稳定转染重组STAT3基因对喉癌Hep-2细胞增殖及侵袭的影响

发布时间：2018-02-28 03:50

本文关键词： 喉癌 RNA干扰 STAT3 稳定转染细胞增殖细胞侵袭　出处：《河北医科大学》2010年硕士论文　论文类型：学位论文

【摘要】： 目的:应用RNAi(RNA干扰)技术沉默STAT3(信号转导子和转录激活子3),筛选出能稳定表达siRNA-STAT3基因的细胞系,观察对Hep-2细胞增殖力及侵袭力的影响,在蛋白水平上进一步探讨STAT3作用于Hep-2细胞增殖和侵袭可能的机制。以寻找抑制喉癌增殖和侵袭的新方法,为临床通过基因沉默技术治疗喉癌提供实验支持和理论依据。方法:构建重组质粒真核表达载体PGPU6/GFP/Neo-siRNA-STAT3,将重组表达质粒用脂质体LipofactaminTM2000转染人喉癌Hep-2细胞,通过G418持续单克隆筛选,Western-blot印迹法鉴定,进而筛选出稳定表达siRNA-STAT3的细胞系。实验分为三组:siRNA-STAT3组、siRNA-shNC组及空白对照组。应用流式细胞术检测Cyclin Dl、P21、ICAM-1、VEGF蛋白水平的表达情况及细胞周期的变化;采用MTT法观察接种培养板1d、2d、3d、4d、5d、6d后喉癌Hep-2细胞增殖状态;MTT法观察其粘附性的变化;细胞划痕法观察其迁移性;Transwell法检测其侵袭能力的变化。结果: (1)构建了重组pGPU6/GFP/Neo-siRNA-STAT3质粒载体通过模板退火和载体的线性化等步骤,构建了重组质粒表达载体。酶切结果表明,所有质粒均为阳性重组载体。 (2)细胞转染与G418筛选荧光显微镜下观察瞬时转染6小时后较多Hep-2细胞内有绿色荧光蛋白(GFP)表达,24小时后GFP表达明显增强。G418筛选结果显示空白对照组的细胞全部死亡,而siRNA-STAT3转染组和siRNA-shNC转染组的细胞出现明显而且数量较多的细胞克隆集落形成。 (3)Western-blot鉴定p-STAT3的表达结果显示siRNA-STAT3组的p-STAT3的表达明显减少,而siRNA-shNC组和空白对照组细胞p-STAT3的表达未见有下降。 (4)MTT检测细胞增殖状态Hep-2细胞在稳定转染siRNA-STAT3后,同空白对照组和转染siRNA-shNC无关质粒组比,2天后就开始表现出较明显的抑制效应(P0.05),即使6天后在对照组细胞生长变缓时,siRNA-STAT3组Hep-2细胞生长抑制仍较明显(P0.05),故其抑制效应随时间延长愈明显,并表现出一定的时间抑制效应关系。 (5)平板克隆形成试验2周后,观察三组均有一定的单克隆集落形成。结果显示siRNA- STAT3组Hep-2细胞克隆形成率为(15.17±2.32)%明显低于siRNA-shNC组(24.33±1.97)%及空白对照组(26.33±2.16)%(P0.05)。 (6)流式细胞术(FCM)检测细胞周期应用FCM检测细胞周期分布,转染重组siRNA-STAT3质粒的Hep-2细胞G1期比率由(60.7±0.9)%上升至(66.0±2.0)%(P0.05),S期所占比率由(34.8±1.1)%降至(24.6±1.9)%(P0.05)。 (7)FCM检测Cyclin Dl、P21、ICAM-1、VEGF蛋白的表达观察siRNA-STAT3组Cyclin Dl、ICAM-1、VEGF蛋白表达荧光指数分别为0.71±0.08(P0.05)、0.83±0.06(P0.05)、0.74±0.02(P0.05),均低于siRNA-shNC组,siRNA-STAT3组P21蛋白表达荧光指数(1.39±0.10)则高于siRNA-shNC组(P0.05)。 (8)细胞黏附试验应用MTT法检测细胞在Matrigel基质上的粘附性,接种96孔培养板20分钟后观察各组细胞的黏附率相差不大(P0.05),但有一定的趋势性,40分钟后siRNA-STAT3组的黏附率小于siRNA-shNC组和空白对照组(P0.05),这种抑制效应在60分钟后更加明显(P0.05)。 (9)细胞迁移试验在培养板生长48小时后,荧光显微镜下见空白对照组和siRNA-shNC组的Hep-2细胞向划痕区生长速度明显快于siRNA-STAT3组,而空白对照组和siRNA-shNC组的Hep-2细胞向划痕区生长速度差异不明显。 (10)体外侵袭试验各组细胞接种Transwell小室上室内24小时后,400×光学显微镜下随机5个视野中观察siRNA-STAT3组穿过Matrigel基质和滤膜的细胞(85.20±4.92)明显少于空白对照组(122.8±7.50)和siRNA-shNC组(126.4±8.73)(P0.05)。结论: 1、成功构建了重组pGPU6/GFP/Neo-siRNA-STAT3质粒载体。 2、应用G418筛选出稳定表达siRNA-STAT3基因的喉癌Hep-2细胞系。 3、抑制STAT3的活性后Hep-2细胞周期形成G1期阻滞,其增殖能力和单克隆形成能力均明显下降。说明喉癌细胞具有无限增殖能力与STAT3的高表达有一定的关联。 4、本实验应用RNA干扰技术沉默STAT3活性发现能抑制喉癌Hep-2细胞的粘附、迁移及侵袭能力。推测表达STAT3基因的喉癌细胞因提高了其黏附、迁移和降解基质的能力而增加了侵袭到达周围的组织及远处转移并定植生长的机会。说明STAT3在喉癌的侵袭及转移中起着重要的作用。 5、阻断STAT3信号通路后,STAT3下游调控蛋白CyclinD1、VEGF、ICAM-1的表达降低,而P21蛋白的表达升高。提示CyclinD1、P21、VEGF、ICAM-1蛋白可能在STAT3的诱导下参与了STAT3信号通路对喉鳞癌细胞增殖、侵袭及浸润转移的作用。
[Abstract]:Objective: to apply RNAi (RNA interference) silencing STAT3 (signal transducer and activator of transcription 3), screened stable expression of siRNA-STAT3 gene in cell lines, to observe its effect on Hep-2 cell proliferation and invasiveness, at the protein level to further explore the mechanism of STAT3 on proliferation and invasion of Hep-2 cells. New method for inhibiting the proliferation and invasiveness of laryngeal carcinoma, and provide experimental and theoretical basis for the gene silencing technique for clinical treatment of laryngeal cancer.
Methods: to construct the recombinant plasmid eukaryotic expression vector PGPU6/GFP/Neo-siRNA-STAT3, the recombinant expression plasmid by liposome transfection of LipofactaminTM2000 human laryngeal carcinoma Hep-2 cells, sustained by G418 monoclonal screening, Western-blot blotting, and then screened siRNA-STAT3 stable expression cell lines. The experiment was divided into three groups: siRNA-STAT3 group, siRNA-shNC group and control group by flow. Detection of Cyclin Dl, P21 ICAM-1, cytometry, change the expression level of VEGF protein and cell cycle; MTT assay was used to observe the inoculated plates 1D, 2D, 3D, 4D, 5D, Hep-2 cell proliferation in laryngeal carcinoma after 6D; observe the changes of adhesion of MTT; cell scratch test was used to observe the movement change; the invasion ability was detected by Transwell.
Result:
(1) a recombinant plasmid vector was constructed by template annealing and vector linearization. The recombinant plasmid expression vector was constructed. The results of pGPU6/GFP/Neo-siRNA-STAT3 digestion showed that all plasmids were positive recombinant vectors.
(2) cells transfected with G418 were observed under fluorescence microscope 6 hours after transient transfection of Hep-2 cells with more green fluorescent protein (GFP) expression, GFP expression was significantly enhanced.G418 test showed the control group cells all died 24 hours after siRNA-STAT3 transfection group and siRNA-shNC transfection group the cell clone and obvious a large number of colony formation.
(3) the expression of p-STAT3 was identified by Western-blot. Results showed that the expression of p-STAT3 in siRNA-STAT3 group was significantly reduced, while the expression of p-STAT3 in siRNA-shNC group and blank control group did not decrease.
(4) MTT detection of cell proliferation in Hep-2 cells stably transfected with siRNA-STAT3, blank control group and transfection of siRNA-shNC plasmid to group, 2 days later began to exhibit obvious inhibitory effect (P0.05), even after 6 days in the control group the cell growth slowed, siRNA-STAT3 group was still significantly inhibited the growth of Hep-2 cells (P0.05), the inhibition effect increased with time is more obvious, and show some time inhibition effect relationship.
(5) after 2 weeks, the colony formation of three groups was observed. The results showed that the colony formation rate of Hep-2 cells in siRNA- STAT3 group was (15.17 + 2.32)%, which was significantly lower than that in siRNA-shNC group (24.33 + 1.97)% and blank control group (26.33 + 2.16)% (P0.05).
(6) flow cytometry (FCM) was used to detect cell cycle. The cell cycle distribution was detected by FCM. The G1 phase ratio of Hep-2 cells transfected with recombinant siRNA-STAT3 plasmid increased from (60.7 + 0.9)% to (66 + 2)% (P0.05), and the proportion of S phase decreased from (34.8 + 1.1)% to (24.6 + 1.9)% (P0.05).
(7) FCM Cyclin Dl P21, ICAM-1 assay, and the expression of VEGF protein was observed in the siRNA-STAT3 group Cyclin Dl, ICAM-1, VEGF protein expression and fluorescence index were 0.71 + 0.08 (P0.05), 0.83 + 0.06, 0.74 + 0.02 (P0.05) (P0.05), siRNA-shNC group was higher than that in siRNA-STAT3 group the expression of P21 protein fluorescence index (1.39 + 0.10) was higher than that of siRNA-shNC group (P0.05).
(8) adhesion to detect cell adhesion test using MTT method on Matrigel matrix, inoculated in 96 well culture plates for 20 minutes were observed after the cell adhesion rate difference (P0.05), but there is a certain trend, 40 minutes after the adhesion rate of siRNA-STAT3 group was lower than siRNA-shNC group and blank control group (P0.05) this inhibitory effect is more obvious, in 60 minutes (P0.05).
(9) cell migration test in culture plates after 48 h of growth under fluorescence microscope showed that the control group and the siRNA-shNC group of Hep-2 cells to wound area faster than the growth rate of siRNA-STAT3 group and blank control group and siRNA-shNC group of Hep-2 cells to wound area growth rate was not significantly different.
(10) in vitro invasion assay cells were inoculated with Transwell cells on the indoor 24 hours later, 400 x optical microscope 5 random field observation group siRNA-STAT3 through Matrigel matrix and cell membrane (85.20 + 4.92) were significantly less than the control group (122.8 + 7.50) and siRNA-shNC group (126.4 + 8.73) (P0.05).
Conclusion:
1, the recombinant plasmid vector of pGPU6/GFP/Neo-siRNA-STAT3 was successfully constructed.
2, using G418 to screen the Hep-2 cell line of the larynx cancer that stably expressed the siRNA-STAT3 gene.
3, after inhibiting STAT3 activity, the Hep-2 cell cycle formed G1 phase arrest, and its proliferative capacity and monoclonal ability were significantly decreased. This indicates that the proliferation ability of laryngeal carcinoma cells is correlated with the high expression of STAT3.
4, we used RNA interference technology to silence STAT3 activity that can inhibit the adhesion of laryngeal carcinoma Hep-2 cells, migration and invasion ability. It is suggested that the expression of STAT3 gene in laryngeal carcinoma cells by improving the adhesion, migration and degradation ability of matrix and increased the invasion to the surrounding tissue and distant metastasis and opportunity for colonization and growth. It shows that STAT3 plays an important role in the invasion and metastasis of laryngeal carcinoma.
5, after blocking the STAT3 pathway downstream of STAT3 regulatory protein CyclinD1, VEGF, decreased the expression of ICAM-1, while the expression of P21 protein increased. P21, VEGF, CyclinD1, ICAM-1 in the induction of STAT3 protein may be involved in STAT3 signaling pathway on the proliferation of laryngeal squamous cell carcinoma, invasion and metastasis effect.

【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R739.6

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