海南医学
海南醫學
해남의학
HAINAN MEDICAL JOURNAL
2015年
1期
18-21
,共4页
黄维%陈洁仪%黄俊勇%周成宇
黃維%陳潔儀%黃俊勇%週成宇
황유%진길의%황준용%주성우
TRPM8%肾癌%侵袭%迁移
TRPM8%腎癌%侵襲%遷移
TRPM8%신암%침습%천이
TRPM8%Renal carcinoma%Invasion%Migration
目的:探讨瞬时受体电位M8(TRPM8)是否通过上皮-间充质转化(EMT)影响人肾癌细胞的侵袭及迁移能力。方法利用荧光定量PCR来检测人肾癌细胞A498、786-0以及GRC-1中TRPM8的表达水平;设计并合成靶向TRPM8的特异性shRNA,通过脂质体转染法转染TRPM8表达最高的肾癌细胞A498以构建稳定低表达TRPM8细胞株,通过Western blot和定量PCR来验证shRNA的干扰效率;通过Transwell法检测干扰后细胞的迁移及侵袭能力,Western blot法检测EMT相关指标E-cadherin和N-cadherin以及其上游信号分子Snail和WNT-5a的变化。结果 TRPM8-shRNA转染后,能够有效抑制A498细胞中TRPM8的表达;Transwell法检测细胞侵袭能力结果显示,A498组、Control组和shRNA组的穿膜细胞数分别为(102.56±11.41)、(105.67±10.83)、(74.62±8.65),A498/shRNA组细胞侵袭能力受到显著抑制(F=49.105,P<0.01);Transwell法检测细胞迁移能力结果显示,A498组、Control组和shRNA组的穿膜细胞数分别为(115.45±10.31)、(109.33±7.53)、(76.21±13.28),A498/shRNA组细胞迁移能力受到显著抑制(F=36.168,P<0.01);Western blot法结果发现,TRPM8干扰组细胞的E-cadherin表达增加,N-cadherin的表达降低;此外,TRPM8干扰组细胞的Snail以及WNT-5a表达较对照组显著下降。结论运用RNA干扰技术能够有效沉默A498细胞的TRPM8基因,并诱导其迁移侵袭能力的下降,其可能的机制是通过调节EMT的上游信号分子Snail、WNT-5a的表达来调控EMT,从而影响肾癌细胞的迁移及侵袭。以上提示TRPM8在肾癌的发生发展中起重要作用,抑制TRPM8的表达可能成为一种治疗肾癌的新方法。
目的:探討瞬時受體電位M8(TRPM8)是否通過上皮-間充質轉化(EMT)影響人腎癌細胞的侵襲及遷移能力。方法利用熒光定量PCR來檢測人腎癌細胞A498、786-0以及GRC-1中TRPM8的錶達水平;設計併閤成靶嚮TRPM8的特異性shRNA,通過脂質體轉染法轉染TRPM8錶達最高的腎癌細胞A498以構建穩定低錶達TRPM8細胞株,通過Western blot和定量PCR來驗證shRNA的榦擾效率;通過Transwell法檢測榦擾後細胞的遷移及侵襲能力,Western blot法檢測EMT相關指標E-cadherin和N-cadherin以及其上遊信號分子Snail和WNT-5a的變化。結果 TRPM8-shRNA轉染後,能夠有效抑製A498細胞中TRPM8的錶達;Transwell法檢測細胞侵襲能力結果顯示,A498組、Control組和shRNA組的穿膜細胞數分彆為(102.56±11.41)、(105.67±10.83)、(74.62±8.65),A498/shRNA組細胞侵襲能力受到顯著抑製(F=49.105,P<0.01);Transwell法檢測細胞遷移能力結果顯示,A498組、Control組和shRNA組的穿膜細胞數分彆為(115.45±10.31)、(109.33±7.53)、(76.21±13.28),A498/shRNA組細胞遷移能力受到顯著抑製(F=36.168,P<0.01);Western blot法結果髮現,TRPM8榦擾組細胞的E-cadherin錶達增加,N-cadherin的錶達降低;此外,TRPM8榦擾組細胞的Snail以及WNT-5a錶達較對照組顯著下降。結論運用RNA榦擾技術能夠有效沉默A498細胞的TRPM8基因,併誘導其遷移侵襲能力的下降,其可能的機製是通過調節EMT的上遊信號分子Snail、WNT-5a的錶達來調控EMT,從而影響腎癌細胞的遷移及侵襲。以上提示TRPM8在腎癌的髮生髮展中起重要作用,抑製TRPM8的錶達可能成為一種治療腎癌的新方法。
목적:탐토순시수체전위M8(TRPM8)시부통과상피-간충질전화(EMT)영향인신암세포적침습급천이능력。방법이용형광정량PCR래검측인신암세포A498、786-0이급GRC-1중TRPM8적표체수평;설계병합성파향TRPM8적특이성shRNA,통과지질체전염법전염TRPM8표체최고적신암세포A498이구건은정저표체TRPM8세포주,통과Western blot화정량PCR래험증shRNA적간우효솔;통과Transwell법검측간우후세포적천이급침습능력,Western blot법검측EMT상관지표E-cadherin화N-cadherin이급기상유신호분자Snail화WNT-5a적변화。결과 TRPM8-shRNA전염후,능구유효억제A498세포중TRPM8적표체;Transwell법검측세포침습능력결과현시,A498조、Control조화shRNA조적천막세포수분별위(102.56±11.41)、(105.67±10.83)、(74.62±8.65),A498/shRNA조세포침습능력수도현저억제(F=49.105,P<0.01);Transwell법검측세포천이능력결과현시,A498조、Control조화shRNA조적천막세포수분별위(115.45±10.31)、(109.33±7.53)、(76.21±13.28),A498/shRNA조세포천이능력수도현저억제(F=36.168,P<0.01);Western blot법결과발현,TRPM8간우조세포적E-cadherin표체증가,N-cadherin적표체강저;차외,TRPM8간우조세포적Snail이급WNT-5a표체교대조조현저하강。결론운용RNA간우기술능구유효침묵A498세포적TRPM8기인,병유도기천이침습능력적하강,기가능적궤제시통과조절EMT적상유신호분자Snail、WNT-5a적표체래조공EMT,종이영향신암세포적천이급침습。이상제시TRPM8재신암적발생발전중기중요작용,억제TRPM8적표체가능성위일충치료신암적신방법。
Objective To investigate the effect of down-regulation of TRPM8 on the invasion and migration of renal carcinoma cells A498. Methods The expression of TRPM8 in human renal carcinoma cell A498, 786-0 and GRC-1 were detected by fluorescence quantitative PCR. shRNA targeting TRPM8 was designed and synthe-sized, and then transfected into the A498 cells via Lipofectamine 2000 mediation. The interference efficiency of shR-NA was evaluated by Western blot and quantitative PCR. The migration ability and invasion ability of A498 were de-tected by using transwell assay. Expression of E-cadherin, N-cadherin, WNT-5a and Snail were detected by using Western blot. Results TRPM8-targeted shRNA could down-regulate the TRPM8 expression of A498. Transwell Cell number of invasion was (102.56±11.41), (105.67±10.83), (74.62±8.65) in A498 group, control group and shRNA group, respectively, which indicated that cell invasion ability were significantly inhibited in shRNA group (F=49.105, P<0.01). Transwell Cell number of migration was (115.45±10.31), (109.33±7.53), (76.21±13.28) in A498 group, con-trol group and shRNA group, respectively, which indicated that cell migration ability were significantly inhibited in shRNA group (F=36.168, P<0.01). In addition, the expression of E-cadherin was increased, while that of N-cadherin, WNT-5a and Snail was decreased in shRNA interference group. Conclusion Down-regulation of TRPM8 can induce inhibition of invasion and migration in human renal carcinoma cells A498 via regulating epithelial mesenchymal tran-sitions (EMT), specifically, the upstream signal molecule Snail, WNT-5a of EMT. It could be regarded as a novel tar-get for clinical diagnosis and gene therapy for renal carcinoma.