中华妇产科杂志
中華婦產科雜誌
중화부산과잡지
CHINESE JOUNAL OF OBSTETRICS AND GYNECOLOGY
2011年
9期
678-683
,共6页
韩肖燕%钱海利%杨隽钧%张雪燕%付明%梁萧%林晨%向阳
韓肖燕%錢海利%楊雋鈞%張雪燕%付明%樑蕭%林晨%嚮暘
한초연%전해리%양준균%장설연%부명%량소%림신%향양
宫颈肿瘤%肿瘤浸润%肿瘤转移%组蛋白脱乙酰基酶类%阻遏蛋白质类
宮頸腫瘤%腫瘤浸潤%腫瘤轉移%組蛋白脫乙酰基酶類%阻遏蛋白質類
궁경종류%종류침윤%종류전이%조단백탈을선기매류%조알단백질류
Uterine cervical neoplasms%Neoplasm invasiveness%Neoplasm metastasis%Histone deacetylases%Repressor proteins
目的：探讨MTA1基因表达与宫颈癌细胞侵袭转移的关系。方法将真核细胞表达载体pcDNA3质粒、MTA1基因表达载体pcDNA3-MTA1质粒、MTA1基因RNA干扰载体pSilencer3.1-MTA1-siRNA质粒稳定转染宫颈癌细胞株CaSki细胞（分别命名为对照组、MTA1组、MTA1 -siRNA组），逆转录(RT) -PCR技术和蛋白印迹法检测CaSki细胞中MTA1 mRNA和蛋白的表达，四甲基偶氮唑蓝(MTT)比色法及平板集落形成实验检测CaSki细胞的增殖情况，划痕实验、穿膜实验检测CaSki细胞的迁移能力，基质黏附实验检测CaSki细胞的黏附能力，细胞侵袭实验检测CaSki细胞的侵袭能力，流式细胞仪检测CaSki细胞的细胞周期比例。将3组CaSki细胞接种于裸鼠腋下，观察其在裸鼠体内的生长情况。结果 MTA1组细胞中MTA1 mRNA和蛋白的表达强度均明显高于对照组，而MTA1 -siRNA组细胞中MTA1 mRNA和蛋白的表达强度均明显低于对照组。MTT比色法检测显示，与对照组比较，自第2天起，MTA1组细胞的生长速度加快，而MTA1-siRNA组细胞的生长速度减慢，分别比较，差异均有统计学意义(P＜0.05)；平板集落形成实验显示，对照组、MTA1组、MTA1 -siRNA组的克隆数分别为( 133±6)、(169±10)和(57±5)个，MTA1组明显多于对照组（P＜0.05），而MTA1-siRNA组明显少于对照组(P＜0.05)。划痕实验显示，MTA1组细胞的迁移能力明显增强，而MTA1 -siRNA组细胞的迁移能力明显减弱；穿膜实验显示，对照组、MTA1组、MTA1-siRNA组穿膜细胞数分别为( 153±17)、( 247±38)和(82±10)个，MTA1组明显多于对照组(P＜0.05)，而MTA1-siRNA组明显少于对照组(P＜0.05)。基质黏附实验显示，孵育90 min时，对照组、MTA1组、MTA1-siRNA组细胞的黏附率分别为(69.3±3.6)％、(80.4±5.6)％、(39.2±7.4)％，MTA1组明显高于对照组(P＜0.05)，而MTA1 -siRNA组明显低于对照组（P＜0.05）。细胞侵袭实验显示，对照组、MTA1组、MTA1 -siRNA组穿膜细胞数分别为(231±19)、(354±36)和(76±7)个，MTA1组明显多于对照组(P＜0.05)，而MTA1-siRNA组明显少于对照组(P＜0.05)。流式细胞仪检测显示，对照组、MTA1组、MTA1-siRNA组细胞的G1、S、G2期细胞比例分别比较，差异均无统计学意义(P＞0.05)。裸鼠接种CaSki细胞30d后，对照组、MTA1组、MTA 1-siRNA组裸鼠肿瘤体积分别为(519 ±236)、(2245±780)、(111±65) mm3，肿瘤质量分别为(0.41 ±0.19)、(2.10±0.39)、(0.11±0.08)g，MTA1组均明显高于对照组(P＜0.05)，而MTA1 -siRNA组均明显低于对照组(P＜0.05)。结论MTA1基因促进宫颈癌细胞的侵袭转移，沉默MTA1基因表达能使宫颈癌细胞生长减慢，细胞侵袭及转移能力下降，从而逆转宫颈癌细胞的恶性表型，为进一步研究MTA1基因的作用机制以及应用RNA干扰技术进行以MTA1基因为靶点的治疗打下了一定的基础。
目的：探討MTA1基因錶達與宮頸癌細胞侵襲轉移的關繫。方法將真覈細胞錶達載體pcDNA3質粒、MTA1基因錶達載體pcDNA3-MTA1質粒、MTA1基因RNA榦擾載體pSilencer3.1-MTA1-siRNA質粒穩定轉染宮頸癌細胞株CaSki細胞（分彆命名為對照組、MTA1組、MTA1 -siRNA組），逆轉錄(RT) -PCR技術和蛋白印跡法檢測CaSki細胞中MTA1 mRNA和蛋白的錶達，四甲基偶氮唑藍(MTT)比色法及平闆集落形成實驗檢測CaSki細胞的增殖情況，劃痕實驗、穿膜實驗檢測CaSki細胞的遷移能力，基質黏附實驗檢測CaSki細胞的黏附能力，細胞侵襲實驗檢測CaSki細胞的侵襲能力，流式細胞儀檢測CaSki細胞的細胞週期比例。將3組CaSki細胞接種于裸鼠腋下，觀察其在裸鼠體內的生長情況。結果 MTA1組細胞中MTA1 mRNA和蛋白的錶達彊度均明顯高于對照組，而MTA1 -siRNA組細胞中MTA1 mRNA和蛋白的錶達彊度均明顯低于對照組。MTT比色法檢測顯示，與對照組比較，自第2天起，MTA1組細胞的生長速度加快，而MTA1-siRNA組細胞的生長速度減慢，分彆比較，差異均有統計學意義(P＜0.05)；平闆集落形成實驗顯示，對照組、MTA1組、MTA1 -siRNA組的剋隆數分彆為( 133±6)、(169±10)和(57±5)箇，MTA1組明顯多于對照組（P＜0.05），而MTA1-siRNA組明顯少于對照組(P＜0.05)。劃痕實驗顯示，MTA1組細胞的遷移能力明顯增彊，而MTA1 -siRNA組細胞的遷移能力明顯減弱；穿膜實驗顯示，對照組、MTA1組、MTA1-siRNA組穿膜細胞數分彆為( 153±17)、( 247±38)和(82±10)箇，MTA1組明顯多于對照組(P＜0.05)，而MTA1-siRNA組明顯少于對照組(P＜0.05)。基質黏附實驗顯示，孵育90 min時，對照組、MTA1組、MTA1-siRNA組細胞的黏附率分彆為(69.3±3.6)％、(80.4±5.6)％、(39.2±7.4)％，MTA1組明顯高于對照組(P＜0.05)，而MTA1 -siRNA組明顯低于對照組（P＜0.05）。細胞侵襲實驗顯示，對照組、MTA1組、MTA1 -siRNA組穿膜細胞數分彆為(231±19)、(354±36)和(76±7)箇，MTA1組明顯多于對照組(P＜0.05)，而MTA1-siRNA組明顯少于對照組(P＜0.05)。流式細胞儀檢測顯示，對照組、MTA1組、MTA1-siRNA組細胞的G1、S、G2期細胞比例分彆比較，差異均無統計學意義(P＞0.05)。裸鼠接種CaSki細胞30d後，對照組、MTA1組、MTA 1-siRNA組裸鼠腫瘤體積分彆為(519 ±236)、(2245±780)、(111±65) mm3，腫瘤質量分彆為(0.41 ±0.19)、(2.10±0.39)、(0.11±0.08)g，MTA1組均明顯高于對照組(P＜0.05)，而MTA1 -siRNA組均明顯低于對照組(P＜0.05)。結論MTA1基因促進宮頸癌細胞的侵襲轉移，沉默MTA1基因錶達能使宮頸癌細胞生長減慢，細胞侵襲及轉移能力下降，從而逆轉宮頸癌細胞的噁性錶型，為進一步研究MTA1基因的作用機製以及應用RNA榦擾技術進行以MTA1基因為靶點的治療打下瞭一定的基礎。
목적：탐토MTA1기인표체여궁경암세포침습전이적관계。방법장진핵세포표체재체pcDNA3질립、MTA1기인표체재체pcDNA3-MTA1질립、MTA1기인RNA간우재체pSilencer3.1-MTA1-siRNA질립은정전염궁경암세포주CaSki세포（분별명명위대조조、MTA1조、MTA1 -siRNA조），역전록(RT) -PCR기술화단백인적법검측CaSki세포중MTA1 mRNA화단백적표체，사갑기우담서람(MTT)비색법급평판집락형성실험검측CaSki세포적증식정황，화흔실험、천막실험검측CaSki세포적천이능력，기질점부실험검측CaSki세포적점부능력，세포침습실험검측CaSki세포적침습능력，류식세포의검측CaSki세포적세포주기비례。장3조CaSki세포접충우라서액하，관찰기재라서체내적생장정황。결과 MTA1조세포중MTA1 mRNA화단백적표체강도균명현고우대조조，이MTA1 -siRNA조세포중MTA1 mRNA화단백적표체강도균명현저우대조조。MTT비색법검측현시，여대조조비교，자제2천기，MTA1조세포적생장속도가쾌，이MTA1-siRNA조세포적생장속도감만，분별비교，차이균유통계학의의(P＜0.05)；평판집락형성실험현시，대조조、MTA1조、MTA1 -siRNA조적극륭수분별위( 133±6)、(169±10)화(57±5)개，MTA1조명현다우대조조（P＜0.05），이MTA1-siRNA조명현소우대조조(P＜0.05)。화흔실험현시，MTA1조세포적천이능력명현증강，이MTA1 -siRNA조세포적천이능력명현감약；천막실험현시，대조조、MTA1조、MTA1-siRNA조천막세포수분별위( 153±17)、( 247±38)화(82±10)개，MTA1조명현다우대조조(P＜0.05)，이MTA1-siRNA조명현소우대조조(P＜0.05)。기질점부실험현시，부육90 min시，대조조、MTA1조、MTA1-siRNA조세포적점부솔분별위(69.3±3.6)％、(80.4±5.6)％、(39.2±7.4)％，MTA1조명현고우대조조(P＜0.05)，이MTA1 -siRNA조명현저우대조조（P＜0.05）。세포침습실험현시，대조조、MTA1조、MTA1 -siRNA조천막세포수분별위(231±19)、(354±36)화(76±7)개，MTA1조명현다우대조조(P＜0.05)，이MTA1-siRNA조명현소우대조조(P＜0.05)。류식세포의검측현시，대조조、MTA1조、MTA1-siRNA조세포적G1、S、G2기세포비례분별비교，차이균무통계학의의(P＞0.05)。라서접충CaSki세포30d후，대조조、MTA1조、MTA 1-siRNA조라서종류체적분별위(519 ±236)、(2245±780)、(111±65) mm3，종류질량분별위(0.41 ±0.19)、(2.10±0.39)、(0.11±0.08)g，MTA1조균명현고우대조조(P＜0.05)，이MTA1 -siRNA조균명현저우대조조(P＜0.05)。결론MTA1기인촉진궁경암세포적침습전이，침묵MTA1기인표체능사궁경암세포생장감만，세포침습급전이능력하강，종이역전궁경암세포적악성표형，위진일보연구MTA1기인적작용궤제이급응용RNA간우기술진행이MTA1기인위파점적치료타하료일정적기출。
Objective To investigate the relationship between metastasis-associated gene 1 ( MTA1 )expression and invasive and metastatic ability of cervical cancer cell. Methods Three kinds of plasmids pcDNA3( control group), pcDNA3-MTA1 ( MTA1 group) and pSilencer3. 1-MTA1-siRNA ( MTA1-siRNAgroup) were transfected into human cervical cancer cell line CaSki cells. Reverse transcription (RT)-PCR and western blot were used to detected MTA1 mRNA and protein expressions. The effects of MTA1 expression on CaSki cell growth and proliferation, cell migration, adhesion and invasion, and cell cycles were tested by methyl thiazolyl tetrazolium (MTT), clone formation experiment, wound-healing assay, transwell assay, adhesion assay and flow cytometry, respectively. In animal experiment, three groups of cells were inoculated to BALB/c nude mouse subcutaneously to observe tumor formation ability. Results Compared with control group, MTA1 mRNA and protein were significantly overexpressed in MTA1 group, while MTA1-siRNA group showed lower MTA1 expression. Compared with control group, MTA1 group showed significantly accelerated cell growth; while MTA1-siRNA group showed decreased cell growth since the second day (P＜0. 05). Clone formation number in control, MTA1 and MTA1-siRNA group were 133 ±6, 169 ± 10 and 57 ±5,respectively. MTA1 group showed accelerated cell formation, while MTA1-siRNA group showed the reverse effect compared with that in control group(P ＜ 0. 05 ). At 24, 48 and 72 hours after wounding, the healing ability of MTA1-siRNA group significantly lagged behind that in the control group, while MTA1 group showed accelerated cell healing ability. The adhesion rate of control, MTA1 and MTA1-siRNA group were (69. 3 ± 3. 6) ％, ( 80. 4 ± 5. 6 ) ％ and ( 39. 2 ± 7.4 ) ％ separately at 90 minutes after cell seeding. In contrast with control group, MTA1 group promoted the adhesion of CaSki cell to matrigel matrix, while MTA1-siRNA group inhibited the adhesion process (P ＜0. 05 ). In the migration assay, the number of cells migrated to the bottom side of the membrane in control,MTA1 and MTA1-siRNA group were 153 ± 17,247 ± 38 and 82 ± 10, respectively. The number of cells in the invasion assay were 231 ± 19,354 ± 36 and 76 ± 7, respectively. Compared with the control group, MTA1 group significantly increased the migration and invasion ability, while MTA 1-siRNA group showed lower cell migration and invasion ability (P ＜ 0. 05 ). In cell cycle experiment, no significant differences of cell proportions including G1, S and G2 stage were found among three groups (P ＞ 0.05).In animal experiment, compared with control group, MTA1 group showed accelersted tumor formation and growth, while the MTA1-siRNA group showed the reverse effect ( P ＜ 0. 05 ). Conclusions MTA1 may play its roles to promote cervical cancer cell invasion, migration, adhesion, as well as cell growth and colony formation, while RNA interference against MTA1 may decrease the malignant phenotypes. This study shows that it will be an effective beginning to explore metastasis mechanisms and cancer gene therapy strategy targeting MTA1 in cervical cancer.
