Expression Of Cyclin Research Articles

6-Methoxyflavone induces S-phase arrest through the CCNA2/CDK2/p21CIP1 signaling pathway in HeLa cells

ABSTRACT This study aimed to elucidate the specific anticancer mechanism of 6-methoxyflavone in HeLa cells. A total of 178 putative targets of 6-methoxyflavone were obtained from the PharmMapper database. Microarray analyses, transcriptome sequencing analyses, functional enrichment analyses, and gene set enrichment analyses were performed to preliminarily explore the roles and mechanisms of the 178 targets in cervical cancer. Cell counting kit-8, cell cycle assays, polymerase chain reactions, and western blotting were used to clarify the mechanism of action of 6-methoxyflavone. Molecular docking and noncovalent interaction analyses were performed to further confirm the mechanism of action in three-dimensional structures. Functional enrichment analyses and gene set enrichment analyses indicated that high mRNA expression of cyclin A2 (CCNA2) and cyclin-dependent kinase 2 (CDK2) stimulated cell cycle progression in cervical cancer. Cell proliferation and cycle assays, transcriptome sequencing, polymerase chain reactions, and western blotting revealed that 6-methoxyflavone inhibited HeLa cell proliferation and induced S-phase arrest via the CCNA2/CDK2/ cyclin-dependent kinase inhibitor 1A (p21CIP1) pathway. Molecular docking and noncovalent interaction analyses showed that 6-methoxyflavone had the strongest affinity toward, inhibitory effect on, and noncovalent interactions with CDK2, and that the combination of CDK2 and CCNA2 enhanced these effects. An analysis of clinical characteristics showed that 6-methoxyflavone might be related to six clinicopathological parameters of cervical cancer patients. 6-Methoxyflavone induces S-phase arrest in HeLa cells via the CCNA2/CDK2/p21CIP1 pathway.

MiR-29b Derived from Bone Marrow Stromal Cell (BMSC) Exosomes Improves Laryngeal Carcinoma by Inhibiting Forkhead Box Protein P1 (FOXP1) to Decrease Cyclin E2 Transcription

This study intends to investigate whether miR-29b derived from BMSC exosomes (BMSC-exos) affects laryngeal cancer progression. RT-qPCR detected miR-29b level in BMSCs and BMSC-exos. After miR-29b was overexpressed in BMSCs, exos were extracted from BMSCs and used to treat laryngeal cancer cells, followed by CCK-8 assay and soft agar assay. When cells were treated with FOXP1 inhibitor or cyclin E2 vector, Western blot analyzed the expression of related proteins and flow cytometry assessed cell cycle distribution. In vivo experiment was conducted to assess miR-29b’s effect on tumor growth. miR-29b was upregulated in BMSC-exos, but lowly expressed in cancer cells. miR-29b upregulation inhibited the proliferation of laryngeal cancer cells and delayed tumor progression In vivo by inducing cell cycle arrest. Importantly, miR-29b bound 3′UTR of FXOP1 to inhibit its expression, and further reduced cyclin E2 level. sh-FXOP1 or cyclin E2 vector can restore the cell cycle and proliferation caused by miR-29b. In conclusion, miR-29b enriched in BMSC-exo can down-regulate cyclin E2 expression through targeted inhibition of FXOP1, thereby inhibiting the progression of laryngeal cancer.

Effects of 4-vinylcyclohexene diepoxide on the cell cycle, apoptosis, and steroid hormone secretion of goat ovarian granulosa cells.

4-Vinylcyclohexene diepoxide (VCD) is a potentially hazardous industrial chemical that may enter a goat's body in various ways during industrial breeding. Ovarian granulosa cells (GCs) play a critical role in supporting follicle development and hormone synthesis. However, there are few studies on the effect of VCD on goat ovarian GCs. In this study, goat ovarian GCs were isolated and treated with VCD. The results showed that treatment with VCD increased the proportion of S phase and G2/M cells, but decreased the proportion of G1 phase. VCD treatment significantly inhibited the expression of cyclin A and cyclin-dependent kinase 2 (CDK2). But the expression levels of p21 and p27 were increased. VCD could induce an apparent increase in the proportion of apoptosis and the level of cleaved caspase 3. Treatment with VCD significantly reduced the progesterone and estrogen concentration in the medium in which goat ovarian GCs were cultured. Correspondingly, the expression level of steroidogenic acute regulatory protein (STAR) was significantly downregulated. Treatment with 0.25 and 0.5mM VCD, the protein expression level of insulin-like growth factor 1 receptor (IGF1R) and Akt were significantly decreased. Moreover, treatment with 0.25mM VCD significantly inhibited the phosphorylation of Akt. In conclusion, VCD exposure had cytotoxic effects such as decreased cell viability, disordered cell cycle, increased apoptosis, and interference with steroid hormone synthesis on goat GCs. These cytotoxic effects of VCD on goat GCs may be due to the downregulation of IGF1R and the inhibition of IGF1R/Akt signaling pathway.

The pro-tumorigenic activity of p38\u03b3 overexpression in nasopharyngeal carcinoma

It is urgent to identify and validate biomarkers for early diagnosis and efficient treatment of nasopharyngeal carcinoma (NPC). Recent studies have proposed p38 gamma (p38γ) as a cyclin-dependent kinase (CDK)-like kinase that phosphorylates retinoblastoma (Rb) to promote cyclins expression and tumorigenesis. Here the Gene Expression Profiling Interactive Analysis (GEPIA) database and results from the local NPC tissues demonstrate that p38γ is significantly upregulated in NPC tissues, correlating with poor overall survival. Furthermore, p38γ mRNA and protein expression is elevated in established NPC cell lines (CNE-1 HONE-1 and CNE-2) and primary human NPC cells, but low expression detected in human nasal epithelial cells. In established and primary NPC cells, p38γ depletion, using the shRNA strategy or the CRISPR/Cas9 gene-editing method, largely inhibited cell growth, proliferation and migration, and induced significant apoptosis activation. Contrarily, ectopic p38γ overexpression exerted opposite activity and promoted NPC cell proliferation and migration. Retinoblastoma (Rb) phosphorylation and cyclin E1/A expression were decreased in NPC cells with p38γ silencing or knockout, but increased after p38γ overexpression. Moreover, mitochondrial subcellular p38γ localization was detected in NPC cells. Significantly, p38γ depletion disrupted mitochondrial functions, causing mitochondrial depolarization, reactive oxygen species production, oxidative injury and ATP depletion in NPC cells. In vivo, intratumoral injection of adeno-associated virus-packed p38γ shRNA potently inhibited primary human NPC xenograft growth in nude mice. In p38γ shRNA virus-injected NPC xenograft tissues, p38γ expression, Rb phosphorylation, cyclin E1/A expression and ATP levels were dramatically decreased. Taken together, we conclude that p38γ overexpression is required for NPC cell growth, acting as a promising therapeutic target of NPC.

Repression of Mafb promotes foreskin fibroblast proliferation through upregulation of CDK2, cyclin E and PCNA.

Mafb plays a significant role in the development and differentiation of various organs, tissues and cells. Nevertheless, its role in the control of external genital cell proliferation and function in the mechanism of hypospadias remains unknown. In this study, the expression of Mafb in foreskin fibroblasts was inhibited by siRNA. The Cell Count Kit-8 (CCK-8) assay showed cell proliferation increased after transfection, and the number of cells entered the S phase significantly increased via flow cytometry. Both mRNA and protein levels of cyclin E, cyclin-dependent kinase 2 (CDK2) and proliferating cell nuclear antigen (PCNA) were significantly upregulated in the siRNA group. Meanwhile, twenty-five prepuce tissue samples were collected from hypospadias repair surgery. These samples were divided into two groups: the severe and mild groups. Normal prepuce tissue specimens were obtained during circumcision as the normal control. The upregulated expression of cyclin E, CDK2 and PCNA and downregulated Mafb expression were observed in the hypospadias group. This study reveals for the first time that the reduction in Mafb promotes the foreskin fibroblast proliferation. Thus, downregulated Mafb expression may cause hypospadias by upregulating CDK2, cyclin E and PCNA. These findings can shed new light on the embryonic development of the urethra.

The anti-cancer role of microRNA-143 in papillary thyroid carcinoma by targeting high mobility group AT-hook 2

ABSTRACT Papillary thyroid carcinoma (PTC), a common thyroid cancer (TC) subtype, rapidly increases in occurrence. MicroRNAs (miRNAs), which are non-coding small RNAs, have been demonstrated to play a role in cancer pathogenic mechanisms. Although miR-143 is involved in suppressing certain malignant tumor progression, its biological role is unknown in PTC. The present study found that miR-143 levels were strongly lower in PTC patient samples and cell lines, implying that miR-143 may play a biological role in PTC. Down-regulation of miR-143 resulted in the increased expression of HMGA2. Furthermore, HMGA2 was found to be a direct target of miR-143. A dual-luciferase assay confirmed a direct binding site for miR-143 was confirmed on HMGA2 using a dual-luciferase assay. Next, over-expression of miR-143 suppressed PTC cell growth as analyzed by MTT, clone formation, and Ki-67 immunofluorescence staining assays. miR-143 mimics transfection downregulated the expression of PCNA, CDK4, CDK1, and Cyclin E1. In addition, wound healing and trans-well assays revealed that miR-143 up-regulation inhibited PTC cells invasion and migration. Co-transfection of HMGA2 expression vector restored HMGA2 expression and rescued PTC cells proliferation capability in miR-143 mimics transfected PTC cells, indicating that miR-143 inhibited PTC cells proliferation via HMGA2. These observations were also obtained in xenografts experiments in nude mice. Altogether, our study shed light on miR-143ʹs anti-cancer biological functions in PTC progression through targeting HMGA2, suggesting that restoration of miR-143 could be a potential therapeutic approach for PTC treatment.

Alachlor breaks down intracellular calcium homeostasis and leads to cell cycle arrest through JNK/MAPK and PI3K/AKT signaling mechanisms in bovine mammary gland epithelial cells

Alachlor is a widely used herbicide for the cultivation of various grains employed as food for cattle. The mechanisms leading to the toxic effects of alachlor on epithelial cells of the bovine mammary gland are not well known. Thus, this study was conducted to clarify the toxicological effects of alachlor on the immortalized epithelial cell line of the bovine mammary gland (MAC-T) cells. After treatment, many factors related to cell viability, proliferation, and cellular homeostasis were evaluated. Alachlor arrested cell cycle progression by blocking the expression of cyclin and cyclin-dependent kinases, and induced the breakdown of Ca2+ homeostasis. The cytosolic and mitochondrial levels of Ca2+ were also abnormally increased after the treatment of cells with alachlor, ultimately leading to the depolarization of mitochondrial membrane potential in MAC-T cells. The signaling cascade was found to be dysregulated by the abnormal phosphorylation of signaling molecules involved in PI3K/AKT (AKT, p70S6K, and S6) and MAPK/JNK (JNK and c-Jun) pathways. In these mechanisms, exposure to alachlor led to a reduction in the viability and proliferation of MAC-T cells. Altogether, the toxic effects of alachlor can lead to abnormal conditions in epithelial cells of the bovine mammary gland, which might hinder these cells from performing their main role, such as producing milk.

Extracellular Polysaccharide from Rhizopus nigricans Inhibits Hepatocellular Carcinoma via miR-494-3p/TRIM36 Axis and Cyclin E Ubiquitination.

Background and AimsThis study was designed to uncover the mechanism for extracellular polysaccharide (EPS1-1)-mediated effects on hepatocellular carcinoma (HCC) development.MethodsHCC cells were treated with EPS1-1, miR-494-3p mimic, sh-TRIM36, and pcDNA3.1-TRIM36. The levels of miR-494-3p and TRIM36 were measured in normal hepatocytes, THLE-2, and HepG2 and HuH7HCC cell lines, along with the protein expression of cyclin D/E and p21. The proliferation, cell cycle, and apoptosis of HCC cells were assayed. The interactions between miR-494-3p and TRIM36, and between TRIM36 and cyclin E were assessed. Finally, the expression and localization of TRIM36 and cyclin E were monitored, and tumor apoptosis was detected, in tumor xenograft model.ResultsEPS1-1 suppressed HCC cell proliferation and cyclin D/E expression and promoted apoptosis and p21 expression. miR-494-3p was upregulated and TRIM36 was downregulated in HCC cells. Transfection with miR-494-3p mimic or sh-TRIM36 facilitated HCC cell proliferation and the expression of cyclin D/E protein but they inhibited apoptosis and p21 expression in the presence of EPS1-1. Overexpression of TRIM36 further consolidated EPS1-1-mediated inhibition of HCC proliferation, cyclin D/E, and the promotion of apoptosis and p21 expression. Those effects were reversed by miR-494-3p overexpression. TRIM36 was a target gene of miR-494-3p, and TRIM36 induced cyclin E ubiquitination. EPS1-1 suppressed cyclin E expression, promoted TRIM36 expression and tumor apoptosis, all of which were abrogated by increasing the expression of miR-494-3p in vivo.ConclusionsEPS1-1 protected against HCC by limiting its proliferation and survival through the miR-494-3p/TRIM36 axis and by inducing cyclin E ubiquitination.

Effect of human decidua mesenchymal stem cells-derived exosomes on the function of high glucose-induced senescent human dermal fibroblasts and its possible mechanism

Effect of human decidua mesenchymal stem cells-derived exosomes on the function of high glucose-induced senescent human dermal fibroblasts and its possible mechanism

Abstract P2-07-05: Activation of the AKT/mTOR signaling pathway is associated with response to the combination of endocrine therapy and CDK4/6 inhibitor in HR+/HER2- metastatic breast cancer

Abstract Background: As part of a multicenter study designed to identify markers of response or resistance to the combination of endocrine therapy (ET) and a cyclin dependent kinase 4/6 inhibitor (CDK 4/6i) as standard 1st-line therapy in patients with hormone receptor-positive, HER2-negative (HR+/HER2-), metastatic breast cancer (MBC), we assessed whether pretreatment levels of expression and phosphorylation of CDK 4/6 substrates and downstream molecules can predict response to this treatment. We then conducted an exploratory analysis to identify kinase-driven, pathway-centered mechanisms associated with response in pretreatment tumor samples. Methods: Tumor epithelia were isolated and enriched from the surrounding microenvironment using laser capture microdissection followed by downstream analysis using the Reverse Phase Protein Microarray (RPPA). Unmodified or post-transitionally modified residues were measured for 120 signaling proteins including 8 pre-specified qualifying proteins/phosphoproteins that are direct substrates of CDK 4/6 or whose activity is controlled by CDKI 4/6 activation as predictive markers of response to ET plus a CDK4/6i as 1st-line treatment in HR+/HER2- MBC. These 8 qualifying markers were: total Rb, phospho-Rb (S780), total Cyclin D1, phospho-Cyclin D1 (S286), total p16INK, total p27KIP, phospho-p27KIP (T187), and phospho-FOXM1 (T600). A total of 20 samples were available for the exploratory analysis. Specimens with expression or phosphorylation of the 120 biomarkers above or below the population median were classified as high or low, respectively. Chi-square analysis was used to compare proportion of patients with high and low expression between responders (CR, PR, or SD for a minimum of 12 months) and non-responders (PD within 12 months). Results: Pretreatment phosphorylation levels of Rb at the S780 residue were higher in non-responders compared to responders (p=0.025) and a similar trend was also detected for unmodified Rb, FoxM1 (T600), p27 Kip1 (T187), and Cyclin D1 (T289). Of the 120 proteins measured, there were statistically significant difference between responders and non-responders in 61. Non-responders were characterized by increased expression and phosphorylation of Rb regulators like CDK 4 (T172), as well as expression of CDK2, Cyclin E1, and Cyclin E2. Non-responders also presented with a broad activation of the PI3K/AKT/mTOR pathway, including phosphorylated PDK1 (S241) and AKT (S473 and T308), along with the AKT substrates FoxO1 (S256), FoxO1/FoxO3 (T24/T32), mTOR (S2448), and the mTOR regulator PRAS40 (T246). Finally, the mTORC1 complex downstream substrates p70S6K (T389) and 4EBP1 (S65 and T37/46) were also increased in non-responders. Conclusion: Taken together our data suggest that expression of Rb regulators along with the activation of the PI3K/AKT/mTOR signaling axis may modulate response to ET in combination with a CDK 4/6i. Targeting these pathways may be a novel therapeutic opportunity to enhance and prolong response to this treatment. Citation Format: Maysa Abu-Khalaf, Christos Hatzis, K. Alex Hodge, Elisa Baldelli, William Sikov, Monica Mita, Frances Valdes-Albini, Bryant Dunetz, Emanuel Petricoin, Mariaelena Pierobon. Activation of the AKT/mTOR signaling pathway is associated with response to the combination of endocrine therapy and CDK4/6 inhibitor in HR+/HER2- metastatic breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-07-05.

SGLT2 inhibition restrains thyroid cancer growth via G1/S phase transition arrest and apoptosis mediated by DNA damage response signaling pathways

BackgroundAlthough the prognosis for most patients with papillary thyroid cancer (PTC) is good, the present treatment is ineffective for 5–10% patients. Several studies found sodium–glucose cotransporter 2 (SGLT2) inhibitors may inhibit the growth of tumors. However, whether SGLT2 inhibitors have therapeutic effect on thyroid cancer remains unclear.Materials and methodsThe levels of SGLT2 in PTC and normal thyroid tissue were assessed by immunohistochemistry and clinical dataset analysis. Cell growth was detected by the CCK-8 and colony formation. Glucose uptake into thyroid cancer cell was evaluated by 2-DG uptake assay. Glycolysis were analyzed by Seahorse XF Extracellular Flux Analysis. RNA-seq were used to screen differentially expressed genes of cells treated with/without canagliflozin (a SGLT2 inhibitor). Furthermore, flow cytometry, western blot, and gene set enrichment analysis were employed to elucidate cell cycle, apoptosis and the underlying mechanism of the anticancer effect of canagliflozin. The effect of canagliflozin on thyroid cancer growth was further confirmed in vivo through xenograft formation assay.ResultsSGLT2 inhibition attenuated the growth of thyroid cancer cells in vitro and in vivo. Canagliflozin inhibited glucose uptake, glycolysis and AKT/mTOR signaling activation, and increased AMPK activation in thyroid cancer cell. Furthermore, canagliflozin inhibited G1/S phase transition and cyclin D1, cyclin D3, cyclin E1, cyclin E2, and E2F1 expression levels in thyroid cancer cell. In addition, canagliflozin increased apoptosis of thyroid cancer cell. Further investigation revealed that canagliflozin could increase γ-H2AX expression levels and DNA damage response signaling ATM/CHK2 activation. In thyroid cancer patients, SGLT2 was increased in thyroid cancer and positively related to cyclin D3.ConclusionsSGLT2 inhibition may limit glucose uptake resulting in energetic crisis, following oxidative stress mediated DNA damage and cell cycle arrest, which resulted to the increased cell apoptosis and decreased proliferation of thyroid cancer cells, suggesting a potential use for SGLT2 inhibitors as thyroid cancer therapeutics.

Growth and Cyclin-E Expression in the Stony Coral Species Orbicella faveolata Post-Microfragmentation.

AbstractCoral growth is critical to reef health, resilience under rapidly changing environmental conditions, and restoration efforts. Although fragmenting coral has been occurring for many years in an effort to restore reefs, recently it was discovered that microfragmenting, the process of cutting one piece of coral into many small pieces (about three to five polyps), induces exponential growth. Our study investigates the process by which microfragments of nine different genotypes from the stony coral species Orbicella faveolata grow and exhibit Cyclin-E expression. Microfragments were examined by using a high-powered dissecting microscope with a camera to document the precise areas of tissue exhibiting exponential growth. We found that new polyp formation occurs only on the microfragment edges and that edge polyp growth rates varied between different genotypes. We then extracted tissue from both the edge and the center of five genotypes for genetic analysis. We chose to analyze Cyclin-E expression because it is involved with stimulating mitotic division and is a conserved signaling pathway that is known to exist in Drosophila, mammals, and Cnidaria. Two primers for Cyclin-E were utilized to examine the level of expression for center and edge tissue. We found that Cyclin-E is expressed differentially between O. faveolata polyps, with a tendency for increased expression of the Cyclin-E in edge versus center tissue in each of five genotypes, although this result was not significant. Despite consistently higher levels of Cyclin-E expression within an organism's edge tissue, genotypes varied significantly in the degree of increased expression. This variation positively correlated with growth rate, suggesting the potential for molecular selection in aid of more rapid reef restoration. Future work will focus on deciphering the specific growth pathways involved in microfragmented coral growth and analyzing expression patterns in injured tissues.

Cryptotanshinone inhibits proliferation and induces apoptosis of breast cancer MCF-7 cells via GPER mediated PI3K/AKT signaling pathway.

G protein-coupled estrogen receptor (GPER) was reported to be a potential target in the breast cancer therapy. This study aimed to illuminate the function of GPER and its mediated PI3K/AKT pathway in cryptotanshinone (CPT) inducing cell apoptosis and antiproliferation effect on GPER positive breast cancer MCF-7 cells. Cell proliferation was tested by MTT assay. Apoptosis rates were tested by Annexin V-FITC/PI double staining and the cell cycle was researched by flow cytometry. Autodock vina was applied to make molecular docking between CPT or estradiol and GPER. siRNA technique and GPER specific agonist G-1 or antagonist G-15 were applied to verify the mediated function of GPER. Apoptosis and cell cycle related proteins, as well as the key proteins on PI3K/AKT signaling pathway were detected by western blot. The results indicated that CPT could exert antiproliferation effects by arresting cell cycle in G2/M phase and downregulating the expression of cyclin D, cyclin B and cyclin A. Besides, apoptosis induced by CPT was observed. CPT might be a novel GPER binding compounds. Significantly, suppression of PI3K/AKT signal transduction by CPT was further increased by G-1 and decreased by G-15. The study revealed that the effect of antiproliferation and apoptosis treating with CPT on MCF-7 cells might be through the downregulation of PI3K/AKT pathway mediated by activated GPER.

Mollugin induced oxidative DNA damage via up-regulating ROS that caused cell cycle arrest in hepatoma cells

Mollugin has been proven to have anti-tumor activity. However, its potential anti-tumor mechanism remains to be fully elaborated. Herein, we investigated the growth inhibition of HepG2 cells, as well as the anti-tumor effect of mollugin and its molecular mechanism on H22-tumor bearing mice. In vitro, mollugin was shown to have a strong inhibitory effect on HepG2 cells in a concentration-dependent manner. Mollugin induced S-phase arrest of HepG2 cells, and increased intracellular reactive oxygen species (ROS) levels. Comet assay demonstrated that mollugin induced DNA damage in HepG2 cells, as well as an increase in the expression of p-H2AX. In addition, mollugin induced changes in cyclin A2 and CDK2. However, the addition of antioxidant glutathione (GSH) was able to reverse the effect of mollugin. In vivo, mollugin significantly inhibited tumor growth and reduced the tendency of tumor volume growth in mice. The tumor cell density was found to be decreased in the administration group, and the content of ROS in the tumor tissue significantly increased. The expression of p-H2AX, cyclin A2 and CDK2 were consistent with in vitro results. Mollugin demonstrated anti-hepatocellular carcinoma activity in vitro and in vivo, and its anti-hepatocellular carcinoma activity was found to be related to DNA damage and cell cycle arrest induced by excessive ROS production in cells.

Regulation of Liver Regeneration by Hepatocyte O-GlcNAcylation in Mice.

Background & AimsThe liver has a unique capacity to regenerate after injury in a highly orchestrated and regulated manner. Here, we report that O-GlcNAcylation, an intracellular post-translational modification regulated by 2 enzymes, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), is a critical termination signal for liver regeneration following partial hepatectomy (PHX).MethodsWe studied liver regeneration after PHX on hepatocyte specific OGT and OGA knockout mice (OGT-KO and OGA-KO), which caused a significant decrease (OGT-KO) and increase (OGA-KO) in hepatic O-GlcNAcylation, respectively.ResultsOGA-KO mice had normal regeneration, but the OGT-KO mice exhibited substantial defects in termination of liver regeneration with increased liver injury, sustained cell proliferation resulting in significant hepatomegaly, hepatic dysplasia, and appearance of small nodules at 28 days after PHX. This was accompanied by a sustained increase in expression of cyclins along with significant induction in pro-inflammatory and pro-fibrotic gene expression in the OGT-KO livers. RNA-sequencing studies revealed inactivation of hepatocyte nuclear 4 alpha (HNF4α), the master regulator of hepatic differentiation and a known termination signal, in OGT-KO mice at 28 days after PHX, which was confirmed by both Western blot and immunohistochemistry analysis. Furthermore, a significant decrease in HNFα target genes was observed in OGT-KO mice, indicating a lack of hepatocyte differentiation following decreased hepatic O-GlcNAcylation. Immunoprecipitation experiments revealed HNF4α is O-GlcNAcylated in normal differentiated hepatocytes.ConclusionsThese studies show that O-GlcNAcylation plays a critical role in the termination of liver regeneration via regulation of HNF4α in hepatocytes.

Potent inhibition of TCP transcription factors by miR319 ensures proper root growth in Arabidopsis.

Proper root growth depends on the clearance of TCP transcripts from the root apical meristem by microRNA miR319. The evolutionarily conserved microRNA miR319 regulates genes encoding TCP transcription factors in angiosperms. The miR319-TCP module controls cell proliferation and differentiation in leaves and other aerial organs. The current model sustains that miR319 quantitatively tunes TCP activity during leaf growth and development, ultimately affecting its size. In this work we studied how this module participates in Arabidopsis root development. We found that misregulation of TCP activity through impairment of miR319 binding decreased root meristem size and root length. Cellular and molecular analyses revealed that high TCP activity affects cell number and cyclin expression but not mature cell length, indicating that, in roots, unchecking the expression of miR319-regulated TCPs significantly affects cell proliferation. Conversely, tcp multiple mutants showed no obvious effect on root growth, but strong defects in leaf morphogenesis. Therefore, in contrast to the quantitative regulation of the TCPs by miR319 in leaves, our data suggest that miR319 clears TCP transcripts from root cells. Hence, we provide new insights into the functions of the miR319-TCP regulatory system in Arabidopsis development, highlighting a different modus operandi for its action mechanism in roots and shoots.

G6PD upregulates Cyclin E1 and MMP9 to promote clear cell renal cell carcinoma progression.

Background: Clear cell renal cell carcinoma (ccRCC) is a cell metabolic disease with high metastasis rate and poor prognosis. Our previous studies demonstrate that glucose-6-phosphate dehydrogenase (G6PD), the first and rate-limiting enzyme of the pentose phosphate pathway, is highly expressed in ccRCC and predicts poor outcomes of ccRCC patients. The aims of this study were to confirm the oncogenic role of G6PD in ccRCC and unravels novel mechanisms involving Cyclin E1 and MMP9 in G6PD-mediated ccRCC progression.Methods: Real-time RT-PCR, Western blot and immunohistochemistry were used to determine the expression patterns of G6PD, Cyclin E1 and MMP9 in ccRCC. TCGA dataset mining was used to identify Cyclin E1 and MMP9 correlations with G6PD expression, relationships between clinicopathological characteristics of ccRCC and the genes of interest, as well as the prognosis of ccRCC patients. The role of G6PD in ccRCC progression and the regulatory effect of G6PD on Cyclin E1 and MMP9 expression were investigated by using a series of cytological function assays in vitro. To verify this mechanism in vivo, xenografted mice models were established.Results: G6PD, Cyclin E1 and MMP9 were overexpressed and positively correlated in ccRCC, and they were associated with poor prognosis of ccRCC patients. Moreover, G6PD changed cell cycle dynamics, facilitated cells proliferation, promoted migration in vitro, and enhanced ccRCC development in vivo, more likely through enhancing Cyclin E1 and MMP9 expression.Conclusion: These findings present G6PD, Cyclin E1 and MMP9, which contribute to ccRCC progression, as novel biomarkers and potential therapeutic targets for ccRCC treatment.

Synchronization of HeLa Cells to Various Interphases Including G1, S, and G2 Phases.

The typical cell cycle in eukaryotes is composed of four phases including the G1, S, G2, and M phases. G1, S, and G2 together are called interphase. Cell synchronization is a process that brings cultured cells at different stages of the cell cycle to the same phase, which allows the study of phase-specific cellular events. While interphase cells can be easily distinguished from mitotic cells by examining their chromosome morphology, it is much more difficult to separate and distinguish the interphases from each other. Here, we describe drug-derived protocols for synchronizing HeLa cells to various interphases of the cell cycle: G1 phase, S phase, and G2 phase. G1 phase synchronization is achieved through serum starvation, S phase synchronization is achieved through a double thymidine block, and G2 phase synchronization is achieved through the release of the double thymidine block followed by roscovitine treatment. Successful synchronization can be assessed using flow cytometry to examine the DNA content and Western blot to examine the expression of various cyclins.

Ranunculus Japonicus Induces Cell Cycle Arrest, Apoptosis, Autophagy and Targeting the Akt/Mapks Pathways in Human Neuroblastoma SH-SY5Y Cells

Objective: Ranunculus japonicus thumb. has been used as a traditional medicine for the treatment of asthma, malaria, tuberculosis, hepatitis, jaundice and so on. The aim of this study was to investigate the anti-cancer effect and action mechanism of the petroleum ether extract of Ranunculus japonicus Thumb. (RJTPEE) on human neuroblastoma SH-SY5Y cells. Methods: Cell viability was detected using MTT assay. FCM and Hoechst33258 staining was used to examine the cell cycle as well as apoptosis, and in addition, Western blot was also used to examine the expression of a range of related proteins. Results: MTT assay showed that RJTPEE could inhibit SH-SY5Y cells proliferation in concentration- and time-dependent manners. FCM analysis showed that RJTPEE could induce S and G2/M phase arrest, cell apoptosis, cell autophagy, the mitochondrial membrane potential loss (ΔΨm) and increase the production of intracellular ROS of SH-SY5Y cells. Topical morphological changes of apoptotic body formation were also observed by Hoechst 33258 staining. Western blot analysis indicated that the expressions of cell cycle-related protein Cyclin B1/D1 and CDK1/2/4/6 were obviously decreased. Meanwhile, obvious increase of apoptosis-related protein expressions, such as Bax, cytochrome c, caspase-3, caspase-9, PARP-1, and decrease of Bcl-2 protein expression were observed. Furthermore, Akt/MAPKs pathway-related proteins were also detected. Moreover, the decreased expression of autophagy-related proteins LC3 I protein indicated that RJTPEE could also enhance the autophagy ability of cells. Conclusion: RJTPEE could exert the antitumor activity on SH-SY5Y cells by inducing cell cycle arrest, cell autophagy, the mitochondrion-dependent cell apoptosis and targeting the Akt/MAPKs signaling pathway. RJTPEE has a promising therapeutic potential for neuroblastoma.

Anticancer Mechanism of Parthenolide Analogue PTLA-1 on Adriamycin Resistant Human Chronic Myeloid Leukemia Cell Line K562/ADR

Objective: To investigate the anticancer effect and mechanism of parthenolide analogue (PTLA-1) on adriamycin resistant human chronic myeloid leukemia cell line K562/ADR cells. Methods K562/ADR cells were cultured in vitro. The inhibitory effects of PTLA-1 on cell proliferation were detected by MTT assay, and the effects of PTLA-1 on K562/ADR cell cycle, apoptosis, mitochondrial membrane potential (ΔΨm) and reactive oxygen species were detected by flow cytometry. The effects of PTLA-1 on K562/ADR cell cycle and apoptosis related proteins expressions were detected by Western blot. Results PTLA-1 had higher inhibitory activity on K562/ADR cells than parthenolide. It inhibited the expressions of Cyclin B1/D1 and CDK1/2/4/6, resulting in the arrest of K562/ADR cells in G0/G1 phase; PTLA-1 induced apoptosis of K562/ADR cells in mitochondrial pathway. PTLA-1 could induce mitochondrial membrane potential (ΔΨm) and down regulate the expressions of reactive oxygen species, Bcl-2 and p-Akt proteins, and up regulate the expressions of Bax, cyto c, caspase 9/3 proteins. The chromatin of K562/ADR cells contracted and apoptotic bodies appeared. Conclusion Parthenolide analogue PTLA-1 could inhibit cell proliferation by causing K562/ADR cells to block in G0/G1 phase. Secondly, it could induce K562/ADR cells to apoptosis through caspase dependent mitochondrial pathway, which was related to Akt signal pathway. It had certain anticancer therapeutic potential for chronic myeloid leukemia.