Proliferating Cell Nuclear Antigen mRNA Levels Research Articles

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.

In ovo betaine injection improves breast muscle growth in newly hatched goslings through FXR/IGF-2 pathway

Betaine has been shown to enhance growth performance and increase breast muscle yield in ducks and broilers through various mechanisms, including the modification of DNA methylation. However, the impact of in ovo betaine injection on muscle growth in newly hatched goslings remains unclear. In this study, fifty eggs were injected with saline or betaine at 7.5 mg/egg prior to incubation, and the subsequent effects on breast muscle growth in the newly hatched goslings were investigated. Betaine significantly increased (P < 0.05) the hatch weight, breast muscle weight, and breast muscle index, accompanied by an augmentation in muscle bundle cross-sectional area. Concurrently, betaine significantly upregulated (P < 0.05) the expression levels of myogenic regulatory factors, including myogenin (MyoG) and paired box 7 (Pax7) both mRNA and protein, while downregulating (P < 0.05) the mRNA and protein levels of myostatin (MSTN). Histological analysis revealed a higher abundance of proliferating cell nuclear antigen (PCNA) and Pax7 immune-positive cells in the breast muscle of the betaine group, consistent with elevated PCNA and Pax7 mRNA and protein levels. Additionally, significantly increased (P < 0.05) contents of insulin-like growth factor 1 (IGF-1) and insulin-like growth factor 2 (IGF-2) were observed in the breast muscle of the betaine group, so was mRNA expression of IGF-1, IGF-2, and insulin-like growth factor 1 receptor (IGF-1R). Betaine also significantly in8creased (P < 0.05) global DNA methylation of the breast muscle, accompanied by enhanced mRNA and protein levels of methionine cycle and DNA methylation-related enzymes, Interestingly, the promoter regions of IGF-1, IGF-2, and IGF-1R genes were significantly hypomethylated (P < 0.05). Moreover, in ovo betaine injection significantly upregulated (P < 0.05) the protein level of farnesoid X receptor (FXR) in breast muscle and FXR binding to the promoter of IGF-2 gene. These findings suggest that in ovo betaine injection promotes breast muscle growth during embryonic development in goslings through the FXR-mediated IGF-2 pathway, ultimately improving hatch weight and breast muscle weight.

Effect of Cryptorchidism on the Histomorphometry, Proliferation, Apoptosis, and Autophagy in Boar Testes.

Simple SummaryBody temperature has detrimental effects on sperm quality in mammalian species, including pigs. However, the molecular mechanism of this is not yet well understood. Cryptorchidism is when one or both testes fail to descend into the scrotum, which leads the testes to be exposed to the body temperature. The aims of present study were to investigate the effect of body temperature on the histomorphometry, apoptosis and the expression of the proliferation-associated protein PCNA and the autophagy-associated protein LC3 in spontaneous unilateral cryptorchid boar testes. Our findings showed that cryptorchidism had no evident influence on the number of Sertoli cells in boars. In cryptorchid testes, spermatogonia markedly decreased and the seminiferous tubule contained only a few spermatogonia, but did not contain post-meiotic germ cells. The altered seminiferous epithelium of the cryptorchid testis showed a low proliferation of its spermatogonia, with apoptosis and autophagy like that of scrotal testis, which probably entailed a gradual degeneration of the epithelium and the impossibility of its recovery. Although the number of Sertoli cells did not change, it was likely that their functionality was altered and that this affected the proliferation capacity of spermatogonia, causing the arrest of spermatogenesis.Spontaneous unilateral cryptorchid boars have one testis in the abdomen or inguinal canal, causing its temperature to be at or near the body temperature, which impairs spermatogenesis, although the histomorphometry and molecular mechanisms underlying this process remain unclear. The aim of the present study was to determine the histomorphometry, proliferation, apoptosis, and autophagy alterations in spermatogonia and Sertoli cells in unilateral cryptorchid, scrotal (contrascrotal), and preweaning piglet (preweaning) testes. Histomorphometrical analysis of cryptorchid testes showed that the seminiferous tubules contained only Sertoli cells and a few spermatogonia, but did not contain post-meiotic germ cells. The number of spermatogonia markedly decreased, and the number of Sertoli cells did not change remarkably in cryptorchid testes. TUNEL assay results showed that apoptosis signals were predominantly observed in spermatogonia. In cryptorchid and contrascrotal testes, proliferating cell nuclear antigen (PCNA) and LC3 were located in spermatogonia. The number of PCNA-positive, TUNEL-positive, and LC3-positive germ cells was low, and the protein and mRNA levels of PCNA and LC3 were significantly decreased in cryptorchid testes. Taken together, the number of Sertoli cells did not change remarkably, whereas the number of germ cells decreased in the cryptorchid testes, compared with that in the contrascrotal testes. Insufficient proliferation, excessive apoptosis, and autophagy were involved in the regulation of the decrease in spermatogonia in cryptorchid boar testes.

Effect of fenofibrate on proliferation of SMMC-7721 cells via regulating cell cycle.

Liver cancer is a malignant cancer with great harmfulness. Fenofibrate is a peroxisome proliferation activated receptor (PPARα) agonist widely used in the treatment of dyslipidemia. Previous studies have shown that fenofibrate may promote cell proliferation, but the underlying mechanism has not been fully characterized. The aim of this study was to investigate the role of PPARα agonist fenofibrate in cell proliferation of SMMC-7721 cells compared with that of THLE-2 cells. SMMC-7721 and THLE-2 cells were treated with different concentrations of fenofibrate. Cell proliferation was analyzed by MTT, using flow cytometry for cell cycle analysis, and CyclinD1, Cyclin-dependent kinases2 (CDK2) and Proliferating Cell Nuclear Antigen (PCNA) were analyzed by Western blotting. RT-qPCR method was used to assess CDK2, CyclinD1 and PCNA mRNA levels. The results showed that 10-9-10-4 mol/L fenofibrate could induce cell growth and 10-4, 10-5, 10-6 mol/L fenofibrate could reduce the number of G0/G1 phase cells and increased in the number of cells in S and G2/M phase of cell cycle in SMMC-7721 cells. Furthermore, fenofibrate could significantly increase the expression of cell cycle related protein (CyclinD1, CDK2)and cell proliferation related proteins (PCNA). The use of PPARα inhibitor MT886 inhibited cell cycle progression and promote tumor cell apoptosis. But fenofibrate had no obvious effect on THLE-2 cells. These results revealed the effect of fenofibrate on the cell cycle of liver cancer cells, and provided a reasonable explanation for studying how fenofibrate promotes cell proliferation.

Asiaticoside inhibits epithelial-mesenchymal transition and stem cell-like properties of pancreatic cancer PANC-1 cells by blocking the activation of p65 and p38MAPK.

To analyze the inhibitory effects of Asiaticoside (ATS) on the epithelial-mesenchymal transition (EMT) and stem cell-like properties of a pancreatic cancer cell line (PANC-1) by blocking the activation of p65 and p38 mitogen-activated protein kinase (p38MAPK). ATS concentrations were set at 0, 10, 25, and 50 µmol/L. The survival rate of PANC-1 cells in each group was detected by CCK-8, and CD133 and CD44 positive cells were detected by flow cytometry. The levels of Ki67 and proliferating cell nuclear antigen (PCNA) mRNA were detected by RT-PCR. The expression of E-cadherin, N-cadherin, vimentin, sex-determining region Y-box2 (SOX2), and octamer-binding transcription factor 4 (OCT4) proteins, and the phosphorylation levels of p65 and p38MAPK were detected by western blot. Nude mouse xenograft models of the tumor were established by subcutaneous injection of PANC-1 cells (1×106-1×108/mL), and they were randomly divided into the control group (0 mg/kg), and low-dose, medium-dose, and high-dose ATS groups (2.5, 5, 10 mg/kg). Apoptosis in xenograft tissue was detected by TUNEL, and the expression of vimentin and SOX2 proteins was detected by immunohistochemistry. As the ATS concentration increased to 25 µmol/L, cell survival rate, levels of Ki67 and PCNA mRNA, expression of N-cadherin, vimentin, SOX2, OCT4, p-p65/p65, and p-p38MAPK/p38MAPK proteins, and the proportions of CD44+ and CD133+ positive cells significantly decreased (P<0.05), while the expression of E-cadherin protein significantly increased (P<0.05). The results of tumor formation in nude mice showed that with the increase of ATS concentration, at 5 mg/kg the volume of the xenograft significantly decreased (P<0.05), the apoptosis rate significantly increased (P<0.05), and positive expression rates of vimentin and SOX2 proteins significantly decreased (P<0.05). ATS may inhibit the proliferation, EMT, and stem cell-like properties of pancreatic cancer cells by blocking the phosphorylation of p38MAPK and nuclear factor-κB (NF-κB)/p65 in PANC-1 cells.

Hepatocyte-derived MANF is protective for rifampicin-induced cholestatic hepatic injury via inhibiting ATF4-CHOP signal activation

Rifampicin (RFP) has been known to be potentially hepatotoxic and often used as an inducer of cholestatic hepatic injury. Here we found that mesencephalic astrocyte-derived neurotrophic factor (MANF), an endoplasmic reticulum (ER) stress inducible protein, is a protector in RFP-induced liver injury. In cholestatic hepatic injury mice induced by RFP, the liver/body ratio and the levels of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), total bile acid (TBA), total bilirubin (TBIL), and direct bilirubin (DBIL) were significantly increased. Meanwhile, the protein and mRNA levels of MANF were remarkably elevated in the liver injury mice. In hepatocyte-specific MANF knockout (HKO) mice, an extra increase in the liver/body ratio and serum ALT, AST, ALP, TBA, TBIL, and DBIL levels was detected after treatment with RFP. In addition, recombinant human MANF (rhMANF) treatment efficiently reduced the liver/body ratio and serum ALT, AST, ALP, TBA, TBIL, and DBIL levels in RFP-induced liver injury mice. Furthermore, we found there is an increase in the number of the apoptotic cells, detected by TUNEL staining in the liver tissues of HKO mice. Meanwhile, the protein levels of C/EBP-homologous protein (CHOP), Ki67, and the proliferating cell nuclear antigen (PCNA), as well as the mRNA level of Ki67 were elevated after treated with RFP, and these parameters were increased more significantly in HKO mice than that in wild type (WT) controls in RFP-induced liver injury. The rhMANF treatment can rescue the cell apoptosis and reduce the protein and mRNA levels of CHOP, Ki67, and PCNA elevated by MANF deletion and RFP. In HKO mice, immunoglobulin heavy chain binding protein (BIP) and activating transcription factor 4 (ATF4) were predominantly increased after treatment with RFP, which were reduced by rhMANF treatment. Therefore, we conclude that hepatocyte-derived MANF is protective for RFP-induced cholestatic hepatic injury via inhibiting ATF4-CHOP signal activation and subsequent cell apoptosis.

Clinical significance of high expression of proliferating cell nuclear antigen in non-small cell lung cancer.

Although proliferating cell nuclear antigen (PCNA) plays an important role in tumor proliferation and its expression level is closely related to the biological activity of tumor cells, PCNA expression in non-small cell lung cancer (NSCLC) has been seldom reported. In this study, we aimed to investigate the significance of PCNA expression in NSCLC tissues. PCNA expression in NSCLC and adjacent tissues were assessed by immunohistochemistry (IHC), western blotting, and reverse transcription polymerase chain reaction. Single factor analysis was used to study the relationship between the expression of PCNA and clinicopathological features of NSCLC. Multi-factor Cox survival analysis was used to evaluate the relationship between the expression of PCNA and overall survival of postoperative NSCLC patients. The areas under the receiver operating characteristics were calculated to evaluate the value of PCNA expression level in predicting the 3-year survival of NSCLC patients. IHC analysis showed that the positive expression rates of PCNA protein in NSCLC and adjacent tissues were 91.79% (257/280) and 25.83% (31/120), respectively. Western blotting confirmed that PCNA protein level was significantly higher in NSCLC tissues than in the adjacent tissues (P < .05). Reverse transcription polymerase chain reaction showed that the positive rate of PCNA mRNA in NSCLC was 88.93% (249/280), which was significantly higher than that in adjacent tissues 29.17% (35/120) (P < .05). Both PCNA mRNA and protein levels were correlated with tumor differentiation, size, metastasis, and stage in NSCLC. Patients exhibiting higher PCNA protein expression had a significantly shorter disease-specific survival rate than the other patients. PCNA protein level and tumor pathological type, metastasis, differentiation degree, and stage were independent factors affecting the overall survival of postoperative patients. The areas under the receiver operating characteristics of PCNA mRNA for predicting the 3-year survival of NSCLC patients was 0.89 (0.79–0.98), with a sensitivity and specificity of 0.84 and 0.76, respectively. In conclusion, high PCNA protein and mRNA levels may be associated with the occurrence, development, and prognosis of NSCLC.

Adipokine Chemerin Stimulates Progression of Atherosclerosis in ApoE-/- Mice.

Background Vascular remodeling is the most critical pathogenesis of atherosclerosis. Adipokine chemerin was known for its relationship with obesity as well as metabolism. Most recently, chemerin was found to play a crucial role in the pathologic process of cardiovascular diseases including coronary heart disease. In this study, we surveyed the role of chemerin in progression of atherosclerosis in ApoE−/− mice. Objective To investigate the relationship between chemerin and progression of atherosclerosis in ApoE−/− mice and its mechanism. Methods 8-week-old ApoE−/− mice were fed with high-fat diet to induce the atherosclerosis model. Adenoviruses were transfected for knockdown or overexpression of chemerin gene into aorta. Serums and aortic tissues of ApoE−/− mice were obtained after feeding high-fat diet for 16 weeks. HE staining and oil red staining were performed to evaluate aortic plaque. ELISA was performed to explore serum levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and transforming growth factor-β1 (TGF-β1). Real-time PCR and western blotting were carried out to investigate the mRNA and protein levels of chemerin, nuclear factor-κB p65 (NF-κBp65), proliferating cell nuclear antigen (PCNA), phosphorylated p38 mitogen-activated protein kinase (p-p38-MAPK), phosphorylated c-Jun N-terminal kinase (p-JNK), and phosphorylated extracellular signal regulated kinase 1/2 (p-ERK 1/2). Result Aortic plaque formation was significantly induced by high-fat diet in ApoE−/− mice. Simultaneously, elevated serum levels of TNF-α and IL-1β and elevated mRNA and protein levels of chemerin, NF-κBp65, PCNA, p-p38-MAPK, p-JNK, and p-ERK 1/2 were found in ApoE−/− mice. After aortic chemerin gene was inhibited by adenovirus, aortic atherosclerosis induced by high-fat diet was significantly meliorated, serum levels of TNF-α and IL-1β decreased, mRNA and protein levels of NF-κBp65, PCNA, p-p38-MAPK, p-JNK, and p-ERK 1/2 decreased simultaneously. Conclusion Our study revealed that chemerin stimulated the progression of atherosclerosis in ApoE−/− mice.

Histone Deacetylase 4 Promotes Osteosarcoma Cell Proliferation and Invasion by Regulating Expression of Proliferating Cell Nuclear Antigen.

Background/Aims: Osteosarcoma (OS) is commonly characterized by lower survival rates and high incidences of local recurrence due to its highly aggressive nature and metastatic tendencies. Studies have shown that histone deacetylase 4 (HDAC4) and proliferating cell nuclear antigen (PCNA) are highly expressed in cancers. Nevertheless, the roles of HDAC4 and PCNA in osteosarcoma (OS) remain unclear. This research aimed to study the expression of HDAC4 and PCNA and their relation to cell proliferation and invasion in human OS.Methods: The levels of HDAC4 and PCNA mRNA and protein were tested in human OS and osteochondroma (OC) tissues. The overexpression and knockdown of HDAC4 in OS cell lines were used to determine the effect of HDAC4 on the expression and degradation of PCNA. The effect of HDAC4 on cell proliferation, invasion and apoptosis was also detected. Additionally, we explored the interaction between HDAC4 and PCNA.Results: The results showed that both HDAC4 and PCNA were increased in human OS tissues. Overexpression of the HDAC4 protein increased the protein level of PCNA, had no effect on the PCNA mRNA level, and decreased the level of ubiquitinated PCNA. We found that overexpression of HDAC4 promoted cell proliferation and invasion and inhibited apoptosis. The opposite effects were observed when HDAC4 was knocked down. The results also showed that HDAC4 could bind to PCNA directly.Conclusions: Our findings suggest that HDAC4 could promote OS cell proliferation and invasion by regulating the expression of PCNA. Thus, our research indicates that HDAC4 may be a potential target for therapy in OS.

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

Urinary bladder organ hypertrophy is partially regulated by Akt1-mediated protein synthesis pathway

AimsThe present study aims to investigate the role of Akt in the regulation of urinary bladder organ hypertrophy caused by partial bladder outlet obstruction (pBOO). Main methodsMale rats were surgically induced for pBOO. Real-time PCR and western blot were used to examine the levels of mRNA and protein. A phosphoinositide 3-kinase (PI3K) inhibitor LY294002 was used to inhibit the activity of endogenous Akt. Key findingsThe urinary bladder developed hypertrophy at 2 weeks of pBOO. The protein but not mRNA levels of type I collagen and α-smooth muscle actin (αSMA) were increased in pBOO bladder when compared to sham control. The phosphorylation (activation) levels of Akt1 (p-Ser473), mammalian target of rapamycin (mTOR), p70S6 kinase (p70S6K), and 4E-BP1 were also increased in pBOO bladder. LY294002 treatment reduced the phosphorylation levels of Akt1 and 4E-BP1, and the protein levels of type I collagen and αSMA in pBOO bladder. The mRNA and protein levels of proliferating cell nuclear antigen (PCNA) were increased in pBOO bladder, and PCNA up-regulation occurred in urothelial not muscular layer. LY294002 treatment had no effect on the mRNA and protein levels of PCNA in pBOO bladder. LY294002 treatment partially reduced the bladder weight caused by pBOO. SignificancepBOO-induced urinary bladder hypertrophy is attributable to fibrosis, smooth muscle cellular hypertrophy, and urothelium cell hyper-proliferation. Akt1-mediated protein synthesis in pBOO bladder contributes to type I collagen and αSMA but not PCNA up-regulation. Target of Akt1 is necessary but not sufficient in treatment of urinary bladder hypertrophy following pBOO.

ECRG2 enhances the anti-cancer effects of cisplatin in cisplatin-resistant esophageal cancer cells via upregulation of p53 and downregulation of PNCA

AIMTo explore the anti-tumor effects of esophageal cancer-related gene 2 (ECRG2) in combination with cisplatin (DDP) in DDP-resistant esophageal cancer cells (EC9706/DDP).METHODSA drug-resistant cell model was established, with EC9706/DDP cells being treated with ECRG2 and/or DDP. Cell viability was examined by MTT assay. The rate of cell apoptosis was determined by flow cytometry. The mRNA expression levels of proliferating cell nuclear antigen (PCNA), metallothionein (MT), and p53 were determined by RT-PCR and PCNA, while MT and p53 protein expression levels were determined by western blotting.RESULTSThe anti-proliferative effect of ECRG2 in combination with DDP was superior when compared to ECRG2 or DDP alone. The inhibition rate for the combination reached its peak (51.33%) at 96 h. The early apoptotic rates of the control, ECRG2 alone, DDP alone, and ECRG2 plus DDP groups were 5.71% ± 0.27%, 12.68% ± 0.61%, 14.15% ± 0.87%, and 27.96% ± 0.36%, respectively. Although all treatment groups were significantly different from the control group (P < 0.05), the combination treatment of ECRG2 plus DDP performed significantly better when compared to either ECRG2 or DDP alone (P < 0.05). The combination of ECRG2 and DDP significantly upregulated p53 mRNA and protein levels and downregulated PCNA mRNA and protein levels compared to ECRG2 or DDP alone (P < 0.05). However, no changes were seen in the expression of MT mRNA or protein.CONCLUSIONECRG2 in combination with DDP can inhibit viability and induce apoptosis in esophageal cancer DDP-resistant cells, possibly via upregulation of p53 expression and downregulation of PCNA expression. These findings suggest that the combination of ECRG2 and DDP may be a promising strategy for the clinical treatment of esophageal cancers that are resistant to DDP.

MicroRNA-24 Attenuates Neointimal Hyperplasia in the Diabetic Rat Carotid Artery Injury Model by Inhibiting Wnt4 Signaling Pathway.

The long-term stimulation of hyperglycemia greatly increases the incidence of vascular restenosis (RS) after angioplasty. Neointimal hyperplasia after vascular injury is the pathological cause of RS, but its mechanism has not been elucidated. MicroRNA-24 (miR-24) has low expression in the injured carotid arteries of diabetic rats. However, the role of miR-24 in the vascular system is unknown. In this study, we explore whether over-expression of miR-24 could attenuate neointimal formation in streptozotocin (STZ)-induced diabetic rats. Adenovirus (Ad-miR-24-GFP) was used to deliver the miR-24 gene to injured carotid arteries in diabetic rats. The level of neointimal hyperplasia was examined by hematoxylin-eosin (HE) staining. Vascular smooth muscle cell (VSMC) proliferation in the neointima was evaluated by immunostaining for proliferating cell nuclear antigen (PCNA). The mRNA levels of miR-24, PCNA, wingless-type MMTV integration site family member 4 (Wnt4), disheveled-1 (Dvl-1), β-catenin and cell cycle-associated molecules (Cyclin D1, p21) were determined by Quantitative Real-Time PCR (qRT-PCR). PCNA, Wnt4, Dvl-1, β-catenin, Cyclin D1 and p21 protein levels were measured by Western blotting analysis. STZ administration decreased plasma insulin and increased fasting blood glucose in Sprague-Dawley (SD) rats. The expression of miR-24 was decreased in the carotid artery after a balloon injury in diabetic rats, and adenoviral transfection (Ad-miR-24-GFP) increased the expression of miR-24. Over-expression of miR-24 suppressed VSMC proliferation and neointimal hyperplasia in diabetic rats at 14 days. Furthermore, compared with Sham group, the mRNA and protein levels of PCNA, Wnt4, Dvl-1, β-catenin, and Cyclin D1 were strikingly up-regulated in the carotid arteries of diabetic rats after a balloon injury. Interestingly, up-regulation of miR-24 significantly reduced the mRNA and protein levels of these above molecules. In contrast, the change trend in p21 mRNA and protein levels was opposite after a balloon injury. However, over-expression of miR-24 after gene delivery increased the mRNA and protein levels of p21. We conclude that over-expression of miR-24 could attenuate VSMC proliferation and neointimal hyperplasia after vascular injuries in diabetic rats. This result is possibly related to the regulation of the expression of Cyclin D1 and p21 through the Wnt4/Dvl-1/β-catenin signaling pathway.

Testing stem cell therapy in a rat model of inflammatory bowel disease: role of bone marrow stem cells and stem cell factor in mucosal regeneration.

BackgroundThe gastrointestinal (GI) mucosal cells turnover regularly under physiological conditions, which may be stimulated in various pathological situations including inflammation. Local epithelial stem cells appear to play a major role in such mucosal renewal or pathological regeneration. Less is clear about the involvement of multipotent stem cells from blood in GI repair. We attempted to explore a role of bone marrow mesenchymal stromal cells (BMMSCs) and soluble stem cell factor (SCF) in GI mucosa regeneration in a rat model of inflammatory bowel diseases (IBD).MethodsBMMSCs labelled with the fluorescent dye PKH26 from donor rats were transfused into rats suffering indomethacin-induced GI injury. Experimental effects by BMMSCs transplant and SCF were determined by morphometry of intestinal mucosa, double labeling of PKH26 positive BMMSCs with endogenous proliferative and intestinal cell markers, and western blot and PCR analyses of the above molecular markers in the recipient rats relative to controls.ResultsPKH26 positive BMMSCs were found in the recipient mucosa, partially colocalizing with the proliferating cell nuclear antigen (PCNA), Lgr5, Musashi-1 and ephrin-B3. mRNA and protein levels of PCNA, Lgr5, Musashi-1 and ephrin-B3 were elevated in the intestine in BMMSCs-treated rats, most prominent in the BMMSCs-SCF co-treatment group. The mucosal layer and the crypt layer of the small intestine were thicker in BMMSCs-treated rats, more evident in the BMMSCs-SCF co-treatment group.ConclusionBMMSCs and SCF participate in but may play a synergistic role in mucosal cell regeneration following experimentally induced intestinal injury. Bone marrow stem cell therapy and SCF administration may be of therapeutic value in IBD.

Expression of the long non-coding RNAs MEG3, HOTAIR, and MALAT-1 in non-functioning pituitary adenomas and their relationship to tumor behavior

It is becoming evident that long non-coding RNAs (lncRNAs) participate in diverse biological processes via distinct mechanisms. Many lncRNAs have altered expression and likely to have functional roles in tumorigenesis. Although loss of maternally-expressed gene 3 (MEG3) expression has been detected in non-functioning pituitary adenomas (NFPAs), there are no published reports regarding the association between MEG3 expression and the invasive ability of NFPAs. Moreover, the roles of Hox transcript antisense intergenic RNA (HOTAIR) and metastasis-associated lung adenocarcinoma transcript 1 (MALAT-1) in NFPAs have not been examined. To investigate the role of MEG3, HOTAIR, and MALAT-1 in NFPA development and invasion. MEG3, HOTAIR, MALAT-1 and proliferating cell nuclear antigen (PCNA) were detected in 52 NFPA samples and seven normal human anterior pituitaries using real-time quantitative reverse transcription polymerase chain reaction. MEG3 lncRNA levels gradually decreased whereas HOTAIR lncRNA levels gradually increased from normal anterior pituitaries to non-invasive NFPAs to invasive NFPAs. There was a significant association between MEG3 (P < 0.01) and HOTAIR (P < 0.05) expression and the biological behavior of the tumor. Furthermore, PCNA mRNA levels markedly increased in invasive NFPAs compared to non-invasive ones (P < 0.01). In addition, PCNA mRNA negatively correlated with MEG3 lncRNA levels (P < 0.05). MEG3 and HOTAIR expression may correlate with NFPA development and invasion.

Effect of nimesulide on the growth of human laryngeal squamous cell carcinoma

PurposeTo investigate the effect of nimesulide on the growth of human laryngeal squamous cell carcinoma. Materials and methodsThe effect of NIM on Hep-2 cell proliferation was measured by the MTT assay. Flow cytometry was used to evaluate the cell cycle and apoptosis in Hep-2 cells. A Western blot analysis was used to detect changes in the protein expression levels of COX-2, Survivin and proliferating cell nuclear antigen (PCNA) in Hep-2 cells. A Hep-2 tumor xenograft model was established in nude mice to observe tumor growth. The changes in the xenograft tumors were observed after hematoxylin/eosin staining. The expression levels of COX-2, Survivin and PCNA proteins and mRNA were measured by immunohistochemical analysis and RT-PCR, respectively. ResultsNIM had time- and dose-dependent inhibitory effect on the proliferation of Hep-2 cells. NIM could prevent the progression of the cell cycle. After NIM treatment, COX-2, Survivin and PCNA protein levels were reduced in the Hep-2 cells. The volume and weight of the xenograft tumors in the NIM treatment group were significantly reduced. The NIM treatment group also exhibited significantly reduced expression levels of COX-2, Survivin and PCNA at both the protein and mRNA levels. ConclusionsOur results suggested that NIM has significant inhibitory effects on the growth of Hep-2 cells and xenograft tumors in nude mice. Selective COX-2 inhibitors could potentially become part of a comprehensive treatment for laryngeal squamous cell carcinoma. Additional research and development will provide new and broader prospects for the prevention and treatment of laryngeal squamous cell carcinoma.

Pig oocytes with a large perivitelline space matured in vitro show greater developmental competence after parthenogenesis and somatic cell nuclear transfer

The objective of this study was to examine the developmental competence of pig oocytes in relation to the size of the perivitelline space (PVS) of oocytes matured in vitro. Immature oocytes were matured in medium 199 or porcine zygote medium (PZM)-3 containing 108 or 61.6 mM NaCl. In vitro-matured (IVM) oocytes were examined for intracellular glutathione (GSH) level; cyclin-dependent kinase 1 (CDK1), proliferating cell nuclear antigen (PCNA), and extracellular signal-regulated kinase 2 (ERK2) mRNA levels; and developmental competence after parthenogenesis (PA) and somatic cell nuclear transfer (SCNT). IVM oocytes with a larger PVS had higher (P < 0.05) levels of intracellular GSH (1.00 pixels/oocyte vs. 0.57 pixels/oocyte) and blastocyst formation (54.3% vs. 37.3%) after PA than oocytes with a smaller PVS. Culturing oocytes for maturation in PZM-3 with reduced (61.6 mM) NaCl increased (P < 0.05) the size of the PVS (6.4 µm vs. 2.8 µm) compared to control oocytes that were matured in normal PZM-3 containing 108 mM NaCl. Moreover, oocytes with a larger PVS showed higher CDK1, PCNA, and ERK2 mRNA and intracellular GSH levels (1.6 pixels/oocyte vs. 1.2 pixels/oocyte) and increased blastocyst formation after PA (52.1% vs. 40.6%) and SCNT (31.8% vs. 18.2%) than control oocytes. Our results demonstrate that pig oocytes with a large PVS have greater developmental competence after PA and SCNT, which is attributed to improved cytoplasmic maturation based on the enhanced GSH level and transcription factor expression. Further, enlargement of the PVS by culturing in low-NaCl medium improves the developmental competence of pig oocytes.

Detecting DNA synthesis of neointimal formation after catheter balloon injury in GK and in Wistar rats: using 5-ethynyl-2'-deoxyuridine

BackgroundNeointimal formation plays an important role in the pathogenesis of coronary restenosis after percutaneous coronary intervention (PCI), especially in patients with diabetes mellitus. Recently, some studies have shown that 5-ethynyl-2'-deoxyuridine (EdU) incorporation can serve as a novel alternative to the 5-bromo-2'-deoxyuridine (BrdU) antibody detection method for detection of DNA synthesis in regenerating avian cochlea, chick embryo and the adult nervous system. However, few studies have been performed to assess the suitability of EdU for detecting DNA synthesis in vascular neointima.MethodsThe carotid artery balloon injury model was established in Goto-Kakizaki (GK) and Wistar rats. A Cell-LightTM EdU Kit was used to detect EdU-labeled cell nuclei of common carotid arteries at day 7 after catheter balloon injury. Different methods of injecting EdU were tested. The protein levels of proliferating cell nuclear antigen (PCNA) and p-Akt (Ser473), as well as the mRNA levels of PCNA were evaluated by Western blotting and quantitative real-time PCR (qRT-PCR), respectively. Immunohistochemical staining was also employed to visualize PCNA-positive cells.ResultsAt day 7 after catheter balloon injury, far more EdU-positive and PCNA-positive cells were observed in GK rats. When comparing groups that received different EdU doses, it was found that the percentage of EdU-positive cells at a dose of 100 mg/kg body weight was than at doses of 25 mg/kg and 50 mg/kg. The number of positive cells was significantly higher in the repeated injection group compared to the single injection group. Further, after balloon injury DNA synthesis in GK rats was more notable than in Wistar rats. Neointimal formation in GK rats was more obvious than in Wistar rats. The protein levels of PCNA and p-Akt (Ser473) and the mRNA levels of PCNA were increased in injured rats as compared to uninjured rats, and were significantly higher in GK rats than in Wistar rats.ConclusionBy intraperitoneal injections of EdU at a dose of 100 mg/kg three times, EdU incorporation can detect carotid arterial DNA synthesis caused by neointimal formation in GK rats and Wistar rats at day 7 after balloon injury by the EdU click reaction quickly and effectively. Moreover, more obvious DNA synthesis in the vascular neointima could be observed in GK rats than in Wistar rats.

Effects of bevacizumab on newborn rat retinal proteoglycans gene expression and on cell proliferation, death and differentiation, in vitro

AbstractPurpose VEGF is an important in nervous development, promoting neurogenesis and glial cell growth. Bevacizumab (BVZ), an anti‐VEGF agent, has been extensively used for controlling pathological retinal neovascularization in adult and newborn patients, although its effect on the developing retina remains largely unknown. In this study we investigate BVZ effect on proteoglycans and on cell death, proliferation and differentiation in newborn rat retina.Methods Retinal explants of sixty 2‐day‐old Lister hooded rats were maintained in culture media with or without BVZ for 2 days. Immunohistochemical staining was assessed against proliferating cell nuclear antigen (PCNA), caspase‐3 and beclin‐1 and vimentin and glial fibrillary acidic protein (GFAP). Gene expressions for PCNA, caspase‐3, beclin‐1, vimentin, GFAP and for proteoglycans were quantified by real‐time reverse‐transcription polymerase chain reaction. Results from treatment and control groups were comparedResults No significant difference in staining intensity of PCNA, caspase‐3, beclin‐1 and GFAP or in the mRNA levels of PCNA, caspase‐3, beclin‐1, vimentin, neurocan and phosphacan was observed. However, a significant increase in vimentin levels, decrease in GFAP and in syndecan‐3 mRNA expression were observed in BVZ‐treated retinas compared to controls.Conclusion Bevacizumab did not affect cell death or proliferation in early developing rat retina but appeared to interfere with glial cell maturation (by interfering with vimentin and GFAP). It can also alter syndecan‐3 gene expression. Thus, we suggest anti‐VEGF agents be used with caution in developing retinal tissue.

Aberrant expression of SPARC and its impact on proliferation and apoptosis in ADPKD cyst-lining epithelia

Autosomal dominant polycystic kidney disease (ADPKD) results from a combination of environmental and genetic factors. Secreted protein acidic and rich in cysteine (SPARC) can be expressed by many different cell types and is associated with development, remodelling, cell turnover and tissue repair. The analysis of SPARC would help evaluate the effect of the unique matricellular glycoprotein on renal disease progression in ADPKD. The concentration of SPARC was measured with an enzyme-linked immunosorbent assay (ELISA); distribution and expression levels were measured with in situ hybridization, immunohistochemistry, reverse transcription-polymerase chain reaction (RT-PCR) and western blot assays. Apoptosis was assessed by morphological observation and fluorescence-activated cell sorting (FACS) apoptosis index (AI) analysis. Cell cycle phase was examined by FACS analysis. Cell proliferation was studied using bromodeoxyuridine (BrdU) incorporation ELISA. The SPARC level in the renal cyst fluid of patients with ADPKD was greater than that in patients with simple renal cyst (SRC), and also greater than that found in the plasma and urine of patients with either ADPKD or SRC and normal subjects. SPARC mRNA and protein levels in polycystic renal tissue were greater than that in normal renal tissue. Additionally, SPARC could inhibit cyst-lining epithelial cell proliferation, bring about cell cycle arrest in the G0/G1 phase and induce apoptosis in vitro. SPARC treatment resulted in decreased mRNA levels of PCNA (proliferating cell nuclear antigen), MCM2 (minichromosome maintenance protein 2), ClnD1 and Bcl-2, but an increased mRNA level of p21(Waf1) in cyst-lining epithelial cells. Our findings suggest that the increased SPARC expression in ADPKD renal tissue may provide negative feedback in ADPKD patients.