Proliferating Cell Nuclear Antigen Protein Expression Research Articles

Circ_0000263 improves radiosensitivity of Hela cells by inhibiting the activity of telomerase protein through miR-338-3p/TERT

Objective: To explore the effect and molecular mechanism of circ_0000263 on HeLa cell activity, apoptosis, telomerase activity, and radiosensitivity. Methods: The Hela cells were divided into si-NC, si-circ, vector, circ_0000263, anti-NC, anti-miR-338-3p, miR-NC, miR-338-3p, si-circ+anti-NC, si-circ+ anti-miR-338-3p, si-circ+vector, si-circ+TERT, sh-NC, sh-circ groups. Reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) was used to detect the expressions of circ_0000263 and miR-338-3p. Cell clone formation array was used to detect cell survival; cell counting kit-8 (CCK-8) to detect cell proliferation; flow cytometry to detect apoptosis; western blot method to detect the expressions of proliferating cell nuclear antigen (PCNA), Cleaved-casp3, telomerase reverse transcriptase (TERT) proteins; double luciferase assay to detect the targeting relationships of circ_0000263 and miR-338-3p, miR-338-3p and TERT; telomere repeat amplification enzyme linked immunosorbent assay (TRAR-ELISA) to detect telomerase activity. Results: Circ_0000263 was highly expressed in Hela cells, miR-338-3p was low expressed, and TERT was highly expressed; circ_0000263 was also highly expressed in Hela cells treated with radiation (P＜0.05). Knockdown of circ_0000263 inhibited the clone formation and cell proliferation ability of HeLa cells, and enhanced the radiosensitivity and apoptosis of HeLa cells. In contrast, knockdown of circ_0000263 decreased PCNA protein expression level and enhanced Cleaved-casp3 protein expression level in HeLa cells (P＜0.05). The apoptosis rate in the si-circ group was (13.19±1.12)%, which was higher than (6.80±0.62)% of si-NC group (P＜0.05). The apoptosis rate in the si-circ+4 Gy group was (24.82±1.57)%, which was higher than (17.00±0.96)% of si-NC+4 Gy group (P＜0.05). Circ_0000263 targeted regulated miR-338-3p, and miR-338-3p targeted regulated TERT. MiR-338-3p was lowly expressed in HeLa cells, and knockdown of circ_0000263 elevated miR-338-3p expression level in HeLa cells. Circ_0000263 regulated TERT expression and inhibited telomerase activity through miR-338-3p. MiR-338-3p/TERT can restore the effect of circ_0000263 on the radiosensitivity of Hela cells. The apoptosis rate in the si-circ+anti-NC group was (27.37±0.89)%, which was higher than (18.22±1.18)% of the si-circ+anti-miR-338-3p group (P＜0.05). The apoptosis rate in the si-circ+vector group was (27.55±0.48)%, which was higher than (20.10±0.68)% of si-circ+TERT group (P＜0.05). After 72 hours of radiation by 4 Gy, the cell survival fraction of si-circ+anti-NC group was 0.41±0.02, which was lower than 0.66±0.03 of the si-circ+anti-miR-338-3p group (P＜0.05); the cell survival fraction of si-circ+vector group was 0.42±0.05, which was lower than 0.70±0.03 of si-circ+TERT group (P＜0.05). Conclusion: Inhibiting the expression of circ_0000263 supresses the proliferation of Hela cells by regulating miR-338-3p/TERT, promotes apoptosis, inhibits telomerase activity, increases the radiosensitivity of cancer cells, and provides a theoretical basis for improving the radiosensitivity of Hela cells.

Protective effects of 2-methoxyestradiol against hypoxic pulmonary hypertension in neonatal rats

To investigate the protective effects of 2-methoxyestradiol (2ME) against hypoxic pulmonary hypertension (HPH) in neonatal rats. Ninety-six Wistar neonatal rats were randomly divided into a normoxia group, a hypoxia group, and a hypoxia + 2ME group, with each group further subdivided into 3-day, 7-day, 14-day, and 21-day subgroups, containing eight rats each. The hypoxia and hypoxia + 2ME groups received daily subcutaneous injections of saline and 2ME (240 μg/kg), respectively, while the normoxia group was raised in a normoxic environment with daily saline injections. Right ventricular systolic pressure (RVSP) was measured using the direct pressure method. Pulmonary vascular morphology was assessed using hematoxylin and eosin staining, with metrics including the percentage of medial thickness of small pulmonary arteries relative to the external diameter (MT%) and the cross-sectional area of the media of small pulmonary arteries relative to the total cross-sectional area (MA%). Immunohistochemistry was used to detect the expression levels of hypoxia-inducible factor-1α (HIF-1α) and proliferating cell nuclear antigen (PCNA) proteins, while real-time quantitative PCR was used to to assess HIF-1α and PCNA mRNA levels. Compared to the normoxia group, the hypoxia and hypoxia + 2ME groups showed increased RVSP and upregulated HIF-1α and PCNA protein and mRNA expression levels at 3, 7, 14, and 21 days after hypoxia (P<0.05). Furthermore, at 7, 14, and 21 days after hypoxia, the hypoxia group showed increased MT% and MA% (P<0.05). In comparison to the hypoxia group, the hypoxia + 2ME group exhibited reduced RVSP and downregulated HIF-1α and PCNA protein and mRNA expression levels, along with decreased MT% and MA% at 7, 14, and 21 days after hypoxia (P<0.05). 2ME may protect against HPH in neonatal rats by inhibiting the expression of HIF-1α and PCNA and reducing pulmonary vascular remodeling. Citation:Chinese Journal of Contemporary Pediatrics, 2024, 26(7): 757-764.

AMPK regulates immature boar Sertoli cell proliferation through affecting CDK4/Cyclin D3 pathway and mitochondrial function

Sertoli cell (SC) proliferation plays an important role in sperm production and quality; however, the regulatory mechanism of SC proliferation is not well understood. This study investigated the role of adenosine monophosphate-activated protein kinase (AMPK) in the regulation of immature boar SC activity. Cell counting kit-8, Seahorse XFe96, mitochondrial respiratory enzyme-related assay kits, and transmission electron microscopy were used to detect SC proliferative viability, oxygen consumption rate (OCR), mitochondrial respiratory enzyme activity, and the ultrastructure of primary cultured SCs in vitro from the testes of 21-day-old boars. A dual luciferase reporter assay was performed to determine the miRNA-mRNA target interaction. Western blotting was used to analyze cell proliferation-related protein expression of p38, p21, proliferating cell nuclear antigen (PCNA), Cyclin-dependent kinase 4 (CDK4), Cyclin D3, and phosphorylated retinoblastoma protein (Rb). Each experiment had a completely randomized design, with three replicates in each experiment. The results showed that the AMPK inhibitor (Compound C, 20 μM-24 h) increased cell proliferation viability, ATP production, and maximal respiration of SCs by 0.64-, 0.12-, and 0.08-fold (p < 0.05), respectively; increased the SC protein expression of PCNA, CDK4, Cyclin D3, and p-Rb by 0.13-, 0.09-, 0.88-, and 0.12-fold (p < 0.05), respectively; and decreased the SC protein expression of p38 and p21 by 0.36- and 0.27-fold (p < 0.05), respectively. The AMPK agonist AICAR (2 mM-6 h) significantly inhibited SC ultrastructure, OCR, mitochondrial respiratory enzyme activity, and cell proliferation-related protein levels. AMPK was validated to be a target gene of miR-1285 based on the result in which the miR-1285 mimic inhibited the luciferase activity of wild-type AMPK by 0.54-fold (p < 0.001). MiR-1285 mimic promoted the OCR of SCs, with 0.45-, 0.15-, 0.21-, and 0.30-fold (p < 0.01) increases in ATP production, basal and maximal respiration, and spare capacity, respectively. MiR-1285 mimic increased the mitochondrial respiratory enzyme activity of SCs, with 0.63-, 0.70-, and 0.97-fold (p < 0.01) increases in NADH-Q oxidoreductase, cytochrome c oxidase, and ATP synthase, respectively. Moreover, the miR-1285 mimic increased the protein expression of PCNA, CDK4, Cyclin D3, and p-Rb by 0.24-, 0.30-, 0.22-, and 0.13-fold (p < 0.05), respectively, and reduced the protein expression of p38 and p21 by 0.58- and 0.66-fold (p < 0.001). MiR-1285 inhibitor showed opposite effects on the above indicators and induced numerous autophagosomes and large lipid droplets in SCs. A high dose of estradiol (10 μM-6 h, showed a promotion of AMPK activation in a previous study) significantly inhibited SC ultrastructure, mitochondrial function, and proliferation-related pathways, while these adverse effects were weakened by Compound C treatment or miR-1285 mimic transfection. Our findings suggest that the activation and inhibition of AMPK induced by specific drugs or synthesized targeted miRNA fragments could regulate immature boar SC proliferative activity by influencing the CDK4/Cyclin D3 pathway and mitochondrial function; this helps to provide a basis for the prevention and treatment of male sterility in clinical practice.

Abstract 1850: From proteomic analysis to early investigations of combining inhibitors targeting PCNA and histone deacetylase

Abstract Proteomic changes were investigated in a neuroblastoma cell line, SK-N-AS, treated with a small molecule inhibitor, AOH1996, designed to specifically bind proliferating cell nuclear antigen (PCNA) in cancer. PCNA is crucial to DNA replication, DNA repair, transcription, cell cycle control, and chromatin organization through interactions with different proteins. Pathway analysis of differential protein expressions between treated and untreated cells revealed less G2/M DNA damage checkpoint regulation and decreased assembly of RNA polymerase complex. Phosphoproteomic analysis by PTM Signature Enrichment Analysis (PTM-SEA) identified signatures of kinase activities, signaling pathways, and perturbations. Specifically, the histone deacetylase inhibitors belinostat and vorinostat were identified as enriched perturbation signatures. Both inhibitors were tested in combination with AOH1996 in SK-N-AS as well as cell lines derived from breast, lung, ovarian, and pancreatic cancer, revealing drug synergies at specific dose combinations. Western blot analysis showed that belinostat decreased protein expression of PCNA, suggestive of mechanistic synergies with AOH1996+belinostat combination therapies. Our proteomic analysis reveals a therapeutic potential of combining AOH1996 and histone deacetylase inhibitors for treating cancer with possibly lower drug dose and toxicities compared to single drug treatment. Citation Format: Caroline M. Li, Krystine Garcia-Mansfield, Robert G. Lingeman, Long Gu, Robert J. Hickey, Patrick Pirrotte, Linda H. Malkas. From proteomic analysis to early investigations of combining inhibitors targeting PCNA and histone deacetylase [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 1850.

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.

Enteric glial cells contribute to chronic stress-induced alterations in the intestinal microbiota and barrier in rats

BackgroundEmerging evidence has demonstrated the impact of psychological stress on intestinal microbiota, however, the precise mechanisms are not fully understood. Enteric glia, a unique type of peripheral glia found within the enteric nervous system (ENS), play an active role in enteric neural circuits and have profound effects on gut functions. In the present study, we tested the hypothesis that enteric glia are involved in the alterations in the intestinal microflora and barrier induced by chronic water-avoidance stress (WAS) in the gut. Methods and resultsWestern blotting and immunohistochemical (IHC) staining were used to examine the expression of glial fibrillary acidic protein (GFAP), nitric oxide synthetase (NOS) and choline acety1transferase (ChAT) in colon tissues. 16S rDNA sequencing was performed to analyse the composition of the intestinal microbiota in rats. Changes in the tight junction proteins Occludin, Claudin1 and proliferating cell nuclear antigen (PCNA) in the colon tissues were detected after WAS. The abundance of Firmicutes, Proteobacteria, Lactobacillus and Lachnospiraceae_NK4A136 decreased significantly, whereas the abundance of Actinobacteria, Ruminococcaceae_UCG−005 and Christensenellaceae-R-7 increased significantly in stressed rats. Meanwhile, the expression of Occludin, Claudin1 and PCNA significantly decreased after WAS. Treatment with L-A-aminohexanedioic acid (L-AA), a gliotoxin that blunts astrocytic function, obviously decreased the abundance of Actinobacteria, Ruminococcaceae_UCG−005 and Christensenel-laceae_R-7 in stressed rats and significantly increased the abundance of Proteobacteria, Lactobacillus and Lachnospiraceae_NK4A136. In addition, the protein expression of colon Occludin, Claudin1, and PCNA increased after intraperitoneal injection of L-AA. Furthermore, the expression level of NOS in colon tissues was significantly decreased, whereas that of ChAT was significantly increased following L-AA treatment. ConclusionsOur results showed that enteric glial cells may contribute to WAS-induced changes in the intestinal microbiota and barrier function by modulating the activity of NOS and cholinergic neurones in the ENS.

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 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.

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.

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.

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.

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.

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.

Regeneration of meniscal avascular zone using autogenous meniscal fragments in a rabbit model

BackgroundTo investigate the effects of autologous meniscus fragment (AMF) implantation on injury in the meniscal avascular zone in mature rabbits.MethodsAdult New Zealand white rabbits were randomly divided into two groups. Massive one-piece meniscus tissue was implanted in situ as control. In the experimental group, AMF was used to repair the meniscal injury in the avascular zone. Meniscal damage was assessed by gross observation of the degree of healing and histological semi-quantitative evaluation within 12 weeks postoperatively. The healing of meniscus interface was assessed by gross observation semiquantitative scoring and microscopic examination hematoxylin and eosin (H&E) staining at 2, 4, 8, and 12 weeks after surgery. The expressions of proliferating cell nuclear antigen (PCNA), collagen type I (COL1A1), and collagen type II (COL2) were detected by immunohistochemical staining.ResultsThe degree of healing in the AMF group showed a significant increase over time (P < 0.05); the AMF group showed higher gross scores than the control group at 4, 8, and 12 weeks after surgery (P < 0.05). The histological scores in the AMF group were significantly higher than those in the control group at 4, 8, and 12 weeks after surgery (P < 0.05). The protein expression of PCNA in the AMF group was greater than that in the control group at 2, 4, and 8 weeks after surgery (P < 0.05). In addition, compared with the control group, the protein levels of COL1A1 and COL2 were significantly upregulated at each time-point. At 2 and 4 weeks after surgery, the expression level of COL1A1 increased in both groups followed by a gradual decrease after 8 weeks (P < 0.05). At 2, 4, 8, and 12 weeks after surgery, the expression levels of COL2 showed a gradual decrease in both groups (P < 0.05).ConclusionsOur study demonstrated that the AMF method can promote the repair of rabbit meniscal injury in the avascular zone, and this method may potentially be used for clinical application.

Regulatory effects of bio-intensity electric field on transformation of human skin fibroblasts

Regulatory effects of bio-intensity electric field on transformation of human skin fibroblasts

Zearalenone promotes follicle development through activating the SIRT1/PGC-1α signaling pathway in the ovaries of weaned gilts.

This study aimed to investigate the effect of zearalenone (ZEA) exposure on follicular development in weaned gilts, and its mechanism based on the silent information regulator 1 (SIRT1)/peroxisome proliferator-activated receptor-γ co-activator 1α (PGC-1α) signaling pathway. A total of 32 healthy female weaned piglets (Landrace × Yorkshire × Duroc) with an average body weight of 12.39±0.24kg were randomly allotted to a basal diet supplemented with 0, 0.15, 1.5, or 3.0mg/kg ZEA for a 32-d feeding test. Blood and ovarian samples were obtained at the end of the experiment to determine serum toxin concentrations, ovarian histology, and the expressions of proliferating cell nuclear antigen (PCNA) and SIRT1/PGC-1α signaling pathway-related genes. Results showed that the vulva area, serum concentrations of ZEA, α-zearalenol and β-zearalenol, the thickness of the growing follicular layer, and the diameter of the largest growing follicles, as well as the expressions of SIRT1, PGC-1α, estrogen-related receptor α (ERRα), ATP synthase subunit beta (ATP5B), and PCNA, increased linearly (P < 0.05) with increasing dietary ZEA, whereas the thickness of the primordial follicle layer decreased linearly (P < 0.05). Immunohistochemical analysis showed that the immunoreactive substances of SIRT1 and PGC-1α in the ovaries enhanced with the increasing dietary ZEA (P < 0.05). In addition, the thickness of the growing follicular layer and the diameter of the largest growing follicle were positively correlated with relative mRNA and protein expressions of SIRT1, PGC-1α, ERRα, ATP5B, and PCNA (P < 0.05). However, the thickness of the primordial follicle layer was negatively correlated with the mRNA and protein expression of SIRT1, PGC-1α, ERRα, ATP5B, and PCNA (P < 0.05). Interestingly, the 1.5mg/kg ZEA treatment had highly hyperplastic follicles, whereas 3.0mg/kg ZEA resulted in a large number of follicular atresia, which indicated that low-dose ZEA exposure accelerated follicular proliferation, while high-dose ZEA promoted follicular atresia, although the critical value interval needs further confirmation. Results provide a theoretical basis for finding the therapeutic target of ZEA-induced reproductive disorders in weaned gilts.

Sulfiredoxin-1 Inhibits PDGF-BB-Induced Vascular Smooth Muscle Cell Proliferation and Migration by Enhancing the Activation of Nrf2/ARE Signaling.

Sulfiredoxin1 (Srxn1), an endogenous antioxidant protein, is involved in cardiovascular diseases. In this study, we aimed to investigate the role of Srxn1 in VSMCs and its molecular mechanism. The murine vascular smooth muscle cells MOVAS were treated with different doses of platelet-derived growth factor-BB (PDGF-BB); then, Srxn1 expression was detected using reverse transcription-quantitative polymerase chain reaction and western blot analysis. MTT and wound healing assay were used to examine the effect of Srxn1 on MOVAS cell proliferation and migration. Reactive oxygen species (ROS) production, malondialdehyde (MDA) level, and superoxide dismutase (SOD) activity in MOVAS cells were detected using corresponding commercial kits. Moreover, the expression of proliferating cell nuclear antigen (PCNA), matrix metalloproteinase 2 (MMP-2), and nuclear factor erythroid-2-related factor 2 (Nrf2) /antioxidant response element (ARE) signaling-related proteins was detected using western blot analysis. In our study, PDGF-BB dose-dependently increased Srxn1 expression in MOVAS cells, and Srxn1 expression was increased with time dependence in PDGF-BB-treated MOVAS cells. The knockdown of Srxn1 increased PDGF-BB-induced the proliferation, migration, ROS production, MDA level, and the protein expression of PCNA and MMP-2, as well as decreased SOD activity and the expression of Nrf2/ARE signaling-related proteins in PDGF-BB-stimulated MOVAS cells. However, the overexpression of Srxn1 showed the opposite results to those of knockdown of Srxn1. Moreover, the inhibitory effects of Srxn1 overexpression on PDGF-BB induced proliferation, migration, ROS production, and MDA level and the promotion of Srxn1 overexpression on PDGF-BB induced SOD activity were partially reversed by the knockdown of Nrf2. Srxn1 inhibited PDGF-BB-induced proliferation, migration, and oxidative stress through activating Nrf2/ARE signaling.

595-nm pulsed dye laser combined with fractional CO2 laser reduces hypertrophic scar through down-regulating TGFβ1 and PCNA.

595-nm pulsed dye laser and fractional CO2 laser have been demonstrated effective to treat hypertrophic scar. The underlying mechanism may involve transforming growth factor-beta1 (TGFβ1) and proliferating cell nuclear antigen (PCNA), but remains to be clarified. Our study was performed to investigate how 595-nm pulsed dye laser combined with fractional CO2 laser treats hypertrophic scars in a rabbit model through regulating the expression of TGFβ1 and PCNA. Twenty-four New Zealand white rabbits were randomly divided into control group, pulsed dye laser group, fractional CO2 laser group, and pulsed dye laser + fractional CO2 laser (combination) group. Surgical wounds were made and allowed to grow into hypertrophic scars at day 28. Next, 595-nm pulsed dye laser (fluence: 15J/cm2; square: 7mm; pulse duration: 10ms) was used in pulsed dye laser and combination group, while fractional CO2 laser (combo mode, deep energy: 12.5mJ; super energy: 90mJ) in fractional CO2 laser and combination groups, once every 4weeks for 3 times. The appearance and thickness of hypertrophic scar samples were measured with hematoxylin-eosin and Van Gieson's straining. The expressions of TGFβ1 and PCNA were evaluated by immunohistochemical and western blot analysis. A significant improvement was noted in the thickness, size, hardness, and histopathology of hypertrophic scar samples after laser treatment, especially in combination group. Scar Elevation Index (SEI), fiber density (NA), and collagen fiber content (AA) decreased most significantly in combination group (2.10 ± 0.14; 2506 ± 383.00; 22.98 ± 2.80%) compared to 595-nm pulsed dye laser group (3.35 ± 0.28; 4857 ± 209.40; 42.83 ± 1.71%) and fractional CO2 laser group (2.60 ± 0.25; 3995 ± 224.20; 38.33 ± 3.01%) (P< 0.001). Furthermore, TGFβ1 and PCNA expressions were more suppressed in combination group (8.78 ± 1.03; 7.81 ± 1.51) than in 595-nm pulsed dye laser (14.91 ± 1.68; 15.73 ± 2.53) and fractional CO2 laser alone group (15.96 ± 1.56; 16.13 ± 1.72) (P< 0.001). The combination of 595-nm pulsed dye laser with fractional CO2 laser can improve the morphology and histology of hypertrophic scars in a rabbit model through inhibiting the expression of TGFβ1 and PCNA protein. Our findings can pave the way for new clinical treatment strategies for hypertrophic scars.

Grape seed extract ameliorated Ehrlich solid tumor-induced hepatic tissue and DNA damage with reduction of PCNA and P53 protein expression in mice.

This study evaluated the ameliorative potential of grape seed extract (GSE) against Ehrlich solid tumor (EST)-induced hepatic tissue alterations in mice. The control group was infused with physiological saline. The second group received GSE (50 mg/kg day by day orally) for 2 weeks. The third group was subcutaneously injected with 2.5 million of EST cells. The fourth group was injected with EST cells and treated with GSE extract simultaneously. The fifth group was injected with EST cells and kept for 2 weeks until the appearance of a solid tumor, then treated with GSE for 2 weeks. The phytochemical analysis of GSE revealed the presence of total phenols (17.442 mg GAE/g) and total flavonoid (6.687 mg CE/g) with antioxidant activity of 81.506 mg TE/g DPPH. The Ehrlich solid tumor significantly raised the activities of ALT, AST, and ALP; the level of alpha fetoprotein (AFP) in serum; and the protein expressions of hepatic proliferating cell nuclear antigen (PCNA) and tumor suppressor protein (P53), as well as induced DNA damage and pathological alterations in liver tissue. However, it significantly reduced serum albumin and total protein levels. In contrast, the co- or post-treatment of EST-bearing mice with GSE reduced the activities of ALT, AST, and ALP; the level AFP in serum; and hepatic P53 and PCNA protein expressions. In addition, it reduced EST-induced hepatic DNA damage and pathological alterations, while it increased serum albumin and total protein levels. This study suggested that GSE is a potent hepatoprotective agent and both co- and post-treatment of EST-bearing mice with GSE almost had the same effects.

CIAP2 expression and clinical significance in pigmented villonodular synovitis.

Pigmented villonodular synovitis (PVNS) is a rare hyperplasia disease of the synovium with a predilection for the knee in either a localized (LPVNS) or a diffuse form (DPVNS). But the exact cause is not clear. The aim of this study was to explore the relationship between the expression of cellular inhibitor of apoptosis 2 (cIAP2) and proliferation, apoptosis, invasive growth and postoperative recurrence in PVNS. Clinical significance of cIAP2 expression in synovium from 63 patients' knee joints with PVNS (40 DPVNS; 23 LPVNS) were investigated with 20 normal subjects acting as controls. The cIAP2 gene was screened by Human Cancer Pathway Finder PCR Array and real-time polymerase chain reaction (RT-PCR). We also used immunohistochemistry to detect cIAP2 and proliferating cell nuclear antigen (PCNA) protein expression and analyzed their relationship with PVNS type, invasive growth, and postoperative recurrence. The expression of cIAP2, PCNA, caspase-8, caspase-9 and caspase-3 protein was tested in Western blot. Screening results of Human Cancer Pathway Finder PCR array and RT-PCR showed significantly more cIAP2 mRNA in DPVNS synovium than in normal or LPVNS synovium (P < 0.05). Immunohistochemistry and western blot showed that the cIAP2 protein expression level in DPVNS was significantly higher than in LPVNS tissue (P < 0.01). As cIAP2 expression increased, the expression of PCNA increased (P < 0.05) and expression of cleaved caspase-3, -8, -9 decreased (P < 0.01). cIAP2 and PCNA overexpression were found to be related to ligament and bone erosion in PVNS and to disease recurrence (P < 0.05). This study suggested that cIAP2 overexpression plays an important role in the anti-apoptotic, proliferative and invasive growth of PVNS, which may account for the recurrence and poor prognosis of DPVNS.