【作者】 李素云；

【导师】 贺修胜；

【作者基本信息】 南华大学 ， 基础医学（病理学与病理生理学）， 2015， 博士

【摘要】 [目的]运用生物信息学预测STGC3基因启动子,克隆并鉴定STGC3启动子;在此基础上,预测STGC3基因转录调控元件,分析并证实STGC3基因转录调控元件;进一步探讨STGC3基因相关转录因子对STGC3基因的转录调控作用。定点突变STGC3基因结构位点,通过检测基因突变后对鼻咽癌细胞生物学行为的影响,探讨STGC3基因发挥抑瘤作用的功能性位点,本研究为阐明STGC3基因的结构、功能及转录调控机制提供重要的实验依据。[方法]采用生物信息学预测并分析STGC3基因启动子区,克隆最有可能的启动子,构建双荧光素酶报告基因质粒,质粒经酶和测序鉴定,经脂质体瞬转至细胞,通过报告基因表达产物双荧光素酶活性检测,以鉴定STGC3基因启动子区。在启动子鉴定基础上,预测STGC3基因核心启动子区转录因子特异性结合位点,采用电泳迁移率变动分析法(EMSA)及染色质免疫共沉淀分析(Ch IP)法,验证转录因子特异性结合位点,从而明确该基因转录调控元件。采用RT-PCR和Western blot检测STGC3基因高表达的NP69细胞系和STGC3基因低表达的CNE2细胞系中Sp1的表达水平,然后运用RNAi技术干扰细胞中Sp1的表达,通过q RT-PCR和Western blot,分析Sp1低表达后对STGC3基因的影响,以验证转录因子Sp1的转录调控作用。针对STGC3基因糖基化位点(656 C)、蛋白激酶C磷酸化位点(725 C)和酪蛋白激酶Ⅱ磷酸化位点(913 T),3个可能性功能位点,运用Stratagene定点突变技术,对STGC3蛋白糖基化位点、蛋白激酶C磷酸化位点及酪蛋白激酶Ⅱ磷酸化位点分别进行单碱基定点突变,使656 C突变为G,725 C突变为T,913 T突变为G。构建带His标签的突变重组真核表达质粒,质粒经转化、抽提、酶切及测序鉴定,转染至鼻咽癌CNE2细胞系。通过RT-PCR和Western blot及免疫细胞化学从m RNA和蛋白质水平检测,得到稳定表达突变STGC3基因的CNE2细胞系。通过MTT和胎盼蓝染色细胞计数法检测STGC3基因突变对CNE2细胞生长增殖影响,采用流式细胞术检测STGC3基因突变对CNE2细胞周期以及凋亡影响,采用Western blot对凋亡相关蛋白Bax的影响,以明确STGC3基因的抑瘤功能位点。[结果]生物信息学结果显示,STGC3基因5’端上游的-3046bp至-46bp区域有启动子活性,在-2992bp至-69bp间启动子分值达0.8以上,其中-845bp至-795bp间分值最高,达到1.0。在-1140bp和-774bp处检测到GC盒信号,在-441bp处检测到CAAT盒信号。有两个Cp G岛:分别在-1805bp至-1705bp和-900bp至-684bp间;有两个转录起始位点:分别在-2348bp和-948bp处。经不同长度的片段缺失比对,取最有可能的283bp(-1360bp至-1077bp)、281 bp(-934bp至-653bp)和571 bp(-500bp至+72bp)启动子片段,构建p GL3-en283、p GL3-en281、p GL3-en571三种质粒,与阴性对照p GL3-enhance质粒相比,三种质粒均具有较强的启动子活性,三种质粒中以p GL3-en281质粒的启动子活性最强。认为-934bp至-653bp为STGC3基因核心启动子区。生物信息学结果显示,在STGC3基因核心启动子区-934bp至-653bp区域存在21个转录因子结合位点,其中9个为Sp1转录因子结合位点;当严格限定参数后,结果显示,基因核心启动子区域含有5个转录因子结合位点,其中Sp1转录因子结合位点2个。经EMSA和Ch IP实验证实,结果显示,STGC3基因中存在转录因子Sp1特异性结合位点,从而鉴定出STGC3基因转录调控元件。RT-PCR技术检测结果显示CNE2细胞组(1.12±0.05)中,转录因子Sp1在m RNA水平上,表达较NP69细胞组(0.33±0.02)明显升高,差异有统计学意义(P<0.01)。Western blot结果显示,CNE2细胞组(2.67±0.28)中转录因子Sp1在蛋白水平上,表达高于NP69细胞组(1.17±0.17),差异具有统计学意义(P<0.05)。说明转录因子Sp1有可能负调控STGC3基因。在此基础上,构建3个Sp1-sh RNA干扰质粒,经酶切及测序鉴定,结果显示,片段大小一致,序列正确,表明成功构建了Sp1干扰载体。Sp1-sh RNA干扰载体,经q RT-PCR及western blot筛选。筛选出干扰效率高的Sp1-sh RNA载体,转染至Sp1高表达的CNE2细胞系,q RT-PCR及western blot检测STGC3基因表达,结果显示CNE2细胞系中,干扰Sp1表达后,STGC3表达增高。突变质粒经酶切和测序鉴定,酶切结果显示目的片段大小一致,测序结果显示序列正确,成功构建三个突变型(His-STGC3-C656G、His-STGC3-C725T和His-STGC3-T913G)真核表达质粒。转染至鼻咽癌CNE2细胞系,突变质粒经RT-PCR、Western Blot及免疫细胞化学结果显示,突变质粒均表达STGC3基因m RNA和His-tag-STGC3融合蛋白质,MTT和胎盼蓝染色细胞计数生长曲线结果表明,His-STGC3-C656G和His-STGC3-C725T突变组对细胞的生长增殖抑制能力降低,与野生型组比较,差异有统计学意义(P<0.05);His-STGC3-T913G突变组不影响细胞生长增殖能力。流式细胞术检测结果显示,His-STGC3-C656G组(1.20%±0.13%)和His-STGC3-C725T组(1.50%±0.26%),细胞凋亡率降低,与His-STGC3组,差异有统计学意义(P<0.05),三个突变组对细胞生长周期无影响。Western Blot对凋亡相关蛋白Bax检测,结果显示,His-STGC3-C656G和His-STGC3-C725T突变组Bax蛋白表达降低,与野生型His-STGC3组比较,差异有统计学意义(P<0.05)。[结论](1)-2992bp至-69bp为STGC3基因的启动子区,-934b至-653bp为其核心启动子区;(2)STGC3基因启动子区内有Sp1特异性结合位点,Sp1对STGC3基因表达起负调控作用;(3)STGC3基因656位C和725位C,是其重要功能性位点。更多还原

【Abstract】 [ 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.更多还原