PCNA Levels Research Articles

Long Noncoding RNA JAKMIP2-AS1 Promotes the Growth of Colorectal Cancer and Indicates Poor Prognosis.

BackgroundThe identification of cancer-associated long noncoding RNAs and the investigation of their molecular and biological functions are important for understanding the molecular biology and progression of cancer. JAKMIP2-AS1 has not been reported in the literature, especially in the context of colorectal cancer. The aim of the present study was to examine the expression pattern of JAKMIP2-AS1 in colorectal cancer (CRC) and evaluate its biological role and clinical significance in tumor progression.MethodsJAKMIP2-AS1 expression was analyzed in 56 CRC tissues and nine CRC cell lines by quantitative reverse-transcription polymerase chain reaction (qRT-PCR). Overexpression and RNA interference (RNAi) approaches were used to investigate the biological functions of JAKMIP2-AS1. The effect of JAKMIP2-AS1 on proliferation was evaluated by CCK-8, colony formation, and EdU assays. Subcutaneous injection of cells was used to study proliferation in BALB/c nude male mice. Proliferation-related protein levels were examined by immunohistochemical analysis. Differences between groups were tested for significance using Student’s t-test (two-tailed).ResultsJAKMIP2-AS1 was highly expressed in both CRC samples and cell lines compared with the corresponding normal counterparts. The upregulation of JAKMIP2-AS1 expression promoted the proliferation of colorectal cancer cells. Moreover, patients with high levels of JAKMIP2-AS1 expression had a relatively poor prognosis. Inhibition of JAKMIP2-AS1 by RNAi decreased the proliferation of CRC cells in vitro and impeded cell growth in vivo. Ki-67 and PCNA levels were affected by JAKMIP2-AS1 knockdown or overexpression in vivo.ConclusionOur findings indicate that JAKMIP2-AS1 is significantly upregulated in CRC tissues and regulates CRC cell proliferation. Thus, JAKMIP2-AS1 may represent a new marker of poor prognosis and is a potential therapeutic target for CRC intervention.

Potential Mechanisms of miR-143/Krupple Like Factor 5 Axis in Impeding the Proliferation of Michigan Cancer Foundation-7 Breast Cancer Cell Line

Breast cancer is one of the most prevailing cancers in females, while the cancerous heterogeneity hinders its early diagnosis and subsequent therapy. miR-143-3p is a critical mediator in malignancy development and tumorigenesis as a tumor suppressor. Its role in various tumor entities has been investigated, such as colon cancer and breast cancer. Using MCF-7 breast cancer cell model, we planned to explore the underlying mechanisms of miR-143/KLF-5 axis in retarding breast cancer cells growth. Bioinformatics analysis searched the target KLF5 of miR-143, and the miR-143-targeted mimic and inhibitor were employed to detect the changes of KLF5. After transfection of mimic miR-143, the CCK-8 reagent assessed cell proliferation. Based on optimal stimulation time, miR-143 stimulation model was established, followed by determining expression of KLF5, EGFR and PCNA via western blot and qPCR. Eventually, siRNA-KLF5 was applied to silencing KLF5 level to evaluate its role in MCF-7 cells. The transcription and translation levels of KLF5 were diminished in miR-143-mimic transfected MCF-7 cells, while enhanced in miR-143-inhibitor transfected MCF-7 cells. When MCF-7 cells were transfected with miR-143-mimic at different time points, 48 hours was found to be the optimal transfection time, with reduced transcription and translation levels of KLF5, EGFR and PCNA. The transcription and translation levels of PNCA and EGFR were declined after silencing KLF5 by siRNA. miR-143/KLF5 axis could retard the proliferation of MCF-7 breast cancer cells.

The effect of copper supplementation on in vitro maturation of porcine cumulus-oocyte complexes and subsequent developmental competence after parthenogenetic activation

Copper (Cu) ions have redox activity and act as cofactors of enzymes related to respiration, radical detoxification, and iron metabolism. In this study, we aimed to examine the effects of copper (II) chloride dihydrate (CuCl2·2H2O) on porcine oocytes during in vitro maturation (IVM) and subsequent embryonic development following parthenogenetic activation (PA). Nuclear maturation, intracellular glutathione (GSH) and reactive oxygen species (ROS) levels, cumulus expansion, the mRNA expression levels of various genes, and developmental competence were analyzed. During IVM, the maturation medium was supplemented with various concentrations of Cu (0, 0.7, 1.4, and 2.8 μg/mL). After 42 h of IVM, Cu supplementation significantly increased the number of oocytes in the metaphase II stage. Further, the 1.4 μg/mL Cu group showed significantly higher intracellular GSH levels than the control group. However, Cu supplementation increased intracellular ROS levels regardless of their concentration. Additionally, the mRNA levels of Has-2, the cumulus cell expansion-related gene, were higher in all the Cu-treated groups than in the control group. The cumulus cell expansion index was higher in the 0.7 and 1.4 μg/mL Cu groups than in the other groups. In the 0.7 μg/mL Cu group, the mRNA expression levels of PCNA, Zar1, and NPM2, which are related to developmental competence, were significantly higher than those in the control group. Moreover, increased levels of Sod1 transcript, correlated with the antioxidative response, were observed in the 0.7 and 1.4 μg/mL Cu groups. The apoptosis rate in Cu-treated cumulus cells and oocytes was decreased compared to that in the corresponding control groups. Upon evaluation of subsequent embryonic development after PA, the 0.7 μg/mL Cu group showed significantly improved cleavage and blastocyst formation rate compared to the control group. In conclusion, our results suggest that Cu supplementation at appropriate concentrations in IVM medium improves porcine oocyte maturation and the subsequent embryonic potential of PA embryos by reducing oxidative stress and apoptosis.

Osthole Alleviates Neointimal Hyperplasia in Balloon-Induced Arterial Wall Injury by Suppressing Vascular Smooth Muscle Cell Proliferation and Downregulating Cyclin D1/CDK4 and Cyclin E1/CDK2 Expression.

Percutaneous coronary intervention (PCI) is the most widely used therapy for treating ischemic heart disease. However, intimal hyperplasia and restenosis usually occur within months after angioplasty. Modern pharmacological researchers have proven that osthole, the major active coumarin of Cnidium monnieri (L.) Cusson, exerts potent antiproliferative effects in lung cancer cells, the human laryngeal cancer cell line RK33 and TE671 medulloblastoma cells, and its mechanism of action is related to cell cycle arrest. The goal of the present study was to observe the effect of osthole on vascular smooth muscle cell (VSMC) proliferation using platelet-derived growth factor-BB (PDGF-BB)-stimulated VSMCs isolated from rats and vascular balloon injury as models to further elucidate the molecular mechanisms underlying this activity. We detected the relative number of VSMCs by the MTT assay and EdU staining and examined cell cycle progression by flow cytometry. To more deeply probe the mechanisms, the protein expression levels of PCNA, the cyclin D1/CDK4 complex and the cyclin E1/CDK2 complex in balloon-treated rat carotid arteries and the mRNA and protein expression levels of the cyclin D1/CDK4 and cyclin E1/CDK2 complexes in VSMCs were detected by real-time RT-PCR and western blotting. The data showed that osthole significantly inhibited the proliferation of VSMCs induced by PDGF-BB. Furthermore, osthole caused apparent VSMC cycle arrest early in G0/G1 phase and decreased the expression of cyclin D1/CDK4 and cyclin E1/CDK2. Our results demonstrate that osthole can significantly inhibit PDGF-BB-induced VSMC proliferation and that its regulatory effects on cell cycle progression and proliferation may be related to the downregulation of cyclin D1/CDK4 and cyclin E1/CDK2 expression as well as the prevention of cell cycle progression from G0/G1 phase to S phase. The abovementioned mechanism may be responsible for the alleviation of neointimal hyperplasia in balloon-induced arterial wall injury by osthole.

Molecular Pathological Markers Correlated With the Recurrence Patterns of Glioma.

PurposeGlioma is one of the most common tumors of the central nervous system, and many patients suffer from recurrence even after standard comprehensive treatment. However, little is known about the molecular markers that predict the recurrence patterns of glioma. This study aimed to demonstrate the correlations between molecular markers and glioma recurrence patterns, which included local/nonlocal recurrence and paraventricular/nonparaventricular recurrence.MethodsImmunohistochemical techniques were used to assess the molecular markers of 88 glioma tissues following surgical resection. The recurrence patterns were divided into local recurrence, marginal recurrence, distant recurrence, multirecurrence, and subarachniod recurrence, with the last four recurrence patterns being collectively called nonlocal recurrence. According to whether the recurrence invaded ventricles, the nonlocal recurrence patterns were divided into paraventricular and nonparaventricular recurrence. Then, we compared the different recurrence patterns and their clinical characteristics, focusing on the expression of molecular markers.ResultsMore patients in the nonlocal recurrence group received combined radiotherapy and chemotherapy than patients in the local recurrence group (p=0.019). Sex, age, extent of surgery, time to recurrence, tumor location, size, and WHO grade were not different in the defined groups (P>0.05). Recurrent tumor volume and WHO grade were significantly different between the paraventricular and nonparaventricular recurrence groups (p=0.046 and 0.033). The expression of Ki-67, P53, and PCNA in the nonlocal recurrence group was significantly higher than that in the local recurrence group (p=0.015, 0.009, and 0.037), while the expression of S-100 in the nonlocal recurrence group was significantly lower than that in the local recurrence group (p=0.015). Cox regression indicated hazard ratio (HR) for high expression level of PCNA associated with non-local recurrence was 3.43 (95% CI, 1.15, 10.24), and HR for high expression level of MGMT associated with paraventricular recurrence was 2.64 (95% CI, 1.15,6.08).ConclusionsKi-67, P53, PCNA, and MGMT might be important clinical markers for nonlocal recurrence and paraventricular recurrence.

MicroRNA-1246 regulates proliferation, invasion, and differentiation in human vascular smooth muscle cells by targeting cystic fibrosis transmembrane conductance regulator (CFTR).

MicroRNA (miRNA) plays a key role in the proliferation and invasion of vascular smooth muscle cells (VSMCs). However, the role and underlying mechanism of miRNAs in VSMCs are not fully understood. Therefore, this study was designed to investigate the role and mechanism of microRNA-1246 (miR-1246) in VSMCs. VSMCs were cultured, and the proliferation of VSMCs was stimulated by platelet-derived growth factor (PDGF-BB) or 15% fetal bovine serum (FBS). The quantitative reverse transcription PCR (qRT-PCR) was used to detect the expression levels of miR-1246 and cystic fibrosis transmembrane conductance regulator (CFTR) in VSMCs. The CCK-8 assay and transwell assay were used to detect the proliferation and invasion of VSMCs. Target gene prediction and screening and luciferase reporter assays were used to verify downstream target genes of miR-1246. Western blotting was used to detect the protein expression levels of PCNA, α-SMA, SM-MHC, Collagen-1, and Cyclin D1 in VSMCs. PDGF-BB and FBS treatment induced VSMCs proliferation and the upregulation of miR-1246 expression. Overexpression of miR-1246 promoted VSMCs proliferation, invasion, and differentiation towards synthetic phenotype, while knockdown of miR-1246 had opposite effects. In addition, CFTR was found to be a direct target for miR-1246, and miR-1246 inhibited the expression of CFTR. Moreover, overexpression of CFTR inhibited VSMC proliferation and synthetic differentiation, while overexpression of miR-1246 partly abolished the effects of CFTR overexpression on VSMCs proliferation and differentiation. Our data suggest that MiR-1246 promotes VSMC proliferation, invasion, and differentiation to synthetic phenotype by regulating CFTR. MiR-1246 may be a potential therapeutic target for atherosclerosis.

Antitumor activity of flavonoids from Alpinia officinarum hance on gastric cancer

Introduction: To investigate antitumor activity and mechanism of flavonoids from Alpinia officinarum Hance against gastric cancer. Methods: Transplanted mouse fore-stomach carcinoma (MFC) tumor mice were divided into six groups: control group, model group, low dose (20 mg/kg), middle dose (40 mg/kg), and high dose (80 mg/kg) groups of TFAO and 5-Fu group. Mice were treated with TFAO or 5-Fu for 14 days, except those of control and model group. Tumor inhibitory rate, spleen, and thymus index were calculated. Contents of proliferating cell nuclear antigen, MMP-9, vascular endothelial growth factor, IL-1β, IL-6, and IL-17 in serum were detected. Effect of galangin on BGC-823 cell growth was detected. Cell apoptosis and cell cycle distribution were measured. Enzyme activity of Caspase-3, Caspase-8, and Caspase-9 was detected. Western blot was used to detect STAT3, Bcl-2, Bax, Caspase-3, Caspase-8, Caspase-9, CyclinB1, and CyclinD1 protein expression in BGC-823 cell. Results: Compared with model group, tumor weight of mice decreased significantly ( p < .01) in 5-Fu group, low dose, middle dose, and high dose group of TFAO; thymus index of mice decreased significantly ( p < .05) in 5-Fu group; and spleen index decreased significantly ( p < .05) in low dose and middle dose groups of TFAO. Compared with model group, levels of PCNA, MMP-9, IL-1β, and IL-6 in serum of mice decreased obviously ( p < .01) in all administration groups; levels of VEGF in serum of mice decreased obviously ( p < .01) in low dose and high dose group of TFAO and 5-Fu group; and levels of IL-17 in serum of mice decreased significantly ( p < .01) in low-dose and middle-dose groups of TFAO and 5-Fu group. Galangin could inhibit BGC-823 cell growth; accelerate apoptosis; block cell cycle; increase cell Caspase-3, Caspase-8, and Caspase-9 enzyme activity; upregulate expression of Caspase-3, Caspase-8, Caspase-9, and Bax; and downregulate expression of STAT3, CyclinB1, CyclinD1, and Bcl-2 protein. Conclusion: Flavonoids from A. officinarum showed antitumor activity in gastric cancer. Mechanisms may be associated with inhibition of tumor angiogenesis, tumor cell proliferation, and cancer-associated inflammation.

Upregulation of miR-137 Expression Suppresses Tumor Growth and Progression via Interacting with DNMT3a Through Inhibiting the PTEN/Akt Signaling in HCC.

BackgroundDownregulation of miR-137 regulates tumor growth in hepatocellular carcinoma (HCC). Yet, the underlying molecular mechanisms stay unclear.Materials and MethodsmiR-137 and DNA methyltransferase 3a (DNMT3a) expression levels were detected by Western blot, immunohistochemistry and qRT-PCR assays. Luciferase reporter and Western blot assays were also carried out to explore the correlation of miR-137 and DNMT3a. Flow cytometry assay, MTT analysis, transwell and wound healing assay were used to evaluate cell apoptosis, proliferation, as well as invasive and migratory abilities. Western blot was used to examine the caspase-3, cleaved caspase-3, PCNA, MMP-2, and MMP-7 protein levels, as well as PTEN/Akt signaling alternations. Methylation-specific PCR was applied to detect the PTEN promoter methylation status. Xenograft tumor assay, Western blot and immunohistochemistry analyses were taken to confirm the miR-137 regulation in vivo.ResultsDownregulation of miR-137, upregulation of DNMT3a, as well as an inverse correlation between them were observed in HCC clinical samples and cells. Moreover, miR-137 targeted directly and inhibited DNMT3a in HCC cells, which further retarded cell proliferative, migratory and invasive capabilities, while promoted apoptotic ones. Additionally, miR-137 overexpression inactivated the PTEN/Akt pathway in HCC cell by decreasing DNMT3a expression. Furthermore, miR-137 overexpression inhibited tumor growth in vivo in HCC via interacting with DNMT3a through inhibiting the PTEN/Akt cascades.ConclusionOur findings suggested that miR-137 inhibited HCC tumor growth and progression via interacting with DNMT3a and suppressing the PTEN/Akt signaling in vitro and in vivo.

Crocin treatment improves testosterone induced benign prostatic hyperplasia in rats

Background and objective: Benign prostatic hyperplasia (BPH) is a typical nonmalignant growth of the prostate in the elderly. Crocin, a bioactive component of Crocus sativus L., commonly known as saffron, is known to have an anti-proliferative activity against numerous types of cancer, including prostate cancer. This study investigated the effects of crocin on testosterone-induced BPH development in rats. Materials and methods: The study sample included three groups of adult male rats (3 months old, weighed 250 g): the control group received corn oil only, the second and the third groups were injected with testosterone (3 mg/kg dissolved in corn oil) subcutaneously. The second group was considered as testosterone-induced BPH (untreated) while the third groups were assigned as testosterone-induced BPH-crocin treated group (100 mg/kg orally for 14 days). Results: After animal sacrifice, histopathological analysis of the prostate tissues was performed in parallel with gene expression of proliferation (PCNA), inflammation (IL-6), and vascularization (VEGF-A) markers, analyzed by qRT-PCR. Crocin treatment significantly reduced prostate index and the thickness of the epithelial layer in rats with BPH. Additionally, the mRNA expression levels of PCNA, a marker of cell proliferation; IL-6, an inflammatory cytokine; and VEGF-A, an angiogenesis marker, were significantly down-regulated in the BPH group that were treated with crocin. Conclusions: The present study indicates that crocin can effectively prevent the development of experimentally induced BPH through inhibition of prostatic cellular proliferation, inflammation, and angiogenesis.

Confirmation of differentiation clusters’ and endoglin markers preset in porcine buccal mucosa cells

Abstract Several genes, namely CD44, CD90, CD105 and PCNA may be important in differentiation of porcine mucosa cell cultures. These genes are, inter alia, responsible for cell adhesion to extracellular matrix and its constituent secretion, cytoskeleton organization, epithelial to mesenchymal transition or proper course of DNA replication. A total of 20 pubertal crossbred Landrace gilts bred on commercial farms were used to produce buccal mucosa cultures, which were harvested on the 7th, 15th and 30th day after initiation of the culture. Expression levels of CD44, CD90, CD105 and PCNA were evaluated employing Real-Time Quantitative Polymerase Chain Reaction. CD44, CD90 and PCNA showed an unchanged expression pattern. Expression of CD44 on day 7 was the highest of all factors measured. The greatest difference between the measurement on 7th and 30th day was found in the PCNA gene. These results broaden the understanding of the transcriptome changes in porcine buccal mucosa cells for the duration of in vitro cultivation. Nevertheless, it is very important to consider that the in vitro conditions do not fully reflect the changes taking place in the living organism. It appears that tissues of the oral cavity possess high regenerative potential, and constitute suitable model for wound healing investigation. Running title: Confirmation of differentiation clusters’ and endoglin markers preset in porcine buccal mucosa cells

Kinesin Superfamily Member 18B (KIF18B) Promotes Cell Proliferation in Colon Adenocarcinoma.

BackgroundThe role of kinesin superfamily proteins (KIFs) has been reported in a variety of tumors and KIFs contributed to the proliferation of cancer cells. But few studies were focus on colon adenocarcinoma.MethodsThrough bioinformatics analysis and immunohistochemistry (IHC) assays, the expression of KIF18B in colon adenocarcinoma tissues was determined. Stable KIF18B-depleted cell lines were constructed using lentivirus-mediated shRNA of KIF18B. Cell colony formation assay and CCK8 assay were performed to assess cell proliferation degree, and the expression level of KI67 and PCNA was used to indicate cell proliferation in vitro and verified using xenograft tumors in vivo.ResultsKIF18B is highly expressed in colon adenocarcinoma tissues and has a negative correlation with the prognosis and tumor grade of colon adenocarcinoma. Interfering with KIF18B inhibits cell proliferation in vitro and in vivo.ConclusionKIF18B can be used as a prognostic marker for colon adenocarcinoma and may be a therapeutic target for colon adenocarcinoma treatment.

Combination of canstatin and arsenic trioxide suppresses the development of hepatocellular carcinoma.

Complication of arsenic trioxide (ATO) and other drugs in cancer treatment has attracted much focus, but is limitedly investigated in hepatocellular carcinoma (HCC). This study aimed to explore the role of ATO combined with canstatin in HCC. HepG2 cells were treated with different concentrations of ATO with or without canstatin, CCK-8, flow cytometry, Transwell assays were conducted to determine cell proliferation, apoptosis, adhesion, migration, and invasion abilities. Besides, the protein expression or mRNA level of caspase-3, PCNA, and MMP-2 was measured using western blotting or qRT-PCR. BALB/c-nu/nu mice were used to establish nude mouse transplantation tumor model, and received ATO or canstatin treatment for 3 weeks. The results showed that ATO inhibited cell proliferation, adhesion, migration and invasion, and promoted cell apoptosis with a concentration-dependent way. Canstatin had a significantly inhibitory effect on cell proliferation, but had limited effects on the other cellular behaviors. Besides, combination with ATO and canstatin strengthened the effects of ATO alone on cell proliferation inhibition and cell apoptosis promotion. Moreover, both of ATO and canstatin increased the protein expression of caspase-3, while decreased PCNA and MMP-2, which was further strengthened upon their combination. Furthermore, both of ATO and canstatin inhibited tumor growth in vivo, which was also strengthened upon their combination. Collectively, we found that combined canstatin and ATO significantly inhibited cell proliferation, migration and adhesion abilities, and promoted cell apoptosis, and inhibited tumor growth, thus suppressed the progression of HCC.

Exosomes derived from idiopathic gingival fibroma fibroblasts regulate gingival fibroblast proliferation and apoptosis.

Exosomes have been proved to play an essential role in intercellular information transmission. However, few researches focused on exosomes derived from gingival fibroblasts (GFs) of IGF. The aim of this study is to investigate the effect of exosomes derived from GFs of IGF (IGF-GFs) on the proliferation and apoptosis of normal gingival fibroblasts (N-GFs). Gingival fibroblasts were cultured and identified using immunocytochemistry. Exosomes were isolated with exosomes extraction kit and characterized by transmission electron microscopy (TEM) and flow cytometry. PKH67 labeling was further used to trace the intracellular distribution of the exosomes. And MTS assay was used to test the effective concentration and time course of IGF-GFs-derived exosomes. Furthermore, the expression of PCNA, Ki67, Bcl-2, and Bax in N-GFs was analyzed by qRT-PCR and Western blot. Exosomes were isolated from IGF-GFs; the identification of exosomes and gingival fibroblasts was successfully finished. Moreover, we found that N-GFs co-cultured with exosomes showed a great increase in PCNA and Bcl-2 levels, and a moderate increase in Ki67 levels. By contrast, the levels of Bax were significantly reduced. These results suggest that exosomes derived from idiopathic gingival fibroma fibroblasts are involved in the regulation of gingival fibroblast proliferation and apoptosis.

Transmembrane protein ADAM29 facilitates cell proliferation, invasion and migration in clear cell renal cell carcinoma

Abnormal expression of ADAM29 has been frequently reported in several cancers, however, its role in clear cell renal cell carcinoma (ccRCC) has not evaluated in detail. Herein, we attempt to determine the biological role and the action mechanism of ADAM29 in ccRCC. Bioinformatics analysis based on the ccRCC RNA-Seq dataset from TCGA database revealed that ADAM29 was up-expressed in ccRCC tissues by comparison with normal tissues. And a significant increase of ADAM29 expression was also observed in 3 ccRCC cell lines (UT33A, Caki-1, and786-O) in comparison with normal cell line. Besides, high level of ADAM29 was found to be connected with the poor prognosis and could be considered as an independent prognosticator for patients with ccRCC. Furthermore, functional experiments in vitro demonstrated that ADAM29 promoted the growth, invasion and migration of ccRCC cells. Moreover, Western blot assays indicated that ADAM29 was positively correlated with the level of proliferation-related proteins Cyclin D1 and PCNA and motion-related proteins MMP9 and Snail. Our data indicate that ADAM29 acts as an oncogene that increases tumour cells proliferation, invasion and migration partly by regulating the expression of Cyclin D1/PCNA/MMP9/Snail, suggesting that ADAM29 may become a prognosticator and therapeutic candidate for ccRCC.

LncRNA AFAP1-AS1 modulates the sensitivity of paclitaxel-resistant prostate cancer cells to paclitaxel via miR-195-5p/FKBP1A axis

ABSTRACT LncRNA AFAP1-AS1 has been corroborated to function in diverse cancers. Our aim was to investigate the molecular mechanism of AFAP1-AS1 in PTX resistance in PCa. The levels of AFAP1-AS1, miR-195-5p, and FKBP1A were checked by qRT-PCR. 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-Diphenyltetrazolium Bromide (MTT) assay was employed to assess the resistance of PTX-resistant PCa cells to PTX. Flow cytometry was introduced to evaluate cell apoptosis. The protein levels of C-caspase 3 were determined by western blot. The starBase was used to predict the interaction between miR-195-5p and AFAP1-AS1. Xenograft tumor model was established to investigate the biological role of AFAP1-AS1 in PTX resistance in vivo. The levels of AFAP1-AS1 and FKBP1A were upregulated in PCa tissues and cells, as well as PTX-resistant PCa cells, while the expression of miR-195-5p was declined. Knockdown of AFAP1-AS1 promoted the sensitivity of PTX-resistant PCa cells to PTX, induced apoptosis of PTX-resistant PCa cells, whereas the impacts could be reversed by reducing the expression of miR-195-5p. FKBP1A overexpression could rescue the effects of miR-195-5p-mediated enhancement on the sensitivity of PTX-resistant PCa cells to PTX, promotion on apoptosis of PTX-resistant PCa cells. AFAP1-AS1 interacted with miR-195-5p and miR-195-5p could bind to the 3ʹUTR of FKBP1A. AFAP1-AS1 silencing inhibited the tumor growth in mice implanted with PC3-TXR cell. The protein level of PCNA was decreased in PC3-TXR cells transfected with sh-AFAP1-AS1, while the expression of C-caspase 3 was upregulated. AFAP1-AS1 silencing attenuated the resistance of PTX-resistant PCa cells to PTX by downregulating FKBP1A via sponging miR-195-5p.

CircB3GNTL1 and miR-598 regulation effects on proliferation, apoptosis, and glutaminolysis in gastric cancer cells

This study was performed to research circB3GNTL1 control, miR-598 and its mechanism for cell proliferation, apoptosis and glutamine breakdown. For this purpose, CirB3GNTL1 and miR-598 expressions were detected by qRT-PCR in gastric and cell lines; MTT tests were performed to detect proliferation; flow cytometry was established in flow; glutamine decomposition was evaluated with glutamine, glutamic acid and α-keto-glutaric acid (α-KG) expression; Bcl-2, PCNA, ASCT2 and GLS1 expression levels were calculated; Methods of expression were calculated. The results showed that CircB3GNTL1 expression was up-regulated and miR-598 expression was up-regulated in gastric cancer tissues and cell lines; Knockdown circB3GNTL1 prevented proliferation and glutamine decomposition of the gastric cancer cells and induced apoptosis compared with normal para-cancers and gastric cancer cell lines. Results circB3GNTL1 can target and control miR-598 expression, and miR-598 can reverse the proliferation, apoptosis, and decomposition of glutamine from gastric cancer cells by knockdown circB3GNTL1. It was concluded that CirB3GNTL1 prevents decomposition of glutamines and induces apoptosis by controlling miR-598 in gastric cancer cells.

The oxidative stress responses caused by phthalate acid esters increases mRNA abundance of base excision repair (BER) genes in vivo and in vitro

The base excision repair (BER) pathway is an important defense response to oxidative DNA damage. It is known that exposures to phthalate esters (PAEs), including Dibutyl phthalate (DBP), Mono-(2-ethylhexyl) phthalate (MEHP), and Di-(2-ethylhexyl) phthalate (DEHP), cause reactive oxygen species-induced DNA damage and oxidative stress. Here, we determined the mRNA levels of BER pathway-related genes (ogg1, nthl1, apex1, parp1, xrcc1, lig3, ung, pcna, polb, pold, fen1, and lig1), pro-apoptotic gene (bax), and apoptotic suppressor gene (bcl2) in different PAEs-exposed zebrafish larvae and HEK293T cells. Further investigations were performed to examine reactive oxygen species (ROS) accumulation, superoxide dismutase (SOD) activity, developmental toxicity, and cell viability after PAEs exposure in vivo and in vitro. The results showed that PAEs exposure can induce developmental abnormalities in zebrafish larvae, and inhibit cell viability in HEK293T cells. Additionally, we found that PAEs exposure results in the accumulation of ROS and the inhibition of SOD activation in vivo and in vitro. Notably, the mRNA levels of BER pathway-related genes (OGG1, NTHL1, APEX1, XRCC1, UNG, POLB, POLD, FEN1) were significantly upregulated after DBP or MEHP exposure, whereas the mRNA levels of NTHL1, UNG, POLB, POLD, and FEN1 were significantly altered in DEHP-treated HEK293T cells. In zebrafish, the mRNA levels of ogg1, pcna, fen1 and lig1 genes were increased after DBP or DEHP exposure, whereas the mRNA levels of nthl1, apex1, parp1, lig3, pcna and polb were decreased after MEHP exposure, respectively. Thus, our findings indicated that PAEs exposure can induce developmental toxicity, cytotoxicity, and oxidative stress, as well as activate BER pathway in vivo and in vitro, suggesting that BER pathway might play critical roles in PAEs-induced oxidative stress through repairing oxidative DNA damage.

Follicle inhibition at the primordial stage without increasing apoptosis, with a combination of everolimus, verapamil.

The aim of the present study was to test whether inhibition of ovarian primordial follicles and subsequent activation can be achieved by transient mTOR inhibition. In this preclinical investigation, forty-five female immature Wistar rats were randomized in 5 groups. The control group received subcutaneous saline injections. The other groups received Everolimus, Everolimus plus Verapamil, Everolimus plus Fisetin, and Fisetin alone. Primary and secondary outcomes were measured in the left ovary after a treatment period of 8weeks. Ten days later, animals received 35IU FSH for 4days and 35IU of hCG on the 5th day. The same parameters were examined in the right ovary. AMH, estradiol, and progesterone levels were assessed at the end of both interventions. Significantly, more primordial and less atretic follicles were observed in the Everolimus plus Verapamil group. AMH and progesterone levels were substantially lower in the Everolimus group. Interestingly, after ovarian stimulation higher levels of AMH and progesterone were observed in the Everolimus plus Verapamil group. Immunoblot analysis of ovarian extracts revealed that the administration of Everolimus led to a significant reduction in the mTORC1-mediated phosphorylation of the 70-kDa ribosomal protein S6 kinase 1. This decrease was reversed in the presence of FSH after stopping drug administration. The expression of the anti-apoptotic molecule Bcl2 as well as of LC3-II and ATG12 was increased after removal of the Everolimus plus Verapamil combination, indicating reduced apoptosis and increased autophagy, whereas the levels of the proliferation marker PCNA in the granulosa cells were elevated, consistent with initiation of follicular growth.Thus, the combination of Everolimus plus Verapamil is capable of increasing the number of competent primordial follicles while reducing atresia.

PCNA antagonizes cohesin-dependent roles in genomic stability.

PCNA sliding clamp binds factors through which histone deposition, chromatin remodeling, and DNA repair are coupled to DNA replication. PCNA also directly binds Eco1/Ctf7 acetyltransferase, which in turn activates cohesins and establishes cohesion between nascent sister chromatids. While increased recruitment thus explains the mechanism through which elevated levels of chromatin-bound PCNA rescue eco1 mutant cell growth, the mechanism through which PCNA instead worsens cohesin mutant cell growth remains unknown. Possibilities include that elevated levels of long-lived chromatin-bound PCNA reduce either cohesin deposition onto DNA or cohesin acetylation. Instead, our results reveal that PCNA increases the levels of both chromatin-bound cohesin and cohesin acetylation. Beyond sister chromatid cohesion, PCNA also plays a critical role in genomic stability such that high levels of chromatin-bound PCNA elevate genotoxic sensitivities and recombination rates. At a relatively modest increase of chromatin-bound PCNA, however, fork stability and progression appear normal in wildtype cells. Our results reveal that even a moderate increase of PCNA indeed sensitizes cohesin mutant cells to DNA damaging agents and in a process that involves the DNA damage response kinase Mec1(ATR), but not Tel1(ATM). These and other findings suggest that PCNA mis-regulation results in genome instabilities that normally are resolved by cohesin. Elevating levels of chromatin-bound PCNA may thus help target cohesinopathic cells linked that are linked to cancer.

Atorvastatin Reduces Accumulation of Vascular Smooth Muscle Cells to Inhibit Intimal Hyperplasia via p38 MAPK Pathway Inhibition in a Rat Model of Vein Graft.

ResumoFundamento:A taxa de falha de enxerto de veia safena um ano após a cirurgia de revascularização do miocárdio varia de 10% a 25%. O objetivo deste estudo foi de investigar se a atorvastatina pode reduzir o acúmulo de células musculares lisas vasculares para inibir a hiperplasia intimal por meio da inibição da via p38 MAPK.Métodos:Quarenta e cinco ratos Sprague-Dawley foram randomizados em três grupos. Trinta ratos foram submetidos à cirurgia de enxerto de veia e randomizados para tratamento com veículo ou atorvastatina; quinze ratos foram submetidos à cirurgia sham. Detectamos a hiperplasia intimal por meio de coloração com hematoxilina-eosina e a expressão de proteínas relacionadas por meio de análise imuno-histoquímica e Western blot. Foram realizadas as comparações por análise de variância de fator único e pelo teste da diferença mínima significativa de Fisher, com p < 0,05 considerado significativo.Resultados:A íntima analisada pela coloração com hematoxilina-eosina era dramaticamente mais espessa no grupo controle que no grupo atorvastatina e no grupo sham (p < 0,01). Os resultados da coloração imuno-histoquímica de α-SMA demonstraram que a porcentagem de células positivas para α-SMA no grupo controle era mais alta que no grupo atorvastatina (p < 0,01). Nós também avaliamos α-SMA, PCNA, p38 MAPK e fosforilação de p38 MAPK após o tratamento com estatina por meio de análise de Western blot e os resultados indicaram que a atorvastatina não levou à redução de p38 MAPK (p < 0,05); no entanto, resultou na inibição da fosforilação de p38 MAPK (p < 0,01) e reduziu significativamente os níveis de α-SMA e PCNA, em comparação com o grupo controle (p < 0,01).Conclusão:Nós demonstramos que a atorvastatina pode inibir o acúmulo de células musculares lisas vasculares por meio da inibição da via p38 MAPK e é capaz de inibir a hiperplasia intimal em modelos de enxerto de veia em ratos.