Differential expression of metallothionein and p21 in gastric cancer and some precursor lesions.

OBJECTIVE: Gastric cancer (GC) is a heterogeneous disease with molecular diversity between and within tumors; therefore, searching for altered genes within this cancer is mandatory to reach the proper individualized targeted therapy. Expressions of Metallothionein (MT) and p21 are not uniform in various types of cancers and their predictive value in GC is controversial. This study aimed to assess the role of MT and p21 in intestinal-type GC and some of its precursor lesions. MATERIALS AND METHODS: Immunohistochemical staining for MT and p21 was applied on paraffin blocks belonging to 30 GCs and 51 benign gastric lesions/precancerous lesions [33 chronic gastritis and 18 chronic gastritis with gastric intestinal metaplasia (GIM)]; 27 of them were associated with H. pylori infection. RESULTS: MT expression was dramatically increased while p21 expression was dramatically decreased from chronic gastritis to GIM to GC. In precancerous lesions, H. pylori-positive cases had significantly higher MT expression and lower p21 expression compared to H. pylori-negative cases. In GCs, decreased expression of both MT and p21 was associated with high-grade and advanced-stage cancers. CONCLUSIONS: Both MT and p21 may have a role in the development and progression of GC, and both proteins may be useful for selecting targeted therapy for GC patients.

Fluorizoline Blocks the Interaction between Prohibitin-2 and γ-Glutamylcyclotransferase and Induces p21Waf1/Cip1 Expression in MCF7 Breast Cancer Cells.

Prohibitin-2 (PHB2) is a scaffold protein that has pleiotropic functions, which include interacting with γ-glutamylcyclotransferase (GGCT) in the cytoplasm and repressing the transcriptional activities of the p21Waf1/Cip (p21) gene in the nucleus. The cytotoxic drug fluorizoline binds to PHB1/2 and exerts antiproliferative actions on cancer cells. However, the precise mechanism underlying the antiproliferative effects of fluorizoline is not fully elucidated. In the present study, we first show that fluorizoline induces p21 expression in several human cancer cell lines, including MCF7 breast cancer cells. Treatment of MCF7 cells with fluorizoline suppressed proliferation and prevented cells from entering into the DNA synthesis phase. Knockdown of p21 rescued the suppressed proliferation, indicating that fluorizoline inhibited MCF7 cell growth via the induction of p21. Overexpression of PHB2 in MCF7 cells prevented the induction of p21 expression by fluorizoline and restored the antiproliferative effects and blockade of cell cycle progression. Moreover, treatment of MCF7 cells with fluorizoline inhibited the interaction between endogenous PHB2 and GGCT proteins and reduced the level of nuclear localization of PHB2 proteins. These results indicate that targeting PHB2 with fluorizoline induces the expression of p21 and consequently blocks proliferation of cancer cells. SIGNIFICANCE STATEMENT: This study shows that fluorizoline may be a promising novel anticancer drug candidate that induces p21 expression and blocks cell-cycle progression in human cancer cell lines. In addition, we show that fluorizoline inhibits the interaction between PHB2 and GGCT and reduces the nuclear localization of PHB2 proteins.

Knockdown of the prognostic cancer stem cell marker Musashi-1 decreases radio-resistance while enhancing apoptosis in hormone receptor-positive breast cancer cells via p21WAF1/CIP1.

PURPOSE: While the stem cell marker Musashi-1 (MSI-1) has been identified as a key player in a wide array of malignancies, few findings exist on its prognostic relevance and relevance for cancer cell death and therapy resistance in breast cancer. METHODS: First, we determined prognostic relevance of MSI-1 in database analyses regarding multiple survival outcomes. To substantiate findings, MSI-1 was artificially downregulated in MCF-7 breast cancer cells and implications for cancer stem cell markers, cell apoptosis and apoptosis regulator p21, proliferation and radiation response were analyzed via flow cytometry and colony formation. Radiation-induced p21 expression changes were investigated using a dataset containing patient samples obtained before and after irradiation and own in vitro experiments. RESULTS: MSI-1 is a negative prognostic marker for disease-free and distant metastasis-free survival in breast cancer and tends to negatively influence overall survival. MSI-1 knockdown downregulated stem cell gene expression and proliferation, but increased p21 levels and apoptosis. Similar to the MSI-1 knockdown effect, p21 expression was strongly increased after irradiation and was expressed at even higher levels in MSI-1 knockdown cells after irradiation. Finally, combined use of MSI-1 silencing and irradiation reduced cancer cell survival. CONCLUSION: MSI-1 is a prognostic marker in breast cancer. MSI-1 silencing downregulates proliferation while increasing apoptosis. The anti-proliferation mediator p21 was upregulated independently after both MSI-1 knockdown and irradiation and even more after both treatments combined, suggesting synergistic potential. Radio-sensitization effects after combining radiation and MSI-1 knockdown underline the potential of MSI-1 as a therapeutic target.

Sgpl1 deletion elevates S1P levels, contributing to NPR2 inactivity and p21 expression that block germ cell development.

Sphingosine phosphate lyase 1 (SGPL1) is a highly conserved enzyme that irreversibly degrades sphingosine-1-phosphate (S1P). Sgpl1-knockout mice fail to develop germ cells, resulting in infertility. However, the molecular mechanism remains unclear. The results of the present study showed that SGPL1 was expressed mainly in granulosa cells, Leydig cells, spermatocytes, and round spermatids. Sgpl1 deletion led to S1P accumulation in the gonads. In the ovary, S1P decreased natriuretic peptide receptor 2 (NPR2) activity in granulosa cells and inhibited early follicle growth. In the testis, S1P increased the levels of cyclin-dependent kinase inhibitor 1A (p21) and apoptosis in Leydig cells, thus resulting in spermatogenesis arrest. These results indicate that Sgpl1 deletion increases intracellular S1P levels, resulting in the arrest of female and male germ cell development via different signaling pathways.

ZFP36L1 plays an ambiguous role in the regulation of cell expansion and negatively regulates CDKN1A in chronic myeloid leukemia cells.

The mRNA-destabilizing proteins ZFP36L1 and ZFP36L2 are described as mediators of quiescence and play a pivotal role in hematopoietic malignancies. Both genes are mainly classified as tumor suppressor genes as they posttranscriptionally downregulate the expression of oncogenes and contribute to cellular quiescence. Here, we analyzed the role of ZFP36L1 and ZFP36L2 in chronic myeloid leukemia (CML). We found ZFP36L1 and ZFP36L2 expression to be deregulated in patients with CML. By use of in vitro models of tyrosine kinase inhibitor resistance, an increase in ZFP36L1 and ZFP36L2 expression was detected during the development of imatinib resistance. CRISPR/Cas9-derived knockout of ZFP36L1, but not of ZFP36L2, in imatinib-sensitive cells led to decreased proliferation rates in response to tyrosine kinase inhibitor treatment. This effect was also observed in untreated ZFP36L1 knockout cells, albeit to a lower extent. Genomewide gene expression analyses of ZFP36L1 knockout cells revealed differential expression of cell cycle regulators, in particular upregulation of the cell cycle inhibitor CDKN1A. In addition, the 3' untranslated region of CDKN1A was proven to be a direct target of ZFP36L1. This indicates that tumor suppressor genes can also be targeted by ZFP36L1. Hence, ZFP36L1 cannot unambiguously be regarded as a tumor suppressor gene.

EZH2-mediated long-chain non-coding RNA LINC00963 promotes proliferation and invasion of glioma cells through inhibiting p21 expression.

PURPOSE: In the early stage, bioinformatics analysis revealed that the expression of long-chain non-coding RNA LINC00963 in glioma tissues was remarkably increased, but its biological effects on glioma and the potential molecular mechanisms have not been reported. This study aimed to conduct a preliminary discussion on the impact of LINC00963 on glioma, so as to provide new ideas for the treatment of this cancer. METHODS: GEPIA database was consulted to determine the expression level of LINC00963 in gliomas. In addition, the interplay between LINC00963 expression and the prognosis of glioma patients was analyzed by Kaplan-Meier method. Effects of LINC00963 on the proliferation and migration of glioma cells were determined using Cell Counting Kit (CCK-8) and Transwell assay. The subcellular localization of LINC00963 was determined by nuclear separation experiments. At the same time, the regulation of LINC00963 on p21 expression was verified through qRT-PCR and Western blot experiments. RESULTS: By analyzing the GEPIA database, we found that LINC00963 was highly expressed in glioma tissues. Meanwhile, qRT-PCR results revealed that LINC00963 level in glioma tissues and cell lines was remarkably higher than that in the normal control group. Kaplan-Meier and log-rank test revealed that there was no statistically significant association between the expression level of LINC00963 and the prognosis of patients with glioma. In addition, in vitro cell assay results indicated that downregulation of LINC00963 markedly suppressed the proliferation and invasiveness of glioma cells. Finally, the related mechanism analysis revealed that LINC00963 may inhibit p21 expression through modulation of EZH2. CONCLUSIONS: LINC00963 can inhibit p21 expression through EZH2 and thus enhance the proliferative and invasive capacities of glioma cells. Consequently, LINC00963 may be a potential therapeutic target for gliomas.

The Impact of Chlorambucil and Valproic Acid on Cell Viability, Apoptosis and Expression of p21, HDM2, BCL2 and MCL1 Genes in Chronic Lymphocytic Leukemia.

Malignant cells in chronic lymphocytic leukemia (CLL) show resistance to apoptosis, as well as to chemotherapy, which are related to deletions or mutations of TP53, high expression of MCL1 and BCL2 genes and other abnormalities. Thus, the main goal of the present study was to assess the impact of chlorambucil (CLB) combined with valproic acid (VPA), a known antiepileptic drug and histone deacetylation inhibitor, on apoptosis of the cells isolated from 17 patients with CLL. After incubation with CLB (17.5 µM) and VPA (0.5 mM), percentage of apoptosis, as well as expression of two TP53 target genes (p21 and HDM2) and two genes from Bcl-2 family (BCL2 and MCL1), were tested. As a result, an increased percentage of apoptosis was observed for CLL cells treated with CLB and VPA, and with CLB alone. Under the treatment with the drug combination, the expression of p21 gene was visibly higher than under the treatment with CLB alone. At the same time, the cultures under CLB treatment showed visibly higher expression of BCL2 than the cultures with VPA alone. Thus, the present study strongly suggests further investigations on the CLB and VPA combination in CLL treatment.

MXD/MIZ1 transcription regulatory complexes activate the expression of MYC-repressed genes.

MXDs are transcription repressors that antagonize MYC-mediated gene activation. MYC, when associated with MIZ1, acts also as a repressor of a subset of genes, including p15 and p21. A role for MXDs in regulation of MYC-repressed genes is not known. We report that MXDs activate transcription of p15 and p21 in U2OS cells. This activation required DNA binding by MXDs and their interaction with MIZ1. MXD mutants deficient in MIZ1 binding interacted with the MYC-binding partner MAX and were active as repressors of MYC-activated genes but failed to activate MYC-repressed genes. Mutant MXDs with reduced DNA-binding affinity interacted with MAX and MIZ1 but neither repressed nor activated transcription. Our data show that MXDs and MYC have a reciprocally antagonistic potential to regulate transcription of target genes.

LMNB2 promotes the progression of colorectal cancer by silencing p21 expression.

Colorectal cancer is the second common cause of death worldwide. Lamin B2 (LMNB2) is involved in chromatin remodeling and the rupture and reorganization of nuclear membrane during mitosis, which is necessary for eukaryotic cell proliferation. However, the role of LMNB2 in colorectal cancer (CRC) is poorly understood. This study explored the biological functions of LMNB2 in the progression of colorectal cancer and explored the possible molecular mechanisms. We found that LMNB2 was significantly upregulated in primary colorectal cancer tissues and cell lines, compared with paired non-cancerous tissues and normal colorectal epithelium. The high expression of LMNB2 in colorectal cancer tissues is significantly related to the clinicopathological characteristics of the patients and the shorter overall and disease-free cumulative survival. Functional analysis, including CCK8 cell proliferation test, EdU proliferation test, colony formation analysis, nude mouse xenograft, cell cycle, and apoptosis analysis showed that LMNB2 significantly promotes cell proliferation by promoting cell cycle progression in vivo and in vitro. In addition, gene set enrichment analysis, luciferase report analysis, and CHIP analysis showed that LMNB2 promotes cell proliferation by regulating the p21 promoter, whereas LMNB2 has no effect on cell apoptosis. In summary, these findings not only indicate that LMNB2 promotes the proliferation of colorectal cancer by regulating p21-mediated cell cycle progression, but also suggest the potential value of LMNB2 as a clinical prognostic marker and molecular therapy target.

Caffeic Acid Enhances the Anti-Leukemic Effect of Imatinib on Chronic Myeloid Leukemia Cells and Triggers Apoptosis in Cells Sensitive and Resistant to Imatinib.

Among the phenolic acids tested on the K562 cell line, a model of chronic myeloid leukemia (CML), caffeic acid (CA) was biologically active on sensitive and imatinib (IM)-resistant cells at micro-molar concentration, either in terms of reduction of cell proliferation or triggering of apoptosis. The CA treatment provoked mitochondrial membrane depolarization, genomic DNA fragmentation and phosphatidylserine exposure, hallmarks of apoptosis. Cell cycle analysis following the treatment with comparable cytotoxic concentrations of IM or CA showed marked differences in the distribution profiles. The reduction of cell proliferation by CA administration was associated with increased expression of two cell cycle repressor genes, CDKN1A and CHES1, while IM at a cytotoxic concentration increased the CHES1 but not the CDKN1A expression. In addition, CA treatment affected the proliferation and triggered the apoptosis in IM-resistant cells. Taken together, these data suggested that CA induced the anti-proliferative effect and triggered apoptosis of CML cells by a different mechanism than IM. Finally, the combined administration of IM and CA at suboptimal concentrations evidenced a synergy of action in determining the anti-proliferative effect and triggering apoptosis. The ability of CA to potentiate the anti-leukemic effect of IM highlighted the nutraceutical potential of CA in CML.

AUF1 ligand circPCNX reduces cell proliferation by competing with p21 mRNA to increase p21 production.

Mammalian circRNAs can influence different cellular processes by interacting with proteins and other nucleic acids. Here, we used ribonucleoprotein immunoprecipitation (RIP) analysis to identify systematically the circRNAs associated with the cancer-related protein AUF1. Among the circRNAs interacting with AUF1 in HeLa (human cervical carcinoma) cells, we focused on hsa_circ_0032434 (circPCNX), an abundant target of AUF1. Overexpression of circPCNX specifically interfered with the binding of AUF1 to p21 (CDKN1A) mRNA, thereby promoting p21 mRNA stability and elevating the production of p21, a major inhibitor of cell proliferation. Conversely, silencing circPCNX increased AUF1 binding to p21 mRNA, reducing p21 production and promoting cell division. Importantly, eliminating the AUF1-binding region of circPCNX abrogated the rise in p21 levels and rescued proliferation. Therefore, we propose that the interaction of circPCNX with AUF1 selectively prevents AUF1 binding to p21 mRNA, leading to enhanced p21 mRNA stability and p21 protein production, thereby suppressing cell growth.

Identification of a druggable protein-protein interaction site between mutant p53 and its stabilizing chaperone DNAJA1.

The TP53 gene is the most frequently mutated gene in human cancers, and the majority of TP53 mutations are missense mutations. As a result, these mutant p53 (mutp53) either directly lose wildtype p53 (wtp53) tumor suppressor function or exhibit a dominant negative effect over wtp53. In addition, some mutp53 have acquired new oncogenic function (gain of function). Therefore, targeting mutp53 for its degradation may serve as a promising strategy for cancer prevention and therapy. Based on our previous finding that farnesylated DNAJA1 is a crucial chaperone in maintaining mutp53 stabilization, and by using an in silico approach, we built 3D homology models of human DNAJA1 and mutp53R175H proteins, identified the interacting pocket in the DNAJA1-mutp53R175H complex, and found one critical druggable small molecule binding site in the DNAJA1 glycine/phenylalanine-rich region. We confirmed that the interacting pocket in the DNAJA1-mutp53R175H complex was crucial for stabilizing mutp53R175H using a site-directed mutagenesis approach. We further screened a drug-like library to identify a promising small molecule hit (GY1-22) against the interacting pocket in the DNAJA1-mutp53R175H complex. The GY1-22 compound displayed an effective activity against the DNAJA1-mutp53R175H complex. Treatment with GY1-22 significantly reduced mutp53 protein levels, enhanced Waf1p21 expression, suppressed cyclin D1 expression, and inhibited mutp53-driven pancreatic cancer growth both in vitro and in vivo. Together, our results indicate that the interacting pocket in the DNAJA1-mutp53R175H complex is critical for mutp53's stability and oncogenic function, and DNAJA1 is a robust therapeutic target for developing the efficient small molecule inhibitors against oncogenic mutp53.

Musashi2 promotes the progression of pancreatic cancer through a novel ISYNA1-p21/ZEB-1 pathway.

Our previous studies found overexpression of Musashi2 (MSI2) conduced to the progression and chemoresistance of pancreatic cancer (PC) by negative regulation of Numb and wild type p53 (wtp53). Now, we further investigated the novel signalling involved with MSI2 in PC. We identified inositol-3-phosphate synthase 1 (ISYNA1) as a novel tumour suppressor regulated by MSI2. High MSI2 and low ISYNA1 expression were prevalently observed in 91 PC tissues. ISYNA1 expression was negatively correlated with MSI2 expression, T stage, vascular permeation and poor prognosis in PC patients. What's more, patients expressed high MSI2 and low ISYNA1 level had a significant worse prognosis. And in wtp53 Capan-2 and SW1990 cells, ISYNA1 was downregulated by p53 silencing. ISYNA1 silencing promoted cell proliferation and cell cycle by inhibiting p21 and enhanced cell migration and invasion by upregulating ZEB-1. However, MSI2 silencing upregulated ISYNA1 and p21 but downregulated ZEB-1, which can be rescued by ISYNA1 silencing. Moreover, reduction of cell migration and invasion resulting from MSI2 silencing was significantly reversed by ISYNA1 silencing. In summary, MSI2 facilitates the development of PC through a novel ISYNA1-p21/ZEB-1 pathway, which provides new gene target therapy for PC.

ETV4 promotes late development of prostatic intraepithelial neoplasia and cell proliferation through direct and p53-mediated downregulation of p21.

BACKGROUND: ETV4 is one of the ETS proteins overexpressed in prostate cancer (PC) as a result of recurrent chromosomal translocations. In human prostate cell lines, ETV4 promotes migration, invasion, and proliferation; however, its role in PC has been unclear. In this study, we have explored the effects of ETV4 expression in the prostate in a novel transgenic mouse model. METHODS: We have created a mouse model with prostate-specific expression of ETV4 (ETV4 mice). By histochemical and molecular analysis, we have investigated in these engineered mice the expression of p21, p27, and p53. The implications of our in vivo findings have been further investigated in human cells lines by chromatin-immunoprecipitation (ChIP) and luciferase assays. RESULTS: ETV4 mice, from two independent transgenic lines, have increased cell proliferation in their prostate and two-thirds of them, by the age of 10 months, developed mouse prostatic intraepithelial neoplasia (mPIN). In these mice, cdkn1a and its p21 protein product were reduced compared to controls; p27 protein was also reduced. By ChIP assay in human prostate cell lines, we show that ETV4 binds to a specific site (-704/-696 bp upstream of the transcription start) in the CDKN1A promoter that was proven, by luciferase assay, to be functionally competent. ETV4 further controls CDKN1A expression by downregulating p53 protein: this reduction of p53 was confirmed in vivo in ETV4 mice. CONCLUSIONS: ETV4 overexpression results in the development of mPIN but not in progression to cancer. ETV4 increases prostate cell proliferation through multiple mechanisms, including downregulation of CDKN1A and its p21 protein product: this in turn is mediated through direct binding of ETV4 to the CDKN1A promoter and through the ETV4-mediated decrease of p53. This multi-faceted role of ETV4 in prostate cancer makes it a potential target for novel therapeutic approaches that could be explored in this ETV4 transgenic model.

The protective effect of 1,25(OH)2D3 against cardiac hypertrophy is mediated by the cyclin-dependent kinase inhibitor p21.

As a critical regulator of the cell cycle, cyclin-dependent kinase (CDK) inhibitor p21 or p21 is involved in the development of cardiac hypertrophy and heart failure. Calcitriol, or 1,25(OH)2D3, the bioactive form of vitamin D (VD), can activate p21 expression and attenuate cardiac hypertrophy. To simulate cardiac hypertrophy in vitro and ex vivo, respectively, mice and cardiomyocytes were treated with isoproterenol (ISO). Moreover, the p21 signaling pathway was examined in ISO + VD and ISO + VD p21 inhibitor-treated cardiomyocytes. We found that calcitriol treatment led to a significant decrease in cardiac size and the mRNA levels of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) in ISO-treated mice. Furthermore, the surface area of cardiomyocytes and the expression of ANP and BNP were decreased, and the expression of p21 was increased in the ISO + VD group compared with those in the ISO group. Furthermore, the surface area of cardiomyocytes and the expression of ANP and BNP were markedly upregulated in the ISO + VD p21 inhibitor group relative to the ISO + VD group, whereas the difference was not statistically significant compared with those of the ISO p21 inhibitor group. Therefore, our findings indicate that 1,25(OH)2D3 protects against cardiac hypertrophy in mice through upregulating p21 expression.

Identification of nagilactone E as a protein synthesis inhibitor with anticancer activity.

Norditerpenoids and dinorditerpenoids represent diterpenoids widely distributed in the genus Podocarpus with notable chemical structures and biological activities. We previously reported that nagilactone E (NLE), a dinorditerpenoid isolated from Podocarpus nagi, possessed anticancer effects against lung cancer cells in vitro. In this study we investigated the in vivo effect of NLE against lung cancer as well as the underlying mechanisms. We administered NLE (10 mg·kg-1·d-1, ip) to CB-17/SCID mice bearing human lung cancer cell line A549 xenograft for 3 weeks. We found that NLE administration significantly suppressed the tumor growth without obvious adverse effects. Thereafter, RNA sequencing (RNA-seq) analysis was performed to study the mechanisms of NLE. The effects of NLE on A549 cells have been illustrated by GO and pathway enrichment analyses. CMap dataset analysis supported NLE to be a potential protein synthesis inhibitor. The inhibitory effect of NLE on synthesis of total de novo protein was confirmed in Click-iT assay. Using the pcDNA3-RLUC-POLIRES-FLUC luciferase assay we further demonstrated that NLE inhibited both cap-dependent and cap-independent translation. Finally, molecular docking revealed the low-energy binding conformations of NLE and its potential target RIOK2. In conclusion, NLE is a protein synthesis inhibitor with anticancer activity.

The KLF6 Super Enhancer Modulates Cell Proliferation via MiR-1301 in Human Hepatoma Cells.

BACKGROUND: Recent studies have attempted to elucidate the function of super enhancers by means of microRNAs. Although the functional outcomes of miR-1301 have become clearer, the pathways that regulate the expressions of miR-1301 remain unclear. OBJECTIVE: The objective of this paper was to consider the pathway regulating expression of miR- 1301 and miR-1301 signaling pathways with the inhibition of cell proliferation. METHODS: In this study, we prepared the cell clones that the KLF6 super enhancer was deleted by means of the CRISPR/Cas9 system-mediated genetic engineering. Changes in miR-1301 expression after the deletion of the KLF6 super enhancer were evaluated by RT-PCR analysis, and the signal pathway of miR-1301 with inhibition of the cell proliferation was examined using RNA interference technology. RESULTS: The results showed that miR-1301 expression was significantly increased after the deletion of the KLF6 super enhancer. Over-expression of miR-1301 induced by deletion of the KLF6 super enhancer also regulated the expression of p21 and p53 in human hepatoma cells. functional modeling of findings using siRNA specific to miR-1301 showed that expression level changes had direct biological effects on cellular proliferation in Human hepatoma cells. Furthermore, cellular proliferation assay was shown to be directly associated with miR-1301 levels. CONCLUSION: As a result, it was demonstrated that the over-expression of miR-1301 induced by the disruption of the KLF6 super enhancer leads to a significant inhibition of proliferation in HepG2 cells. Moreover, it was demonstrated that the KLF6 super enhancer regulates the cell-proliferative effects which are mediated, at least in part, by the induction of p21and p53 in a p53-dependent manner. Our results provide the functional significance of miR-1301 in understanding the transcriptional regulation mechanism of the KLF6 super enhancer.

A novel member of Prame family, Gm12794c, counteracts retinoic acid differentiation through the methyltransferase activity of PRC2.

Embryonic stem cells (ESCs) fluctuate among different levels of pluripotency defined as metastates. Sporadically, metastable cellular populations convert to a highly pluripotent metastate that resembles the preimplantation two-cell embryos stage (defined as 2C stage) in terms of transcriptome, DNA methylation, and chromatin structure. Recently, we found that the retinoic acid (RA) signaling leads to a robust increase of cells specifically expressing 2C genes, such as members of the Prame family. Here, we show that Gm12794c, one of the most highly upregulated Prame members, and previously identified as a key player for the maintenance of pluripotency, has a functional role in conferring ESCs resistance to RA signaling. In particular, RA-dependent expression of Gm12794c induces a ground state-like metastate, as evaluated by activation of 2C-specific genes, global DNA hypomethylation and rearrangement of chromatin similar to that observed in naive totipotent preimplantation epiblast cells and 2C-like cells. Mechanistically, we demonstrated that Gm12794c inhibits Cdkn1A gene expression through the polycomb repressive complex 2 (PRC2) histone methyltransferase activity. Collectively, our data highlight a molecular mechanism employed by ESCs to counteract retinoic acid differentiation stimuli and contribute to shed light on the molecular mechanisms at grounds of ESCs naive pluripotency-state maintenance.

lncRNA SNHG16 Exerts Oncogenic Functions in Promoting Proliferation of Glioma Through Suppressing p21.

Glioma is a malignant brain tumor that accounts for 30% of all brain tumors and 80% of malignant brain tumors. This poor clinical outcome makes the study of molecular mechanisms in glioma as an urgent subject. However, the certain mechanism remains unclear. Long non-coding RNAs (lncRNAs) plays a key role in glioma development and progression. In the present study, we aimed to explore the potential mechanisms of lncRNA SNHG16 in glioma. The levels of lncRNA SNHG16 were qualified in both glioma tissues and cell lines using qRT-PCR assay. The ability of cell proliferation was tested via CCK-8 and colony formation assays. Transfections were performed to knockdown SNHG16 and its target gene p21. The cell cycles and cell apoptosis were evaluated using flow cytometry, and the expression of SNHG16, p21 and apoptosis biomarkers were qualified with qRT-PCR and western blot assays. The expression of SNHG16 were up-regulated in both glioma tissues and cell lines. Knockdown of SNHG16 was associated with poor proliferation, decreased monoclonal formation rates, but increased apoptosis rates, which also caused the high expression of p21. Moreover, p21 could mediate cell proliferation and monoclonal formation, promote cell apoptosis in glioma, which was negatively correlated with lncRNA SNHG16. The molecule mechanism experiments revealed that SNHG16 could not only inhibit the expression of p21 but also suppressed the level of caspase 3 and 9, while promoted cyclinD1 and cyclinB1 expression. lncRNA SNHG16 could promote the cell proliferation and inhibit the apoptosis of glioma through suppressing p21, indicating that lncRNA SNHG16 might be quite vital for the diagnosis and progression of glioma and could even be a novel therapeutic target for glioma.

NANOG Attenuates Hair Follicle-Derived Mesenchymal Stem Cell Senescence by Upregulating PBX1 and Activating AKT Signaling.

Stem cells derived from elderly donors or harvested by repeated subculture exhibit a marked decrease in proliferative capacity and multipotency, which not only compromises their therapeutic potential but also raises safety concerns for regenerative medicine. NANOG-a well-known core transcription factor-plays an important role in maintaining the self-renewal and pluripotency of stem cells. Unfortunately, the mechanism that NANOG delays mesenchymal stem cell (MSC) senescence is not well-known until now. In our study, we showed that both ectopic NANOG expression and PBX1 overexpression (i) significantly upregulated phosphorylated AKT (p-AKT) and PARP1; (ii) promoted cell proliferation, cell cycle progression, and osteogenesis; (iii) reduced the number of senescence-associated-ß-galactosidase- (SA-ß-gal-) positive cells; and (iv) downregulated the expression of p16, p53, and p21. Western blotting and dual-luciferase activity assays showed that ectopic NANOG expression significantly upregulated PBX1 expression and increased PBX1 promoter activity. In contrast, PBX1 knockdown by RNA interference in hair follicle- (HF-) derived MSCs that were ectopically expressing NANOG resulted in the significant downregulation of p-AKT and the upregulation of p16 and p21. Moreover, blocking AKT with the PI3K/AKT inhibitor LY294002 or knocking down AKT via RNA interference significantly decreased PBX1 expression, while increasing p16 and p21 expression and the number of SA-ß-gal-positive cells. In conclusion, our findings show that NANOG delays HF-MSC senescence by upregulating PBX1 and activating AKT signaling and that a feedback loop likely exists between PBX1 and AKT signaling.