临床肿瘤学杂志
臨床腫瘤學雜誌
림상종류학잡지
CHINESE CLINICAL ONCOLOGY
2015年
8期
679-684
,共6页
小干扰RNA%核糖体蛋白L6%宫颈癌%HeLa细胞%增殖%凋亡
小榦擾RNA%覈糖體蛋白L6%宮頸癌%HeLa細胞%增殖%凋亡
소간우RNA%핵당체단백L6%궁경암%HeLa세포%증식%조망
Small interfering RNA%Ribosomal protein L6%Cervical cancer%HeLa cells%Proliferation%Apoptosis
目的：探讨小干扰RNA（ siRNA）靶向抑制核糖体蛋白L6（ RPL6）基因表达对人宫颈癌HeLa细胞增殖及凋亡的影响。方法优化siRNA转染条件，将2条靶向抑制RPL6基因的siRNA载体片段（ siRPL6?1、siRPL6?2）分别高效转染人宫颈癌HeLa细胞（抑制组），同时设转染无义序列（siControl）的空转染组及不进行任何处理的对照组。分别于转染24、48、72、96 h后采用实时定量PCR（ qRT?PCR）检测RPL6表达水平以筛选抑制率高的siRPL6载体用于后续实验。噻唑蓝比色（ MTT）法检测各组转染24、48、72、96 h后的增殖抑制率，流式细胞术检测各组转染48、96 h后的细胞凋亡和细胞周期情况， Western blotting检测各组转染96 h后的cyclin A、CDK2和p21CIP1表达情况。结果抑制组的RPL6 mRNA水平均低于空转染组和对照组（ P＜0?05），siRPL6?2片段的干扰效率高于siRPL6?1，故后续实验选择siRPL6?2片段；与其余两组相比，抑制组转染后的增殖抑制率、凋亡率及caspase?3活化率均升高，且G0／G1期细胞比例升高，但S期和G2／M期细胞比例均降低，以上差异均有统计学意义（ P＜0?05）；抑制组转染后的p21CIP1水平升高，cyclin A和CDK2水平降低，与空转染组和对照组的差异有统计学意义（ P＜0?05）。空转染组和对照组以上指标的差异均无统计学意义（ P＞0?05）。结论通过siRNA抑制RPL6基因表达可抑制增殖并诱导凋亡和细胞周期阻滞，对宫颈癌防治有一定价值。
目的：探討小榦擾RNA（ siRNA）靶嚮抑製覈糖體蛋白L6（ RPL6）基因錶達對人宮頸癌HeLa細胞增殖及凋亡的影響。方法優化siRNA轉染條件，將2條靶嚮抑製RPL6基因的siRNA載體片段（ siRPL6?1、siRPL6?2）分彆高效轉染人宮頸癌HeLa細胞（抑製組），同時設轉染無義序列（siControl）的空轉染組及不進行任何處理的對照組。分彆于轉染24、48、72、96 h後採用實時定量PCR（ qRT?PCR）檢測RPL6錶達水平以篩選抑製率高的siRPL6載體用于後續實驗。噻唑藍比色（ MTT）法檢測各組轉染24、48、72、96 h後的增殖抑製率，流式細胞術檢測各組轉染48、96 h後的細胞凋亡和細胞週期情況， Western blotting檢測各組轉染96 h後的cyclin A、CDK2和p21CIP1錶達情況。結果抑製組的RPL6 mRNA水平均低于空轉染組和對照組（ P＜0?05），siRPL6?2片段的榦擾效率高于siRPL6?1，故後續實驗選擇siRPL6?2片段；與其餘兩組相比，抑製組轉染後的增殖抑製率、凋亡率及caspase?3活化率均升高，且G0／G1期細胞比例升高，但S期和G2／M期細胞比例均降低，以上差異均有統計學意義（ P＜0?05）；抑製組轉染後的p21CIP1水平升高，cyclin A和CDK2水平降低，與空轉染組和對照組的差異有統計學意義（ P＜0?05）。空轉染組和對照組以上指標的差異均無統計學意義（ P＞0?05）。結論通過siRNA抑製RPL6基因錶達可抑製增殖併誘導凋亡和細胞週期阻滯，對宮頸癌防治有一定價值。
목적：탐토소간우RNA（ siRNA）파향억제핵당체단백L6（ RPL6）기인표체대인궁경암HeLa세포증식급조망적영향。방법우화siRNA전염조건，장2조파향억제RPL6기인적siRNA재체편단（ siRPL6?1、siRPL6?2）분별고효전염인궁경암HeLa세포（억제조），동시설전염무의서렬（siControl）적공전염조급불진행임하처리적대조조。분별우전염24、48、72、96 h후채용실시정량PCR（ qRT?PCR）검측RPL6표체수평이사선억제솔고적siRPL6재체용우후속실험。새서람비색（ MTT）법검측각조전염24、48、72、96 h후적증식억제솔，류식세포술검측각조전염48、96 h후적세포조망화세포주기정황， Western blotting검측각조전염96 h후적cyclin A、CDK2화p21CIP1표체정황。결과억제조적RPL6 mRNA수평균저우공전염조화대조조（ P＜0?05），siRPL6?2편단적간우효솔고우siRPL6?1，고후속실험선택siRPL6?2편단；여기여량조상비，억제조전염후적증식억제솔、조망솔급caspase?3활화솔균승고，차G0／G1기세포비례승고，단S기화G2／M기세포비례균강저，이상차이균유통계학의의（ P＜0?05）；억제조전염후적p21CIP1수평승고，cyclin A화CDK2수평강저，여공전염조화대조조적차이유통계학의의（ P＜0?05）。공전염조화대조조이상지표적차이균무통계학의의（ P＞0?05）。결론통과siRNA억제RPL6기인표체가억제증식병유도조망화세포주기조체，대궁경암방치유일정개치。
Objective To explore the effect of siRNA targeting RPL6 gene on the proliferation and apoptosis of human cervi?cal cancer HeLa cells. Methods After the optimization of the transfection conditions, 2 siRNA vector fragments ( siRPL6?1, siRPL6?2) targeting RPL6 gene were effectively transfected into HeLa cells of human cervical cancer(inhibition group), respectively. Mean?while, there were also empty transfection group whose cells were transfected with the antisense sequence ( siControl) and control group without any treatment. The RPL6 expression levels were detected by real?time quantitative PCR ( qRT?PCR) at 24, 48, 72 and 96 h after transfection, and the siRPL6 vector with the higher inhibition rate was used for the following experiments. Thiazolyl blue ( MTT) was used to detect the proliferation inhibition rate at 24, 48, 72, 96 h after transfection. The apoptosis and cell cycle of the cells were detected by flow cytometry after 48 and 96 h. The expressions of CDK2, cyclin A and p21CIP1 after 96 h were detected by Western blot?ting. Results The level of mRNA RPL6 in the inhibition group was lower than that in the empty transfection group and the control group ( P<0?05) . Since the interference efficiency of the siRPL6?2 fragment was higher than that of the siRPL6?1, so the subsequent trials chose the siRPL6?2 fragment. Compared with other two groups, the proliferation inhibitory rate, apoptosis rate and caspase?3 acti?vation rate together with the proportion of cells in G0/G1 phase increased, but the proportion of cells in S phase and G2/M phase de?creased in inhibition group with statistically significant difference ( P<0?05);After transfection, the levels of p21CIP1 increased but the levels of CDK2 and cyclin A were decreased in inhibition group versus other groups ( P<0?05) . Conclusion The inhibition of RPL6 gene expression by siRNA can inhibit the proliferation and induce apoptosis and cell cycle arrest, and it can be valuable for the preven?tion and treatment of cervical cancer.
