小干扰RNA抑制核因子-κB增强^131I治疗甲状腺癌疗效的研究

Synergistic effects of nuclear factor-kappa B inhibition by small interferece RNA on ^131I therapy in differentiated thyroid cancer cells

目的探讨小干扰RNA（siRNA）抑制核因子-κB（NF—KB）对^131I致DTC细胞凋亡能否产生协同作用。方法2×10^4 MBq／L^131I作用人甲状腺乳头状癌细胞株KTC-124h后做DNA结合实验，48h后做细胞存活分析。Westernblot鉴定^131I作用6h后细胞NF—KBp65的变化，24h后凋亡抑制因子[X-染色体相关凋亡抑制蛋白（XIAP）、细胞凋亡抑制因子1（eIAPl）、B细胞淋巴瘤因子大亚基（Bel—xL）]、凋亡关键因子[半胱氨酸蛋白酶（caspase3）和多聚腺苷二磷酸核糖聚合酶（PARP）]的变化。p65和凋亡抑制因子的Westernblot检测分4组：未转染（A）组、未转染＋^131I（B）组、转染对照siRNA＋^131I（c）组和转染p65siRNA＋^131I（D）组；其余实验分为6组：未转染（1）组、转染对照siRNA（2）组、转染p65siRNA（3）组、未转染＋^131I（4）组、转染对照siRNA＋^131I（5）组和转染p65siRNA＋^131I（6）组。多组间均数比较采用单因素方差分析，均数两两比较采用q检验。结果1至6组DNA结合率分别为（100．00±11．65）％、（96．00±17．98）％、（9．28-I-5．01）％、（322．72±50．81）％、（311．36±44．81）％和（36．96±15．66）％，差异有统计学意义（F=137．74，P〈0．01）；^131I作用后KTC-1细胞NF—KB活性均增强（q4：1自=10．90，q5：2g=11．38，均P〈0．01）；p65siRNA可抑制NF—KB功能（q㈦目=18．25，q46目=13．71，均P〈0．01）。6组细胞存活率分别为（100．00±11．65）％、（96．32±9．44）％、（70．88±7．41）％、（64．16±9．50）％、（62．24±9．37）％和（28．64±6．74）％（F=52．76，P〈0．01）；3、4和6组比，q=10．76和7．79，均P〈0．Ol。Westernblot结果显示A、B、C和D组p65相对表达水平分别为（56．60±7．37）％、（111．07±13．31）％、（113．16±15．04）％和（12．46±2．74）％，差异有统计学意义（F=60．17，P〈0．01）；^131I作用后p65浓度增高（qB：A组=6．20，qC：A组=5．85，均P〈0．01）；p65siRNA可抑制其浓度增高（q帅自=12．57，qC：D组=11．41，均P〈0．01）。4组XIAP、cIAPl和Bel—xL分别为（17．59±1．96）％、（16．45±1．85）％和（19．92-4-2．22）％，（98．37±17．92）％、（109．81±19．16）％和（95．59±22．20）％，（98．43±18．71）％、（98．86±15．88）％和（100．99±21．70）％，（7．00±0．95）％、（5．86±0．35）％和（9．52±0．90）％，差异均有统计学意义（F=44．22、56．51和29．11，均P〈0．01）；^131I作用后三者表达增加（qB：A组=Ⅷ=7．76、8．40和5．88，均P〈0．01）；055siRNA可抑制三者表达（qBD自=8．82、9．40和6．71，均P〈0．01）。6组caspase3亚基p19和p17、PARP活性蛋白p116和失活产物p89差异均有统计学意义（F=39．03、48．45、32．56和52．20，均P〈0．01）；3、4和6组比q=3．18～9．98，均P〈0．05。结论^131I通过活化NF—KB导致甲状腺癌细胞内凋亡抑制因子表达升高，p65siRNA可抑制这种变化；联合使用p65siRNA对^131I致DTC细胞凋亡产生协同效应。

Objective To study the effect of nuclear factor-kappa B （NF-KB） inhibition by small interference RNA （siRNA） on the apoptosis of DTC cells treated by ^131I. Methods DNA binding assay was performed at 24 h after ^131I treatment （2 × 10^4 MBq/L） on KTC-1 ceils. The cell survival assay was conducted at 48 h after ^131I treatment. Western blot was used to detect the changes of NF-KB p65 at 6 h after ^131 I treatment, and the changes of anti-apoptotic factors and apoptotic key factors at 24 h after %131 I treatment. The anti-apoptotic factors included in this study were X chromosome-linked inhibitor of apoptosis （ XIAP）, cellular inhibitor of apoptosis 1 （ cIAP1 ） and B-cell lymphoma extra large （ Bcl-xL）, and the apoptotic key factors were caspase 3 and poly-ADP-ribose polymerase （PARP）. A total of 4 groups were studied for the detection of p65 and anti-apoptotic factors by Western blot： no oligonucleotide transfection control group （A）, no oligonucleotide transfection ＋ ^131I group （B）, scrambled oligonucleotides transfection ＋ ^131I group （C） and p65 siRNA transfection ＋ ^131I group （D）. Another 6 groups of studies were ： oligonucleotide transfection control group （1） , scrambled oligonucleotides transfection group （2）, p65 siRNA transfection group （3）, no oligonucleotide transfection ＋ ^131I group （4）, scrambled oligonucleotides transfection ＋ ^131I group （5） and p65 siRNA transfection ＋ ^131I group （6）. One-way analysis of variance and q test were performed for statistical analysis. Results The results of DNA binding assays for the 6 groups （ 1, 2, 3, 4, 5, 6） were （100.00＋11.65）%, （96.00±17.98）%, （9.28 ±5.01）%, （322.72±50.81）%, （311.36 ＋44.81）% and （36.96 ± 15.66）%, respectively （F = 137.74, P 〈0.01 ）. NF-KB functions were strengthened with 1311 treatment （qgroup4：1 = 10.90, qgroup5：2 = 11.38, both P 〈0.01）. However, NF-KB p65 siRNA transfection could inhibit NF-KB functions （qgoupl：3 = 18. 25, qgroup4：6 = 13. 71, both P 〈 0. 01 ）. Cell survival rates of the 6 groups were （ 100.00 ± 11.65 ） % , （96.32 ± 9.44 ） % , （ 70. 88 ± 7.41 ） %, （64.16 ± 9.50） %, （62.24 ± 9.37） % and （28.64 ± 6.74） % （F = 52.76, P 〈 0.01 ）. There were significant differences between groups 3 and 6, groups 4 and 6 （ q = 10.76 and 7.79, both P 〈 0.01 ）. Western blot results showed that the expression of NF-KB p65 in the 4 groups （A, B, C, D） were （56.60 ± 7.37）%, （111.07 ± 13.31）%, （113.16 ± 15.04）% and （12.46 ±2.74）%, respectively （F=60.17, P 〈0. 01）. The p65 levels increased with 13tI treatment （qsroup S：A = 6. 20, qsroup C：A = 5. 85, both P 〈 0. 01 ）; while decreased significantly using NF-KB p65 siRNA transfection （qsroup B：D = 12. 57; qsroup C：D = 11.41, both P 〈0. 01 ）. Western blot results showed that XIAP, clAP1 and Bcl-xL in the 4 groups were （17.59±1.96）%, （16.45 ±1.85）% and （19.92 ±2.22）%; （98.37±17.92）%, （109.81 ± 19. 16）% and （95.59 ±22.20）% ; （98.43 ±18.71）%, （98.86 ± 15.88）% and （100.99 ±21.70）% ; （7.00 ± 0. 95 ） %, （5.86 ± 0.35 ） % and （9.52 ± 0.90） % , respectively （ F = 44.22, 56.51 and 29.11, all P 〈 0.01 ）.^131I treatment induced higher expression of all the 3 genes （qroup B： A = 7. 76, 8.40 and 5.88, all P 〈0.01 ） , while NF-KB p65 siRNA transfection, on the contrary, reduced the expression of all the 3 genes （qsroup B：D = 8. 82, 9. 40 and 6.71, all P 〈 0.01 ）. There were significant differences of p19, p17, p116 and p89 in the 6 groups（F = 39.03, 48.45, 32.56, 52.20, all P 〈 0. 01 ） , especially among group 3, 4 and 6 （q = 3.18 - 9.98, all P 〈 0.05 ）. Conclusions ^131I could activate NF-KB function and enhance the expressions of anti-apoptotie factors. NF-KB p65 siRNA transfection could effectively suppress this effect and therefore magnify ^131I induced apoptosis in DTC cells.