钙对HaCaT细胞整合素β1启动子活性和表达及细胞迁移的影响

Effect of calcium on the activity and expression of integrin β1 promoter in HaCaT cells and cell migration

目的 了解钙离子对人永生化KC株HaCaT细胞整合素β1启动子活性、蛋白表达及细胞迁移的影响.方法 （1）体外培养HaCaT细胞（12孔板）,按随机数字表法分为5组,每组18孔.分别用pGL3启动子（阳性对照组）、pGL3空载体（阴性对照组）及pGL3-1756 bp（全长启动子组）、pGL3-1442 bp（远端启动子组）、pGL3-261 bp（近端启动子组）报告质粒转染HaCaT细胞,转染分别在含0.00、0.03、0.09、0.30、0.80、1.20 mmol/L钙离子的无血清RPMI 1640培养液中进行（每组每种浓度3孔）.24 h后用双荧光素酶报告基因检测系统检测荧光素酶相对活性.（2）取本室构建的稳定转染小干扰RNA-整合素β1载体（简称稳定转染）的HaCaT细胞,常规培养后按随机数字表法将细胞分为6部分,用前述6种浓度钙离子处理,每种浓度3个样本.采用蛋白质印迹法检测整合素β1蛋白表达水平.（3）于6孔板中培养正常HaCaT细胞和稳定转染的HaCaT细胞,每种细胞18孔.按常规进行划痕处理后,用含前述6种浓度钙离子的培养液（按随机数字表法划分.每种浓度3孔细胞）培养12 h.观察并检测细胞迁移率.（4）对各实验结果进行单因素方差分析和独立样本t检验.结果 （1）全长启动子组细胞在钙离子浓度由0.00 mmol/L升至0.09、0.30 mmol/L时,荧光素酶相对活性由0.16±0.09升至0.39±0.09、0.35±0.05（t值分别为3.143、3.140,P值均小于0.05）;当钙离子浓度升至0.80、1.20 mmol/L时,荧光素酶活性下降.远端启动子组细胞荧光素酶相对活性随钙离子浓度变化的趋势与全长启动子组相似,但在0.09、0.30 mmol/L时,该酶活性分别为0.56±0.32和0.64±0.06,明显高于全长启动子组（t值分别为0.887、6.122,P值均小于0.05）.各种浓度钙离子对近端启动子组荧光素酶相对活性无明显影响.（2）当钙离子浓度为0.00 mmol/L时,稳定转染的HaCaT细胞整合素β1蛋白表达量为0.53±0.10.当钙离子浓度为0.03、0.09、0.30、0.80、1.20 mmol/L时,整合素β1表达量分别为0.58±0.09、1.40±0.29、1.41±0.09、0.99±0.10、1.16±0.15,与0.00 mmol/L时比较均明显升高（t值分别为0.687、4.880、11.210、5.578、6.199,P值均小于0.05）.（3）与钙离子浓度为0.00 mmol/L比较,当钙离子浓度为0.09、0.30 mmol/L时,正常HaCaT细胞迁移率明显增高;0.80、1.20 mmol/L时,迁移率明显降低.稳定转染的HaCaT细胞经各种浓度钙离子处理后,细胞迁移率无明显改变.结论 整合素β1远端启动子是调节人表皮细胞整合素β1转录的主要区域,钙离子对整合素β1启动子活性、蛋白表达和细胞迁移具有调节作用.

Objective To study the effect of calcium on the activity and protein expression of integrin P, promoter in human immortal keratinocyte colony HaCaT cell and cell migration. Methods （1） HaCaT cells were cultured in vitro （12-slot plate） and divided into 5 groups according to the random number table, with 18 slots in each group： reporter plasmid pGL3 promoter （positive control group, PC） , pGL3 empty vector （negative control group, NC） , pGL3-1756 bp （total length promoter group, TL） , pGL3-1442 bp （distal promoter group, D） , and pGL3-261 bp （proximal promoter group, P） was respectively used to transfect HaCaT cells in non-serum RPMI 1640 culture medium with 0. 00, 0. 03, 0. 09, 0. 30, 0. 80, or 1. 20 mmol/L calcium （3 slots in each group with each concentration）. Luciferase activity was detected with dual-luciferase reporter assay system 24 hours after transfection. （2） HaCaT cells steadily transfected with small interfering RNA-integrin β1 vector （steadily transfected in brief） constructed in our laboratory were normally cultured and divided into 6 parts according to the random number table. And then they were treated with former 6 different concentrations of calcium, with 3 samples for each concentration. Expression level of integrin β1 protein was determined with Western blot. （3） Normal and steadily transfected HaCaT cells were cultured in 6-slot plate, 18 slots for each kind of cells. They were cultured with former 6 kinds of calcium culture media （divided according to the random number table, with 3 slots of cells for each concentration） for 12 hours after scratch test. Cell migration rate was observed and determined. （4） Data were processed with one-way analysis of variance and independent samples t test. Results （1） The luciferase activity of cells in TL group increased from 0. 16 ± 0. 09 to 0. 39 ± 0. 09 and 0. 35 ± 0. 05 （with t value respectively 3. 143, 3. 140, P values all below 0.05） as calcium concentration increasing from 0.00 mmol/L to 0.09 and 0. 30 mmol/L, and it decreased as calcium concentration increased to 0. 80 and 1. 20 mmol/L. The change pattern of luciferase activity of cells along with calcium concentration in D group was similar to that in TL group, but its activity （0.56 ±0.32, 0.64 ±0.06） at the concentration of 0.09, 0.30 mmol/L was respectively higher than that in TL group （with t value respectively 0. 887, 6. 122, P values all below 0. 05）. There was no obvious influence of calcium in either concentration on the luciferase activity of cells in P group. （2） The expression amount of integrin β1 of steadily transfected HaCaT cells cultured with 0.03, 0.09, 0.30, 0.80, 1.20 mmol/L calcium （0.58±0.09, 1.40±0.29, 1.41 ±0.09, 0.99 ±0.10, 1.16± 0. 15） were all increased as compared with that cultured with 0.00 mmol/L calcium （0.53 ±0. 10, with t value respectively 0.687, 4.880, 11.210, 5.578, 6. 199, P values all below 0.05）. （3） Migration speed of normal HaCaT cells cultured with 0.09, 0. 30 mmol/L calcium increased obviously as compared with that cultured with 0. 00 mmol/L calcium, and it slowed down when cultured with 0. 80, 1.20 mmol/L calcium. There was no obvious difference of migration rate among steadily transfected HaCaT cells treated with different concentration of calcium. Conclusions Distal promoter region of integrin β1 plays a vital role in regulating integrin β1 transcription in human epidermal cells. And calcium regulates activity, protein expression of integrin β1 promoter and cell migration.