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Objective:To screen key genes of renal clear cell carcinoma based on bioinformatics methods, identify possible microRNA (miRNA)-mRNA action axis, and explore the expression of related genes in clear cell renal cell carcinoma tissues and cells.Methods:Gene expression profiles of GSE40435 and GSE71302 datasets were obtained from the Gene Expression Omnibus (GEO) database. TCGA-KIRC datasets were obtained from The Cancer Genome Atlas (TCGA) database. R software was used to identify the differentially expressed mRNA and miRNA, and the functional enrichment analysis was performed. STRING database and Cytoscape software were used to perform the protein interaction analysis. The prognosis-related differentially expressed miRNA was evaluated by the Oncomir database. The potential targeted genes regulated by miRNA were determined by using TargetScan and miRDB targeted gene prediction tools. The tissue samples and clinicopathological features of 34 patients with clear cell renal cell carcinoma in the First Hospital of Shanxi Medical University from June to December 2021 were collected, and normal renal cell line 293T and clear cell renal cell carcinoma cell line 786O were selected. The real-time fluorescence quantitative polymerase chain reaction (qRT-PCR), was used to detect the relative expression of genes; Western blotting and immunohistochemical staining were used to detect the expression levels of the targeted proteins. The dual luciferase reporter gene assay was carried out to verify the targeting relationship between genes.Results:A total of 1 351 differentially expressed mRNA and 50 differentially expressed miRNA were screened and identified. The result of functional enrichment analysis suggested that the fatty acid metabolism pathway and xenobiotic metabolism pathway were suppressed in clear cell renal cell carcinoma, while the apoptosis and immune response pathways were activated. Protein interaction analysis suggested that the signal transduction and protein ubiquitination pathways might play a key role in clear cell renal cell carcinoma. The screening results showed that miRNA-224-5p (miR-224-5p) was most closely associated with clear cell renal cell carcinoma progression and was highly expressed in tumor tissues, and its prognosis-related target gene was NEDD4L. The relative expression of NEDD4L mRNA in clear cell renal cell carcinoma tissues and paraneoplastic tissues were 0.138±0.103 and 1.000±0.026 ( t = 46.23, P < 0.05), and the relative expression of miR-224-5p was 1.000±0.043 and 0.129±0.108 ( t = 45.28, P < 0.05). The differences of NEDD4L mRNA and miR-224-5p expressions in different grades and stages of clear cell renal cell carcinoma tissues were statistically significant (all P < 0.05). The expression of NEDD4L protein was decreased in clear cell renal cell carcinoma. The relative expression of NEDD4L gene in 293T and 786O cells were 1.000±0.125 and 0.210±0.044 ( t = 17.52, P < 0.05); the relative expressions of miR-224-5p gene were 0.209±0.049 and 1.000±0.234 ( t = 10.61, P < 0.05). The relative expressions of NEDD4L mRNA in miRNA mimic group and negative control group were 0.236±0.062 and 1.000±0.024, and the difference was statistically significant ( t = 43.56, P < 0.05). NEDD4L protein expression was reduced in the miRNA mimic group. Dual luciferase reporter gene assay suggested that NEDD4L was a direct target gene of miR-224-5p. Conclusions:In clear cell renal cell carcinoma, miR-224-5p targets and regulates NEDD4L expression, and this mechanism may be related to carcinogenesis and progression of clear cell renal cell carcinoma.
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Objective:To evaluate the effect of c-CBL overexpression on activation of astrocytes in the spinal cord of rats with neuropathic pain and the relationship with Kindlin-1.Methods:Eighteen clean-grade healthy male Sprague-Dawley rats, aged 10-12 weeks, weighing 250-280 g, were divided into 3 groups ( n=6 each) using a random number table method: sham operation group (S group), neuropathic pain group (NP group) and c-CBL overexpression group (c-CBL group). The model of neuropathic pain was developed by chronic compression of the sciatic nerve in anesthetized rats.On 1 day before operation, c-CBL overexpression vector 10 μl was intrathecally injected in group c-CBL, while blank vehicle 10 μl was intrathecally injected in S and NP groups.The mechanical paw withdrawal threshold (MWT) and thermal paw withdrawal latency (TWL) were measured on 1 day before operation (T 0) and 1, 4 and 7 days after operation (T 1-3). The rats were sacrificed by decapitation after measurement of the pain threshold at T 3, and the spinal cord of L 4-6 was taken for determination of the expression of Kindlin-1, glial fibrillary acidic protein (GFAP), tumor necrosis factor-alpha (TNF-α), and interleukin-1beta (IL-1β) (by Western blot) and co-expression of Kindlin-1 with c-CBL (by co-immunoprecipitation). Results:Compared with group S, the MWT was significantly decreased and TWL was shortened at T 2, 3 in group NP and group c-CBL, the expression of Kindlin-1, GFAP, TNF-α and IL-1β was significantly up-regulated in group NP, and the expression of c-CBL was significantly up-regulated in group c-CBL ( P<0.05). Compared with group NP, the MWT was significantly increased and TWL was prolonged at T 2, 3, the expression of Kindlin-1, GFAP, TNF-α and IL-1β was down-regulated, and the expression of c-CBL was up-regulated in group c-CBL ( P<0.05). The results of co-immunoprecipitation showed that there was a protein interaction and co-expression relationship between Kindlin-1 and c-CBL in group NP, and the co-expression of Kindlin-1 with c-CBL was enhanced in group c-CBL when compared with group NP. Conclusions:The overexpression of c-CBL can inhibit activation of astrocytes by down-regulating the expression of Kindlin-1 in the spinal cord, thus reducing inflammatory responses and relieving neuropathic pain in rats.
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Objective To investigate the expression of the E3 ubiquitin ligase neural precursor cell expressed developmentally downregulated 4-1 (NEDD4-1) in pancreatic cancer tissue and its clinical significance. Methods Clinical data were collected from 58 patients who underwent surgical treatment in Xuzhou Central Hospital from January 2017 to December 2019 and were diagnosed with pancreatic ductal adenocarcinoma based on pathological examination. Immunohistochemistry was used to measure the expression of NEDD4-1 in pancreatic cancer tissue samples, and the association between the expression of NEDD4-1 and the clinicopathological features of pancreatic cancer was analyzed. Western blot was used to measure the protein expression level of NEDD4-1 in normal pancreatic ductal epithelial HPDE6-C7 cells and pancreatic cancer SW1990, BxPC-3, and PANC-1 cells. The t -test was used for comparison of continuous data between two groups, and the chi-square test was used for comparison of categorical data between two groups. The Kaplan-Meier method was used to plot survival curves, and the log-rank test was used for survival analysis. The Cox proportional-hazards regression model was used to investigate the factors associated with prognosis. Results The expression level of NEDD4-1 in pancreatic cancer tissue was significantly higher than that in adjacent tissue (79.31% vs 19.05%, χ 2 =35.614, P < 0.01), and the protein expression of NEDD4-1 in pancreatic cancer cells was significantly higher than that in normal pancreatic ductal epithelial cells ( P < 0.01). In the patients with pancreatic cancer, the expression of NEDD4-1 was associated with distant metastasis ( χ 2 =5.089, P =0.040), tumor differentiation ( χ 2 =9.071, P =0.003), and TNM stage ( χ 2 =8.882, P =0.003). The patients with high NEDD4-1 expression had a significantly shorter mean survival time than those with low expression (13.61±0.95 months vs 22.22±2.20 months, P =0.001). The Cox regression analysis showed that NEDD4-1 expression (hazard ratio [ HR ]=2.312, 95% confidence interval [ CI ]: 1.010-5.295, P =0.047), degree of tumor differentiation ( HR =2.981, 95% CI : 1.556-5.712, P =0.001), and lymph node metastasis ( HR =2.144, 95% CI : 1.155-3.979, P =0.016) were independent risk factors for the prognosis of patients with pancreatic cancer. Conclusion There is a significant increase in the expression of NEDD4-1 in pancreatic cancer tissue and cells, and the high expression of NEDD4-1 is associated with poor prognosis. Therefore, it can be used as a prognostic biomarker and a therapeutic target for pancreatic cancer.
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The occurrence and development of tumors is a complex process with multiple factors and multiple steps. Ubiquitination refers to a multi-step cascade of protein modification processes including ubiquitin, ubiquitin-activating enzyme, ubiquitin-binding enzyme, ubiquitin ligase and proteasome, which is important for maintaining eukaryotic homeostasis. mechanism. The E3 ubiquitin ligases family is an important component of the ubiquitin-proteasome system. This family includes many proteins that catalyze the ubiquitination of various protein substrates and promote their degradation by the proteasome system. Up to date, E3 ubiquitin ligases has played an important role in a variety of tumor cell biology processes, including cell proliferation, apoptosis and cycle regulation. HECT-type E3 ubiquitin ligases, one of the earliest studies of E3 ubiquitin ligases, is involved in the ubiquitination of transcriptional regulation of protein translation. This article reviews the recent research progress of HECT-type E3 ubiquitin ligases and its role in tumors.
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The occurrence and development of tumors is a complex process with multiple factors and multiple steps.Ubiquitination refers to a multi-step cascade of protein modification processes including ubiquitin,ubiquitin-activating enzyme,ubiquitin-binding enzyme,ubiquitin ligase and proteasome,which is important for maintaining eukaryotic homeostasis.mechanism.The E3 ubiquitin ligases family is an important component of the ubiquitin-proteasome system.This family includes many proteins that catalyze the ubiquitination of various protein substrates and promote their degradation by the proteasome system.Up to date,E3 ubiquitin ligases has played an important role in a variety of tumor cell biology processes,including cell proliferation,apoptosis and cycle regulation.HECT-type E3 ubiquitin ligases,one of the earliest studies of E3 ubiquitin ligases,is involved in the ubiquitination of transcriptional regulation of protein translation.This article reviews the recent research progress of HECT-type E3 ubiquitin ligases and its role in tumors.
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Objective To evaluate the effect of ultraviolet (UV) irradiation and all-trans retinoic acid (ATRA) on expression of Hrd1 in human skin and fibroblasts,and to explore their mechanisms.Methods From December 2017 to June 2018,12 human skin tissue samples were collected from Department of Dermatology,The First Affiliated Hospital of Nanjing Medical University,including 3 sun-exposed and 3 non-sun-exposed skin tissue samples of patients aged 30-40 years,and 3 sun-exposed and 3 non-sun-exposed skin tissue samples of patients aged 60-70 years.Immunohistochemicai examination was performed to determine the expression of Hrd 1 in the above samples.A total of 40 BALB/c mice were randomly classified into 4 groups:UV group treated with UVA irradiation at 10 J/cm2 and UVB irradiation at 30 mJ/cm2 every day,ATRA group topically treated with 0.1 ml of ATRA 0.1％ cream once a day on the shaved back,UV + ATRA group treated with topical ATRA 0.1％ cream before the above UV irradiation,and control group receiving no treatment.After 14 weeks,these mice were sacrificed,skin tissues were excised from the back,and the expression of Hrd 1 was determined by immunohistochemical examination.In vitro cultured human fibroblasts were divided into 4 groups:UV group and ATRA + UV group covered with phosphate buffer saline (PBS) followed by UVA irradiation at 10 J/cm2 or UVB irradiation at 30 mJ/cm2,ATRA group treated with culture media containing 1.μmol/L ATRA for 24 hours,and ATRA + UV group also treated with culture media containing 1 μmol/L ATRA for 24 hours after the ultraviolet irradiation.Western blot analysis was performed to determine the expression of Hrd 1 in fibroblasts in the above groups,fluorescence microscopy to detect the levels of reactive oxygen species (ROS) in the above groups.Statistical analysis was carried out by one-way analysis of variance (ANOVA) for comparison among groups,and least significant difference (LSD)-t test for multiple comparisons.The difference was considered to be statistically significant when the P value was less than the significant level of 0.05.Results In both the groups of 30-40 years and 60-70 years,the expression of Hrd1 was significantly higher in the sun-exposed skin tissues (0.307 ± 0.256,0.486 ± 0.579,respectively) than in the non-sun-exposed skin tissues (0.196 ± 0.330,0.199 ± 0.375,respectively;t =5.486,10.579 respectively,both P ＜ 0.05).In the in vivo experiment,the expression of Hrd1 in the skin tissues of mice significantly differed among the control group,UV group,ATRA group and ATRA + UV group (0.189 ± 0.015,0.288 ± 0.017,0.187 ±0.020,0.226 ± 0.021 respectively,F =19.553,P ＜ 0.001),and the UV group showed significantly higher Hrd1 expression compared with the control group (t =5.337,P =0.033)and ATRA + UV group (t =4.891,P =0.039).In the in vitro experiment,the level of Hrd1 in the fibroblasts significantly differed among the 4 groups after the UVA or UVB irradiation (F =120.704,102.119,both P ＜ 0.001).The effect of the UVA and UVB irradiation on the expression of Hrd1 was basically consistent,and the Hrd1 level was significantly higher in the UV group than in the control group and ATRA + UV group (both P ＜ 0.05).After the UV irradiation,the ROS level was significantly higher in the UV group than in the control group and ATRA + UV group (both P ＜ 0.05).Conclusion ATRA can inhibit ultraviolet-induced Hrd1 expression in skin fibroblasts,likely by inhibiting the generation of cellular ROS.
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Objective To evaluate the role of autophagy in Parkin over-expression-induced reduction of cardiac hypertrophy in diabetic rats.Methods Thirty-two healthy SPF male Sprague-Dawley rats,aged 2 months,weighing 200-230 g,were divided into 4 groups (n =8 each) using a random number table method:non-diabetes mellitus (DM) group (group Non-DM),DM group,DM plus AAV9-CMV-Parkin group (group DM+P) and DM puls AAV9-CMV-Parkin puls autophagy inhibitor 3-methyladenine (3-MA) group (group DM+P+MA).DM was induced by intraperitoneal streptozotocin (STZ) 60 mg/kg and confirmed by blood glucose level ＞16.7 mmol/L,and Parkin overexpression was achieved with adeno-associated virus serotype 9 as a vector.AAV9-CMV-Parkin 1 × 1012 mg/kg was injected into the tail vein at 3 weeks after STZ injection in group DM+P.In group DM+P+MA,AAV9-CMV-Parkin 1× 1012 mg/kg was injected into the tail vein at 3 weeks after STZ injection,and 3 weeks later 3-MA 15 mg/kg was injected into the tail vein,once a week for 6 weeks in total.Cardiac diastolic and systolic function was detected using Bmode ultrasonography.Cardiomyocytes were obtained for microscopic examination of the cross-sectional area (with a light microscope) and for determination of the expression of microtubule-associated protein 1 light chain 3,P62 and Parkin.Results Compared with group Non-DM,the cross-sectional area of cardiomyocytes was significantly increased,and the diastolic and systolic function was decreased in DM group (P＜0.05).Compared with group DM,the cross-sectional area of cardiomyocytes was significantly decreased,the diastolic and systolic function was improved,the expression of microtubule-associated protein 1 light chain 3 and Parkin in myocardial tissues was up-regulated,and the expression of P62 was down-regulated in group DM+P (P＜0.05).Compared with group MD+P,the cross-sectional area of cardiomyocytes was significantly increased,the diastolic and systolic function was decreased,the expression of microtubule-associated protein 1 light chain 3 was down-regulated,and the expression of P62 was up-regulated in group DM+P+MA (P＜0.05) Conclusion Autophagy is involved in Parkin over-expression-induced reduction of cardiac hypertrophy in diabetic rats.
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Objective To study the role of ubiquitin ligase FBW7 in the sensitivity of glioma to temozolomide and its mechanism in glioma cells. Methods FBW7 overexpression lentivirus was constructed. Glioma cell line U251 was divided into 4 groups: the control group, temozolomide group, FBW7 overexpression group, and FBW7 overexpression + temozolomide group. Compared with the intervention on U251 cell lines, the differences of cell inhibitory rates in 4 different groups were analyzed by using contrast microscopy and methyl thiazolyl tetrazolium (MTT) colorimetric assay after 36 h and 72 h respectively. Flow cytometry (FC) was used to determine the cell cycle and apoptosis rate. Results The survival number of U251 cells in the three treatment groups was increased compared with the control group at both 36 h and 72 h. The inhibitory rates of temozolomide group, FBW7 overexpression group, and FBW7 overexpression +temozolomide group at 36 h were (17.6±0.8) ％, (10.4±0.6) ％, (18.6±0.6) ％ respectively compared with the control group (F=67.02, P<0.01); while at 72 h, the inhibitory rates of the three treatment groups were (25.1 ±0.4) ％, (16.7 ±0.7) ％, (29.0 ±0.9) ％ respectively compared with the control group (F= 74.61, P<0.001). Moreover, FBW7 overexpression + temozolomide group presented much greater inhibitory rate than temozolomide group (P<0.01). The G2/M arrest ratio and the cell apoptotic rate at 72 h in the three treatment groups were higher than those in the control group (F=41.63, P<0.001;F=42.30, P<0.01). The increased degree of G2/M arrest ratio and the cell apoptotic rate in FBW7 overexpression + temozolomide group were more obvious compared with temozolomide group (P<0.05, P<0.01). Conclusion FBW7 could enhance the sensitivity of glioma cells to temozolomide treatment, which is associated with G 2/M arrest and the increased apoptosis rate induced by FBW7.
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Ubiquitinating enzyme damaged-DNA binding protein 2 (DDB2) is a rind of DDB1 and CUL4-associated factors (DCAF), and identifies belonging to the family of ubiquitinating E3 enzymes. DDB2 combines with CUL4-DDB1 to form the ubiquitin ligase complex, and identifies targets protein substrate specificity to make the substrate ubiquitin and degradation. It affects the development of tumors through various pathways, such as DNA damage repair, cell cycle regulation and apoptosis, cell invasion and metastasis, cell premature senescence, cell proliferation and cancer stem cell population. This paper reviews the progress of the relationship between DDB2 and the development, treatment and prognosis judgment of tumors.
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Objective To analyze differentially expressed proteins in A375 melanoma cells before and after short hairpin RNA (shRNA)-mediated Cbl-b gene silencing.Methods The label-free quantitative proteomics approach was performed to identify differentially expressed proteins between A375 cells transfected with lentiviral vectors containing Cbl-b shRNA (Cbl-b shRNA group) and those with control lentiviral vectors (control group).Then,the properties of differentially expressed proteins were analyzed by gene ontology (GO) and Kyoto Encyclopedia of Genes and Genome (KEGG) enrichment analysis.Western blot analysis was conducted to determine the expression of differential proteins (EphA2 and GSK3β) and phosphorylated protein kinase (p-AKT) after shRNA-mediated Cbl-b gene silencing.Statistical analysis was carried out by t test of two independent-samples for comparison of protein expression abundance between the two groups with SPSS 23.0 software.Additionally,the results of GO and KEGG enrichment analysis were analyzed by Fisher's exact test.Results A total of 3 449 proteins were identified and quantified,and 74 of them were differentially expressed between the Cbl-b shRNA group and control group.Compared with the control group,52 proteins were up-regulated and 22 were down-regulated in the Cbl-b shRNA group.GO enrichment analysis of differential proteins revealed that the top five significantly enriched biological processes were integrin-mediated cell adhesion,single-organism metabolic process,regulation of integrin-mediated cell adhesion,regulation of protein-targeting mitochondria and nucleic acid metabolic process.The top five significantly enriched molecular functions included DNA binding,2-iron,2-sulfur cluster binding,signaling receptor activity,cadherin binding and cell adhesion molecule binding.The top five significantly enriched cell components included nucleosome,DNA packaging complex,photoreceptor connecting cilium,DNA-protein complex and extracellular region part.KEGG enrichment analysis demonstrated that the top five significantly enriched melanoma-related signaling pathways were folate biosynthesis,axon guidance,extracellular matrix-receptor interaction,adherens junction and Wnt signaling pathways.As Western blot analysis revealed,the Cbl-b shRNA group showed lower protein expression of EphA2 (0.369),but higher protein expression of GSK3β (3.524) compared with the control group (1),which were consistent with the results of proteomics analysis.Additionally,the protein expression of p-AKT was down-regulated in Cbl-b shRNA group (0.453) compared with the control group (1).Conclusion Cbl-b may be involved in the occurrence of melanoma through a variety of biological pathways,and the EphA2/PI3K/AKT signaling pathway may be one important pathway.
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Objective To evaluate the effect of Iduna protein overexpression on poly(ADP-ribose) polymerase 1 ( PARP-1)∕apoptosis-inducing factor ( AIF) pathway during oxygen-glucose deprivation (OGD)-induced damage to hippocampal neurons in neonatal rats. Methods Primarily cultured hippocam-pal neurons of healthy Sprague-Dawley rats born within 24 h were isolated and cultured. Hippocampal neu-rons were divided into 4 groups (n = 40 each) using a random number table: control group (group C), OGD group ( group O), negative control lentivirus group ( group NL) and Iduna protein overexpression group (group IO). OGD was induced by incubating the neurons in glucose-free medium and hypoxic envi-ronment. Negative control lentivirus and recombinant lentivirus overexpressing Iduna were added at 48 h be-fore establishing the model in NL and IO groups, respectively. Neurons were cultured in the normal culture medium for 24 h in group C. The cell survival rate was detected using methyl thiazolyl tetrazolium assay, the lactic dehydrogenase (LDH) leakage rate was measured using colorimetric method, the comet assay was used to detect DNA content in the tail of neurons, and the expression of PARP-1, cytochrome C (Cyt c) and AIF in the nucleus was detecteded by Western blot. Results Compared with group C, the survival rate of neurons was significantly decreased, the LDH leakage rate and DNA content in the tail of neurons were increased, and the expression of PARP-1, Cyt c and AIF was up-regulated in the other three groups (P<0. 05). Compared with group O, the survival rate of neurons was significantly increased, the LDH leakage rate and the DNA content in the tail of neurons was decreased, and the expression of PARP-1, Cyt c and AIF was down-regulated in group IO (P<0. 05), and no significant change was found in group NL (P>0. 05). Compared with the group NL, the survival rate of neurons was significantly increased, and the LDH leakage rate and DNA content in the tail of neurons were decreased, and the expression of PARP-1, AIF and Cyt c was down-regulated in the group IO (P<0. 05). Conclusion Iduna protein overexpression reduces OGD-induced damage to hippocampal neurons through inhibiting PARP-1∕AIF pathway in neonatal rats.
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Objective To study the correlation of the prognosis of Icotinib administration with the expression levels of F-box and WD repeat domain-containing 7(FBW7) and myeloid cell leukemia-1 (MCL-1) in peripheral blood in elderly patients with advanced non-small-cell lung cancer.Methods A total of 76 patients aged 60 years or over diagnosed with non-small-cell lung cancer(NSCLC) with EGFR-sensitive mutations and under Icotinib treatment were enrolled in this study.FBW7 and MCL-1 mRNA expression levels in peripheral blood were detected by real-time quantitative PCR(RT-QPCR).The correlation of FBW7 and MCL-1 expression levels with clinical and histological parameters,overall survival (OS),and progression-free-survival (PFS) was analyzed.Results The FBW7 expression level and the MCL-1 expression level were negative correlated(r =-0.37,P ＜0.001).High FBW7 expression levels and low MCL-1 expression levels in peripheral blood were associated with improved therapeutic efficacy of Icotinib (P＜0.001) and extended OS and PFS.Cox regression analysis showed that the expression levels of FBW7 and MCL-1 in peripheral blood were independent influencing factors for OS and PFS.Conclusions Patients with high FBW7 expression levels and low MCl-1 expression levels are more likely to benefit from Icotinib treatment.Expression levels for either factor can be used as a predictive indicator for the effectiveness of Icotinib and provide guidance for its clinical use.
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Objective To investigate the mechanism of Smad ubiquitination-related factor 2 (Smurf2)neddylation. Methods The Smurf2 protein level was tested by overexpression of Nedd8,while the method of immunoprecipitation(IP) and Western blotting were used to analyz Smurf2-Nedd8 modification.The GST-pulldown experiment was conducted to prove protein interactions.The protein was obtained by grinding mouse tissue and Western blotting was used to test the protein expression level.Results Over expression of Nedd8 could lead to the down regulation of the Smurf2′s protein level.Smurf1 and Smurf2 could interact in the GST-pulldown experiment. Smurf1 could enhance Smurf2-Nedd8 modification.The Smurf2′s protein level was up-regulated in mouse tissue of Smurf1 C426A.Conclusion There is some relationship and also difference between Smurf1 and Smurf2.Smurf1 can enhance Smurf2-Nedd8 modification.
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Objective To investigate the relationship between single nucleotide polymorphism (SNP) of BARD 1 gene and BRCA1 gene in epithelial ovarian cancer (EOC).Methods Nineteen EOC patients with BRCA1 gene mutation and 50 EOC cases without BRCA1 gene mutation between January 2016 and October 2016 were collected,and all EOC were diagnosed by pathological method.BARD1 gene variants were detected by next generation sequencing (NGS).The SNP of BARD1 gene was analyzed by Pearson linear correlation.Logistic regression analysis was used to research the clinicopathologic features and BRCA1 gene mutation associated with BARD1 gene SNP.Pearson's chi-square test was used to analyze the association between BARD1 gene Val507Met,Arg378Ser and Pro24Ser with different clinicopathologic features and BRCA1 gene mutation risk.Results (1) Eight BARD1 gene variants were found in 69 ovarian cancer patients,in which Val507Met,Arg378Ser and Pro24Ser were common variants,and the rate of mutation were all 54％ (37/69).(2) There was a significant linear correlation among Val507Met,Arg378Ser and Pro24Ser (all P＜0.01).(3) Obvious differences were found in Val507Met,Arg378Ser and Pro24Ser of BARD1 gene between BRCA1+ and BRCA1 (all P＜0.05).(4) No differences were found between BARD1 gene Val507Met,Arg378Ser and Pro24Ser and the clinicopathologic features (all P＞0.05),while obvious differences were found in BRCA1 gene mutation compared to the controls group.The risk of BRCA1 mutation in Val507Met and Arg378Ser were more evident in subjects with negative family history,positive menopause history,negative tubal ligation,onset age (≤60 years old) and sensitivity to platinum-based chemotherapy in EOC (all P＜0.05),while Pro24Ser was only more evident in positive menopause history of EOC (P＜0.05).Conclusions BARD1 Val507Met,Arg378Ser and Pro24Ser are the common genotypes,which are associated with BRCA1 mutation in EOC.The family history,menopause history,tubal ligation,onset age and sensitivity to platinum-based chemotherapy have effects on BARD1 SNP in the risk of BRCA1 gene mutation.
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Objective To investigate the correlation between RNF213 gene polymorphisms (rs112735431,rs138130613,and rs148731719) and the susceptibility of intracranial vascular stenosis disease.Methods The literature of studies on the correlation between RNF213 gene polymorphisms and intracranial vascular stenosis disease was collected according to the related databases.Using the Stata 12.0 software and selecting suitable genetic model,the heterogeneity was analyzed and the pooled odds ratio (OR) and its 95％ confidence interval (CI) were calculated.Results A total of 12 articles were included after screening.The result of meta-analysis showed that the rs112735431 polymorphism had a significant correlation with the susceptibility of moyamoya disease (MMD) in all genetic models,especially the most significant dominant model (AA + GA genotype vs.GG genotype:OR 101.46,95％ CI 59.41-173.27;P ＜0.001),at the same time,the polymorphism of this site also had significant correlation with the nonMMD intracranial large artery stenosis/occlusion (AA + GA genotype vs.GG genotype:OR 13.82,95％ CI 4.48-42.61;P＜ 0.001);the rs138130613 polymorphism had significant correlation with the susceptibility of MMD in Chinese population (OR 5.01,95％ CI 1.57-15.98;P=0.006);and no correlation between the rs148731719 polymorphism and the susceptibility of MMD was observed.Conclusions The RNF213 gene rs112735431 polymorphism is a susceptible factor of MMD,at the same time,the polymorphism of this site is also associated with the formation of non-MMD intracranial large artery stenosis.Systematic study on the molecular function of RNF213 may have important significance for diagnosis and treatment of such vascular stenosis diseases.
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Objective To construct a eukaryotic expression plasmid vector encoding Cbl-b gene-specific short hairpin RNAs (shRNAs),and to evaluate its interference effect,so as to lay a foundation for further study on the role of Cbl-b in the immunotherapy of malignant melanoma.Methods According to the sequence of Cbl-b cDNA,4 pairs of shRNAs targeting the Cbl-b gene were designed and synthesized,and then inserted into the plasmid PGPU6/GFP/Neo to construct recombinant plasmids.After identification by DNA sequencing,the 4 shRNA expression vectors were cotransfected into 293T cells with the Cbl-b gene eukarytic expresson plasmid,respectively.The knockdown efficiency of these shRNA expression plasmids on Cbl-b expression was evaluated by real-time (RT) fluorescence-based quantitative PCR and Western blot at 48 hours aftert transfection.Results Sequencing analysis revealed that all the 4 pairs of shRNAs were successfully inserted into the eukarytic expression vector PGPU6/GFP/Neo.As RT-PCR and Western blot showed,all the 4 shRNA-expressing vectors could downregulate Cbl-b expession,and the NO.1 shRNA-expressing vector displayed the strongest interference effect(P ＜ 0.05).Conclusions A eukaryotic expression plasmid vector was successfully constructed for Cbl-b gene-specific shRNAs,and the most effective shRNA was selected in this study.
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Objective To investigate the SENP1 and c-myb gene expression and their correlations in bone marrow specimens in the patients with acute lymphoblastic leukemia (ALL ) to provide the basis for expounding the role ,mechanism and prognosis of SENP1 and c-myb in ALL .Methods 31 patients diagnosed with ALL (22 cases of B-ALL ,1 case of T-ALL and 8 cases of uncate-gorized ALL ;6 cases in the low/medium risk group ,25 cases in the high risk group) and 31 patients with proliferative bone marrow and hyperplastic anemia diagnosed by the morphology were taken as the control group .The real-time PCR and immunocytochemical staining(SP method) were adopted to detect the mRNA and protein expressions of SENP1 and c-myb in the bone marrow specimens of the ALL patients and the control group .Results The expression of SENP1 and c-myb were both increased in the bone marrow specimens and smears of ALL patients ,which showed the statistical difference compared with the control group (P< 0 .05) ,the Pearson correlation analysis found that the high expression of SENP1 and c-myb had correlation .The expression of SENP1and c-myb in the low/medium risk group were lower than that in the high risk group ,but the difference had no statistical significance . Conclusion The high expression of SENP1 and c-myb exists in the bone marrow specimens of the ALL patients ,SENP1 and c-myb could possibly have the correlation with the occurrence and development of ALL ;but now the differences of SENP1 and c-myb ex-pression among different risk groups of ALL patients are yet to be proven .
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A hallmark of group/species A rotavirus (RVA) replication in MA-104 cells is the logarithmic increase in viral mRNAs that occurs four-12 h post-infection. Viral protein synthesis typically lags closely behind mRNA synthesis but continues after mRNA levels plateau. However, RVA non-structural protein 1 (NSP1) is present at very low levels throughout viral replication despite showing robust protein synthesis. NSP1 has the contrasting properties of being susceptible to proteasomal degradation, but being stabilised against proteasomal degradation by viral proteins and/or viral mRNAs. We aimed to determine the kinetics of the accumulation and intracellular distribution of NSP1 in MA-104 cells infected with rhesus rotavirus (RRV). NSP1 preferentially localises to the perinuclear region of the cytoplasm of infected cells, forming abundant granules that are heterogeneous in size. Late in infection, large NSP1 granules predominate, coincident with a shift from low to high NSP1 expression levels. Our results indicate that rotavirus NSP1 is a late viral protein in MA-104 cells infected with RRV, presumably as a result of altered protein turnover.
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Animals , Guinea Pigs , Capsid Proteins/metabolism , Gene Expression Regulation, Viral , Rotavirus/metabolism , Viral Nonstructural Proteins/metabolism , Cell Line , RNA, Viral/genetics , Rotavirus/physiology , Virus ReplicationABSTRACT
Pirh2 (p53-induced protein with a Ring-H2 domain),one of the ubiquitin-protein E3 ligases,can promote p53 degradation via ubiquitin-proteasome pathway,and repress the biological functions of p53.Researchers have found that the Pirh2 is overexpressed in many cancers,which suggests Pirh2 is probably correlated with tumorigenesis and cancer development,and it may become a promising cancer therapeutic target.
ABSTRACT
Background : The MIB-1 labeling index (LI) has proved to be useful in assigning grading and prognosis to astrocytomas. The purpose of our study was to analyze the utility of MIB-1 LI in differentiating astrocytomas of varying grades and the possible relationships of MIB-1 LI with clinical parameters like age and sex. We also wanted to study the prognostic role of MIB-1 index in predicting behavior of astrocytomas. Materials and Methods : Our study included 145 patients with astrocytic tumors of varying grades. Immunolabeling for all patients was done using MIB-1 antibody. Survival data could be obtained for 64 patients. A Mann-Whitney U test was used to test the difference in MIB-1 LI between different histological grades. The univariate analysis was done by the Kaplan-Meier method, and the multivariate analysis for survival was performed using the Cox proportional hazard model. Results : Significant differences were noted in mean MIB-1 LI of high-grade and low-grade diffuse astrocytomas. MIB-1 LI did not vary significantly with age and sex. Univariate analysis showed favorable prognostic factors for low histopathological grade, young patient age and low MIB-1 LI; however, multivariate analysis showed that only histopathological grade had independent prognostic significance. Conclusions : Our study proves that MIB-1 LI is not dependent on factors like age and sex and is solely dependent on histological grade. Though the average level of MIB-1 LI varies considerably in the different grades of astrocytomas, considerable overlap can be observed between them. MIB-1 LI is a very useful adjunct to the histopathological diagnosis and can be of great help in situations where the clinical and radiological findings do not correlate with histological diagnosis.