摘要:目的: 探讨激活转录因子(ATF1)在血管紧张素Ⅱ(AngⅡ)诱导血管平滑肌细胞(VSMCs)中NOX1基因表达增加的作用。 方法 :体外培养大鼠主动脉VSMCs,用荧光实时定量逆转录PCR(Realtime RTPCR)检测NOX1基因表达的量,Western Blot检测ATF1蛋白在AngⅡ的刺激是否引起NOX1基因的高表达并用RNA干扰(RNAi)技术转染VSMCs使ATF1基因沉默来观察NOX1的表达。 结果 :AngⅡ能够诱导 NOX1基因的表达增加以及增强ATF1的磷酸化及活性,ATF1基因沉默反过来可抑制AngⅡ诱导的NOX1基因表达的增加。 结论 :在大鼠的VSMCs中,ATF1是介导NOX1基因表达的一个必须的转录因子。Abstract: Objective: To detect the role of activating transcription factor (ATF1) involved in angiotensinⅡ(AngⅡ) stimulated NOX1 gene expression.Methods :Rat aortic vascellum smooth muscle cells(VSMCs) were cultured in vitro.Use Realtime RTPCR to measure the expression of NOX1 gene.Western Blot Analysis was carried out to test the activity of ATF1 protein. RNA interference was used and transfected into VSMCs to knockdown ATF1 gene expression, and then measured NOX1 gene expression.Results : AngⅡ stimulated NOX1 gene expression and phosphorylation of ATF1 Gene silencing of ATF1 attenuated the upregulation of NOX1 mRNA by AngⅡ. Conclusion :ATF1 is an essential transcription factor that mediates expression of NOX1 gene in VSMCs by AngⅡ.
ObjectiveTo detect the expression of Krüppel like factor 8 (KLF8) in breast cancer tissues and cells and to explore the clinical significance of KLF8.Methods① The Oncomine database was used to analyze the differential expression of KLF8 mRNA in the breast cancer tissues. The Kaplan-Meier Plotter database was used to analyze the relationship between KLF8 mRNA expression and prognosis (relapse free survival, overall survival, post-progression survival, and distant metastasis-free survival) of patients with breast cancer. ② The quantitative real-time PCR (qRT-PCR) and Western blot were used to detect the KLF8 expression levels in the 16 clinical patients with breast cancer and 7 breast cancer cell lines (MDA-MB-231, MCF-12A, Hs-578T, MCF-7, BT-474, MDA-MB-453, ZR-75-30) and normal breast epithelial cell lines MCF-10A, and the immunofluorescence was used to further detect the localization of KLF8 expression in the 2 breast cancer cell lines with higher KLF8 expression level. ③ The immunohistochemistry was used to detect the expression of KLF8 protein in 135 cases of breast cancer tissue microarrays, and the relationships between KLF8 protein expression and clinicopathologic characteristics or overall survival were analyzed.Results① The Oncomine database showed that KLF8 mRNA expression in the breast cancer tissues was higher than that in the normal breast tissues (P<0.001). The median KLF8 mRNA expression level was taken as the cut-off point for high or low KLF8 expression. The results of Kaplan-Meier Plotter data analysis showed that the prognosis (relapse free survival, overall survival, postprogression survival, and distant metastasis-free survival) of patients with low KLF8 mRNA expression were better than those of patients with high KLF8 mRNA expression (P<0.05). ② The results of qRT-PCR and Western blot all showed that the KLF8 mRNA and protein expression levels in the breast cancer tissues were higher than those in the adjacent normal tissues (P=0.002, P<0.001). In addition, the Western blot results showed that the expression of KLF8 protein in the 7 breast cancer cell lines was higher than that in the normal breast epithelial cell lines MCF-10A respectively, and KLF8 protein mainly expressed in the cytoplasm of breast cancer cells and highly expressed in the nuclear of a few cells. ③ There were 63 cases of high KLF8 expression and 72 cases of low KLF8 expression by the immunohistochemical analysis of 135 patients with breast cancer tissue microarray (the H-score of the immunohistochemical test results was 75 as the cut-off point, H-score >75 was the high KLF8 expression and H-score ≤75 was the low KLF8 expression), the differences of statuses of estrogen receptor (ER) and progesterone receptor (PR) between the patient with high KLF8 expression and low KLF8 expression were significant (P<0.05). The Kaplan-Meier survival curve analysis showed that the prognosis of patients with high KLF8 expression was worse than that of patients with low KLF8 expression (P=0.002). The univariate analysis showed that the TNM stage, statuses of ER and PR, and KLF8 expression were related to the prognosis of patients with breast cancer (P<0.05), further multivariate Cox proportional hazards regression analysis indicated that the later stage of TNM and high KLF8 expression were the independent risk factors (P<0.05).ConclusionsThe results of this study suggest that KLF8 highly expresses in both breast cancer tissues and breast cancer cells, which is related to the statuses of ER and PR and prognosis of patients with breast cancer. KLF8 might be involved in the progression of breast cancer as an oncogenic gene, or it might provide a new direction for prognosis judgment and molecular targeted therapy of breast cancer.
Objective To investigate the inhibitive effect of E2F decoy oligodeoxynucleotides (E2F decoy ODNs) on cultured human retinal pigment epithelial (HRPE) cells.Methods E2F decoy ODNs or scramble decoy ODNs at varied concentrations were put into the HRPE cells mediated by lipofectamineTM2000. The proliferative activity of HRPE was detected by methythiazolyl-terazollium assay, and the competitive combinative activity of E2F decoy ODNs and transcription factor E2F was detected by electrophoresis mobility-shift assay. Results The proliferation of HRPE was inhibited markedly by E2F decoy ODNs at the concentration of 0.2 μmol/L (P=0.002) in a dose-dependent manner but not by scrambled decoy. The results of electrophoresis mobility-shift assay showed that the combinative activity of transcription factor E2F was abolished completely by E2F decoy ODNs. Conclusions E2F decoy ODNs may sequence-specifically inhibit the combinative activity of transcripti on factor E2F,and inhibit the proliferation of HRPE cells.(Chin J Ocul Fundus Dis，2004，20：182-185)
Objective To elucidate the role of the transcription factor liver activator protein (LAP, a member of the C/EBP family) in the expression of α1(I) collagen gene in activated hepatic stellate cells (HSCs). Methods Rat HSCs were prepared from SD rats by in situ perfusion and singlestep density Nycodenz gradient. Two chimeric luciferase reporter gene plasmids containing the human collagen α1(I) gene promoter fragments (-804～+1 452 or -804～+222) were constructed. Culture-activated HSCs were co-transfected with the reporter gene contructs and mammalian vector expressing LAP using the cationic-liposome mediated method, and the promoter activity was determined by measuring luciferase activity. Results The luciferase reporter gene construct containing the first intron of α1(I) collagen gene (-804～+1 452, was called as PGL3-col) had a higher level of gene expression, as compared with the construct lacking the first intron 〔was called as PGL3-col (△intron)-in activated HSCs (315±45 U/mg protein vs 220±70 U/mg protein, Ｐ＜0.05). Transient transfection of the vector expressing LAP significantly increased basal transcription from PGL3-col and PGL3-col (△intron) reporter gene vectors (587±62 U/mg protein vs 315±45 U/mg protein and 326±52 U/mg protein vs 220±70 U/mg protein respectively, both Ｐ＜0.05). Conclusion The transcription factor LAP transactivates collagen α1(I) gene in activated HSCs, and the first intron is important for α1(I) collagen gene transcription activity in activated HSCs.
Objective To investigate the expression of suppressor gene Runt-related transcription factor 3 (Runx3) in gastric carcinoma and its relationship with clinicopathologic parameters. Methods RT-PCR and Western blot were used to determine the mRNA expression and protein expression of Runx3 gene in primary tumor and corresponding normal tissues respectively in 52 patients with gastric carcinoma. The relationship between Runx3 expression and clinicopathologic parameters was analyzed. Results RT-PCR and Western blot analysis in 52 patients with gastric carcinoma showed down-regulation of Runx3 mRNA and Runx3 protein in 59.6% (31/52) and 48.1% （25/52） of the primary tumors tested, and in none of the normal tissues (P＜0.05) respectively. There was a significant negative correlation between the expression level of Runx3 gene and the clinicopathologic parameters such as tumor size, differentiation, infiltrative depth, lymph node metastasis and TNM stage (P＜0.05， P＜0.01). Runx3 gene transcription was coincident with its protein expression (r=0.840, P＜0.01）. Conclusion The expression of Runx3 gene is down-regulated in gastric carcinoma, which suggests that Runx3 gene plays an important role in carcinogenesis and the progression of gastric carcinoma. It may be a new target of diagnosis and treatment of gastric carcinoma.
Objective To explore the expression and activation of transcription factor E2F1 in cultured human retinal pigment epithelium (RPE) cells. Methods Cultured human RPE cells were divided into two groups after synchronization: one was cultured in Dulbecco′s modified Eagle′s medium (DMEM) without serum; the other was cultured in DMEM supplemented with 20% serum of newborn calf. The expressions of E2F1 protein in two groups were detected by Western blot analysis. The E2F1-DNA binding activities were measured by gel mobility-shift assay(EMSA). Results E2F1 protein of 60 000 molecular weight was detected in the nuclear extract of human RPE cells, and serum stimulation could increase its expression（P＜0.001）. EMSA exhibited the increased binding activity of E2F1 in the serum-stimulated RPE cells with DNA. Conclusions E2F1 is expressed in the nuclei of human RPE cells. Serum stimulation can increase its protein expression as well as binding activity, so as to play a regulation role of gene transcription. (Chin J Ocul Fundus Dis, 2002, 18: 224-226)
Objective To investigate the effects of simvastatin on lung tissue in septic rats by observing the protein expression of nuclear factor kappa B ( NF-κB) and pathologic changes in lung tissue at different time points. Methods 90 healthy male Sprague-Dawley rats were randomly divided into three groups ( n =30 in each group) . All the rats received administration by caudal vein and capacity volume is 2 mL. The rats in the control group were treated with saline ( 2 mL) . The rats in the LPS group were treated with LPS ( 5 mg/kg ) . The rats in the simvastatin group were treated with LPS ( 5 mg/kg) and simvastatin ( 20 mg/kg) . Six rats in each group were killed randomly at 2, 4, 6, and 12 hours after the injection, and the right middle lobe of lung was taken out. Pathological changes of lung tissue wee investigated under light microscope. The expression of NF-κB in lung tissue was determined by immunohistochemistry ( IHC) method. Results Microscopic studies showed that there were not pathological changes in the lung tissue of rats in the control group. While in the LPS group, the alveolar spaces were narrowed and the alveolar wall were thickened. Furthermore, severe interstitial edema of lung and proliferation of epithelial cells were observed. In the simvastatin group, the degree of the infiltration of leukocytes and the lung interstitial edema were less severe than those in the simvastatin group. In the control group, the expression of NF-κB protein in most of lung tissue was negative. In the LPS group, the expression of NF-κB protein was detected at 2h, andreached the peak at 6h, then decreased at 12h. In the Simvastatin group, the NF-κB expression was significantly lower than that in the LPS group at all time points ( P lt; 0. 01) . Conclusion Simvastatin can ameliorate pathological lesions and decrease expression of NF-κB in lung tissue of septic rats.
Objective To investigate the expression of transcriptional factors zinc finger Krüppel like transcription factor 2 ( Klf2) and NF-E2 related factor 2 ( Nrf2) /BTB CNC homology 1 ( Bach1) in rat bronchial epithelial cells stimulated by cigarette smoke extract ( CSE) , and explore the regulatingmechanism of γ-glutamylcysteine synthetase ( γ-GCS) expression in the oxidative condition. Methods Rat bronchial epithelial cells were harvested using enzyme digestion method, and intervened by 10% CSE for 6 hours. Then γ-GCS activity was detected by two enzymes method, and the nuclear transfer of Nrf2 /Bach1 in cells was detected by immunohistochemistry. Western blot and reverse transcription-polymerase chain reaction ( RT-PCR) techniques were used for detecting the protein and mRNA expressions of Klf2, Nrf2, Bach1, and γ-GCS in the cells. Results The γ-GCS activity was elevated in the CSE group. Immunohistochemical results showed that Nrf2 translocated from cytoplasm to nucleus in response to stimulation by CSE. On the contrary, Bach1 translocated from nucleus to cytoplasm in the same condition. Western blot results showed that protein levels of Klf2, Nrf2, Bach1, and γ-GCS were higher in the CSE group than those in the control group ( Plt;0.05) . RT-PCR results were the same as the Western blot results ( Plt;0.05) . Linear correlation analysis showed that γ-GCS expression was positively correlated with Klf2, Nrf2, and Bach1 ( Plt;0. 05) . Conclusion CSE might regulate the expression of γ-GCS through the transcription factors of Klf2, Nrf2, and Bach1.
Objective A series of bioinformatics methods were used to identify ferroptosis related biomarkers in lupus nephritis (LN). Methods We retrieved sequencing data of GSE112943 from the GEO (Gene Expression Omnibus) database and screened LN differentially expressed genes. We searched for ferroptosis-related gene (FRG) through FerrDb database, and screened LN-FRG. We conducted enrichment analysis on the LN-FRGs using David online bioinformatics database and screened the core LN-FRG using cytoHubba. We used external data sets to verify the core LN-FRGs, constructed competing endogenous RNA networks, and conducted molecular docking analysis. Results A total of 37 LN-FRGs were selected through screening. These genes are mainly enriched in inflammation, immune regulation and ferroptosis related signaling pathways. Through the cytoHubba and external dataset validation, the key core LN-FRG of ATF3 (activating transcription factor 3) was ultimately identified, and its expression was significantly increased in LN (P<0.05). Molecular docking analysis showed that ATF3 was closely bound to SLC7A11 and NRF2, and may participate in the occurrence and development of LN through the microRNA-27-ATF3 regulation axis. Conclusion The pivotal gene ATF3 may participate in the inflammation and immune injury of LN through ferroptosis.
Objective To investigate the expression of E26 transformation-specific-1（E26ts-1），matrix metalloproteinase-1（MMP-1）and tissue inhibitor of matrix metalloproteinase-1 （TIMP-1） in choroidal melanoma and the correlation with the tumorprime;s infiltration and metastasis. Methods Immunohistochemistry was used to detect the expression of E26ts-1，MMP-1 and TIMP-1 in 78 cases of choroidal melanoma who were divided into shuttle-cells，paraepithelial-cells and mixed-cells type according to the configuration of tumor cells.The patients were followed up and their average existing time was calculated.The results were statistically computed with statistic SPSS 10.0 package. Results In the 78 cases，shuttle-cells type was found in 21，paraepithelial-cells type in 34，and mixed-cells type in 23. Expression of TIMP-1was low in uveal melanoma，while expression of E26ts-1 and MMP-1 was obviously found in the three types of choroidal melanoma;the sequence of expression intensity was shuttle-cells，mixed-cells and paraepithelial-cells type.Among 37 cases who had been followed up，the shuttle-cells type was in 18 with the average existing time of （78.33plusmn;24.69）months，the mixed-cells type was in 10 with the average existing time of（61.44plusmn;20.46）months，and the paraepithelial-cells type was in 9 with the average existing time of（36.76plusmn;12.19）months.The existing time was negative correlated with the intensity of expresion of E26ts-1 and MMP-1. Conclusion The high expression of E26ts-1 and MMP-1and low expression of TIMP-1may relate to the choroidal melanomaprime;s infiltration and metastasis. (Chin J Ocul Fundus Dis, 2006, 22: 174-176)