Proliferating Cell Nuclear Antigen Gene Research Articles

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.

The PCNA GENE EXPRESSION AS A MARKER OF ALZHEIMER'S DISEASE IN THE BRAIN OF LONG-TAILED MACAQUES (Macaca fascicularis)

Early diagnosis of Alzheimer's is still difficult to do, thus it is important to carry out further research to find biomarkers that can be used to detect early Alzheimer's disease. Proliferating Cell Nuclear Antigen (PCNA) is known as a proliferation marker, which has the potential to detect neurogenesis in the brain. This study aimed to identify PCNA gene expression in the brain as a marker of Alzheimer’s disease in Macaca fascicularis. Macaca fascicularis was used in this study because of their similarities with humans in terms of their behavioral complexity, as well as high cognitive abilities and the formation of a pathological characteristic of Alzheimer's disease in the brain. This study used the brains of 7 monkeys in the hippocampus and cortical regions. Monkeys previously have been divided into old and adult age groups. The detection of PCNA gene expression was done using RT-qPCR method. The results showed the gene expression tended to be higher in the adult group and the hippocampus region, although based on statistical analysis showed no significant differences

The Effect of Pantoprazole on Tumor Growth and Apoptosis in Gastric Cancer Stem-Like Cells

Background: Gastric cancer is a daunting global problem with unsatisfactory treatment. Due to the key role of Gastric Cancer Stem-like Cells (GCSCs) in all stages of gastric cancer and the failure of contemporary anticancer therapies, many research efforts are focusing on these treatmentresistant cells. Pantoprazole, as recently considered antitumor agent with well-documented effects on tumorigenesis inhibition, has seldom been investigated in GCSCs in previous studies. We aimed to study the influence of pantoprazole on cell proliferation, apoptosis, and the transcription of genes involved in the cell proliferation and apoptosis pathways. Materials and Methods: Herein, we isolated GCSCs from MKN-45 cell line, on a non-adherent surface and then evaluated the effect of pantoprazole on cell growth and apoptosis of GCSCs by means of MTT, DNA laddering and quantitative real-time RT-PCR techniques. Results: Our findings showed that treated cells with pantoprazole decreased cell proliferation and induced apoptosis. PCNA (Proliferating Cell Nuclear Antigen) and antiapoptotic Bcl2 genes were downregulated and Bax and CASP3 proapoptotic genes, as well as tumor suppressor p53 gene, were overexpressed. Conclusion: Our results revealed that pantoprazole induced apoptosis and declined tumor growth and support the idea that pantoprazole played as a promising breakthrough in gastric cancer therapy.

Grain-Sized Moxibustion Heightens the AntiTumor Effect of Cyclophosphamide in Hepa1-6 Bearing Mice.

Objective The side effects of chemotherapy as a treatment of liver cancer cannot be ignored. Grain-sized moxibustion, a characteristic external therapy, has been shown to reduce the toxic and side effects of chemotherapy and regulate the immune function. The purpose of this study was to explore the synergistic antitumor activity of grain-sized moxibustion combined with cyclophosphamide (CTX). Methods A hepatoma 1–6 (Hepa1-6)-bearing mouse model was established by injecting mice with Hepa1-6 cancer cells. CTX and grain-sized moxibustion on Dazhui (DU14), Zusanli (ST36), and Sanyinjiao (SP6) were used for treatment, and mouse survival status, body weight, and tumor growth, weight, and volume were measured. White blood cells (WBCs) and bone marrow nucleated cells (BMNCs) were quantified. The spleens and livers of Hepa1-6-bearing mice were pathologically examined and scored. Serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels were measured with enzyme-linked immunosorbent assay (ELISA) kits, and protein and mRNA expression levels of Ki67 and proliferating cell nuclear antigen (PCNA) in tumor tissues were measured with immunohistochemistry and real-time quantitative polymerase chain reaction (RT-qPCR) techniques. Results Both grain-sized moxibustion and CTX could restrain the growth of Hepa1-6 tumors, reducing both tumor volume and weight; the combined treatment had a greater effect. Grain-sized moxibustion down-regulated the expression of proliferation genes Ki67 and PCNA, weakened the proliferation ability of Hepa1-6 tumor cells, inhibited tumor growth, and enhanced the antitumor effect of CTX. In addition, grain-sized moxibustion significantly improved the signs of CTX-induced toxicity (including weight loss, leukopenia, bone marrow suppression, and hepatotoxicity), down-regulated serum AST and ALT levels, reduced spleen and liver inflammation, and improved liver and spleen indices. Conclusion Grain-sized moxibustion can synergize with CTX to enhance the antitumor effect of CTX and alleviate its toxic and side effects. It may be a promising adjuvant therapy to chemotherapy.

Can PCNA and LIM15 gene expression levels predict sperm retrieval success in men with non-obstructive azoospermia?

Introduction and objectivesIt is necessary to be able to predict sperm retrieval before microdissection testicular sperm extraction (mTESE) in azoospermic men. This study established the importance of proliferating cell nuclear antigen (PCNA) and LIM15 gene expression levels in predicting the success of sperm retrieval by mTESE. Materials and methodsOne hundred and forty-three men who were diagnosed with non-obstructive azoospermia (NOA) were included in the study. Patients’ age, total testosterone and follicle stimulating hormone values, testicular volume and testicular histology were recorded by prospectively. PCNA and LIM15 gene expression levels were determined by real-time PCR in the materials from both ejaculate and testicular specimens. ResultsTestis volume and histology were the most important factors in predicting the sperm retrieval rate (SRR). The PCNA and LIM15 gene expression levels measured in testicular tissues and the LIM15 gene expression levels measured in ejaculate significantly correlated with the SRR in mTESE (p=0.038, p=0.022, and p=0.004, respectively). Although the PCNA gene expression level measured in ejaculate was higher in men with successful sperm retrieval, the difference was not statistically significant (p=0.061). According to the multivariate logistic regression analysis, testicular volume and LIM15 gene expression level in ejaculate were independent predictive parameters for sperm retrieval. ConclusionThe data showed that LIM15 gene expression level in ejaculate is a useful molecular marker to predict the SRR before mTESE.

Manipulation of viral protein production using the PCNA of halovirus фCh1 via alternative start codon usage

• Proliferating Cell Nuclear Antigen (PCNA) is a scaffold protein principally found at the centre of replication, coordinating with a wide array of interaction partners. фCh1, unusually for a virus, encodes a putative PCNA. • Natrialba magadii, the only known host of фCh1, also encodes a putative PCNA of high sequence similarity, differing majorly in the presence of an alternate GTG start codon in the фCh1 variant 5’ of the ATG start codon resulting in a larger protein. Homologous recombination was used to delete the viral PCNA gene. • Complementation and overexpression strains with plasmid expressed viral PCNA variants were created and analyzed for growth and lytic behavior, viral protein levels, and virus titer. • Deletion of фCh1-ORF59, encoding PCNA ΦCh1 resulted in a significant reduction in the virus titer, reduced viral protein load, and reduced lysis. • Complementation and overexpression with WT, and mutant variants of ORF59revealed that the 5’ GTG to ATG sequence enables фCh1 to control its equilibrium with the host. The different variants have a broad range of variable effects on each of the phenotypes observed. • Experiments with a halophilic beta-galactosidase assay revealed a cis/trans mediated interaction between the viral PCNA and the origin of replication of φCh1 modulated by the 5’ GTG to ATG sequence. The virally encoded PCNA is not essential, but is crucial, to the behavior of the virus фCh1. The 5’ region upstream of the primary ATG codon and its regulation with an alternate start codon is essential to the homeostasis of the virus-host relationship. Proliferating Cell Nuclear Antigen (PCNA) is a scaffold protein principally found at the centre of replication, coordinating with a wide array of interaction partners. фCh1, unusually for a virus, encodes a putative PCNA. Natrialba magadii, the only known host of фCh1, also encodes a putative PCNA of high sequence similarity, differing in the presence of an alternate GUG start codon 5’ of the AUG start codon resulting in a larger protein. Homologous recombination was used to delete the viral PCNA gene. Complementation and overexpression strains with plasmid expressed viral PCNA variants were created and analyzed for growth and lytic behaviour, viral protein levels, and virus titer. Deletion of фCh1 ORF59, encoding PCNA ФCh1 resulted in a significant reduction in the virus titer, reduced viral protein load, and reduced lysis. Complementation and overexpression with WT and mutant variants of ORF59 revealed that modification of the GTG start codon to ATG changes the life cycle in terms of production of progeny virus particles. The different variants have a broad range of variable effects on each of the phenotypes observed. Experiments with a halophilic beta-galactosidase assay revealed a cis/trans mediated interaction between the viral PCNA and the origin of replication of фCh1 modulated by the 5’GTG to ATG sequence. The virally encoded PCNA is not essential, but is crucial, to the life cycle of the virus фCh1. The 5’ region upstream of the primary AUG codon and its regulation with an alternate start codon is essential to the homeostasis of the virus-host relationship.

Ex Vivo Human Colon Tissue Exposure to Pristine Graphene Activates Genes Involved in the Binding, Adhesion and Proliferation of Epithelial Cells.

Toxicology studies on pristine graphene are limited and lack significant correlations with actual human response. The goal of the current study was to determine the response of total colonic human tissue to pristine graphene exposure. Biopsy punches of colon tissues from healthy human were used to assess the biological response after ex vivo exposure to graphene at three different concentrations (1, 10, and 100 µg/mL). mRNA expression of specific genes or intestinal cytokine abundance was assessed using real-time PCR or multiplex immunoassays, respectively. Pristine graphene-activated genes that are related to binding and adhesion (GTPase and KRAS) within 2 h of exposure. Furthermore, the PCNA (proliferating cell nuclear antigen) gene was upregulated after exposure to graphene at all concentrations. Ingenuity pathway analysis revealed that STAT3 and VEGF signaling pathways (known to be involved in cell proliferation and growth) were upregulated. Graphene exposure (10 µg/mL) for 24 h significantly increased levels of pro-inflammatory cytokines IFNγ, IL-8, IL-17, IL-6, IL-9, MIP-1α, and Eotaxin. Collectively, these results indicated that graphene may activate the STAT3–IL23–IL17 response axis. The findings in this study provide information on toxicity evaluation using a human-relevant ex vivo colon model and serve as a basis for further exploration of its bio-applications.

Resistance Analysis of CRISPR/Cas9 Genome-Edited Chili M2 Mutant Lines against Pepper Yellow Leaf Curl Viral Disease

<p>Pepper yellow leaf curl virus (PepYLCV) infection transmitted by silverleaf whitefly (Bemisia tabaci [Gennadius]) can decrease chili pepper yield up to 100%. At this moment, there is no chili pepper variety resistant to PepYLCV available. Genome editing approach through CRISPR/Cas9 is an effort to develop variety resistance to the viral infection. The purpose of this study was to obtain M2 lines developed by CRISPR/Cas9 system on proliferating cell nuclear antigen (PCNA) gene for resistance to PepYLCV. A total of four M2 lines (C47-7, L84-2, L84-23, and L120-19) consisting of 60 chili plants were tested for their resistance to PepYLCV. PCR analysis was performed to detect the presence (infection) of the virus. The results showed that a total of 35 plants derived from the four lines were resistant to PepYLCV. They consisted of 7 plants from C47-7 line, 11 plants from L84-2 line, 9 plants from L84-23 line, and 8 plants from L120-19 line. PCR analysis confirmed that the resistant plants obtained from this study were negatively infected by the virus. Since not all tested plants were resistant to virus infection, the PCNA gene allele in these resistant lines were most likely heterozigotes. Sequencing of PCNA gene of the resistant lines is needed to confirm that the resistance phenotypes obtained was due to mutation of the gene. Therefore, further selection needs to be performed to obtain stable and PepYLCV-resistant lines.</p>

Protective Effect of Hydroalcoholic Extract of Orange Peel on PCNA and FSH-R Gene Expression in Histological Damage and Oxidative Stress Due to Ovarian Torsion in Adult Rats

Objectives: The aim of this study was to evaluate the protective effect of the hydroalcoholic extract of orange peel on proliferating cell nuclear antigen (PCNA) and follicle-stimulating hormone receptor (FSH-R) gene expression in histological injuries and acid stress caused by ovarian torsion in adult rats. Materials and Methods: In this experimental study, 32 adult female rats were randomly divided into 4 groups. In group 1 (Sham), the abdominal wall was cut without applying torsion and in group 2, ovarian torsion was performed for 2 hours, followed by detorsion for 2 weeks. The hydro-alcoholic extract of orange peel was added to their diet for two weeks in group 3, followed by ovarian torsion for 2 hours and detorsion for 2 hours. Group 4 received the orange peel extract for two weeks and after then ovarian resection for the evaluation of histological damage and blood sampling to examine the serum level of antioxidant enzymes, as well as the expression of PCNA and FSH-R genes in the ovarian tissue. Results: Histological changes in the ovary tissue of rats showed that torsion and detorsion have destructive effects on the ovarian tissue, and torsion/detorsion led to a reduction in the expression of PCNA and FSH-R (P < 0.05). Based on biochemical and hormonal results, the ovarian torsion resulted in an imbalance in the oxidative stress markers and hormone profile of rats. Finally, the administration of the hydroalcoholic extract of orange peel due to its high antioxidant properties improves these effects. Conclusions: In general, administering an appropriate dose of the hydroalcoholic extract of orange peel for two consecutive weeks in the diet had a protective effect on the ovarian tissue at the risk of torsion/detorsion.

Editing of PCNA Gene by CRISPR/Cas9 Technology to Improve the Red Chili Resistance to Yellow Leaf Curl Disease

<p>Yellow leaf curl disease caused by Pepper yellow leaf curl virus (PepYLCV), member of geminiviruses group, is responsible for heavy yield losses for chili pepper production. Resistant genes which can cause immunity to the disease have not been found in germplasm collection. The aim of the research was to edit proliferating cell nuclear antigen (PCNA) gene by using CRISPR/Cas9 technology for developing plant resistance against geminivirus in chili pepper. A CRISPR/Cas9 plasmid cassette construct harboring the guide RNA of PCNA gene was constructed by Golden Gate cloning strategy. The construct was then introduced into chili genome via in planta method using Agrobacterium tumefaciens EHA105. The transformed plants were bioassayed by virus inoculation and confirmed using PCR and DNA sequencing to identify a mutagenesis event in PCNA gene target. The results showed that CRISPR/Cas9 plasmid cassette harboring gRNA of PCNA gene was successfully constructed. In planta transformation using A. tumefaciens vector harboring CRISPR/Cas9-gRNA PCNA construct resulted in 307 and 193 transformed plants from chili var. Lingga and Ciko, respectively. Bioassay by using virus inoculation to the transformed plants obtained 6 and 14 lines of Lingga and Ciko, respectively, which were resistant to geminivirus (no symptom observed). The resistant lines of chili pepper var. Lingga and Ciko were mutated in PCNA gene with one base insertion or deletion mutation types. These results exhibit that the CRISPR/Cas9 genome editing can be used to induce mutant of PCNA gene in chili pepper. Further investigation is necessary to evaluate the selected chili lines resistant to PepYLCV infection.</p>

Interferon regulatory factor 7 inhibits rat vascular smooth muscle cell proliferation and inflammation in monocrotaline-induced pulmonary hypertension

AimsAlthough interferon regulatory factor 7 (IRF7) has known roles in regulating the inflammatory response, vascular smooth muscle cell proliferation, and apoptosis, its role in the pathogenesis of pulmonary hypertension (PH) is unclear. We hypothesized that IRF7 overexpression could inhibit pulmonary vascular remodeling and slow the progression of PH. Main methodsIRF7 mRNA and protein levels in the lung samples and pulmonary artery smooth muscle cells (PASMCs) isolated from monocrotaline (MCT)-induced PH rats were assessed. We evaluated the effects of IRF7 on inflammation, proliferation, and apoptosis using an in vivo MCT-induced PH rat model and in vitro methods. Key findingsWe noted decreased IRF7 mRNA and protein levels in the pulmonary vasculature of MCT-induced PH rats. IRF7 upregulation attenuated pulmonary vascular remodeling, decreased the pulmonary artery systolic pressure, and improved the right ventricular (RV) structure and function. Our findings suggest that nuclear factor kappa-Bp65 (NF-κBp65) deactivation could confer pulmonary vasculature protection, reduce proinflammatory cytokine (tumor necrosis factor-α, interleukin 6) release, and decrease PASMC proliferation and resistance to apoptosis via deactivating transcription factor 3 (ATF3) signaling. ATF3 deactivation induced the downregulation of the proliferation-dependent genes proliferating cell nuclear antigen (PCNA), cyclin D1, and survivin, coupled with increased levels of B cell lymphoma-2-associated X protein (Bax)/B cell lymphoma-2 (Bcl2) ratio, and cleaved caspase-3 in PASMCs. SignificanceOur findings showed that IRF7 downregulation could initiate inflammation via NF-κBp65 signaling, causing PASMC proliferation via ATF3 signaling pathway activation. Therefore, IRF7 could be a potential molecular target for PH therapy.

Evaluating the Effects of Different Concentrations of Human Follicular Fluid on Growth, Development, and PCNA Gene Expression of Mouse Ovarian Follicles

Follicle culture in vitro provides a method for investigating stages of folliculogenesis that can lead to preserving fertility through cryopreservation techniques. This study aims to assess the effects of various concentrations of human follicular fluid (hFF) on growth, development, and expression of the proliferating cell nuclear antigen (PCNA) gene in mouse ovarian follicles in vitro. Preantral follicles were isolated from 14-day NMRI mouse ovaries. The follicles were cultured in basic media enriched with FBS, FSH, and insulin-transferrin-selenium, and supplemented with different concentrations of hFF (10, 20, and 30%) for 12 days. During the culture period, survival rate and follicular maturation, follicular diameter, levels of estrogen and progesterone secretion, and PCNA gene expression rate were evaluated. Survival rate, maturation, and antrum formation were significantly higher in the 10% hFF group than in the 20 and 30% hFF groups. On day 4, follicle diameter in the 10% hFF group was also higher than in the 20 and the 30% hFF group. In comparison with other groups, significantly higher estrogen and progesterone production levels were measured in the 10% hFF group. PCNA gene expression was also higher with 10 than 20 and 30% hFF concentrations. The present study suggests that addition of 10% hFF to mice ovarian preantral follicle culture media enhances follicle growth and oocyte maturation.

Effects of probiotics on apoptosis and proliferation of intestinal epithelial cells in rats with necrotizing enterocolitis

Objective To study the effect of compound Lactobacillus acidophilus on the apoptosis of intestinal epithelial cells and expression of proliferating cell nuclear antigen (PCNA) in rats with necrotizing enterocolitis, so as to provide some theoretical reference for the application of compound Lactobacillus acidophilus in the prevention and treatment of necrotizing enterocolitis(NEC) for newborn infants. Methods Thirty-six 48-hour old newborn SD rats were randomly divided into normal group, model group and intervention group, with 12 rats in each group.The NEC model was established by hypoxia and cold stimulation for 3 days, and the intervention group was given the compound Lactobacillus acidophilus at the same time; after the model was established, all the rats were sacrificed after 12 hours of fasting and their intestinal tissues were excised by terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL) method to determine the condition of apoptosis and to detect expression of the PCNA gene in the intestinal tissues by fluorescent quantitative PCR. Results (1) The apoptosis rates of the intestinal tissues: in the model group, the normal group and the intervention group were (88.33±2.77)%, (4.75±0.75)% and (44.41±4.81)%, respectively, and the differences had statistical significance(all P<0.05). (2) The PCNA expression in the intestinal tissue by real-time PCR in the model group, normal group and intervention group were (9.28±3.26)×10-4, (15.35±1.91)×10-4 and (12.09±3.06)×10-4, respectively, and the differences had statistical significance(all P<0.05). Conclusions The apoptosis rate of intestinal epithelial cells increases and PCNA expression decreases when NEC occurs.Compound Lactobacillus acidophilus can reduce apoptosis, promote intestinal epithelial cell proliferation, and thus help to reduce the risk and severity of NEC in neonatal rats. Key words: Neonatal necrotizing enterocolitis; Apoptosis; Proliferating cell nuclear antigen; Compound Lactobacillus acidophilus

Cloning and comparative studies of proliferating cell nuclear antigen (PCNA) genes for nine dinoflagellates

Proliferating cell nuclear antigen (PCNA), a co-factor of DNA polymerases δ and e, is associated with active cell proliferation, particularly with the S phase of the eukaryotic cell cycle, and the expression of this gene has been therefore proposed as a molecular marker for estimating phytoplankton growth rate and even population dynamics. In this study, the full-length cDNA sequences of PCNA for nine dinoflagellate species (ten strains) were obtained, analyzed, and further characterized with the expression in response to alterations of growth stage and life cycle. All the ten genes have a 777 or 780 bp ORF and encode a protein of 258 or 259 amino acids, similar to PCNAs from vertebrates and plants. The deduced amino acid sequences include conservative motif characteristic to the PCNA gene family, implying conserved functions of PCNA among organisms of different taxa. Alignment analysis in combination with other sequences available in NCBI database exhibited high amino acid similarities (79.2–96.9%) among PCNAs from 17 dinoflagellate species. Phylogenetic tree derived from 37 PCNA sequences of dinoflagellates was consistent with the taxonomic affiliation of these taxa. Then, we used the cosmopolitan, toxic, and resting cyst-producing dinoflagellate Scrippsiella trochoidea as a representative of HAB-forming dinoflagellates to investigate PCNA (StPCNA) expression at different stages of growth and life cycle and the results of real-time quantitative PCR revealed clearly a stage-dependent pattern in the transcription of StPCNA. The transcripts dramatically reduced from an exponential to a stationary growth stage and then further significantly decreased from a stationary stage to dormant stage (resting cysts), a pattern in accordance with that previously reported from the dinoflagellates Alexandrium catenella and Prorocentrum donghaiense. In terms of its minimal implication, this work enhances the previous perception about the function and possible application of PCNA gene, and more importantly, our results indicated that PCNA is not silent during the dormant stage of dinoflagellates.

Relationship between microRNA-95 and proliferation, apoptosis of gastric cancer cells and underlying mechanism

Objective To investigate the role of microRNA (miRNA, miR)-95 in the proliferation and apoptosis of gastric cancer cells and the possible mechanism. Methods Real-time quantitative polymerase chain reaction (Real-time PCR) was used to detect the expression of miR-95 in tumor tissues and gastric mucosal tissues from 70 patients with gastric cancer. The patient’s clinicopathological features were recorded and the relationship between miR-95 and these clinicopathologic features was analyzed. The human gastric cancer cell line BGC823 was transfected with anti-miR-95 and the cell viability, cell cycle and apoptosis rate were examined before and after transfection. Simultaneously, Real-time PCR and Western blotting were used to detect the expression of proliferation- and apoptosis-related genes proliferating cell nuclear antigen (PCNA), Cyclin D1, p21, Survivin, Livin and Bad. Results The miR-95 level in gastric cancer was significantly higher than that in gastric mucosal tissues (t=-3.399, P<0.05). Its expression was related to the degree of tumor differentiation and lymph node metastasis. The level of miR-95 in poorly differentiated tumors and in tumors with lymph node metastasis was significantly higher than that in well-differentiated tumors and without lymph node metastasis (t=-3.021, P<0.05; t=3.348, P<0.05). In gastric cancer cell lines AGS, SGC7901, MKN45 and BGC823, and gastric epithelial cell line GES-1, the miR-95 expression was higher in gastric cancer cell lines than GES-1, and highest in BGC823 cells (F=5.998, P<0.05). After transfection of anti-miR-95, the activity of the cells significantly decreased, and the ratio of G0/G1 phase increased (F=39.758, P<0.01), but the ratio of G2/M (F=10.349, P<0.05) and S phases decreased (F=15.118, P<0.01); apoptosis rate increased significantly after anti-miR-95 transfection (F=25.182, P<0.01). The mRNA and protein expression levels of PCNA, Cyclin D1 and Survivin significantly decreased, and those of p21 and Bad increased (P<0.05). Conclusion The up-regulated expression of miR-95 in gastric cancer is associated with tumor differentiation and lymph node metastasis, which may be involved in the progression of gastric cancer by regulating the expression of some proliferative and apoptotic genes. Key words: Gastric cancer; MicroRNA-95; Proliferation; Apoptosis; Clinicopathological features

Effect of targeted silencing Notch1 on proliferation and apoptosis of human non-small cell lung cancer stem cells

Objective To investigate the effect of targeted silencing Notch1 on proliferation and apoptosis of human non-small cell lung cancer stem cells. Methods Lung cancer A549 cells and SPC-A-1 cells were selected and divided into control group, Nc-shRNA group and Notch1-shRNA group. The Nc-shRNA group was a negative control RNAi lentivirus group, and the Notch1-shRNA group was a Notch1 inhibitory RNAi lentivirus group. The lentiviral-mediated shRNA interference technology was used to target the silencing of Notch1. The silencing effect of Notch1 gene was verified by quantitative real time polymerase chain reaction (qRT-PCR) and Western blotting. Cell proliferation was detected by methyl thiazolyl tetrazolium (MTT) and sarcosphere formation assay. Apoptosis was detected by Annexin V/7-AAD double staining. Western blotting was used to detect the expression of proliferating cell nuclear antigen (PCNA), B-cell lymphoma-2 (Bcl-2) and Notch1 downstream gene Hes-1. Results The results of qRT-PCR showed that the relative expression levels of Notch1 in control group, Nc-shRNA group and Notch1-shRNA group in A549 cells and SPC-A-1 cells were 1.000±0.000, 0.937±0.025, 0.490±0.036 and 1.000±0.000, 1.077±0.070, 0.373±0.038, with statistically significant differences (F=359.707, P<0.001; F=210.455, P<0.001), further paired comparison, the relative expression of Notch1 in Notch1-shRNA group was significantly lower than that in Nc-shRNA group (all P<0.05). Western blotting showed that the expressions of Notch1 protein in A549 cells and SPC-A-1 cells were consistent with the mRNA results. MTT assay showed that the 24 h A values of A549 cells in control group, Nc-shRNA group and Notch1-shRNA group were 0.209±0.005, 0.219±0.009, 0.159±0.006, 48 h A values were 0.293±0.004, 0.302±0.004, 0.205±0.005, 72 h A values were 0.450±0.003, 0.430±0.012, 0.348±0.017, with statistically significant differences (F=79.487, P<0.001; F=508.664, P<0.001; F=57.156, P<0.001), further paired comparison, the proliferation ability of Notch1-shRNA group was significantly lower than that of Nc-shRNA group at 24, 48, 72 h (all P<0.05). The 48 h A values of SPC-A-1 cells in control group, Nc-shRNA group and Notch1-shRNA group were 0.438±0.022, 0.412±0.015, 0.364±0.010, 72 h A values were 0.540±0.016, 0.519±0.009, 0.438±0.019, with statistically significant differences (F=15.667, P=0.004; F=37.299, P<0.001), further paired comparison, the proliferation ability of Notch1-shRNA group was significantly lower than that of Nc-shRNA group at 48 h and 72 h (all P<0.05). The sphere sizes of control group, Nc-shRNA group and Notch1-shRNA group in A549 cells were (149.667±6.506)μm, (136.667±7.095)μm, (86.676±7.638)μm, with statistically significant difference (F=65.940, P<0.001). The sphere sizes of the three groups in SPC-A-1 cells were (118.667±6.658)μm, (128.000±7.000)μm, (60.675±4.509)μm, with statistically significant difference (F=105.372, P<0.001). Further paired comparison, the sphere size of Notch1-shRNA group was significantly smaller than that of Nc-shRNA group in the two kinds of cells (all P<0.05). The apoptosis rates of control group, Nc-shRNA group and Notch1-shRNA group in A549 cells and SPC-A-1 cells were (0.489±0.014)%, (0.633±0.021)%, (1.683±0.221)% and (1.323±0.194)%, (1.690±0.188)%, (3.017±0.356)%, with statistically significant differences (F=77.660, P<0.001; F=32.200, P=0.001), further paired comparison, the apoptosis rate of Notch1-shRNA group was significantly higher than that of Nc-shRNA group in the two kinds of cells (all P<0.05). Western blotting showed that the expressions of PCNA, Bcl-2 and Hes-1 in control group, Nc-shRNA group and Notch1-shRNA group in A549 cells were statistically significant (F=155.343, P<0.001; F=22.576, P=0.002; F=70.108, P<0.001), and the expressions of PCNA, Bcl-2 and Hes-1 in the three groups in SPC-A-1 cells were statistically significant (F=49.419, P<0.001; F=28.090, P=0.001; F=12.040, P=0.007). Further paired comparison, the expressions of PCNA, Bcl-2 and Hes-1 in Notch1-shRNA group were significantly lower than those in Nc-shRNA group in the two kinds of cells, and the differences were statistically significant (all P<0.05). Conclusion Targeted silencing of Notch1 can reduce the proliferation activity of lung cancer stem cells and promote apoptosis, which may be related to the down-regulation of its downstream gene Hes-1. Key words: Receptor, Notch1; Neoplastic stem cells; Carcinoma, non-small-cell lung; Cell proliferation; Apoptosis

FAL1 regulates endothelial cell proliferation in diabetic arteriosclerosis through PTEN/AKT pathway.

This study aims to investigate the role of FAL1 in the occurrence and progression of diabetic arteriosclerosis and its underlying mechanism. FAL1 expression in coronary artery disease (CAD) tissues, normal artery tissues, and tumor necrosis factor-α (TNF-α)-induced endothelial cells was detected by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). The regulatory effects of FAL1 on cell proliferation, migration, and cell cycle were examined by cell counting kit-8 (CCK-8) assay, transwell assay, and flow cytometry, respectively. Western blot was used to detect protein expressions of proliferation-related gene PCNA (proliferating cell nuclear antigen), cell cycle-related genes cyclin D1, PTEN (phosphatase and tensin homolog deleted on chromosome ten) and AKT (protein kinase B) in HUVECs. Subsequently, rescue experiments were performed to assess whether PTEN/AKT signaling pathway is activated during the process of FAL1-regulated proliferation and migration of HUVECs. FAL1 was highly expressed in CAD tissues and TNF-α-induced endothelial cells compared with that of controls. Overexpression of FAL1 in HUVECs promoted cell cycle, proliferation, and migration. FAL1 activated PTEN/AKT pathway in HUVECs, which was partially reversed by PTEN overexpression. Highly expressed FAL1 can promote proliferation and migration of endothelial cells through activating PTEN/AKT signaling pathway.

Molecular cloning and characterization of proliferating cell nuclear antigen; its potential involvement in germ cell development of stony corals

To better understand the molecular mechanisms underlying germ cell development in stony corals, this study focused on proliferating cell nuclear antigen (PCNA), which is an auxiliary protein of DNA polymerases and is principally responsible for DNA replication and repair. We performed the molecular identification and characterization of the complementary DNA for the pcna gene in the stony coral Euphyllia ancora. Quantitative reverse transcription-polymerase chain reaction analysis and Western blotting with anti-E. ancora PCNA antibody demonstrated that E. ancora PCNA gene products (transcripts and proteins) were present in all polyp tissues, including the testis and ovaries. Immunohistochemical analysis of spermatogenesis showed that intensive immunoreactivity of PCNA (irPCNA) was detected in the nuclei of spermatogonia. The irPCNA was also weakly detected in spermatocytes, and became almost undetectable in the spermatids and sperm. In contrast, in female germline cells, the irPCNA was detected in the nuclei of oogonia and all stage of oocytes. These results suggest that PCNA plays an important role in both the mitotic and meiotic processes of the coral germ cells. To the best of our knowledge, this study is the first to show the expression profile of the PCNA protein and its subcellular localization in coral germ-cell development.

Platelet-rich plasma induces post-natal maturation of immature articular cartilage and correlates with LOXL1 activation

Platelet-rich plasma (PRP) is used to stimulate the repair of acute and chronic cartilage damage even though there is no definitive evidence of how this is achieved. Chondrocytes in injured and diseased situations frequently re-express phenotypic biomarkers of immature cartilage so tissue maturation is a potential pathway for restoration of normal structure and function. We used an in vitro model of growth factor-induced maturation to perform a comparative study in order to determine whether PRP can also induce this specific form of remodeling that is characterised by increased cellular proliferation and tissue stiffness. Gene expression patterns specific for maturation were mimicked in PRP treated cartilage, with chondromodulin, collagen types II/X downregulated, deiodinase II and netrin-1 upregulated. PRP increased cartilage surface cell density 1.5-fold (P < 0.05), confirmed by bromodeoxyuridine incorporation and proportionate increases in proliferating cell nuclear antigen gene expression. Atomic force microscopy analysis of PRP and growth factor treated cartilage gave a 5-fold increase in stiffness correlating with a 10-fold upregulation of lysyl oxidase like-1 gene expression (P < 0.001). These data show PRP induces key aspects of post-natal maturation in immature cartilage and provides the basis to evaluate a new biological rationale for its activity when used clinically to initiate joint repair.

Age-associated prognostic and predictive biomarkers in patients with breast cancer.

To date, no comprehensive prognostic or predictive marker profiling analysis has been performed in association with the age of patients with breast cancer. In the present study, 632 breast cancer tissue samples were analyzed for expression of estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2), B-cell lymphoma (Bcl)-2 protein, HER2 gene amplification, proliferation [as evaluated by proliferating cell nuclear antigen (PCNA) and Ki-67 index], tumor grade, histological type and molecular subtype. The data revealed correlations with the age of patients. A statistically significant positive correlation was identified between patient age and expression of ER (P<0.0001). There was no significant association between patient age and PR, HER2 protein expression, HER2 gene amplification or PCNA. A significant negative correlation between age and Ki-67 expression (P<0.0001) as well as grade of tumor (P=0.007) was identified. The spectrum of molecular subtypes differed according to age (P=0.0003). The highest incidence of aggressive triple-negative and HER2-positive breast cancer was present in patients aged between 20 and 39 years. Luminal A subtype was the most frequent cancer subtype in patients from age 40 onwards, where proliferation activity declined with age and expression of hormone receptors increased along with Bcl-2 expression. Aggressive forms of breast cancer were more common in younger patients. Prognostic and predictive markers have a complex age-specific distribution. The findings of less aggressive luminal A and B subtypes in older patients, and the positive correlation with ER, PR and Bcl-2 expression reveal the potential efficacy of Bcl-2 as a marker of hormone responsiveness in these patients.