STC-1蛋白对肾癌细胞生长平衡影响机制探讨

Influence of STC-1 and HIF-1α on growth balance in renal carcinoma cells

目的:研究斯钙素1(stanniocalcin1,STC-1)与缺氧诱导因子1α(hypoxia-inducible factor 1α,HIF-1α)的相互作用,是否借助调节Ca2+水平参与肾癌细胞生长调控。方法:构建高表达HIF-1α的肾癌细胞模型,使用不同浓度STC-1蛋白干预转染后的肾癌细胞(转染组)和单纯肾癌细胞(非转染组)。MTT法检测各组细胞增殖情况;RT-PCR及ELISA法检测细胞内HIF-1α、STC-1基因及蛋白表达;荧光分光光度计检测细胞内Ca2+水平。结果:非转染组和转染组肾癌细胞HIF-1α蛋白表达分别为8.3±1.2和15.1±0.9,t=24.18,P<0.001;STC-1蛋白表达分别为7.2±0.9和10.8±1.1,t=8.90,P=0.003;提示转染HIF-1α的肾癌细胞内HIF-1α和STC-1表达显著提高。不同浓度STC-1溶液干预非转染组肾癌细胞24h后,对照组HIF-1α蛋白表达为8.3±0.5,低剂量组为7.8±0.7,中剂量组为5.3±0.4,高剂量组为4.2±0.3,F=171.726,P<0.001,对照组STC-1蛋白表达为7.1±0.4,低剂量组为6.8±0.3,中剂量组为4.1±0.2,高剂量组为3.3±0.4,F=257.174,P<0.001;对照组细胞内Ca2+含量为95.7±8.6,低剂量组为60.3±5.5,中剂量组为48.6±6.3,高剂量组为33.7±4.2,F=198.931,P<0.001,对照组细胞增殖活性为0.226±0.021,低剂量组为0.343±0.024,中剂量组为0.293±0.018,高剂量组为0.252±0.023,F=23.615,P<0.001;不同浓度STC-1溶液干预转染组肾癌细胞24h后,对照组HIF-1α蛋白表达为15.3±1.6,低剂量组为14.6±1.1,中剂量组为10.1±0.9,高剂量组为8.6±0.8,F=135.696,P<0.001,对照组STC-1蛋白表达为11.2±0.4,低剂量组为10.9±0.5,中剂量组为7.4±0.7,高剂量组为5.6±0.8,F=105.101,P<0.001;对照组细胞内Ca2+含量为65.5±6.7,低剂量组为40.1±3.4,中剂量组为30.7±4.6,高剂量组为17.7±3.3,F=136.621,P<0.001;对照组细胞增殖活性为0.295±0.033,低剂量组为0.446±0.025,中剂量组为0.379±0.015,高剂量组为0.313±0.022,F=45.571,P<0.001。提示STC-1蛋白可促进单纯肾癌细胞和转染后肾癌细胞的增殖,但对两种细胞的促增殖作用却随着STC-1剂量的增加而逐渐下降,此时细胞内HIF-1α、STC-1的表达及Ca2+水平进行性下降。结论:STC-1蛋白可能通过调节HIF-1α和Ca2+的水平,促进肾癌细胞增殖,但该促增殖作用又因STC-1对HIF-1α的逐渐抑制而逐渐减弱,从而调控肾癌细胞的生长平衡。

OBJECTIVE： To research the regulation of proliferation and discuss whether Stanniocalcinl （STC-1）, Hypoxia-inducible factor 1α （HIF-1α） and Ca2＋ were participated in proliferation mechanism of renal carcinoma cells. METHODS： After successfully constructed the HIF-I~ highly expressing cell models, different concentrations of STC-1 solutions were added to the culture medium, then, proliferation of cells, expressions of HIF-la, STC-1 and levels of Ca2＋ were detected by MTT, RT-PCR, ELISA and Fluorescence Speetrophotometer respectively. RESULTS： HIF-1α protein ex pression in non-transfected group and transferred group cells were 8.3 ± 1.2 and 15.1±0.9 respectively （t = 24. 18, P〈 0. 001）. STC-1 protein expression in non-transfeeted group and transferred group cells were 7.2 ± 0. 9 and 10.8 ± 1. 1 respectively （t= 8.90, P= 0. 003）. The results showed stable transfection of HIF-1α/pcDNA3.0 into renal carcinoma cells resulted in efficiently rised expression of HIF 1α and STC-1. Non-transfected group cells were exposed with STC-1 solutions of different concentration （0,0.1,0.5 and 1.0 nmol/L） for 24 hours,the expression of HIF-1α was 8.3±0.5,7.8± 0. 7,5. 3±0. 4 and 4.2±0.3 （F=171. 726,P〈0. 001） ,the expression of STC-1 was 7.1±0.4,6.8±0.3,4. 1±0.2 and 3.3±0.4 （F=257.174,P〈0.001）,the levels of Ca2＋ was 95.7±8.6,60.3±5.5,48.6±6.3 and 33.7±4.2 （F= 198. 931,P〈0. 001）,the cells proliferative activity was 0. 226±0. 021,0. 343±0. 024,0. 293±0. 018 and 0. 252±0. 023 respectively （F= 23. 615,P〈0. 001）. Transfected group ceils were exposed with STC-1 solutions of different concentra- tion （0,0.1,0.5 and 1.0 nmol/L） for 24 hours,the expression of HIF-la was 15.3±1.6,14.6±1.1,10.1±0.9 and 8.6±0.8 （F=135. 696,P〈0. 001） ,the expression of STC-1 was 11.2±0.4,10.9±0.5,7.4±0.7 and 5.6±0.8 （F= 105. 101,P〈0. 001） ,the levels of Ca2＋ was 65.5±6.7,40.1±3.4,30.7±4.6 and 17.7±3.3 （F=136. 621,P〈0. 001）, the cells proliferative activity was 0. 295 ± 0. 033,0. 446 ±0. 025,0. 379 ± O. 015 and O. 313 ± 0. 022 respectively （F = 45. 571, P〈0. 001）. The results showed that the cells treated with STC-1 protein exhibited characteristics of proliferation, while proliferation of cells,expressions of HIF-1α and STC-1 ,levels of Ca2＋ were down-regulated by STC-1 protein. CON- CLUSION： STC-1 protein may participate in malignant proliferation of renal carcinoma ceils through depressing HIF-1α or down-regulation Ca2＋ ,which could be attenuated when HIF-1α inhibited by redundant STC-l,thus to regulate the growth balance of renal carcinoma cells.