| [ Objectives] Using bioinformatics to predict the promoter of STGC3 gene, then clone and identify it. calculating the transcriptional regulatory elements of STGC3 gene, while analyze and confirm it to investigate the transcriptional regulation function of Sp1 to STGC3 gene.The structrual sites of STGC3 gene were mutated by site-specific mutations of Stratagene, through detecting the effects of biological behavior of nasopharyngeal carcinoma cells(CNE2) after the gene mutation to explore the functional sites of STGC3 gene which play a role of tumor inhibition, This study will provide an important experimental basis for elucidating the structure, function and transcriptional regulation mechanism of STGC3 gene.[Methods] The bioinformatics was used to predict and analyze the promoter region of STGC3 gene, and the most likely promoters were cloned. We structured the dual-luciferase reporter gene plasmid and instant transfected it into cells by lipofictamine 2000, then the enzyme activity was identified to affirm the promoters of STGC3 gene.Based on promoter identification, the transcription factor specific binding sites of STGC3 gene core promoter region were predicted and verified by electrophoretic mobility analysis(EMSA) and chromatin immunoprecipitation assay(Ch IP) to clarify the transcriptional regulatory elements of STGC3 gene.The expression of Sp1 in NP69 cells with high expression of STGC3 gene and CNE2 cells with low expression of STGC3 gene were detected by RT-PCR and western blot. We knocked down of Sp1 in cells through RNAi,then the expression of STGC3 gene in cells were analyzed by q RT-PCR and Western blot to confirmate transcriptional regulation function of Sp1 to STGC3 gene.We mutated the STGC3 gene in glycosylation site(656 C), protein kinase C phosphorylation site(725 C), and casein kinase II phosphorylation site(913 T) with the method of site-specific mutagenesis.The recombinant eukaryotic expression plasmids with His tag were constructed and transfected into CNE2 cells. The CNE2 cells line with stable expression of STGC3 gene was obtained after detecting by RT-PCR, Western blot and immunohistochemistry.Then we detected the cells growth and proliferation by MTT and trypan blue staining. The cell cycle and apoptosis was detected by flow cytometry. The effect of protein Bax was detected by Western blot. The functional sites of STGC3 gene were determined finaly.[Results] The bioinformatics results showed that there are promoter activity from-3046 bp to-46 bp. The activity value of-2992 bp to-69 bp is 0.8 and-845 bp to-795 bp is 1.0. There is a GC box in-1140 bp to-774 bp and a CAAT box in-441 bp. Two Cp G islands were detected in- 1805 bp to- 1705 bp and- 900 bp to- 684 bp. Two transcription start sites(-2348 bp and-948bp) were found. We choiced the most possible fragments 283bp(-1360 bp to-1077bp), 281 b P(-934 bp to- 653bp) and 571 b P(- 500 bp to- 72bp) to constructe plasmids p GL3-en283, p GL3-en281 and p GL3-en571 and transfected them into cells. Compared with the negative control plasmid p GL3 enhance, The result showed that the plasmid p GL3-en281 was the one with strongest promoter activity in the three plasmids. It illustrated that-934 bp to-653 bp were the core promoter region of STGC3 gene.The bioinformatics results showed that there were twenty-one transcription factor binding sites in the core promoter region including nine transcription factor Sp1 binding sites. It revealed that there were only two transcription factor Sp1 binding sites in the core promoter after setting parameter. The Sp1 specific binding site in the STGC3 gene was confirmed by EMSA and Ch IP. We identified the transcription regulatory elements of STGC3 gene.The m RNA expression of Sp1 in CNE2 cells(1.12 ±0.05) was significantly higher than in NP69 cells(0.3 ± 0.02) by RT-PCR. Thedifference was statistically significant(P<0.01). The protein expression of Sp1 in CNE2 cells(2.67 ±0.28) was higher than in NP69 cells(1.17±0.17) by Western blot. The difference was statistically significant(P<0.05). This result indicated that Sp1 is likely to negatively regulated STGC3 gene. Then three different Sp1-sh RNA interference plasmids were successfully constructed. The Sp1-sh RNA vector with high interference efficiency was selected by q RT-PCR and Western blot, and then transfected it into CNE2 cell line. The expression of STGC3 gene was detected by q RT-PCR and western blot. The result showed the expression of STGC3 gene was increased in CNE2 cells after knocking down of Sp1.The three mutants plasmids His-STGC3-C656 G, His-STGC3-C725 T and His-STGC3-T913 G were successfully constructed and transfected into CNE2 cells. These mutant plasmids stably expressed STGC3 gene by RT-PCR,Western blot and immunocytochemistry. The effects of growth and proliferation were detected by MTT and trypan blue staining, and the result showed that the inhibition ability of His-STGC3-C656 G and His-STGC3-C725 T were significantly lower than that of wild type His-tag-STGC3. The difference was statistically significant(P<0.05). The apoptosis rate was examed by flow cytometry, and the result showed that His-STGC3-C656G(1.20% ± 0.13%) and His-STGC3-C725T(1.50% ±0.26%) were decreased comparing with the wild-type His-STGC3.Thedifference was statistically significant(P<0.05). The three groups had no effect on cell growth cycle. The Bax protein was detected by Western blot, and the result showed that the expression of Bax protein in His-STGC3-C656 G and His-STGC3-C725 T were decreased comparing with the wild-type His-STGC3. The difference was statistically significant(P<0.05).[Conclusion](1) There is promoter region of STGC3 gene in-2992 bp to- 69 bp, and the core promoter region of STGC3 gene is located in-934 b to-653 bp.(2) There are some transcription factor Sp1 specific binding sites in STGC3 gene core promoter region,and the transcription factor Sp1 negatively regulate STGC3 gene expression in nasopharyngeal carcinoma CNE2 cells.(3) The 656 C and 725 C are important functional sites in STGC3 gene for its negative regulation on cell growth. |
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