ABSTRACT
OBJECTIVE@#To investigate the effect of inhibitor of growth protein-2 (Ing2) silencing on angiotensin Ⅱ (AngⅡ)-induced cardiac remodeling in mice and explore the underlying mechanism.@*METHODS@#An adenoviral vector carrying Ing2 shRNA or empty adenoviral vector was injected into the tail vein of mice, followed 48 h later by infusion of 1000 ng · kg-1 · min-1 Ang Ⅱ or saline using a mini-osmotic pump for 42 consecutive days. Transthoracic echocardiography was used to assess cardiac geometry and function and the level of cardiac hypertrophy in the mice. Masson and WGA staining were used to detect myocardial fibrosis and cross-sectional area of cardiomyocytes, and myocardial cell apoptosis was detected with TUNEL assay. Western blotting was performed to detect myocardial expressions of cleaved caspase 3, ING2, collagen Ⅰ, Ac-p53(Lys382) and p-p53 (Ser15); Ing2 mRNA expression was detected using real-time PCR. Mitochondrial biogenesis, as measured by mitochondrial ROS content, ATP content, citrate synthase activity and calcium storage, was determined using commercial assay kits.@*RESULTS@#The expression levels of Ing2 mRNA and protein were significantly higher in the mice with chronic Ang Ⅱ infusion than in saline-infused mice. Chronic infusion of AngⅡ significantly increased the left ventricular end-systolic diameter (LVESD) and left ventricular end-diastolic diameter (LVEDD) and reduced left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) in the mice. Ing2 silencing obviously alleviated AngⅡ-induced cardiac function decline, as shown by decreased LVEDD and LVESD and increased LVEF and LVFS, improved myocardial mitochondrial damage and myocardial hypertrophy and fibrosis, and inhibited cardiomyocyte apoptosis. Chronic AngⅡ infusion significantly increased myocardial expression levels of Ac-p53(Lys382) and p-p53(Ser15) in the mice, and Ing2 silencing prior to AngⅡ infusion lessened AngⅡ- induced increase of Ac-p53(Lys382) without affecting p53 (ser15) expression.@*CONCLUSION@#Ing2 silencing can inhibit AngⅡ-induced cardiac remodeling and dysfunction in mice by reducing p53 acetylation.
Subject(s)
Animals , Mice , Angiotensin II , Tumor Suppressor Protein p53 , Acetylation , Stroke Volume , Ventricular Remodeling , Ventricular Function, Left , Myocytes, CardiacABSTRACT
Abstract The Inhibitor of Growth (ING) gene family is a group of tumor suppressor genes that play important roles in cell cycle control, senescence, DNA repair, cell proliferation, and apoptosis. However, inactivation and downregulation of these proteins have been related in some neoplasms. The present study aimed to evaluate the immunohistochemical profiles of ING3 and ING4 proteins in a series of benign epithelial odontogenic lesions. Methods: The sample comprised of 20 odontogenic keratocysts (OKC), 20 ameloblastomas (AM), and 15 adenomatoid odontogenic tumors (AOT) specimens. Nuclear and cytoplasmic immunolabeling of ING3 and ING4 were semi-quantitatively evaluated in epithelial cells of the odontogenic lesions, according to the percentage of immunolabelled cells in each case. Descriptive and statistics analysis were computed, and the p-value was set at 0.05. Results: No statistically significant differences were found in cytoplasmic and nuclear ING3 immunolabeling among the studied lesions. In contrast, AOTs presented higher cytoplasmic and nuclear ING4 labeling compared to AMs (cytoplasmic p-value = 0.01; nuclear p-value < 0.001) and OKCs (nuclear p-value = 0.007). Conclusion: ING3 and ING4 protein downregulation may play an important role in the initiation and progression of more aggressive odontogenic lesions, such as AMs and OKCs.
Resumo Objetivos: A família dos Genes Inibidores de Crescimento (ING) é um grupo de genes supressores tumorais que desempenham papéis importantes no controle do ciclo celular, na senescência, no reparo do DNA, na proliferação celular e na apoptose. No entanto, a inativação e a regulação negativa dessas proteínas têm sido relacionadas em algumas neoplasias. O objetivo do presente estudo foi avaliar o perfil imuno-histoquímico das proteínas ING3 e ING4 em uma série de lesões odontogênicas epiteliais benignas. Métodos: A amostra foi composta por espécimes de 20 ceratocistos odontogênicos (CO), 20 ameloblastomas (AM) e 15 tumores odontogênicos adenomatoides (TOA). A imunoexpressão nuclear e citoplasmática de ING3 e ING4 foram avaliadas semi-quantitativamente nas células epiteliais das lesões odontogênicas, de acordo com a porcentagem de células imunomarcadas em cada caso. As análises descritivas e estatísticas foram computadas, e o valor de p estabelecido foi de 0,05. Resultados: Não foram encontradas diferenças estatisticamente significativas na imunoexpressão citoplasmática e nuclear de ING3 entre as lesões estudadas. Em contrapartida, os TOAs apresentaram maior marcação citoplasmática e nuclear de ING4 em comparação aos AMs (valor de p citoplasmático=0,01; valor de p nuclear <0,001) e COs (valor nuclear de p=0,007). Conclusão: A regulação negativa das proteínas ING3 e ING4 pode desempenhar um papel importante na iniciação e na progressão de lesões odontogênicas mais agressivas, como AMs e COs.
Subject(s)
Humans , Ameloblastoma , Odontogenic Cysts , Odontogenic Tumors , Homeodomain Proteins , Cell Cycle Proteins , Tumor Suppressor Proteins , Cell ProliferationABSTRACT
AIM: To investigate the expression of long non-coding RNA (lncRNA) PCAT19 in nasopharyngeal carcinoma tissues and cell lines, and to explore its molecular mechanism of inhibiting proliferation and invasion of nasopharyngeal carcinoma cells. METHODS: Real-time fluorescent quantitative PCR (qRT-PCR) was used to detect the relative expression of PCAT19 in nasopharyngeal carcinoma tissues and five nasopharyngeal carcinoma cell lines. The lowest expressing nasopharyngeal carcinoma cell line was transfected with empty plasmid (control group) or high expression PCAT19 plasmid (experimental group). qRT-PCR was used to detect transfection efficiency. MTS method and Transwell invasion test were used to detect the effect of overexpressing PCAT19 on the proliferation and invasion ability of nasopharyngeal carcinoma cells. Bioinformatics predicts target genes for PCAT19. qRT-PCR and Western blot were used to detect target gene expression at mRNA and protein levels. RESULTS: Compared with adjacent tissues, the expression of PCAT19 in nasopharyngeal carcinoma tissues was decreased (P<0.01). Compared with immortalized nasopharyngeal epithelial cells, the expression of PCAT19 in five nasopharyngeal carcinoma cell lines was significantly reduced (P<0.05), and the expression was the lowest in SUNE-1 cells (P<0.01). Transfection of high-expressing PCAT19 plasmid could significantly promote the expression of PCAT19 (P<0.01). High expression of PCAT19 could inhibit the proliferation ability (P<0.01) and invasive ability (P<0.01) of nasopharyngeal carcinoma SUNE-1 cells. The target gene of PCAT19 was miR-142-5p, the target gene of miR-142-5p was inhibitor of growth gene 3 (ING3). After high expression of PCAT19, miR-142-5p expression was significantly reduced (P<0.01), and the expression of ING3 at both mRNA and protein levels was significantly increased (P<0.01). CONCLUSION: PCAT19 expression is down-regulated in nasopharyngeal carcinoma tissues and cell lines. Up-regulating PCAT19 can inhibit the proliferation and invasion of nasopharyngeal carcinoma SUNE-1 cells. The mechanism may be that PCAT19 promotes the expression of ING3 gene by adsorbing miR-142-5p.
ABSTRACT
Objective To investigate the expression of miR-650 in osteosarcoma tissue and cell lines and its action mechanism in the osteosarcoma formation.Methods The realtime fluorescence quantitative PCR was used to detect and compare the expres-sions of miR-650 between osteosarcoma tissue and normal tissue or between osteosarcoma cell lines(MG63)and normal human os-teoblast cell lines(hFOB1.19);the MTT experiment was used to investigate the proliferation situation in different groups;Western blot was used to detect the ING4 protein expression.Results The expression of miR-650 in osteosarcoma tissue and MG63 cells was higher than that in normal tissue and human osteoblast cell;after inhibiting miR-650 expression,the proliferation ability of MG63 was significantly decreased.Moreover the expression of inhibitor of growth 4(ING4)was significantly increased when miR-650 was reduced,ING4 mRNA of negative control group,scramble group and miRNA group were 1.00 ± 0.16,1.08 ± 0.14 and 5.35 ± 0.32 respectively;the ING4 protein expressions were 0.62 ± 0.06,0.59 ± 0.12 and 2.45 ± 0.20 respectively;compared with negative control group,the difference was statistically significant(P<0.05);but after suppressing this elevation,the proliferation a-bility of MG63 cells had a certain recovery.Conclusion MiR-650 promotes MG63 proliferation via reducing ING4 expression,which suggests that miR-650 could be a new target of treating osteosarcoma.
ABSTRACT
Objective To investigate the effects of inhibitor of growth 5 (ING5) gene on the proliferation, apoptosis, migration and cell cycle of human breast cancer Bcap-37 cells.Methods The eukaryotic ING5-expressing plasmid and GFP-empty plasmid were steadily transfected in Bcap-37 cells, the expression of green fluorescent protein was measured with fluorescence microscopy, and the high expression of ING5 was measured by real time-PCR. Bcap-37-ING5 cells served as the experimental group, Bcap-37-GFP cells as the mock group and Bcap-37 as the control group. The effects of ING5 on the proliferation were detected by MTT, the cell cycle and apoptosis were detected by Flow cytometry, and the cell migration was detected by cell wound scratch assay and Transwell experiment.Results Bcap-37 cell lines steadily expressing ING5 protein with GFP-tag were acquired by stable transfection. ING5 over-expression inhibited the proliferation and led to G2 arrest of Bcap-37 cells, increased cells apoptosis and decreased the cell migration ability (P<0.05).Conclusion ING5 over-expression may have reverse effect for malignant phenotype of breast cancer cells, and may be employed to indicate the biomarker of prognosis of breast cancer patients and regarded as a target of gene therapy.
ABSTRACT
Objective To investigate the effects of inhibitor of growth 4(ING4)on the proliferation,migration and the expression of angiogenesis related factors such as VEGF,MMP-2,MMP-9 in endothelial cells. Methods Human umbilical vein endothelial cells(HUVECs)were cultured in vitro;ING4 plasmid and siRNA were constructed and transfected to HUVECs;the proliferation of HUVECs was evaluated by MTT assay;the ability of migration was evaluated by Transwell assay;real-time PCR and Western blotting were used to determine the expression of mRNA and pro-tein of angiogenesis related factors such as VEGF,MMP-2,and MMP-9. Results MTT and Transwell assay showed that ING4 has the ability to inhibit the proliferation and migration of HUVECs,and the results of real-time PCR and Western blotting proved that ING4 can inhibit the expres-sion of angiogenesis related factors such as VEGF,MMP-2,and MMP-9. Conclusion ING4 can inhibit the proliferation and migration of HU-VECs,down-regulate the expression of angiogenesis related factors such as VEGF,MMP-2,MMP-9,and inhibit angiogenesis.
ABSTRACT
Objective To investigate the expression and prognostic significance of inhibitor of growth 4 (ING4) and tail-type homeobox transcription factor 2 (CDX2) in colorectal cancer. Methods The expressions of ING4 and CDX2 pro-teins were detected by immunohistochemistry in 99 tissue samples of colorectal cancer and 30 corresponding para-cancer-ous normal tissue samples. The data of clinic outcomes were collected. The correlations between the expressions of ING 4 and CDX2 and clinicopathological parameters were also analyzed. Results The positive expression rates of ING4 and CDX2 were 68.8%and 72.7%in colorectal cancer tissues, which were significantly lower than those of corresponding normal tissue samples (93.3% and 96.7%, P<0.05). There were significant differences in the differentiation, depth of invasion, lymph node metastasis and tumor stage between expressions of ING4 and CDX2 (P<0.05). The 5-year survival rate was significant-ly lower in ING4 negative group (35.5%) compared with that of ING4 positive group (77.9%). The 5-year survival rate was significantly lower in CDX2 negative group (48.1%) than that of CDX2 positive group (70.8%, P<0.05). The expression of ING4 was positively correlated with the expression of CDX2 in colorectal cancer. Conclusion The expressions of ING4 and CDX2 are strongly associated with the carcinogenesis, development and prognosis of the colorectal cancer,which suggests that ING4 and CDX2 might be used as prognostic markers for the colorectal cancer.
ABSTRACT
Objective Inhibitor of growth 1 ( ING1 ) gene has been identified as a novel candidate of tumor suppressor gene .Over-expression of ING1 plays well-established roles in numerous cell processes ,inclu-ding DNA repair and cell apoptosis .Our study is to investigate the clinical significances of expression of ING 1 in colorectal cancer ( CRC) .Methods The mRNA level of ING1 in 82 matched samples comprising primary CRC and paired non-cancerous mucosa were detected and compared by quantitative RT -PCR.Then the correlations of mRNA level of ING1 with the clinicopathological characteristics and prognosis of patients with CRC were ana -lyzed.Results (1)In the same matched tissues,the expression level of ING1 was significantly higher in normal tissues than that in cancer tissues.(2)mRNA expression of ING1 was associated with certain clinical -pathologic variables such as tumor infiltrating level ,lymphatic metastasis,distant metastasis and advancing TNM stage .(3) We obtained the expression levels ratio of cancer tissue and normal tissue and found the lower ratio has a lower Disease-Free Survival(DFS)(P<0.0001).(4)ING1,as a candidate of tumor suppressor gene ,remained a sta-tistically-significant prognostic marker in the Cox regression analysis .Conclusion Down-regulation of ING1 may be correlated tightly with the occurrence and progression of sporadic colorectal cancer .Its expression level can be used to predict prognosis of CRC .
ABSTRACT
Objective To discuss the expressions and clinical significance of p53 and p33ING1b in human psoriasis and basal cell carcinoma (BCC). Methods Immunohistochemistry EnVision technique was used to detect the expressions of p53 and p33ING1b in samples of 36 psoriasis vulgaris, 28 BCC and 14 normal skins. Results The expression of p53 increased while p33ING1b had a degressive expression in the control group, the psoriasis group and the BCC group. It was found significant statistical difference between the two groups (all P < 0.05). Prominent positive correlation between p53 and p33ING1b were found in both psoriasis group and BCC group (all P<0.05). Conclusions p53 coacts with p33ING1b at local lesions of abnormal proliferative diseases . It′s one of the most prominent mechanisms contributing to deviant cell proliferation.
ABSTRACT
Objective To construct a recombinant adenoviral vector carrying and co-expressing human inhibitor of growth 4(ING4) and human interleukin-24(IL-24) mediated by poly( A)-Promoter[Ad-ING4-poly(A)-Promoter-IL-24, referred to as Ad-ING4-IL-24] and explore its effect on the growth of HepG-2 human hepatocellular carcinoma cellsin vitro. Methods The poly(A)-Promoter(hEFl-elF4g) (Sal Ⅰ and Not Ⅰ ), ING4 ( Bgl Ⅱ and Sal Ⅰ ), and IL-24 ( Xho Ⅰ and Xba Ⅰ ) fragments were amplified by PCRusing pORF-mbcl-2α, pcDNA3.0-IL-24, and pcDNA3.0-ING4 plasmids as templates and subcloned into pAdTrack-CMV transfer vector to form pAdTrack-CMV-ING4-poly (A)-Promoter-lL-24, respectively. The pAdTrack-CMV-ING4-poly (A)-Promoter-IL-24 transfer vector linearized with Pme Ⅰ digestion and pAdEasy-1 backbone vector was further cotransformed into the bacteria BJ5183 competent cells for homologous recombination. The resultant pAdEasy-l-pAdTrack-CMV-ING4-poly ( A )-Promoter-IL-24 homologous recombinant plasmids were linearized with Pac Ⅰ digestion and transfected into the human embryonic kidney 293 (QBI-293A) cells by liposome, leading to formation of the recombinant adenoviruses Ad-ING4-IL-24co-expressing ING4 and IL-24. The Ad-ING4-IL-24 were amplified in QBI-293A cells and its titer was up to 3.5 × 109 PFU/ml. Adenovirus-mediated ING4 and IL-24 genes expression in HepG-2 cells was examined by RT-PCR and Western blot. The growth-suppressing and apoptosis-inducingg effect of Ad-ING4-IL-24 coexpressing ING4 and IL-24 on HepG-2 human hepatocellular carcinoma cells was assessed by MTT assay and FCM, respectively. Results DNA sequencing showed that the ING4, poly (A)-Promoter, and IL-24 fragments subcloned into pAdTrack-CMV plasmids were completely identical to those reported in GenBank.ING4 and IL-24 gene mediated by adenovirus could both successfully express in HepG-2 cells. Adenovirusmediated ING4 and IL-24 co-expression significantly suppressed HepG-2 hepatocellular carcinoma cell growth and induced cell apoptosis, and the effect of Ad-ING4-IL-24 group was more significant than AdING4 and Ad-IL-24 group. Conclusion The adenoviral vector co-expressing ING4 and IL-24 mediated by poly(A)-Promoter(Ad-ING4-IL-24) was successfully constructed. Ad-ING4-IL-24 had marked anti-tumor effect in suppressing HepG-2 human hepatocellular carcinoma cell growth and inducing cell apoptosis in vitro. Compared with Ad-ING4 and Ad- IL-24, Ad-ING4-IL-24 enhanced anti-tumor effect.