain a regular menstrual cycle and pregnancy. The luteinizing hormone (LH) surge induces the luteinization of granulosa cells and thecal cells to form the corpus luteum, which is responsible for progesterone synthesis. However, the specific mechanism of how hCG, the analog of LH, regulates progesterone synthesis has yet to be fully discovered. In this study, we found that progesterone level was increased in adult wild-type pregnant mice 2 and 7 days post-coitum, along with a decrease in let-7 expression compared with the estrus stage. Besides, the let-7 expression was negatively correlated with progesterone level in post-delivery day 23 wild-type female mice after being injected with PMSG and hCG. Then, using let-7 transgenic mice and a human granulosa cell line, we found that overexpression of let-7 antagonized progesterone level via targeting p27Kip1 and p21Cip1 and steroidogenic acute regulatory protein (StAR) expression, which is a rate-limiting enzyme in progesterone synthesis. Furthermore, hCG suppressed let-7 expression by stimulating the MAPK pathway. This study elucidated the role of microRNA let-7 in regulating hCG-induced progesterone production and provided new insights into its role in clinical application.

Knockdown of UBE2I inhibits tumorigenesis and enhances chemosensitivity of cholangiocarcinoma via modulating p27kip1 nuclear export.

The asymptomatic nature of cholangiocarcinoma (CCA), particularly during its early stages, in combination with its high aggressiveness and chemoresistance, significantly compromises the efficacy of current therapeutic options, contributing to a dismal prognosis. As a tumor suppressor that inhibits the cell cycle, abnormal cytoplasmic p27kip1 localization is related to chemotherapy resistance and often occurs in various cancers, including CCA. Nevertheless, the underlying mechanism is unclear. SUMOylation, which is involved in regulating subcellular localization and the cell cycle, is a posttranslational modification that regulates p27kip1 activity. Here, we confirmed that UBE2I, as the only key enzyme for SUMOylation, was highly expressed and p27kip1 was downregulated in CCA tissues, which were associated with poor outcomes in CCA. Moreover, UBE2I silencing inhibited CCA cell proliferation, delayed xenograft tumor growth in vivo, and sensitized CCA cells to the chemotherapeutics, which may be due to cell cycle arrest induced by p27kip1 nuclear accumulation. According to the immunoprecipitation result, we found that UBE2I could bind p27kip1, and the binding amount of p27kip1 and SUMO-1 decreased after UBE2I silencing. Moreover, nuclear retention of p27kip1 was induced by UBE2I knockdown and SUMOylation or CRM1 inhibition, further suggesting that UBE2I could cooperate with CRM1 in the nuclear export of p27kip1. These data indicate that UBE2I-mediated SUMOylation is a novel regulatory mechanism that underlies p27kip1 export and controls CCA tumorigenesis, providing a therapeutic option for CCA treatment.

ABL1/2 and DDR1 Drive MEKi Resistance in NRAS-Mutant Melanomas by Stabilizing RAF/MYC/ETS1 and Promoting RAF Homodimerization.

Melanomas harboring NRAS mutations are a particularly aggressive and deadly subtype. If patients cannot tolerate or the melanomas are insensitive to immune checkpoint blockade, there are no effective 2nd-line treatment options. Drugs targeting the RAF/MEK/ERK pathway, which are used for BRAF-mutant melanomas, do little to increase progression-free survival (PFS). Here, using both loss-of-function and gain-of-function approaches, we show that ABL1/2 and DDR1 are critical nodes during NRAS-mutant melanoma intrinsic and acquired MEK inhibitor (MEKi) resistance. In some acquired resistance cells, ABL1/2 and DDR1 cooperate to stabilize RAF proteins, activate ERK cytoplasmic and nuclear signaling, repress p27/KIP1 expression, and drive RAF homodimerization. In contrast, other acquired resistance cells depend solely on ABL1/2 for their survival, and are sensitive to highly specific allosteric ABL1/2 inhibitors, which prevent ß-catenin nuclear localization and destabilize MYC and ETS1 in an ERK-independent manner. Significantly, targeting ABL1/2 and DDR1 with an FDA-approved anti-leukemic drug, reverses intrinsic MEKi resistance, delays acquisition of acquired resistance, and doubles the survival time in a NRAS-mutant mouse model. These data indicate that repurposing FDA-approved drugs targeting ABL1/2 and DDR1 may be a novel and effective strategy for treating patients with treatment-refractory NRAS-driven melanomas.

11-Cl-BBQ, a select modulator of AhR-regulated transcription, suppresses lung cancer cell growth via activation of p53 and p27Kip1.

Aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor and functions as a tumour suppressor in different cancer models. In the present study, we report detailed characterization of 11-chloro-7H-benzimidazo[2,1-a]benzo[de]iso-quinolin-7-one (11-Cl-BBQ) as a select modulator of AhR-regulated transcription (SMAhRT) with anti-cancer actions. Treatment of lung cancer cells with 11-Cl-BBQ induced potent and sustained AhR-dependent anti-proliferative effects by promoting G1 phase cell cycle arrest. Investigation of 11-Cl-BBQ-induced transcription in H460 cells with or without the AhR expression by RNA-sequencing revealed activation of p53 signalling. In addition, 11-Cl-BBQ suppressed multiple pathways involved in DNA replication and increased expression of cyclin-dependent kinase inhibitors, including p27Kip1 , in an AhR-dependent manner. CRISPR/Cas9 knockout of individual genes revealed the requirement for both p53 and p27Kip1 for the AhR-mediated anti-proliferative effects. Our results identify 11-Cl-BBQ as a potential lung cancer therapeutic, highlight the feasibility of targeting AhR and provide important mechanistic insights into AhR-mediated-anticancer actions.

HMGA2 drives the IGFBP1/AKT pathway to counteract the increase in P27KIP1 protein levels in mtDNA/RNA-less cancer cells.

Recent comprehensive analyses of mtDNA and orthogonal RNA-sequencing data revealed that in numerous human cancers, mtDNA copy numbers and mtRNA amounts are significantly reduced, followed by low respiratory gene expression. Under such conditions (called mt-Low), cells encounter severe cell proliferation defects; therefore, they must acquire countermeasures against this fatal disadvantage during malignant transformation. This study elucidated a countermeasure against the mt-Low condition-induced antiproliferative effects in hepatocellular carcinoma (HCC) cells. The mechanism relied on the architectural transcriptional regulator HMGA2, which was preferably expressed in HCC cells of the mt-Low type in vitro and in vivo. Detailed in vitro analyses suggest that HMGA2 regulates insulin-like growth factor binding protein 1 (IGFBP1) expression, leading to AKT activation, which then phosphorylates the cyclin-dependent kinase inhibitor (CKI), P27KIP1, and facilitates its ubiquitin-mediated degradation. Accordingly, intervention in the HMGA2 function by RNAi resulted in an increase in P27KIP1 levels and an induction of senescence-like cell proliferation inhibition in mt-Low-type HCC cells. Conclusively, the HMGA2/IGFBP1/AKT axis has emerged as a countermeasure against P27KIP1 CKI upregulation under mt-Low conditions, thereby circumventing cell proliferation inhibition and supporting the tumorigenic state. Notably, similar to in vitro cell lines, HMGA2 was likely to regulate IGFBP1 expression in HCC in vivo, thereby contributing to poor patient prognosis. Considering the significant number of cases under mt-Low or the threat of CKI upregulation cancer-wide, the axis is noteworthy as a vulnerability of cancer cells or target for tumor-agnostic therapy inducing irreversible cell proliferation inhibition via CKI upregulation in a large population with cancer.

Nanoparticles design considerations to co-deliver nucleic acids and anti-cancer drugs for chemoresistance reversal.

Chemoresistance and hence the consequent treatment failure is considerably challenging in clinical cancer therapeutics. The understanding of the genetic variations in chemoresistance acquisition encouraged the use of gene modulatory approaches to restore anti-cancer drug efficacy. Many smart nanoparticles are designed and optimized to mediate combinational therapy between nucleic acid and anti-cancer drugs. This review aims to define a rational design of such co-loaded nanocarriers with the aim of chemoresistance reversal at various cellular levels to improve the therapeutic outcome of anticancer treatment. Going through the principles of therapeutics loading, physicochemical characteristics tuning, and different nanocarrier modifications, also looking at combination effectiveness on chemosensitivity restoration. Up to now, these emerging nanocarriers are in development status but are expected to introduce outstanding outcomes.

p27kip1 Modulates the Morphology and Phagocytic Activity of Microglia.

p27kip1 is a multifunctional protein that promotes cell cycle exit by blocking the activity of cyclin/cyclin-dependent kinase complexes as well as migration and motility via signaling pathways that converge on the actin and microtubule cytoskeleton. Despite the broad characterization of p27kip1 function in neural cells, little is known about its relevance in microglia. Here, we studied the role of p27kip1 in microglia using a combination of in vitro and in situ approaches. While the loss of p27kip1 did not affect microglial density in the cerebral cortex, it altered their morphological complexity in situ. However, despite the presence of p27kip1 in microglial processes, as shown by immunofluorescence in cultured cells, loss of p27kip1 did not change microglial process motility and extension after applying laser-induced brain damage in cortical brain slices. Primary microglia lacking p27kip1 showed increased phagocytic uptake of synaptosomes, while a cell cycle dead variant negatively affected phagocytosis. These findings indicate that p27kip1 plays specific roles in microglia.

RNF4 promotes tumorigenesis, therapy resistance of cholangiocarcinoma and affects cell cycle by regulating the ubiquitination degradation of p27kip1 in the nucleus.

Among the hallmarks of cholangiocarcinoma (CCA) progression and unresponsiveness to therapy is impaired ubiquitin-dependent degradation of nuclear tumor suppressor protein. In the previous stage, our research group found that as a key tumor suppressor, nuclear dysfunction of p27kip1 is closely related to chemotherapy resistance of CCA, but the specific mechanism is unclear. It was recently shown that p27kip1-driven tumors were strongly dependent on the SUMO pathway. RNF4, as the SUMO-targeted ubiquitin ligase (STUbL), identifies SUMOylated proteins as a substrate through sumo-interacting motifs (SIM) and causes its degradation via the ubiquitin proteasome pathway. Here we described that the expression of RNF4 was upregulated in CCA tissues and related to malignant features. Silencing RNF4 arrested human CCA cells at the G1 phase, which was associated with the upregulation of p27kip1 and the downregulation of its downstream cycle-related proteins. Silencing RNF4 inhibited cell proliferation and migration, increased cell apoptosis, and sensitized CCA cells to treatment of chemotherapeutic drugs in vitro. Immunofluorescence showed that p27kip1 and RNF4 were mainly co-located in the nucleus. Immunoprecipitation and Western blot showed that p27kip1 was a target protein for SUMOylation and high expression of RNF4 decreased the levels of nuclear p27kip1, enhanced the levels of ubiquitinated and SUMOylated p27kip1, indicating that RNF4 could regulate cell cycle progression via recognizing SUMOylated p27kip1 and facilitating its ubiquitination degradation. These data indicate that RNF4-mediated ubiquitination degradation of SUMOylated proteins is a novel regulatory mechanism of p27kip1 dysfunction and CCA tumorigenesis, which provides a potential option for therapeutic intervention of CCA.

Cancer cell cycle dystopia: heterogeneity, plasticity, and therapy.

The mammalian cell cycle has been extensively studied regarding cancer etiology, progression, and therapeutic intervention. The canonical cell cycle framework is supported by a plethora of data pointing to a relatively simple linear pathway in which mitogenic signals are integrated in a stepwise fashion to allow progression through G1/S with coordinate actions of cyclin-dependent kinases (CDK)4/6 and CDK2 on the RB tumor suppressor. Recent work on adaptive mechanisms and intrinsic heterogeneous dependencies indicates that G1/S control of the cell cycle is a variable signaling pathway rather than an invariant engine that drives cell division. These alterations can limit the effectiveness of pharmaceutical agents but provide new avenues for therapeutic interventions. These findings support a dystopian view of the cell cycle in cancer where the canonical utopian cell cycle is often not observed. However, recognizing the extent of cell cycle heterogeneity likely creates new opportunities for precision therapeutic approaches specifically targeting these states.

Nup50 plays more than one instrument.

Nup50 is nuclear pore complex component localized to the nuclear side of the pore and in the nucleoplasm. It has been characterized as an auxiliary factor in nuclear transport reactions. Our recent work indicates that it interacts with and stimulates RCC1, the sole guanine nucleotide exchange factor for the GTPase Ran. Here, we discuss how this interaction might contribute to Nup50 function in nuclear transport but also its other functions like control of gene expression, cell cycle and DNA damage repair.

Neurogenesis and Proliferation of Neural Stem/Progenitor Cells Conferred by Artesunate via FOXO3a/p27Kip1 Axis in Mouse Stroke Model.

Promoting neurogenesis and proliferation of endogenous neural stem/progenitor cells (NSPCs) is considered a promising strategy for neurorehabilitation after stroke. Our previous study revealed that a moderate dose of artesunate (ART, 150 mg/kg) could enhance functional recovery in middle cerebral artery occlusion (MCAO) mice. This study aimed to investigate the effects of ART treatment on neurogenesis and proliferation of NSPCs using a rodent MCAO model. MRI results indicated that the ischemic brain volume of MCAO mice was reduced by ART treatment. The results of diffusion tensor imaging, electron microscopic, and immunofluorescence of Tuj-1 also revealed that ischemia-induced white matter lesion was alleviated by ART treatment. After ischemia/reperfusion, the proportion of Brdu + endogenous NSPCs in the ipsilateral subventricular zone and peri-infarct cortex was increased by ART treatment. Furthermore, the neuro-restorative effects of ART were abolished by the overexpression of FOXO3a. These findings suggested that ART could rescue ischemia/reperfusion damage and alleviate white matter injury, subsequently contributing to post-stroke functional recovery by promoting neurogenesis and proliferation of endogenous NSPCs via the FOXO3a/p27Kip1 pathway.

An Unanticipated Modulation of Cyclin-Dependent Kinase Inhibitors: The Role of Long Non-Coding RNAs.

It is now definitively established that a large part of the human genome is transcribed. However, only a scarce percentage of the transcriptome (about 1.2%) consists of RNAs that are translated into proteins, while the large majority of transcripts include a variety of RNA families with different dimensions and functions. Within this heterogeneous RNA world, a significant fraction consists of sequences with a length of more than 200 bases that form the so-called long non-coding RNA family. The functions of long non-coding RNAs range from the regulation of gene transcription to the changes in DNA topology and nucleosome modification and structural organization, to paraspeckle formation and cellular organelles maturation. This review is focused on the role of long non-coding RNAs as regulators of cyclin-dependent kinase inhibitors' (CDKIs) levels and activities. Cyclin-dependent kinases are enzymes necessary for the tuned progression of the cell division cycle. The control of their activity takes place at various levels. Among these, interaction with CDKIs is a vital mechanism. Through CDKI modulation, long non-coding RNAs implement control over cellular physiology and are associated with numerous pathologies. However, although there are robust data in the literature, the role of long non-coding RNAs in the modulation of CDKIs appears to still be underestimated, as well as their importance in cell proliferation control.

Role of Genetic Variations in CDK2, CCNE1 and p27KIP1 in Prostate Cancer.

BACKGROUND/AIM: Our aim was to investigate possible influences of genetic variants in genes involved in the G1/S transition [cyclin-dependent kinase-2 (CDK2), cyclin E1 (CCNE1) and cyclin-dependent kinase inhibitor 1B (p27KIP1)] on the expression/activity of their corresponding proteins and to assess the functional impact of these variants on the risk of prostate cancer. MATERIALS AND METHODS: We genotyped 530 cases and 562 healthy controls for two relevant single nucleotide polymorphisms (CDK2 rs2069408 and CCNE1 rs997669) by TaqMan genotyping assay. p27KIP1 rs2066827 polymorphisms were studied by polymerase chain reaction-restriction fragment length polymorphism assay. In addition, the expression of CDK2, CCNE1 and p27KIP1 was evaluated by quantitative real-time-polymerase chain reaction and western blotting in 44 prostate cancer tissues and 31 benign prostatic hyperplasia tissues. RESULTS: No association was found between CDK2 rs2069408, CCNE1 rs997669 or p27KIP1 rs2066827 polymorphisms and an increased risk of prostate cancer development. Higher CDK2 expression was more prevalent in those with rs2069408 GG genotype than in AA carriers (p>0.05). We also noted reduced p27KIP1 protein expression in those with the p27KIP1 G109 allele. No difference was observed for CCNE1 expression in relation to the risky genotype (CC). A significant association was detected between CCNE1 mRNA overexpression and development of higher-grade carcinomas (Gleason score >7, p<0.05). CONCLUSION: Polymorphisms CDK2 rs2069408, CCNE1 rs997669 and p27KIP1 rs2066827 have no significant impact on prostate cancer risk nor on the gene and protein expression of CDK2, CCNE1 and p27KIP1, although high CCNE1 expression was significantly associated with a higher tumour grade in patients with prostate cancer.

Apigenin inhibits growth of melanoma by suppressing miR-512-3p and promoting the G1 phase of cell cycle involving the p27 Kip1 protein.

In the present study, we screened multiple melanoma cell lines for treatment of Apigenin and miRNA expression, also studied the role of miR-512-3p in melanoma. RT-PCR analysis was done for screening miRNA in melanoma cell lines (WM1361B, WM983A, WM1341D, SK-MEL-3, SH-4, SK-MEL-24 and RPMI-7951) compared to normal human epidermal melanocytes. Colony formation assay for cell viability studies, cell cycle by flowcytometry and protein expression by immunoblot analysis. For in vivo analysis tumour xenograft mouse model was created. Immunohistochemistry was done for PCNA positive cells. For expression of miR-512-3p in tumour tissues fluorescence in situ hybridization was done. In silico studies were done by molecular docking studies. The WM1361B and WM983A cell lines showed overexpression of miR-512-3p and increased cell proliferation compared to normal human epidermal melanocytes. Treatment of anti-miR-512-3p to WM1361B and WM983A cells halted cell proliferation and also caused G1-phase arrest. We studied the effect of Apigenin on the expression levels of miR-512-3p and associated molecular targets. Apigenin treatment in WM1361B and WM983A cells showed inhibition in expression of miR-512-3p, arrest of G1 phase of cell cycle, cytotoxicity and revival of p27 Kip1. Apigenin treatment significantly suppressed the growth of WM1361B in tumour induced mice, the activity was associated with decreased levels of miR-512-3p, tumour cell proliferation and increased levels of p27 Kip1 protein. Docking studies confirm potential affinity of Apigenin for p27 Kip1. Apigenin acts as an inhibitor of miR-512-3p by suppressing growth of melanoma both in vitro and in vivo targeting the p27 Kip1 axis.

p27, The Cell Cycle and Alzheimer´s Disease.

The cell cycle consists of successive events that lead to the generation of new cells. The cell cycle is regulated by different cyclins, cyclin-dependent kinases (CDKs) and their inhibitors, such as p27Kip1. At the nuclear level, p27Kip1 has the ability to control the evolution of different phases of the cell cycle and oppose cell cycle progression by binding to CDKs. In the cytoplasm, diverse functions have been described for p27Kip1, including microtubule remodeling, axonal transport and phagocytosis. In Alzheimer's disease (AD), alterations to cycle events and a purported increase in neurogenesis have been described in the early disease process before significant pathological changes could be detected. However, most neurons cannot progress to complete their cell division and undergo apoptotic cell death. Increased levels of both the p27Kip1 levels and phosphorylation status have been described in AD. Increased levels of Aß42, tau hyperphosphorylation or even altered insulin signals could lead to alterations in p27Kip1 post-transcriptional modifications, causing a disbalance between the levels and functions of p27Kip1 in the cytoplasm and nucleus, thus inducing an aberrant cell cycle re-entry and alteration of extra cell cycle functions. Further studies are needed to completely understand the role of p27Kip1 in AD and the therapeutic opportunities associated with the modulation of this target.

Functional assessment of miR­1291 in colon cancer cells.

miR­1291 exerts an anti­tumor effect in a subset of human carcinomas, including pancreatic cancer. However, its role in colorectal cancer (CRC) is largely unknown. In the present study, the expression and effect of miR­1291 in CRC cells was investigated. It was identified that miR­1291 significantly suppressed the proliferation, invasion, cell mobility and colony formation of CRC cells. Additionally, miR­1291 induced cell apoptosis. A luciferase reporter assay revealed that miR­1291 directly bound the 3'­untranslated region sequence of doublecortin­like kinase 1 (DCLK1). miR­1291 also suppressed DCLK1 mRNA and protein expression in HCT116 cells that expressed DCLK1. Furthermore, miR­1291 suppressed cancer stem cell markers BMI1 and CD133, and inhibited sphere formation. The inhibitory effects on sphere formation, invasion and mobility in HCT116 cells were also explored and verified using DCLK1 siRNAs. Furthermore, miR­1291 induced CDK inhibitors p21WAF1/CIP1 and p27KIP1 in three CRC cell lines, and the overexpression of DCLK1 in HCT116 cells led to a decrease of p21WAF1/CIP1 and p27KIP1. Intravenous administration of miR­1291 loaded on the super carbonate apatite delivery system significantly inhibited tumor growth in the DLD­1 xenograft mouse model. Additionally, the resultant tumors exhibited significant upregulation of the p21WAF1/CIP1 and p27KIP1 protein with treatment of miR­1291. Taken together, the results indicated that miR­1291 served an anti­tumor effect by modulating multiple functions, including cancer stemness and cell cycle regulation. The current data suggested that miR­1291 may be a promising nucleic acid medicine against CRC.

Differential regulation of p27Kip1 depending on culture conditions and its correlation with status of p14ARF and p53.

p27Kip1 is known as a major cyclin-dependent kinase inhibitor and a tumor suppressor, and often functionally hampered at protein level. p27 protein expression levels are frequently low in various cancers and negatively correlated with malignancy of cancer. However, in our previous study, we discovered that p27 overexpression does not inhibit the proliferation of two cancer cell lines due to a functional suppression of p27 by nucleophosmin isoform 1 (NPM1); that is, a qualitative, not quantitative, suppression of p27 function occurs in these cancer cell lines. To clarify the regulation of p27 in several types of cancer, we investigated p27 function in other cancer cell lines, based on proliferation assays in those cell lines carrying doxycycline-inducible p27, and found that MDAH041 cells which express p14ARF, an antagonist of NPM1, show growth inhibition depending on p27 induction. Moreover, to investigate p27 function under anchorage-independent culture conditions, we performed soft agar colony formation assay and observed that the colony formation of some cell lines carrying wild-type p53, a major tumor suppressor, was inhibited depending on p27 induction. These results suggest that p27 function is regulated differentially among cancer cell types under anchorage-dependent and anchorage-independent culture conditions.