CAJ | 학술논문

目的研究自噬基因beclin 1对宫颈癌细胞株HeLa细胞生长的抑制作用,并探讨其可能的机制.方法实验分为5组:(1)pcDNA3.1(+)-beclin 1组:转染重组载体pcDNA3.1(+)-beclin 1质粒(过度表达beclin 1基因)的HeLa细胞;(2)pSUPER-beclin 1组:转染重组载体pSUPER-beclin 1质粒的HeLa细胞(抑制Beclin 1基因表达);(3)pcDNA3.1(+)组:转染空载体pcDNA3.1(+)质粒的HeLa细胞;(4)pSUPER组:转染空载体pSUPER质粒的HeLa细胞;(5)空白对照组:未转染质粒的HeLa细胞.采用逆转录(RT)PCR技术和蛋白印迹法检测各组细胞中beclin 1 mRNA和蛋白的表达以及凋亡因子--半胱氨酸天冬氨酸蛋白酶9(caspase-9)mRNA及蛋白的表达;四甲基偶氮唑蓝(MTT)比色法检测各组细胞的生长情况;流式细胞仪检测各组细胞的凋亡率;电镜观察和流式细胞仪检测各组细胞的自噬情况.将各组细胞分别接种于裸鼠皮下,观察各组裸鼠体内的成瘤性及肿瘤生长情况.结果 (1)各组细胞中beclin 1、caspase-9 mRNA和蛋白的表达:pcDNA3.1(+)-beclin 1组beclin 1和caspase-9 mRNA的表达水平分别为994.72±468.76和12.88±2.71,pSUPER-beclin 1组分别为0.18±0.63和0.11±0.08,pcDNA3.1(+)组分别为0.57±0.12和4.28±3.25,pSUPER组分别为0.67±0.29和2.77±1.27,空白对照组分别为0.74±0.25和3.67±3.78,pcDNA3.1(+)-beclin 1组均明显高于pcDNA3.1(+)组、pSUPER组和空白对照组,pSUPER-beclin 1组均明显低于pcDNA3.1(+)组、pSUPER组和空白对照组,差异均有统计学意义(P＜0.05).各组细胞中beclin 1和caspase-9蛋白的表达情况与mRNA相似.(2)各组细胞的生长情况:pcDNA3.1(+)-beclin 1组细胞的生长明显慢于空白对照组及pcDNA3.1(+)组,而pSUPER-beclin 1组细胞的生长明显快于空白对照组及pSUPER组,差异均有统计学意义(P＜0.05).(3)各组细胞的凋亡率:pcDNA3.1(+)-beclin 1组为(28.2±2.3)%,pcDNA3.1(+)组为(14.6±4.6)%,pSUPER-beclin 1组为(5.7±2.0)%,pSUPER组为(16.2±3.1)%,空白对照组为(11.2±3.0)%,pcDNA3.1(+)-beclin 1组和pSUPER-beclin 1组分别与另外3组比较,差异均有统计学意义(P＜0.05).(4)各组细胞的自噬情况:pcDNA3.1(+)-beclin 1组细胞内可见自噬体的形成,而其余各组自噬体形成不明显.pcDNA3.1(+)-beclin 1组自噬率为(10.3±1.5)%,pcDNA3.1(+)组为(3.6±0.8)%,pSUPER-beclin 1组为(1.2±0.3)%,pSUPER组为(3.2±1.2)%,空白对照组为(2.2±1.1)%,pcDNA3.1(+)-beclin 1组和pSUPER-beclin 1组分别与另外3组比较,差异均有统计学意义(P＜0.05).(5)各组裸鼠的成瘤情况:pcDNA3.1(+)-beclin 1组裸鼠成瘤时间长于空白对照组、pcDNA3.1(+)组和pSUPER组.pSUPER-beclin 1组裸鼠肿瘤体积第7天开始明显大于空白对照组、pcDNA3.1(+)组和pSUPER组(P＜0.05);pcDNA3.1(+)-beclin 1组第21天开始明显小于空白对照组、pcDNA3.1(+)组和pSUPER组(P＜0.05).接种第28天,pSUPER-beclin 1组肿瘤质量为(0.52±0.08)g,明显高于空白对照组的(0.37±0.12)g和pSUPER组的(0.34±0.24)g(P＜0.05);pcDNA3.1(+)-beclin 1组肿瘤质量为(0.18±0.12)g,明显低于空白对照组和pcDNA3.1(+)组的(0.34±0.18)g(P＜0.05).结论自噬基因beclin 1可以抑制宫颈癌HeLa细胞体内、外的生长,这种作用不仅与自噬调控通路有关,而且可能通过调控caspse-9基因的表达参与内源性细胞凋亡通路的调节,为宫颈癌基因治疗提供了新途径. 目的研究自噬基因beclin 1對宮頸癌細胞株HeLa細胞生長的抑製作用,併探討其可能的機製.方法實驗分為5組:(1)pcDNA3.1(+)-beclin 1組:轉染重組載體pcDNA3.1(+)-beclin 1質粒(過度錶達beclin 1基因)的HeLa細胞;(2)pSUPER-beclin 1組:轉染重組載體pSUPER-beclin 1質粒的HeLa細胞(抑製Beclin 1基因錶達);(3)pcDNA3.1(+)組:轉染空載體pcDNA3.1(+)質粒的HeLa細胞;(4)pSUPER組:轉染空載體pSUPER質粒的HeLa細胞;(5)空白對照組:未轉染質粒的HeLa細胞.採用逆轉錄(RT)PCR技術和蛋白印跡法檢測各組細胞中beclin 1 mRNA和蛋白的錶達以及凋亡因子--半胱氨痠天鼕氨痠蛋白酶9(caspase-9)mRNA及蛋白的錶達;四甲基偶氮唑藍(MTT)比色法檢測各組細胞的生長情況;流式細胞儀檢測各組細胞的凋亡率;電鏡觀察和流式細胞儀檢測各組細胞的自噬情況.將各組細胞分彆接種于裸鼠皮下,觀察各組裸鼠體內的成瘤性及腫瘤生長情況.結果 (1)各組細胞中beclin 1、caspase-9 mRNA和蛋白的錶達:pcDNA3.1(+)-beclin 1組beclin 1和caspase-9 mRNA的錶達水平分彆為994.72±468.76和12.88±2.71,pSUPER-beclin 1組分彆為0.18±0.63和0.11±0.08,pcDNA3.1(+)組分彆為0.57±0.12和4.28±3.25,pSUPER組分彆為0.67±0.29和2.77±1.27,空白對照組分彆為0.74±0.25和3.67±3.78,pcDNA3.1(+)-beclin 1組均明顯高于pcDNA3.1(+)組、pSUPER組和空白對照組,pSUPER-beclin 1組均明顯低于pcDNA3.1(+)組、pSUPER組和空白對照組,差異均有統計學意義(P＜0.05).各組細胞中beclin 1和caspase-9蛋白的錶達情況與mRNA相似.(2)各組細胞的生長情況:pcDNA3.1(+)-beclin 1組細胞的生長明顯慢于空白對照組及pcDNA3.1(+)組,而pSUPER-beclin 1組細胞的生長明顯快于空白對照組及pSUPER組,差異均有統計學意義(P＜0.05).(3)各組細胞的凋亡率:pcDNA3.1(+)-beclin 1組為(28.2±2.3)%,pcDNA3.1(+)組為(14.6±4.6)%,pSUPER-beclin 1組為(5.7±2.0)%,pSUPER組為(16.2±3.1)%,空白對照組為(11.2±3.0)%,pcDNA3.1(+)-beclin 1組和pSUPER-beclin 1組分彆與另外3組比較,差異均有統計學意義(P＜0.05).(4)各組細胞的自噬情況:pcDNA3.1(+)-beclin 1組細胞內可見自噬體的形成,而其餘各組自噬體形成不明顯.pcDNA3.1(+)-beclin 1組自噬率為(10.3±1.5)%,pcDNA3.1(+)組為(3.6±0.8)%,pSUPER-beclin 1組為(1.2±0.3)%,pSUPER組為(3.2±1.2)%,空白對照組為(2.2±1.1)%,pcDNA3.1(+)-beclin 1組和pSUPER-beclin 1組分彆與另外3組比較,差異均有統計學意義(P＜0.05).(5)各組裸鼠的成瘤情況:pcDNA3.1(+)-beclin 1組裸鼠成瘤時間長于空白對照組、pcDNA3.1(+)組和pSUPER組.pSUPER-beclin 1組裸鼠腫瘤體積第7天開始明顯大于空白對照組、pcDNA3.1(+)組和pSUPER組(P＜0.05);pcDNA3.1(+)-beclin 1組第21天開始明顯小于空白對照組、pcDNA3.1(+)組和pSUPER組(P＜0.05).接種第28天,pSUPER-beclin 1組腫瘤質量為(0.52±0.08)g,明顯高于空白對照組的(0.37±0.12)g和pSUPER組的(0.34±0.24)g(P＜0.05);pcDNA3.1(+)-beclin 1組腫瘤質量為(0.18±0.12)g,明顯低于空白對照組和pcDNA3.1(+)組的(0.34±0.18)g(P＜0.05).結論自噬基因beclin 1可以抑製宮頸癌HeLa細胞體內、外的生長,這種作用不僅與自噬調控通路有關,而且可能通過調控caspse-9基因的錶達參與內源性細胞凋亡通路的調節,為宮頸癌基因治療提供瞭新途徑.
목적 연구자서기인beclin 1대궁경암세포주HeLa세포생장적억제작용,병탐토기가능적궤제.방법 실험분위5조:(1)pcDNA3.1(+)-beclin 1조:전염중조재체pcDNA3.1(+)-beclin 1질립(과도표체beclin 1기인)적HeLa세포;(2)pSUPER-beclin 1조:전염중조재체pSUPER-beclin 1질립적HeLa세포(억제Beclin 1기인표체);(3)pcDNA3.1(+)조:전염공재체pcDNA3.1(+)질립적HeLa세포;(4)pSUPER조:전염공재체pSUPER질립적HeLa세포;(5)공백대조조:미전염질립적HeLa세포.채용역전록(RT)PCR기술화단백인적법검측각조세포중beclin 1 mRNA화단백적표체이급조망인자--반광안산천동안산단백매9(caspase-9)mRNA급단백적표체;사갑기우담서람(MTT)비색법검측각조세포적생장정황;류식세포의검측각조세포적조망솔;전경관찰화류식세포의검측각조세포적자서정황.장각조세포분별접충우라서피하,관찰각조라서체내적성류성급종류생장정황.결과 (1)각조세포중beclin 1、caspase-9 mRNA화단백적표체:pcDNA3.1(+)-beclin 1조beclin 1화caspase-9 mRNA적표체수평분별위994.72±468.76화12.88±2.71,pSUPER-beclin 1조분별위0.18±0.63화0.11±0.08,pcDNA3.1(+)조분별위0.57±0.12화4.28±3.25,pSUPER조분별위0.67±0.29화2.77±1.27,공백대조조분별위0.74±0.25화3.67±3.78,pcDNA3.1(+)-beclin 1조균명현고우pcDNA3.1(+)조、pSUPER조화공백대조조,pSUPER-beclin 1조균명현저우pcDNA3.1(+)조、pSUPER조화공백대조조,차이균유통계학의의(P＜0.05).각조세포중beclin 1화caspase-9단백적표체정황여mRNA상사.(2)각조세포적생장정황:pcDNA3.1(+)-beclin 1조세포적생장명현만우공백대조조급pcDNA3.1(+)조,이pSUPER-beclin 1조세포적생장명현쾌우공백대조조급pSUPER조,차이균유통계학의의(P＜0.05).(3)각조세포적조망솔:pcDNA3.1(+)-beclin 1조위(28.2±2.3)%,pcDNA3.1(+)조위(14.6±4.6)%,pSUPER-beclin 1조위(5.7±2.0)%,pSUPER조위(16.2±3.1)%,공백대조조위(11.2±3.0)%,pcDNA3.1(+)-beclin 1조화pSUPER-beclin 1조분별여령외3조비교,차이균유통계학의의(P＜0.05).(4)각조세포적자서정황:pcDNA3.1(+)-beclin 1조세포내가견자서체적형성,이기여각조자서체형성불명현.pcDNA3.1(+)-beclin 1조자서솔위(10.3±1.5)%,pcDNA3.1(+)조위(3.6±0.8)%,pSUPER-beclin 1조위(1.2±0.3)%,pSUPER조위(3.2±1.2)%,공백대조조위(2.2±1.1)%,pcDNA3.1(+)-beclin 1조화pSUPER-beclin 1조분별여령외3조비교,차이균유통계학의의(P＜0.05).(5)각조라서적성류정황:pcDNA3.1(+)-beclin 1조라서성류시간장우공백대조조、pcDNA3.1(+)조화pSUPER조.pSUPER-beclin 1조라서종류체적제7천개시명현대우공백대조조、pcDNA3.1(+)조화pSUPER조(P＜0.05);pcDNA3.1(+)-beclin 1조제21천개시명현소우공백대조조、pcDNA3.1(+)조화pSUPER조(P＜0.05).접충제28천,pSUPER-beclin 1조종류질량위(0.52±0.08)g,명현고우공백대조조적(0.37±0.12)g화pSUPER조적(0.34±0.24)g(P＜0.05);pcDNA3.1(+)-beclin 1조종류질량위(0.18±0.12)g,명현저우공백대조조화pcDNA3.1(+)조적(0.34±0.18)g(P＜0.05).결론 자서기인beclin 1가이억제궁경암HeLa세포체내、외적생장,저충작용불부여자서조공통로유관,이차가능통과조공caspse-9기인적표체삼여내원성세포조망통로적조절,위궁경암기인치료제공료신도경.
Objective To investigate the inhibitory effects and the mechanism of autophagy gene beclin 1 on cervical cancer HeLa cells. Methods The eukaryotic expression vector and short hairpin RNA (shRNA) expression vector of beclin 1 were transfected via lipofectamine into HeLa cells. Experimental cells were classified into 5 groups: pcDNA3. 1 ( + )-beclin 1 group, pSUPER-beclin 1 group, pcDNA3.1 ( + )group, pSUPER group and HeLa group. Real time-PCR and western blot were used for detecting expression of mRNA and protein of beclin 1 and caspase-9 in transfected cells. Flow cytometry was employed to observe the effect of transfection on the apoptosis, and autophagy of HeLa, while proliferation was analyzed by methyl thiazolyl tetrazolium (MTT) assay. The ultrastructural analysis of autophagic vacuoles was under the electron microscope. Five groups cells were seeded subcutaneously on nude mice. The carcinogenic and growth activities of cancer cells in vivo were observed, and immunohistochemistry was used to detect the protein expression of beclin 1 in tumor tissue. Results ( 1 ) The mRNA expression of beclin 1 and caspase-9: pcDNA3. 1 ( + )-beclin 1 group were 994.72 ±468.76 and 12. 88 ±2. 71, pSUPER-beclin 1 group were 0. 18 ± 0. 63 and 0. 11 ± 0. 08, pcDNA3. 1 ( + ) group were 0. 57 ± 0. 12 and 4. 28 ± 3. 25,pSUPER group were 0. 67 ± 0. 29 and 2. 77 ± 1.27, and HeLa group were 0. 74 ± 0. 25 and 3.67 ± 3.78,respectively. The eukaryotic expression vector pcDNA3. 1 ( + ) -beclin 1 significantly improved the expression of mRNA of beclin 1 and caspase-9 in HeLa cells( P ＜0. 05 ), and the shRNA expression vector inhibited the expression of mRNA of beclin 1 and caspase-9 ( P ＜ 0.05 ). (2) The cell proliferations: pcDNA3.1 ( + ) -beclin 1 vector significantly inhibited the growth of HeLa cells, while pSUPER-beclin 1 vector significantly improved the growth of HeLa cells(P ＜0. 05). (3) The rate of apoptosis: pcDNA3.1 ( + )-beclin 1 group was (28.2 ±2.3)%, pcDNA3. 1( + ) group was(14.6 ±4.6)%,pSUPER-beclin 1 group was(5.7 ±2. 0) %, pSUPER group was( 16. 2 ± 3.1 ) %, and HeLa group was( 11.2 ± 3. 0) %. The pcDNA3. 1 ( + ) -beclin 1 vector significantly increased the apoptosis rate, while the pSUPER-beclin 1 vector significantly decreased the apoptosis rate(P ＜0. 05 ). (4)The activity of autophagy: more autophagy cells were identified in pcDNA3.1( + )-beclin 1 group; the rate of autophagy of five group were( 10. 3 ± 1.5)% in pcDNA3. 1 ( + ) -beclin 1 group, ( 3.6 ± 0. 8 ) % in pcDNA3. 1 ( + ) group, ( 1.2 ± 0. 3 ) % in pSUPER-beclin 1 group, (3.2 ± 1.2)% in pSUPER group and (2.2 ± 1. 1)% in HeLa group, there was statistical significances between test groups and control groups( P ＜ . 05 ). (5)Carcinogenic activity of HeLa cells in nude mice: the duration of tumorigenesis was the longest in pcDNA3.1 ( + )-beclin 1 group and the shortest in pSUPER-beclin 1 group among all groups. The tumor size began to grow larger from 7th day after injection in pSUPER-beclin 1 group than in control groups( P ＜ 0. 05 ). The tumor size was smaller from 21st day after injection in pcDNA3.1( + )-beclin 1 group than in control groups(P ＜0. 05). From 28th day after injection,the tumor weigh was (0. 52 ± 0. 08 )g in pSUPER-beclin 1 group, apparently more than HeLa group (0. 37 ±0. 12) g and pSUPER group (0. 34 ± 0. 24 ) g ( P ＜ 0. 05 ). While in pcDNA3. 1 ( + )-beclin 1 group the tumor weighed (0. 18 ±0. 12) g, which was lower than HeLa group and pcDNA3. 1 ( + ) group (0. 34 ± 0. 18 ) g ( P ＜ 0. 05 ) . Conclusions Autophagy gene beclin 1 overexpression can inhibit proliferation and growth of HeLa cells in vitro and in vivo. Beclin 1 not noly participate in the regulation of autophagy signaling, but also play an important role in the regulation of endogenous apoptosis signaling through caspase-9. So it might be one of the new strategies for gene therapy of cervical carcinoma.