rs37973位点多态性对氟替卡松诱导的Raji和THP-1细胞中<i>GLCCI1</i>基因启动子活性影响

doi:10.3877/cma.j.issn.2095-1221.2022.06.005

目的

通过荧光素酶实验评价rs37973位点多态性对氟替卡松诱导条件下Raji和THP-1细胞中人糖皮质激素诱导转录因子1 （GLCCI1）基因启动子活性的影响。

方法

生物信息学预测并调取人GLCCI1基因启动子，构建rs37973-G和rs37973-A对应的荧光素酶报告基因表达载体并转染细胞，转染后细胞再经1 nmol/L氟替卡松继续处理24 h，后检测不同处理细胞中荧光素酶活性。多组间比较采用单因素方差分析，组间两两比较采用LSD法。

结果

成功获取人GLCCI1基因启动子并构建荧光素酶表达载体pGL3- pro （rs37973-G）和pGL3-pro （rs37973-A）。转染后48 h，与转染对照组比较，Raji和THP-1细胞中报告基因表达载体转染组细胞中荧光素酶活性（23.82±2.79比18.03±2.28，24.52±2.62比19.11±2.37）均升高，差异具有统计学意义（P均< 0.05）。与pGL3- pro （rs37973-G）- GLCCI1转染组比较，1 nmol/ L氟替卡松处理24 h能够分别增强转染后Raji和THP-1细胞中荧光素酶活性（30.05±5.23比18.03±2.28，31.12±4.69比19.11±2.37），差异具有统计学意义（P均< 0.05）。与pGL3-pro （rs37973-A）-GLCCI1转染组比较，1 nmol/ L氟替卡松处理24 h能够分别增强转染后Raji和THP-1细胞中荧光素酶活性（50.03±7.28比23.82±2.79，48.25±5.91比24.52±2.62），差异具有统计学意义（P均< 0.05）。与pGL3- pro （rs37973-G）- GLCCI1转染且经1 nmol/L氟替卡松处理24 h组比较，pGL3-pro （rs37973-A）- GLCCI1转染且经1 nmol/ L氟替卡松处理24 h组Raji和THP-1细胞中荧光素酶活性增强（50.03±7.28比31.02±5.23，48.25±5.91比31.12±4.69），差异具有统计学意义（P均< 0.05）。

结论

rs37973位点突变能够影响Raji与THP-1细胞中GLCCI1基因启动子活性，且与rs37973-A比较，rs37973-G能够更明显降低氟替卡诱导的Raji和THP-1细胞内GLCCI1基因启动子活性。

关键词: GLCCI1, 单核苷酸多态性, 荧光素酶, rs37973, 氟替卡松

Objective

To evaluate the effect of rs37973 polymorphism on the promoter activity of GLCCI1 gene in Raji and THP-1 cells induced by fluticasone luciferase method.

Methods

The promoter of the human GLCCI1 gene was predicted by bioinformatics and obtained, and the luciferase vectors corresponding to rs37973-G and rs37973-A were constructed and then transfected into the cells. After treatment with 1 nmol/L fluticasone for 24 h, the luciferase activity in the cells of different treatment groups was detected. One-way ANOVA was used for comparison among multiple groups, and LSD test was used for comparison between groups.

Results

The promoter of the human GLCCI1 gene was successfully obtained, and the luciferase expression vectors pGL3-pro (rs37973-G) and pGL3 pro (rs37973-A) were constructed. 48 h after transfection, compared with the control group, the luciferase activity in Raji and THP-1 cells transfected with reporter vector was statistically significant higher (23.82±2.79 vs 18.03±2.28, 24.52±2.62 vs 19.11±2.37) (P < 0.05) . Compared with pGL3-pro- (rs37973-G) -GLCCI1 transfection group, the luciferase activity in theRaji and THP- 1 cells treated with 1 nmol/L fluticasone for 24 h was significantly increased (30.05±5.23 vs 18.03± 2.28, 31.12±4.69 vs 19.11±2.37) (P < 0.05) . Compared with pGL3-pro (rs37973-A) -GLCCI1 transfection group, the luciferase activity in the Raji and THP-1 cells treated with 1 nmol/ L fluticasone for 24 hours was significantly increased (50.03±7.28 vs 23.82±2.79, 48.25±5.91 vs 24.52± 2.62) (P < 0.05) . Compared with the group transfected with pGL3-pro (rs37973-G) -GLCCI1 and treated with 1 nmol/L fluticasone for 24 h, the luciferase activity in Raji and THP-1 cells transfected with pGL3- pro (rs37973-A) -GLCCI1 and treated with 1 nmol/L fluticasone for 24 h also significantly increased (50.03±7.28 vs 31.02±5.23, 48.25±5.91 vs 31.12±4.69) (P < 0.05) .

Conclusionrs

37973 mutation can affect the promoter activity of GLCCI1, and rs37973-G can significantly reduce activity of GLCCI1 promoter in Raji and THP-1 cells induced by fluticasone.

Key words: GLCCI1, SNP, Luciferase, rs37973, Fluticasone

表1 人GLCCI1rs37973位点PCR及突变引物

基因	引物序列（5'-3'）	扩增产物长度（bp）
rs37973-A PCR	上游TTCGGAAAAGCCAGGGGCGCCCTT	338
	下游ACACATGAGGCAAACTTTAATTTGCAA
rs37973-G PCR	上游TTCGGAAAAGCCAGGGGCGCCCTT	338
	下游ACACATGAGGCAAACTTTAATTTGCAA
rs37973-A	突变上游TAAGAGTATTTTACTTTGTTCAAT	338
	突变下游AATGTCTGGAACCTGCATTGAA
rs37973-G	突变上游ATTTTACTTTGTTCAGTGCAG	338
	突变下游ATGTCTGGAACCTGCACTGAACAA

表1 人GLCCI1rs37973位点PCR及突变引物

基因	引物序列（5'-3'）	扩增产物长度（bp）
rs37973-A PCR	上游TTCGGAAAAGCCAGGGGCGCCCTT	338
	下游ACACATGAGGCAAACTTTAATTTGCAA
rs37973-G PCR	上游TTCGGAAAAGCCAGGGGCGCCCTT	338
	下游ACACATGAGGCAAACTTTAATTTGCAA
rs37973-A	突变上游TAAGAGTATTTTACTTTGTTCAAT	338
	突变下游AATGTCTGGAACCTGCATTGAA
rs37973-G	突变上游ATTTTACTTTGTTCAGTGCAG	338
	突变下游ATGTCTGGAACCTGCACTGAACAA

图1 生信预测获得的人GLCCI1基因启动子序列

图2 PCR扩增GLCCI1基因启动子注：通过琼脂糖凝胶检测扩增产物大小，琼脂糖浓度为2%；M为DL2000 DNA Marker；1为PCR产物1 μL上样；2为PCR产物5 μL上样；3为不加PCR产物

图3 倒置荧光显微镜下观察细胞中GFP表达注：293细胞转染后48h；a、c图为可见光视场；b、d图为紫外视场

图4 rs37973位点突变型荧光素酶报告基因表达载体序列分析

图5 Raji细胞转染效率检测及不同方式处理后细胞内荧光素酶活性检测注：a ~ b图分别为倒置荧光显微镜下观察细胞转染GFP 48 h后的总细胞数及紫外光下GFP表达的细胞数；c图为荧光素酶活性组间差异分析，荧光素酶活性表示为萤火虫荧光素酶与海肾荧光素酶比值，Fluticasone为氟替卡松；与传染pGL3-pro （rs37973-G）-GLCCI1比较，^aP < 0.05，与转染pGL3-pro（rs37973-A）-GLCCI1且经1 nmol/L氟替卡松处理24 h比较，^bP < 0.01

图6 THP-1细胞转染效率检测及不同处理后细胞内荧光素酶活性检测注：a ~ b图分别为倒置荧光显微镜下观察细胞转染GFP 48 h后的总细胞数及紫外光下GFP表达的细胞数；c图为荧光素酶活性组间差异分析，荧光素酶活性表示为萤火虫荧光素酶与海肾荧光素酶比值，Fluticasone，氟替卡松；与THP-1+pGL3-pro（rs37973-G）-GLCCI1比较，^aP < 0.05，与THP- 1+ pGL3-pro（rs37973-A）-GLCCI1且经1 nmol/L氟替卡松处理24 h比较，^bP < 0.01

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Effect of rs37973 polymorphism on GLCCI1 gene promoter activity in Raji and THP-1 cells induced by fluticasone

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Effect of rs37973 polymorphism on GLCCI1 gene promoter activity in Raji and THP-1 cells induced by fluticasone