Proliferating Cell Nuclear Antigen Protein Research Articles

Effects of silencing NLRP3 gene on proliferation of psoriasis-like HaCaT cells and expressions of cytokines.

We aimed to explore the effects of silencing NOD-like receptor protein 3 (NLRP3) on proliferation of psoriasis-like HaCaT cells and expressions of cytokines. HaCaT cells were treated with human keratinocyte growth factor (KGF) and were divided into KGF group, negative control group, NLRP3-RNAi group and control group. Cells proliferation was detected by CCK8, cell clone formation rate was detected by clone formation assay, distribution of cells cycle was detected by flow cytometry, expressions of cyclin B1 (Cyclin B1), cyclin-dependent kinase 2 (CDK2), Ki67 and proliferating cell nuclear antigen (PCNA) proteins were detected by Western blot, and levels of interleukin (IL)-17, IL-23, IL-6 and tumor necrosis factor α (TNF-α) were detected by enzyme-linked immunosorbent assay. Compared with control group, expressions of NLRP3 mRNA and protein, proliferation rate and clonal formation rate were increased in KGF group, percentage of cells in G0/G1 phase was decreased, percentage of cells in S phase was increased, expressions of Cyclin B1, CDK2, Ki67 and PCNA proteins were increased, and levels of IL-17, IL-23, IL-6 and TNF-α were increased. Compared with negative control group, expressions of NLRP3 mRNA and protein, proliferation rate and clonal formation rate were decreased in NLRP3-RNAi group, percentage of cells in G0/G1 phase was increased, percentage of cells in S phase was decreased, expressions of Cyclin B1, CDK2, Ki67 and PCNA proteins were decreased, and levels of IL-17, IL-23, IL-6 and TNF-α were decreased. Silencing NLRP3 gene can inhibit the proliferation of psoriasis-like HaCaT cells, arrest cell cycle, inhibit the expressions of cell proliferation-related proteins and reduce levels of pro-inflammatory factors.

Age-dependent changes in the expression and localization of LYZL4, LYZL6 and PCNA during testicular development in the Ashidan yak

Lysozyme like 4 (LYZL4), lysozyme like 6 (LYZL6) and proliferating cell nuclear antigen (PCNA) are implicated in the regulation of testicular function, but there was no research reported available on the expression patterns of LYZL4, LYZL6 and PCNA genes at different developmental stages of yak testes. In this study, we used the qRT-PCR, western blotting and immunohistochemistry estimated the LYZL4, LYZL6 and PCNA gene expression and protein lo-calization at different developmental stages of yak testes. The qPCR results showed that the mRNA expression of LYZL4, LYZL6 and PCNA genes significantly increased with age in the testes of yaks. Western blot results showed that the protein abundance of LYZL4, LYZL6 and PCNA in yak testes was significantly higher after puberty than before puberty. Furthermore, the results of immunohistochemistry indicated that LYZL4, LYZL6 and PCNA may be involved in the regulation of spermatogonia proliferation and Leydig cell function in immature testis. In adult yak testes, LYZL4, LYZL6 and PCNA may involve in the development of round spermatids and primary spermatocytes during testicular development. Our results indicated that LYZL4, LYZL6 and PCNA may be involved in the development of Sertoli cells, Leydig cells and gonocytes in yak testes.

Abstract 2690: The role of PCNA tyrosine phosphorylation in evading innate immune surveillance

Abstract Cancer cells are continually exposed to intrinsic and extrinsic proliferative stresses that can send the genome stability awry. The aberrant DNA metabolism, evolving through deregulated replicative growth in cancer cells, is increasingly recognized as a major mechanism bridging the crosstalk between the immune microenvironment and tumor tissue, thereby either promoting or suppressing tumor progression. However, the triggering and regulation of these mechanisms remain largely unknown. Proliferating cell nuclear antigen (PCNA) and its homologs are the evolutionarily conserved central components of the DNA replication machinery. In eukaryotic cells, PCNA forms homotrimeric rings encircling the DNA double helix to coordinate the complex processes of DNA replication and damage repair, directed at least in part by differential post-translational modifications of the PCNA protein. In cancer cells, growth signal-stimulated phosphorylation at tyrosine 211 of PCNA (pY211-PCNA) is known to play an important function for active proliferation. Here, we demonstrate that inhibition of pY211 disrupts the processivity of replication forks. This disruption triggers single-stranded DNA (ssDNA) production catalyzed by the MRE11 nuclease, activating the cGAS-STING axis to launch an anti-tumor immune response by natural killer (NK) cells. We further show that the pY211 regulates site-specific post-translational modifications of MRE11, and loss of pY211 promotes the endonuclease mode of MRE11. This causes ssDNA generation in the cytosol, subsequently inducing type I interferon-activated cytotoxicity by NK cells and fostering an anti-tumor immunity that abrogates distant metastasis. Mechanistically, the pY211 determines the recruitment of the writer or eraser of the modification on MRE11, thus tuning the nucleolytic modes of MRE11 and, consequently, ssDNA generation. Therapeutic measures promoting MRE11-dependent ssDNA enhance the response of triple-negative breast cancer to cytotoxic killing mediated by endogenous or allogenic NK cells in mice. In breast cancer patients, the level of cytosolic ssDNA is inversely correlated with the expression of the eraser and pY211-PCNA, which is associated with poor overall survival in cancer patients. Our studies shed new light on the shaping of the tumor immune microenvironment and demonstrate the potential to develop therapeutic approaches that proactively leverage genomic instability and immune activation to target malignant tumors. Citation Format: Chuan-Chun Lee, Wan-Rong Wu, Yi-Chun Shen, Feng-Chi Chung, Yuan-Liang Wang, You-Zhe Lin, Chih-Hao Lu, Wei-Chung Cheng, Han Chang, Liang-Chih Liu, Ji-An Liang, Chang- Fang Chiu, Mien-Chie Hung, Shao-Chun Wang. The role of PCNA tyrosine phosphorylation in evading innate immune surveillance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2690.

Long noncoding RNA GPRC5D-AS1 in renal cell carcinoma: a molecular mechanism study.

Clear cell renal cell carcinoma (RCC) is the most common subtype of RCC. Although targeted therapy can provide superior treatment outcomes, it is prone to drug resistance, and individual responses to immunotherapy vary greatly. Therefore, finding new diagnostic and therapeutic targets for RCC is of considerable importance. Long noncoding RNA (lncRNA) GPRC5D-AS1 can serve as a biomarker in clinical applications and the prognosis of lung squamous cell carcinoma. However, the specific mechanism of action of lncRNA GPRC5D-AS1 in RCC has not yet been clarified. Therefore, this paper explores the expression of lncRNA GPRC5D-AS1 in the renal cancer cell line 786-0, and conducts a preliminary study of its molecular mechanism. Selecting nude mice for tumor experiments is because of the high genomic and physiological similarity between mice and humans. Conducting tumor research on mice allows for better control of experimental conditions, aiding researchers in more accurately observing and analysing tumor characteristics and responses. Small interfering RNA (siRNA) and plasmid cloning DNA (pcDNA) 3.1 were used to transfect renal cancer cell line 786-0 to silence and overexpress the lncRNA GPRC5D-AS1 gene. Quantitative real-time fluorescence polymerase chain reaction was used to detect the difference in lncRNA GPRC5D-AS1 expression in blank control group, negative control group, siGPRC5D-AS1 group and oeGPRC5D-AS1 group. The effects of silence and overexpression of lncRNA GPRC5D-AS11 on the proliferation of 786-0 cells were detected in cell colony formation experiments; the changes in the migration and invasion of 786-0 cells were detected via cell scratch assay and transwell assay, respectively; the differences in tumor growth between groups were determined via tumorigenesis experiments in nude mice; and the expression of proliferation-related protein [β-catenin, Ki67 and proliferating cell nuclear antigen (PCNA)] and invasion-related protein (N-cadherin and E-cadherin) were detected via Western blotting. Compared with blank control group and negative control group, the siGPRC5D-AS1 group showed a significant decrease in the relative expression of lncRNA GPRC5D-AS1 (P<0.05), a significant increase in the number of proliferating cells and migrating cells (P<0.05), a significant increase in the tumor volume of nude mice (P<0.05), a significant increase in β-catenin, Ki67, PCNA and N-cadherin protein expression (P<0.05), and a significant decrease in E-cadherin protein expression (P<0.05); conversely, these results were opposite for the eGPRC5D-AS1 group. Silencing the expression of lncRNA GPRC5D-AS1 can enhance the proliferation, invasion, and migration ability of renal cancer cell line 786-0, which can be weakened by the overexpression of lncRNA GPRC5D-AS1.

Icariin exerts anti-tumor activity by inducing autophagy via AMPK/mTOR/ULK1 pathway in triple-negative breast cancer

BackgroundBreast cancer is the most prevalent female tumor, of which triple-negative breast cancer (TNBC) accounts for about 15%. Characterized by its aggressive nature and limited treatment options, TNBC currently stands as a significant clinical challenge. This study aimed to investigate the effects of icariin (ICA) on TNBC and explore the underlying molecular mechanism.MethodsCell viability was assessed using CCK-8 assay, whereas the impact of ICA on cell proliferation was determined using colony formation assay and detection of proliferating cell nuclear antigen protein. Wound healing and transwell assays were used to evaluate the effects of ICA on cell migration and invasion, respectively. Flow cytometry was used to analyze cell cycle distribution and apoptosis. Transmission electron microscopy and monodansylcaverine staining were performed to detect the induction of autophagy, whereas molecular docking was conducted to predict the potential targets associated with autophagy. The in vivo anti-tumor effects of ICA were evaluated using a TNBC 4T1 xenograft mouse model. Protein expression levels were examined using immunoblotting and immunohistochemistry.ResultsIn vitro, ICA effectively suppressed the viability, proliferation, migration, and invasion of TNBC cells and induced G0/G1 phase cell cycle arrest, apoptosis, and autophagy in TNBC cells by regulating the adenosine monophosphate-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR)/Unc-51-like kinase 1 (ULK1) signaling pathway. The knockdown of AMPK and inhibition of autophagy with 3-methyladenine reversed the effects of ICA, highlighting the importance of AMPK and autophagy in the anti-cancer mechanism of ICA. In vivo, ICA significantly inhibited TNBC growth, promoted autophagy, and regulated AMPK/mTOR/ULK1 pathway.ConclusionsOur findings demonstrated that ICA exerts anti-cancer effects against TNBC and the associated molecular mechanisms. This study will help to facilitate further preclinical and clinical investigations for the treatment of TNBC.

Parental betaine supplementation promotes gosling growth with epigenetic modulation of IGF gene family in the liver.

Betaine is widely used as a feed additive in the chicken industry to promote laying performance and growth performance, yet it is unknown whether betaine can be used in geese to improve the laying performance of goose breeders and the growth traits of offspring goslings. In this study, laying goose breeders at 39wk of age were fed basal (Control, CON) or betaine-supplemented diets at low (2.5g/kg, LBT) or high (5g/kg, HBT) levels for 7wk, and the breeder eggs laid in the last week were collected for incubation. Offspring goslings were examined at 35 and 63 d of age. The laying rate tended to be increased (P = 0.065), and the feed efficiency of the breeders was improved by betaine supplementation, while the average daily gain of the offspring goslings was significantly increased (P < 0.05). Concentrations of insulin-like growth factor 2 (IGF-2) in serum and liver were significantly increased in the HBT group (P < 0.05), with age-dependent alterations of serum T3 levels. Concurrently, hepatic mRNA expression of the IGF gene family was significantly increased in goslings derived from betaine-treated breeders (P < 0.05). A higher ratio of proliferating cell nuclear antigen (PCNA)-immunopositive nuclei was found in the liver sections of the HBT group, which was confirmed by significantly upregulated hepatic expression of PCNA mRNA and protein (P < 0.05). Moreover, hepatic expression of thyroxine deiodinase type 1 (Dio1) and thyroid hormone receptor β (TRβ) was also significantly upregulated in goslings of the HBT group (P < 0.05). These changes were associated with significantly higher levels of global DNA 5-mC methylation, together with increased expression of methyl transfer genes (P < 0.05), including betaine-homocysteine methyltransferase (BHMT), glycine N-methyltransferase (GNMT), and DNA (cytosine-5-)-methyltransferase 1 (DNMT1). The promoter regions of IGF-2 genes, as well as the predicted TRβ binding site on the IGF-2 gene, were significantly hypomethylated (P < 0.05). These results indicate that gosling growth can be improved by dietary betaine supplementation in goose breeders via epigenetic modulation of the IGF gene family, especially IGF-2, in the liver.

The effect and mechanism of low-molecular-weight heparin on the decidualization of stromal cells in early pregnancy

Objective This study aimed to analyze the effect of low-molecular-weight heparin (LMWH) on the decidualization of stromal cells in early pregnancy and explore the effect of LMWH on pregnancy outcomes. Methods Recurrent spontaneous abortion (RSA) mouse model (CBA/J × DBA/2) and normal pregnant mouse model (CBA/J × BALB/c) were established. The female mice were checked for a mucus plug twice daily to identify a potential pregnancy. When a mucus plug was found, conception was considered to have occurred 12 h previously. The pregnant mice were divided randomly into a normal pregnancy control group, an RSA model group, and an RSA + LMWH experimental group (n = 10 mice in each group). Halfway through the 12th day of pregnancy, the embryonic loss of the mice was observed; a real-time quantitative polymerase chain reaction was used to detect the messenger ribonucleic acid (mRNA) expressions of prolactin (PRL) and insulin-like growth factor-binding protein 1 (IGFBP1) in the decidua of the mice. Additionally, the decidual tissues of patients with RSA and those of normal women in early pregnancy who required artificial abortion were collected and divided into an RSA group and a control group. Decidual stromal cells were isolated and cultured to compare cell proliferation between the two groups, and cellular migration and invasion were detected by membrane stromal cells. Western blotting was used to detect the protein expressions of proliferating cell nuclear antigen (PCNA), cyclin D1, matrix metalloproteinase- (MMP) 2, and MMP-7 in stromal cells treated with LMWH. Results Compared with the RSA group, LMWH significantly reduced the pregnancy loss rate in the RSA mice (p < 0.05). Compared with the RSA group, the LMWH + RSA group had significantly higher expression levels of PRL and IGFBP1 mRNA (p < 0.01). LMWH promoted the proliferation, migration, and invasion of human decidual stromal cells; compared with the control group, the expression levels of MMP-2, MMP-7, cyclin D1, and PCNA proteins in the decidual stromal cells of the LMWH group increased (p < 0.05). Conclusions The use of LMWH can improve pregnancy outcomes by enhancing the proliferation and migration of stromal cells in early pregnancy and the decidualization of stromal cells.

Phylogenetic and recombination analysis of Begomoviruses associated with Cotton leaf curl disease and in silico analysis of viral-host protein interactions

Cotton leaf curl disease (CLCuD), caused by numerous begomoviruses (BGVs), is a highly disastrous disease in cotton crops worldwide. To date, several efforts have shown limited success in controlling this disease. CLCuD-associated BGVs (CABs) are known for their high rate of intra and interspecific recombinations, which raises an urgent need to find an efficient and conserved target region to combat disease. In the present study, phylogenetic analysis of selected 11 CABs, along with associated alphasatellites, and betasatellites revealed a close evolutionary relationship among them. Recombination analysis of 1374 isolates of CABs revealed 54 recombination events for the major players of CLCuD in cotton and the Cotton leaf curl Multan virus (CLCuMuV) as the most recombinant CAB. Recombination breakpoints were frequent in all regions except C2 and C3. C3-encoded protein, known as viral replication enhancer (REn), promotes viral replication by enhancing the activity of replicase (Rep) protein. Both proteins were found to contain significantly conserved domains and motifs. The identified motifs were found crucial for their interaction with host protein PCNA (Proliferating cell nuclear antigen), facilitating viral replication. Interruption at the REn-PCNA and Rep-PCNA interactions by targeting the identified conserved motifs is proposed as a prospect to halt viral replication, after suitable experimental validation.

The amelioration of ovarian dysfunction by hesperidin in malathion-treated mice through the overexpression of PCNA and FSHR proteins

ObjectiveMalathion (MAL), a pesticide used for decades, is a highly toxic substance. Several studies have documented the negative effects of such agents on reproductive organ physiology, but the precise mechanism of action in the induction of ovarian dysfunction remains unclear. Therefore, the purpose of this research was to examine the effects of the antioxidant hesperidin (HES) on ovarian damage and toxicity caused by malathion. Materials and methodsIn this experiment, forty adult female bulb/c mice weighing 27–30 g were categorized into four groups, namely hesperidin (20 mg/kg, i.p.), malathion (3 mg/kg, i.p.), malathion + hesperidin, and control groups. Following a period of 35 consecutive days of treatment, mice were euthanized, and their ovarian tissues were gathered for the purposes of histopathological analysis by H&E staining, immunohistochemical assessment via proliferating cell nuclear antigen (PCNA) and follicle-stimulating hormone receptor (FSHR) immunostaining, and biochemical evaluation via measuring the levels of malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), tumor necrosis factor-alpha (TNF-α), and interleukin-1 beta (IL-1β). In addition, serum samples were collected from the blood of mice to perform hormonal analyses, especially 17β-estradiol (E2), progesterone (P4), luteinizing hormone (LH), and follicle-stimulating hormone (FSH). ResultsThe results demonstrated that MAL exposure resulted in the development of abnormalities in the architecture and structure of ovaries. Also, the treatment of mice with MAL led to declined follicular counts at all three stages, namely, primary, secondary, and tertiary, reduced serum levels of sex hormones, decreased immunoreactivity of FSHR and PCNA, and diminished activity of CAT and SOD enzymes. In contrast, it caused an increase in MDA, IL-1β, and TNF-α, as well as the count of atretic follicles. Nonetheless, it was observed that HES exhibited the ability to ameliorate the deleterious impacts of malathion across all the aforementioned parameters. ConclusionTreatment with HES via upregulating the protein expression of PCNA and FSHR and activating antioxidant defense was able to ameliorate the adverse effects of MAL on ovarian tissues.

Effect of quercetin on granulosa cells development from hierarchical follicles in chicken

ABSTRACT 1. The bioflavonoid quercetin is a biologically active component, but its functional regulation of granulosa cells (GCs) during chicken follicular development is little studied. To investigate the effect of quercetin on follicular development in laying hens, an in vitro study was conducted on granulosa cells from hierarchical follicles treated with quercetin. 2. The effect of quercetin on cell activity, proliferation and apoptosis of granulosa cells was detected by CCK-8, EdU and apoptosis assays. The effect on progesterone secretion from granulosa cells was investigated by enzyme-linked immunosorbent assay (ELISA). Expression of proliferating cell nuclear antigen (PCNA) mRNA and oestrogen receptors (ERs), as well as the expression of steroid acute regulatory protein (StAR), cytochrome P450 cholesterol side chain cleavage enzyme (P450scc) and 3β-hydroxysteroid dehydrogenase (3β-HSD) mRNA during progesterone synthesis, were measured by real-time quantitative polymerase chain reaction (RT-qPCR). PCNA, StAR and CYP11A1 protein expression levels were detected using Western blotting (WB). 3. The results showed that treatment with quercetin in granulosa cells significantly enhanced cell vitality and proliferation, reduced apoptosis and promoted the expression of gene and protein levels of PCNA. The levels of progesterone secretion increased significantly following quercetin treatment, as did the expression levels of StAR and CYP11A1 using the Western Blot (WB) method. 4. The mRNA expression levels of ERα were significantly upregulated in the 100 ng/ml and 1000 ng/ml quercetin-treated groups, while there was no significant difference in expression levels of ERβ mRNA.

α2-Macroglobulin Promotes Chondrocyte Proliferation and Cartilage Matrix Synthesis via Inducing PCNA.

α2-Macroglobulin (A2M) can prevent cartilage degeneration by blocking many types of cartilage-degrading enzymes, but the mechanism remains to be clarified. This study aimed to test that A2M protects against cartilage degeneration by promoting chondrocyte proliferation and cartilage matrix synthesis via inducing proliferating cell nuclear antigen (PCNA). The cartilage degeneration of the anterior cruciate ligament transection (ACLT) model was evaluated by Safranin O-fast green staining, and articular cartilage degeneration was graded using the Osteoarthritis Research Society International (OARSI)-modified Mankin criteria. The chondrocyte proliferation was detected by 5-Bromodeoxyuridinc (BrdU), MTT, and Cell Counting Kit-8 (CCK8) methods. The chondrocyte apoptosis was detected by lactate dehydrogenase (LDH) assay and Annexin PI staining with the flow cytometer. The glycosaminoglycan (sGAG) and aggrecan in culture supernatant were measured by enzyme-linked immunosorbent assay (ELISA). Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was used to analyze the type II collagen and aggrecan mRNA expression. The PCNA protein expression was analyzed by western blot and immunofluorescent staining. A2M can attenuate cartilage degeneration in ACLT rats. The OARSI scores for cartilage degeneration in the A2M group were lower than those in the phosphate-buffered saline (PBS) group. A2M can promote chondrocyte proliferation and inhibit chondrocyte apoptosis, promote the cartilage matrix synthesis in chondrocytes (type II collagen and aggrecan), and culture supernatant (sGAG and aggrecan). At the same time, it also up-regulated the PCNA protein expression in chondrocytes. A2M can promote chondrocyte proliferation and cartilage matrix synthesis via inducing PCNA expression.

Circ_0000396 suppresses the proliferation and inflammation of rheumatoid arthritis synovial fibroblasts by targeting miR-574-5p/RSPO1 axis

BackgroundCircular RNAs (circRNAs) are important regulators on the onset and progression of rheumatoid arthritis (RA). Our purpose is to explore the role and underpin mechanism of circ_0000396 in RA progression.MethodsRA patients (n = 39) and healthy volunteers (n = 33) were recruited from the Affiliated Hospital of Shaanxi University of Chinese Medicine for the present work. Circ_0000396, microRNA-574-5p (miR-574-5p) and R-spondin 1 (RSPO1) RNA levels were analyzed by reverse transcription-quantitative polymerase chain reaction. Cell proliferation was analyzed by 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, colony formation assay, and 5-ethynyl-2′-deoxyuridine (EDU) assay. Cell apoptosis was assessed by flow cytometry. Protein expression levels of proliferating cell nuclear antigen (PCNA), Cyclin D1, Cyclin E1, BCL2-associated × protein (Bax), B-cell lymphoma-2 (Bcl2), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α) and RSPO1 were detected by western blot assay. Enzyme-linked immunosorbent assay (ELISA) was conducted to analyze the secretion of pro-inflammatory cytokines including IL-1β and TNF-α. The interaction between miR-574-5p and circ_0000396 or RSPO1 was confirmed by dual-luciferase reporter assay and RNA-pull down assay.ResultsCirc_0000396 expression was notably down-regulated in RA patients compared with healthy controls. Circ_0000396 overexpression suppressed the proliferation and inflammatory response and triggered the apoptosis of RA synovial fibroblasts (RASFs), accompanied by decreases in PCNA, Cyclin D1, Cyclin E1, Bcl2, IL-1β and TNF-α protein expression and an increase in Bax protein expression. Circ_0000396 acted as a molecular sponge for miR-574-5p, and circ_0000396 overexpression-mediated protective effects on RASFs dysfunction were largely reversed by the introduction of miR-574-5p mimics. miR-574-5p interacted with the 3’ untranslated region (3’UTR) of RSPO1, and miR-574-5p negatively regulated RSPO1 expression in RASFs. Circ_0000396 could up-regulate the expression of RSPO1 by sponging miR-574-5p in RASFs. RSPO1 interference largely overturned circ_0000396 overexpression-mediated effects in RASFs.ConclusionCirc_0000396 restrained the proliferation and inflammation and induced the apoptosis of RASFs by mediating miR-574-5p/RSPO1 axis, which provided novel potential targets for RA treatment.

Comparative Analysis of Cluster of Differentiation 57 and Proliferating Cell Nuclear Antigen Expression in Different Grades of Oral Squamous Cell Carcinoma: An Immunohistochemical Study.

The immune defense against tumor cells is mainly mediated by the natural killer (NK) cells. Cluster of differentiation 57 (CD57) is a 110-kd glycoprotein, typically expressed by the NK cells, attacks the cancer cells and inhibits tumor development. Proliferating cell nuclear antigen (PCNA) is a 36-kd auxiliary protein for DNA polymerase delta that correlates with cell proliferation and DNA synthesis. It is an essential component of DNA replication, DNA recombination, and DNA repair. The uncoordinated proliferation of PCNA protein characterizes the biological behavior of malignant lesions. The aim of the present study is to compare and correlate the expression of CD57 and PCNAin different grades of oral squamous cell carcinoma (OSCC) by immunohistochemistry. This retrospective analysis comprises 30 samples of various grades of OSCCsand10 samples of healthy mucosa.Sections of 4-5 µm thickness were done and stained with monoclonal anti-PCNA and anti-CD57 antibodies.The statistical package for social science (SPSS) version 16.0 software (IBM Corp., Armonk, NY) was used to analyze the data in this study. The expression of CD57 and PCNA was compared and correlated between the groups using analysis of variance (ANOVA) post hoc, Dunnet t-test, and Pearson's correlation coefficient test.For statistical significance, a p-value of 0.05 or less was used. A significant decrease in CD57 labeling index was seen from well-differentiated squamous cell carcinoma (16.63 ± 2.33) to poorly differentiated squamous cell carcinoma (5.53 ± 1.20) whereas the significant increase in PCNA labeling index was noted from well-differentiatedsquamous cell carcinoma (45.88 ± 2.20), followed by moderately differentiated and poorly differentiated squamous cell carcinoma (72.77 ± 4.35). The combination of CD57 and PCNA biomarkers appears to be good indicators of the immune status of the patient and the aggressiveness of the lesion.

EZH2 activates Wnt/β-catenin signaling in human uterine fibroids, which is inhibited by the natural compound methyl jasmonate

To investigate the link between EZH2 and Wnt/β-catenin signaling and its role in uterine fibroids (UFs) pathogenesis and explore the potential effect of natural compound methyl jasmonate (MJ) against UFs. EZH2 overexpression or inhibition was achieved in human uterine leiomyoma (HuLM) cells using EZH2-expressing adenovirus or chemical EZH2 inhibitor (DZNep), respectively. The HuLM and normal uterine smooth muscle cells were treated with 0.1-3 mM of MJ, and several experiments were employed. Laboratory study. None. Methyl jasmonate. Protein expression of EZH2, β-catenin, and proliferating cell nuclear antigen (PCNA) was measured by Western blot as well as gene expression alterations of Wnt ligands (Wnt5A, Wnt5b, and Wnt9A), WISP1, CTNNB1, and its responsive gene PITX2 using quantitative real-time polymerase chain reaction. The protein and ribonucleic acid (RNA) levels of several markers were measured in MJ-treated or untreated HuLM cells, including EZH2 and β-catenin, extracellular matrix markers collagen type 1 (COL1A1) and fibronectin (FN), proliferation markers cyclin D1 (CCND1) and PCNA, tumor suppressor marker p21, and apoptotic markers (BAX, cytochrome c, and cleaved caspase 3). EZH2 overexpression significantly increased the gene expression of several Wnt ligands (PITX2, WISP1, WNT5A, WNT5B, and WNT9A), which increased nuclear translocation of β-catenin and PCNA and eventually HuLM cell proliferation. EZH2 inhibition blocked Wnt/β-catenin signaling activation where the aforementioned genes significantly decreased as well as PCNA, cyclin D1, and PITX2 protein expression compared with those in untreated HuLM. Methyl jasmonate showed a potent antiproliferative effect on HuLM cells in a dose- and time-dependent manner. Interestingly, the dose range (0.1-0.5 mM) showed a selective growth inhibitory effect on HuLM cells, not on normal uterine smooth muscle cells. Methyl jasmonate treatment at 0.5 mM for 24 hours significantly decreased both protein and RNA levels of EZH2, β-catenin, COL1A1, FN, CCND1, PCNA, WISP1, and PITX2 but increased the protein levels of p21, BAX, cytochrome, c and cleaved caspase 3 compared with untreated HuLM. Methyl jasmonate-treated cells exhibited down-regulation in the RNA expression of 36 genes, including CTNNB1, CCND1, Wnt5A, Wnt5B, and Wnt9A, and up-regulation in the expression of 34 genes, including Wnt antagonist genes WIF1, PRICKlE1, and DKK1 compared with control, confirming the quantitative real-time polymerase chain reaction results. Our studies provide a novel link between EZH2 and the Wnt/β-catenin signaling pathway in UFs. Targeting EZH2 with MJ interferes with the activation of wnt/β-catenin signaling in our model. Methyl jasmonate may offer a promising therapeutic option as a nonhormonal and cost-effective treatment against UFs with favorable clinical utility, pending proven safe and efficient in human clinical trials.

Silencing of LINC00467 inhibits cell proliferation in testicular germ cell tumors cells.

A significant decrease in LINC00467 expression in testicular germ cell tumors (TGCTs) was found in our previous study in comparison to adjacent tissue. Interestingly, the expression of LINC00467 correlated with the pathological grade of the tumor in TGCT patients. The higher the expression of LINC00467 was, the worse the prognosis of the patients with TGCT was. Despite these findings, the exact role of LINC00467 in the development of TGCTs requires further investigation. LINC00467 expression was downregulated in the NCCIT and TCam-2 cell lines via small interfering RNA (siRNA) silencing. The levels of gene expression were validated using quantitative real-time polymerase chain reaction (qRT-PCR) analyses. Cell proliferation was evaluated by the MTT and Cell Counting Kit-8(CCK8) assays, whereas flow cytometry was used to assess the effects on the cell cycle. Western blotting analysis was used to detect expression levels of protein. Additionally, RNA-sequencing and bioinformatics methods were used to investigate the mechanism of action of LINC00467 in TGCTs. The suppression of LINC00467 expression resulted in decreased cell proliferation and induced S-phase arrest. Furthermore, the suppression of LINC00467 downregulated proliferating cell nuclear antigen (PCNA), a protein related to cell cycle regulation, while it upregulated p21 expression. In other studies involving dihydrotestosterone (DHT) stimulation, it was observed that DHT could upregulate LINC00467 expression. In addition, silencing of the LINC00467 reversed the effect of testosterone on cell proliferation. The Gene Set Enrichment Analysis (GSEA) revealed that LINC00467 regulated the p53 pathway by modulating the expression of CCNG1. Our study found that LINC00467 regulates cell proliferation by inducing S-phase arrest through the cell cycle-related proteins PCNA and p21. These findings contribute to our understanding of non-coding RNAs mechanisms involved in the development of TGCTs.

SUMOylation regulates low-temperature survival and oxidative DNA damage tolerance in Botrytis cinerea.

SUMOylation as one of the protein post-translational modifications plays crucial roles in multiple biological processes of eukaryotic organisms. Botrytis cinerea is a devastating fungal pathogen and capable of infecting plant hosts at low temperature. However, the molecular mechanisms of low-temperature adaptation are largely unknown in fungi. Combining with biochemical methods and biological analyses, we report that SUMOylation regulates pathogen survival at low temperature and oxidative DNA damage response during infection in B. cinerea. The heat shock protein (Hsp70) BcSsb and E3 ubiquitin ligase BcRad18 were identified as substrates of SUMOylation; moreover, their SUMOylation both requires a single unique SUMO-interacting motif (SIM). SUMOylated BcSsb regulates β-tubulin accumulation, thereby affecting the stability of microtubules and consequently mycelial growth at low temperature. On the contrary, SUMOylated BcRad18 modulates mono-ubiquitination of the sliding clamp protein proliferating cell nuclear antigen (PCNA), which is involved in response to oxidative DNA damage during infection. Our study uncovers the molecular mechanisms of SUMOylation-mediated low-temperature survival and oxidative DNA damage tolerance during infection in a devastating fungal pathogen, which provides novel insights into low-temperature adaptation and pathogenesis for postharvest pathogens as well as new targets for inhibitor invention in disease control.

Efeito protetor de Mucuna pruriens (L.) DC. var. extrato de semente de pruriens, em células germinativas apoptóticas em ratos machos etanólicos

Abstract Thai Mucuna pruriens (L.) DC. var pruriens (T-MP) seed containing levodopa (L-DOPA) and antioxidant capacity has been shown to improve sexual behavior and male reproductive parameters in rats treated with ethanol (Eth). However, its protective effect on testicular apoptotic germ cells has never been reported. This study aimed to investigate the potential effects of T-MP seed extract on expressions of caspase, proliferating cell nuclear antigen (PCNA), and dopamine D2 receptor (D2R) proteins in Eth rats. Thirty-six male Wistar rats were divided into four groups (9 animals/group), including control, Eth, T-MP150+Eth, and T-MP300+Eth, respectively. Control rats received distilled water, and Eth rats received Eth (3g/kg BW; 40%v/v). The T-MP groups were treated with T-MP seed extract at a dose of 150 or 300 mg/kg before Eth administration for 56 consecutive days. The results showed that the seminiferous tubule diameter and epithelial height were significantly increased in both T-MP treated groups compared to the Eth group. Additionally, the caspase-9 and -3, and PCNA expressions were decreased, but D2R expression was markedly increased in T-MP groups. It was concluded that T-MP seed extract could protect testicular apoptosis induced by Eth via changes in caspase, PCNA, and D2R protein expressions.

ROS and DNA repair in spontaneous versus agonist-induced NETosis: Context matters

Reactive oxygen species (ROS) is essential for neutrophil extracellular trap formation (NETosis). Nevertheless, how ROS induces NETosis at baseline and during neutrophil activation is unknown. Although neutrophils carry DNA transcription, replication and repair machineries, their relevance in the short-lived mature neutrophils that carry pre-synthesized proteins has remained a mystery for decades. Our recent studies show that (i) NETosis-inducing agonists promote NETosis-specific kinase activation, genome-wide transcription that helps to decondense chromatin, and (ii) excess ROS produced by NADPH oxidase activating agonists generate genome-wide 8-oxy-guanine (8-OG), and the initial steps of DNA repair are needed to decondense chromatin in these cells. These steps require DNA repair proteins necessary for the assembly and nicking at the damaged DNA sites (poly ADP ribose polymerase PARP, apurinic endonuclease APE1 and DNA ligase), but not the enzymes that mediate the repair DNA synthesis (Proliferating cell nuclear antigen (PCNA) and DNA Polymerases). In this study, we show that (i) similar to agonist-induced NETosis, inhibition of early steps of oxidative DNA damage repair proteins suppresses spontaneous NETosis, but (ii) the inhibition of late stage repair proteins DNA polymerases and PCNA drastically promotes baseline NETosis. Hence, in the absence of excessive ROS generation and neutrophil activation, DNA repair mediated by PCNA and DNA polymerases is essential to prevent chromatin decondensation and spontaneous NETosis. These findings indicate that ROS, oxidative DNA damage, transcription and DNA repair differentially regulate spontaneous and agonist-induced NETosis. Therefore, context matters.

Antioxidant and Anticancer Potentials of the Olive and Sesame Mixture against Dimethylhydrazine-Induced Colorectal Cancer in Wistar Rats.

Cancer is one of the leading causes of death worldwide, and natural agents have shown some promise in fighting it. Thus, the present study tried to evaluate the healing potential of an equal combination of olive and sesame extract (MOS) against the colorectal cancerous lesions that were induced by dimethylhydrazine (DMH) in male rats and also compare the anticarcinogenic potential of the MOS and vitamin E with each other. Therefore, the mixture of equal olive and sesame extract (MOS) was used as the main treatment, alongside vitamin E as a parallel treatment. This study examined the red blood cell (RBC) and white blood cell (WBC) levels, biochemical indices, lactate dehydrogenase (LDH), C-reactive protein (CRP), total protein (TP), creatine kinase (CPK), albumin, and the colon tissue pathology, as well as the level of protein expression of the adenomatous polyposis coli (APC), proliferating cell nuclear antigen (PCNA), carcinoembryonic antigen (CEA), and platelet-derived growth factor (PDGF). Also, the tissue stress markers including total antioxidant capacity (TAC), malondialdehyde (MDA), and superoxide dismutase (SOD) were analyzed. Overall, the results represented a significant reduction in the congestion, mitotic index, inflammation, and cell destruction in the MOS group compared to the DMH group. In terms of the oxidative stress level, a significant increase was observed in the DMH group in comparison with the DMH-MOS group (P < 0.05), and the MOS significantly increased TAC level (P < 0.05). Furthermore, the DMH+MOS-exposed group exhibited a significantly lower expression of the PCNA, CEA, and PDGF proteins than those of the DMH group. Overall, the MOS showed that it can effectively prevent DMH-induced colon lesions. This mixture, as a strong antioxidant agent, can be clinically applied for preventing and treating colorectal cancer, the effectiveness of which is higher than that of vitamin E.

The Protective Effects on Ischemia-Reperfusion Injury Mechanisms of the Thoracic Aorta in Daurian Ground Squirrels (Spermophilus dauricus) over the Torpor-Arousal Cycle of Hibernation.

Hibernators are a natural model of vascular ischemia–reperfusion injury; however, the protective mechanisms involved in dealing with such an injury over the torpor–arousal cycle are unclear. The present study aimed to clarify the changes in the thoracic aorta and serum in summer-active (SA), late-torpor (LT) and interbout-arousal (IBA) Daurian ground squirrels (Spermophilus dauricus). The results show that total antioxidant capacity (TAC) was unchanged, but malondialdehyde (MDA), hydrogen peroxide (H2O2), interleukin-1β (IL-1β) and tumor necrosis factor α (TNFα) were significantly increased for the LT group, whereas the levels of superoxide dismutase (SOD) and interleukin-10 (IL-10) were significantly reduced in the LT group as compared with the SA group. Moreover, the levels of MDA and IL-1β were significantly reduced, whereas SOD and IL-10 were significantly increased in the IBA group as compared with the SA group. In addition, the lumen area of the thoracic aorta and the expression of the smooth muscle cells (SMCs) contractile marker protein 22α (SM22α) were significantly reduced, whereas the protein expression of the synthetic marker proteins osteopontin (OPN), vimentin (VIM) and proliferating cell nuclear antigen (PCNA) were significantly increased in the LT group as compared with the SA group. Furthermore, the smooth muscle layer of the thoracic aorta was significantly thickened, and PCNA protein expression was significantly reduced in the IBA group as compared with the SA group. The contractile marker proteins SM22α and synthetic marker protein VIM underwent significant localization changes in both LT and IBA groups, with localization of the contractile marker protein α-smooth muscle actin (αSMA) changing only in the IBA group as compared with the SA group. In tunica intima, the serum levels of heparin sulfate (HS) and syndecan-1 (Sy-1) in the LT group were significantly reduced, but the serum level of HS in the IBA group increased significantly as compared with the SA group. Protein expression and localization of endothelial nitric oxide synthase (eNOS) was unchanged in the three groups. In summary, the decrease in reactive oxygen species (ROS) and pro-inflammatory factors and increase in SOD and anti-inflammatory factors during the IBA period induced controlled phenotypic switching of thoracic aortic SMCs and restoration of endothelial permeability to resist ischemic and hypoxic injury during torpor of Daurian ground squirrels.