中华肿瘤杂志
中華腫瘤雜誌
중화종류잡지
Chinese Journal of Oncology
2015年
9期
664-670
,共7页
李增军%王海鹏%宋宝%孙燕来%徐忠法%韩建军
李增軍%王海鵬%宋寶%孫燕來%徐忠法%韓建軍
리증군%왕해붕%송보%손연래%서충법%한건군
结直肠肿瘤%高迁移率族蛋白B1%LoVo细胞%RNA干扰%肿瘤侵袭%肿瘤转移%裸鼠
結直腸腫瘤%高遷移率族蛋白B1%LoVo細胞%RNA榦擾%腫瘤侵襲%腫瘤轉移%裸鼠
결직장종류%고천이솔족단백B1%LoVo세포%RNA간우%종류침습%종류전이%라서
Colorectal neoplasm%High mobility group box-1,HMGB1%LoVo cells%RNA interference%Neoplasm invasion%Neoplasm metastasis%Nude mice
目的：探讨小分子RNA（ siRNA）干扰高迁移率族蛋白B1（ HMGB1）基因的表达对结直肠癌细胞株LoVo增殖和侵袭的抑制作用。方法采用慢病毒介导的RNA干扰技术沉默HMGB1的表达，以逆转录聚合酶链反应和Western blot法检测干扰后LoVo细胞中HMGB1 mRNA和蛋白的表达情况。采用四甲基偶氮唑蓝（ MTT）法、流式细胞术、Transwell小室实验、细胞划痕实验及裸鼠成瘤实验，分析HMGB1基因沉默对LoVo细胞生长、增殖、侵袭及转移的影响。结果慢病毒介导siRNA可成功转染结直肠癌细胞株LoVo。 HMGB1?siRNA转染组LoVo细胞中HMGB1 mRNA和蛋白的相对表达水平分别为0．24±0．04和0．21±0．03，阴性对照组分别为0．82±0．13和1．15±0．18，空白对照组分别为0．93±0．15和1．21±0．20；HMGB1?siRNA转染组HMGB1 mRNA和蛋白的表达水平均明显低于阴性对照组和空白对照组（ P＜0．05）。 MTT法检测结果显示，HMGB1?siRNA转染组LoVo细胞的生长速度较阴性对照组和空白对照组降低（ P＜0．05）。流式细胞术检测结果显示，HMGB1?siRNA转染组LoVo细胞的增殖指数为38．27±1．32，明显低于阴性对照组（54．66±1．74）和空白对照组（57．43±1．29，P＜0．05）。Transwell小室实验显示，HMGB1?siRNA转染组穿膜细胞数为（14．0±3．5）个／高倍视野，明显低于阴性对照组[（51．0±6．7）个／高倍视野]和空白对照组[（68．0±5．3）个／高倍视野，P＜0．05]。细胞划痕实验显示，划痕后培养48 h时，HMGB1?siRNA转染组LoVo细胞的迁移距离为（83．61±23．21）μm，明显低于阴性对照组[（202．86±46．46）μm]和空白对照组[（214．58±57．38）μm，P＜0．05]。裸鼠成瘤实验显示，21 d后，肿瘤最终体积为（521±34）mm3，明显小于阴性对照组[（763±46）mm3]和空白对照组[（802±51）mm3，P＜0．05]。结论慢病毒介导的RNA干扰技术可有效抑制结直肠癌LoVo细胞中HMGB1基因的表达，HMGB1基因沉默可使结直肠癌细胞生长缓慢，增殖周期延长，侵袭与迁移能力明显下降，裸鼠移植瘤的生长受到明显抑制。
目的：探討小分子RNA（ siRNA）榦擾高遷移率族蛋白B1（ HMGB1）基因的錶達對結直腸癌細胞株LoVo增殖和侵襲的抑製作用。方法採用慢病毒介導的RNA榦擾技術沉默HMGB1的錶達，以逆轉錄聚閤酶鏈反應和Western blot法檢測榦擾後LoVo細胞中HMGB1 mRNA和蛋白的錶達情況。採用四甲基偶氮唑藍（ MTT）法、流式細胞術、Transwell小室實驗、細胞劃痕實驗及裸鼠成瘤實驗，分析HMGB1基因沉默對LoVo細胞生長、增殖、侵襲及轉移的影響。結果慢病毒介導siRNA可成功轉染結直腸癌細胞株LoVo。 HMGB1?siRNA轉染組LoVo細胞中HMGB1 mRNA和蛋白的相對錶達水平分彆為0．24±0．04和0．21±0．03，陰性對照組分彆為0．82±0．13和1．15±0．18，空白對照組分彆為0．93±0．15和1．21±0．20；HMGB1?siRNA轉染組HMGB1 mRNA和蛋白的錶達水平均明顯低于陰性對照組和空白對照組（ P＜0．05）。 MTT法檢測結果顯示，HMGB1?siRNA轉染組LoVo細胞的生長速度較陰性對照組和空白對照組降低（ P＜0．05）。流式細胞術檢測結果顯示，HMGB1?siRNA轉染組LoVo細胞的增殖指數為38．27±1．32，明顯低于陰性對照組（54．66±1．74）和空白對照組（57．43±1．29，P＜0．05）。Transwell小室實驗顯示，HMGB1?siRNA轉染組穿膜細胞數為（14．0±3．5）箇／高倍視野，明顯低于陰性對照組[（51．0±6．7）箇／高倍視野]和空白對照組[（68．0±5．3）箇／高倍視野，P＜0．05]。細胞劃痕實驗顯示，劃痕後培養48 h時，HMGB1?siRNA轉染組LoVo細胞的遷移距離為（83．61±23．21）μm，明顯低于陰性對照組[（202．86±46．46）μm]和空白對照組[（214．58±57．38）μm，P＜0．05]。裸鼠成瘤實驗顯示，21 d後，腫瘤最終體積為（521±34）mm3，明顯小于陰性對照組[（763±46）mm3]和空白對照組[（802±51）mm3，P＜0．05]。結論慢病毒介導的RNA榦擾技術可有效抑製結直腸癌LoVo細胞中HMGB1基因的錶達，HMGB1基因沉默可使結直腸癌細胞生長緩慢，增殖週期延長，侵襲與遷移能力明顯下降，裸鼠移植瘤的生長受到明顯抑製。
목적：탐토소분자RNA（ siRNA）간우고천이솔족단백B1（ HMGB1）기인적표체대결직장암세포주LoVo증식화침습적억제작용。방법채용만병독개도적RNA간우기술침묵HMGB1적표체，이역전록취합매련반응화Western blot법검측간우후LoVo세포중HMGB1 mRNA화단백적표체정황。채용사갑기우담서람（ MTT）법、류식세포술、Transwell소실실험、세포화흔실험급라서성류실험，분석HMGB1기인침묵대LoVo세포생장、증식、침습급전이적영향。결과만병독개도siRNA가성공전염결직장암세포주LoVo。 HMGB1?siRNA전염조LoVo세포중HMGB1 mRNA화단백적상대표체수평분별위0．24±0．04화0．21±0．03，음성대조조분별위0．82±0．13화1．15±0．18，공백대조조분별위0．93±0．15화1．21±0．20；HMGB1?siRNA전염조HMGB1 mRNA화단백적표체수평균명현저우음성대조조화공백대조조（ P＜0．05）。 MTT법검측결과현시，HMGB1?siRNA전염조LoVo세포적생장속도교음성대조조화공백대조조강저（ P＜0．05）。류식세포술검측결과현시，HMGB1?siRNA전염조LoVo세포적증식지수위38．27±1．32，명현저우음성대조조（54．66±1．74）화공백대조조（57．43±1．29，P＜0．05）。Transwell소실실험현시，HMGB1?siRNA전염조천막세포수위（14．0±3．5）개／고배시야，명현저우음성대조조[（51．0±6．7）개／고배시야]화공백대조조[（68．0±5．3）개／고배시야，P＜0．05]。세포화흔실험현시，화흔후배양48 h시，HMGB1?siRNA전염조LoVo세포적천이거리위（83．61±23．21）μm，명현저우음성대조조[（202．86±46．46）μm]화공백대조조[（214．58±57．38）μm，P＜0．05]。라서성류실험현시，21 d후，종류최종체적위（521±34）mm3，명현소우음성대조조[（763±46）mm3]화공백대조조[（802±51）mm3，P＜0．05]。결론만병독개도적RNA간우기술가유효억제결직장암LoVo세포중HMGB1기인적표체，HMGB1기인침묵가사결직장암세포생장완만，증식주기연장，침습여천이능력명현하강，라서이식류적생장수도명현억제。
Objective To inquire into the influence of silencing HMGB1 expression by small interfering RNA ( siRNA) on cell growth, proliferation, invasion and metastasis of colorectal cancer LoVo cells both in vitro and in vivo. Methods Lentivirus?mediated HMGB1 siRNA was transfected into LoVo cells to silence the HMGB1 expression. The HMGB1 mRNA and protein expression after siRNA transfection was detected by RT?PCR and Western blot. MTT assay was used to observe the cell proliferation and to draw a growth curve. Cell cycle was measured by flow cytometry. The ability of invasion and speed of cell migration were evaluated by transwell chamber invasion and cell scratch assay. The influence of HMGB1 silencing on the proliferation of LoVo cells in vivo was observed in LoVo tumor?bearing nude mice. Results Lentivirus?mediated siRNA was successfully transfected into colorectal cancer cell line LoVo. The expression of HMGB1 mRNA and protein in the HMGB1?siRNA group were 0. 24 ± 0. 04 and 0. 21 ± 0. 03, respectively. Compared with the HMGB1?siRNA?Neg group (0.82±0.13, 1.15±0.18) and control group (0.93±0.15, 1.21±0.20), the difference was significant ( P<0.05) . MTT assay showed that the cell proliferation in the HMGB1?siRNA group was significantly inhibited when compared with that in the HMGB1?siRNA?Neg group and control group (P<0.05). Flow cytometry showed that the proliferation index (PI) of HMGB1?siRNA group was 38.27± 1.32, significantly lower than 54.66±1.74 in the HMGB1?siRNA?Neg group and 57.43±1.29 in the control group (P<0.05). The transwell assay showed that the number of penetrated cells in the HMGB1?siRNA group was 14.0±3.5, significantly lower than 51.0±6.7 in the HMGB1?siRNA?Neg group and 68.0±5.3 in the control group (P<0.05). Similarly, the scrape wound recovered significantly slower in the HMGB1?siRNA group (83.61±23.21) μm than that in the other two groups (202.86±46.46) μm and (214.58±57.38) μm (P<0.05). The nude mouse xenograft tumor experiment showed that the final tumor volume was (521±34) mm3 in the HMGB1?siRNA group, significantly smaller than that in the HMGB1?siRNA?Neg group of (763± 46) mm3 and control group of (802±51) mm3(P<0.05). Conclusions Lentivirus?mediated HMGBl?siRNA can effectively inhibit the HMGB1 expression in colorectal cancer LoVo cells both in vitro and in vivo. HMGB1 gene silencing can slow the growth of colorectal cancer cells, extend the cell proliferation cycle, decrease their invasion and migration, and significantly inhibit the growth of xenograft tumor in nude mice.
