ABSTRACT
Drug-induced long QT syndrome (LQTS) has become an important clinical research topic, and the occurrence of acquired long QT syndrome (acLQTS) is mainly caused by drug inhibition of the human ether-α-go-go related gene (hERG) channel. The hERG gene encodes the α subunit of the fast-activating delayed rectifying potassium ion channel (Ikr), which plays an important role in the process of action potential phase 3 repolarization and is also the target of most antiarrhythmic drugs. The purpose of this study was to investigate the effect of hydroxyrutaecarpine (HRU) on the hERG channel and to evaluate its cardiotoxicity. The whole cell patch clamp technique was used to detect the effects of HRU on the current and kinetics of the hERG channel, and to confirm the binding site on the hERG channel. PCR was used to determine the effect of HRU on hERG mRNA expression. Western blotting was used to detect the effects of HRU on the expression of hERG protein and transcription factor Sp1. Immunofluorescence was used to confirm the effects of HRU on localization and expression of hERG protein and transcription factor Sp1. Studies have shown that transient HRU can inhibit hERG current and shorten the inactivation time constant. Its binding sites to the hERG channel are F656 and Y652. After incubation for 24 h, HRU can reduce the expression of hERG protein, inhibit the hERG current, reduce the level of hERG mRNA, and reduce the expression of transcription factor Sp1 in the nucleus and hERG protein in the cytoplasm. Immunofluorescence experiments also showed the same results suggesting that the inhibition of Sp1 expression by HRU is the cause of the decreased expression of hERG mRNA. In conclusion, the acute inhibition of HRU accelerates the channel inactivation process and reduces the inactivation time constant by binding to the F656 and Y652 sites in the hERG channel, thus reducing the hERG current. In addition, HRU also inhibits the expression of hERG protein, mainly by inhibiting the expression of transcription factor Sp1, the transcription function of hERG channel protein is down-regulated, so that the hERG protein is reduced.
ABSTRACT
Objective To identify the expression of transcription factor Sp1 in NK/T-cell lymphoma (NK/TCL) cell lines and to investigate the role of Sp1 in regulation of cell invasion. Methods Real-time PCR, immunofluorescence and Western blot were performed to detect the expression of Sp1 in NK/TCL cell lines SNK-1 and SNK-6 and normal NK cells. Expression levels of IGF-1R and MMP-2 were measured by real-time PCR and Western blot, respectively. Transwell assay was applied to observe the effects of mythramycin A(MIT) on cell invasion. Results Sp1 expression in mRNA and protein were over-expression in NK/TCL cell lines SNK-1 and SNK-6 when compared with normal NK cells. Inhibition of Sp1 by MIT remarkably reduced expression of IGF-1R and MMP-2 in SNK-1, SNK-6 and as a result, or significantly suppressed cell invasion. Expression levels of Sp1 mRNA in SNK-1 and SNK-6 were (9.4±0.3) and (10.6±0.3) foldsincrease as compared with that of control group, respectively (P=0.005 2, P=0.003 7). Levels of Sp1 protein were (5.4±0.3) and (8.6±0.5) foldsincrease times than control groups, respectively (P=0.008 3, P=0.006 9). Inhibition of Sp1 by MIT (100 nmol/L) remarkably reduced expression levels of IGF-1R mRNA by (83.9±3.7) % and (65.8±4.2) % (P = 0.008 2, P = 0.009 7) as compared with controls. Meanwhile, levels of IGF-1R protein were reduced by (51.5±7.1) % and (49.6±9.1) % (P = 0.017 8, P = 0.015 5) as compared with control group. Inhibition of Sp1 by MIT (100 nmol/L) significantly reduced cell invasion and MMP-2 expression in the two cell lines,the cell invasion rates were reduced by (29.6±6.4) % and (37.2±7.6) % (P =0.041 8, P = 0.037 2) in SNK-1 and SNK-6 as compared with control group. The MMP-2 protein levels were found to be (52.7±4.7) % and (29.7±5.6) % (P = 0.028 6, P = 0.020 2) of control group. Conclusion Sp1 is over-expressed in NK/TCL cell lines, and it promotes NK/TCL cell invasion by up-regulating IGF-1R and further increasing MMP-2 expression.
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Objective: To explore the clinical significance of transcription factor Sp1 expression in hepatocellular carcinoma (HCC) and association between Sp1 expression and survival in HCC patients. Methods:With the use of immunoreactivity, Sp1 expres-sion and its correlation with other clinicopathological characteristics were examined in a tissue microarray that contains samples from 98 HCC patients. Results:HCC tissues expressed markedly higher levels of Sp1 than did adjacent normal liver tissues. Sp1 expression was closely associated with microvascular invasion, which suggests that HCC with more microvascular invasion is prone to have in-creased Sp1 expression. Overexpression of Sp1 correlates with significantly shorter overall survival and higher recurrence rates in HCC patients after curative resection. Conclusion:Transcription factor Sp1 may be an independent prognostic factor for both overall surviv-al and cumulative recurrence rate.
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Objective To investigate whether porcine endothelial cells transfected with SP1 de-coy ODNs could resist complement-mediated cytotoxicity during the model of SV-40-PED with human serum in vitro. Methods Immortalized porcine aortic endothelial cells of the line PED were divided in-to three groups. The transfected group was SV-40-PED with SP1 decoy ODNs. The mismatched group was SV-40-PED with scrambled SP1 decoy ODNs. The negative group was cells with oligo-fectamine only. The expression of α1,3-GT mRNA and αGal was detected after 26 h by using fluores-cence microscope, Western blot, RT-PCR and lactate dehydrogenase (LDH) activity assay. Results Fluorescence microscopy observed the decreased fluorescence of αGal after SP1 decoy ODNs transfec-tion. Dotted fluorescent decrease could be observed on some membrane while the mismatched group and negative group with bright green fluorescence. Western blot showed that the average absorbance of the PED cells transfected with decoy ODNs was decreased to 52.6% of the negative group (P<0.05). The expression of α1,3-GT mRNA in the PED cells transfected with decoy ODNs was 0.42±0.20 (isoform 1) and 0.27±0.12 (isoform 2) respectively, significantly lower than in the negative group (isoform 1, 0.72±0.17; isoform 2, 0.50±0.19; both P<0.05). The expression of α1, 3-G Tin the mismatch group was not different from that in the negative group (P>0.05). 20% normal hu-man serum (NHS) and 40 % NHS had cytotoxic effect on both mismatch and negative groups, but de-coy ODNs could confer SV-40-PED protection from the cytolysis effect (P<0.05), which made a re-markable reduction of complement-mediated cytotoxicity towards SV-40--PED. Conclusions SP1 decoy ODNs could confer porcine endothelial cells protection from complement-mediated cytotoxicity effect in vitro.
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LCRG1(laryngeal carcinoma related gene1,LCRG1),a new candidate tumor suppressor gene of laryngeal carcinoma.However,it is known little about the possible regulatory mechanisms of LCRG1 gene expression.Restriction endonuclease digestion was used to obtain a set of the 5',or 3'deletion mutants from the region(-169～+127) of the LCRG1 gene.It has been found that the minimal promoter of the LCRG1 gene is mapped at the region from-169～-57.Linker scanning mutational analysis in the region(-169～+127) of the LCRG1 gene was used to identify the crucial cis-elements within the promoter region,The key cis-elements are within the region from-137～-122.SP1,E2F1/DP1,EKLF and ZF9 transcription factor binding site sites were predicted in the region by bioinformatics analysis.Co-transfection with each of a panel of the expression plasmids of the known transcription factors with the relevant reporter construct indicates Sp1 is potent transcription factor for enhancement of the promoter activity,SP1 can also up-regulate the endogenous expression of LCRG1 gene.Electrophoretic mobility shift assay(EMSA) was applied to verify that the key cis-elements of LCRG1 gene exist sequence of Sp1 binding sites.The findings,which showed that the key cis-elements within the region from 137～-122 play an important role in expression of the LCRG1 gene,provide a novel evidence for further study of the function of LCRG1 gene.
ABSTRACT
AIM: To identify the non-steroid transcription factors upregulating the expression of L-plastin in hormone-independent prostate cancer, and partly elucidate the mechanism of hormone-refractory prostate cancer. METHODS: TF SEARCH software was used to analysis the possible binding sites of transcription factors in the 3’ end of L-plastin promoter that had been identified as important part of regulation response elements. Gel shift assay and supershift assay were used to confirm the transcription factors binding the speculated response elements. PCR site-mutagenesis technique was performed to delete the binding site of transcription factor and luciferase activity assay was carried out after deletion of the binding site. RESULTS: SP-1 respond element GGTGGGGCGGGGA located at -54- -41 of L-plastin promoter was identified with the TF SEARCH software. Gel shift assay and supershift assay confirmed that SP-1 was the transcription factor binding to GGTGGGGCGGGGA. Mutant deleted the SP-1 binding-site had low-luciferase activity than that of the naive. CONCLUSION: SP-1 plays an important role in the up-regulation of L-plastin expression in hormone-independent prostate cancer.