摘要
目的:间皮素(Mesothelin,MSLN)是近年来发现的一种肿瘤分化抗原,通常表达在胸膜、腹膜和心包的正常间皮细胞,但在恶性间皮瘤、卵巢癌、胰腺癌等恶性肿瘤中高表达。MSLN的前体蛋白可被furin蛋白酶水解为Mr分别为40kDa和31kDa的两个片段,其中40kDa的片段通过糖基磷脂酰肌醇(glycosylphosphtidylinositol)与胞膜相连,称之为间皮素(MSLN);31kDa的片段脱落,称为巨核细胞集落刺激因子(megakaryocyte-potentiating factor,MPF)。应用cDNA微阵列方法分析卵巢上皮性癌(Epithelial Ovarian Cancer EOC)和正常卵巢组织的基因表达谱,结果显示具有潜在生物学标记的MSLN显著上调表达。研究发现CA125与膜连接蛋白间皮素之间以某种特殊的方式结合,两者的相互作用介导了细胞之间的粘附,可能与卵巢上皮性癌细胞的腹腔种植,最终导致广泛转移有关,提示MSLN可能是一种很有前途的肿瘤标志物和治疗靶点。但迄今为止MSLN的功能研究还仅仅限于细胞粘附方面,对其他方面的功能了解甚少。
利用慢病毒介导的RNA干扰(RNA interference RNAi)方法,可以长期沉默靶细胞的基因表达,用于基因功能研究和基因治疗等方面。本课题组前期研究构建了间皮素RNA干扰重组慢病毒质粒载体,制备的重组质粒经酶切及测序鉴定证明重组成功,并包装出病毒颗粒,测定的病毒颗粒符合后续实验要求。基于此,本研究利用RNAi技术对MSLN基因表达进行抑制以达到治疗肿瘤的目的,进行了以下研究。
方法:1分别用携带pMSLN-negative、pMSLN- shRNA1、pMSLN- shRNA2、pMSLN- shRNA3、pMSLN- shRNA4质粒的慢病毒液感染SKOV-3细胞96小时后,激光共聚焦显微镜下观察感染效率。2利用Western blotting检测各组细胞中间皮素蛋白的表达,选择干扰效率高的shRNA4序列细胞进行后续实验。3应用间接免疫荧光染色法检测重组慢病毒介导的RNAi对间皮素蛋白表达定位的影响。4应用细胞增殖实验和克隆形成的方法检测间皮素低表达细胞的增殖力变化。5 EDTA介导的细胞分离实验和细胞粘附实验检测间皮素低表达细胞体外粘附能力的变化。6细胞侵袭、迁移实验检测间皮素低表达侵袭能力的变化。
结果:1慢病毒感染SKOV-3细胞96小时后,激光共聚焦显微镜下观察感染细胞中荧光细胞的数量,说明感染效率为80%-90%。
2 Western blotting结果显示,与OVCAR-3细胞相比,OVCAR-3/neg以及OVCAR-3/(shRNA1～shRNA4)的蛋白表达分别下调了10%、9%、53%、84%、90% ,其中OVCAR-3/shRNA4的干扰效率最高为:90%。
3 MSLN在SKOV-3细胞的定位表达,可见MSLN蛋白标记为红色荧光,主要分布在细胞膜, SKOV-3/shRNA组的MSLN蛋白荧光强度明显低于SKOV-3、SKOV-3/neg两组。
4细胞增殖实验结果显示,干扰组(SKOV-3/shRNA),阴性对照组(SKOV-3/neg)和空白对照组(SKOV-3)的细胞个数分别为(9.89±2.0)×105,(18.9±2.24)×105,(19.81±2.5)×105,结果比较差异有统计学意义(F=29.76,P<0.05)。干扰组细胞增殖速度显著慢于阴性对照组和空白对照组(P<0.05),阴性对照组和空白对照组之间增殖速度无明显差别(P=0.531)。
克隆形成实验结果显示,干扰组(SKOV-3/shRNA)克隆形成率为(15.85±2.5)% ,阴性对照组(SKOV-3/neg)为(28.3±2.7)%和空白对照组(SKOV-3)为(29.13±1.98)%,结果比较差异有统计学意义(F=47.62,P<0.05)。干扰组细胞克隆形成率明显低于其他两组(P<0.05),阴性对照组(SKOV-3/neg)和空白对照组(SKOV-3)之间克隆形成率无明显差别(P=0.612)。
5细胞黏附能力的测定
EDTA介导的细胞分离实验:干扰组(SKOV-3/shRNA)各个时相点的脱壁率(13.86±1.05)%,(34.44±2.72)% ,(56.71±3.4)% ,(83.76±4.37)%显著高于阴性对照组(8.32±1.1)% ,(20.04±1.88)% ,(33.07±2.52)% ,(42.73±3.61)%和空白对照组(9.28±0.95)%, (18.12±0.85)% , (29.97±2.15)%, (40.43±2.66)% (P<0.05),阴性对照组和空白对照组之间无明显差别(P>0.05)。
细胞粘附实验结果:干扰组(SKOV-3/shRNA)、阴性对照组(SKOV-3/neg)和空白对照组(SKOV-3)的粘附率分别为(12.12±2.21)%,(49.78±3.2)%及(50.74±2.65)%,结果分析差异有统计学意义(F=327.87,P<0.05),干扰组(SKOV-3/shRNA)的粘附率显著低于阴性对照组(SKOV-3/neg)和空白对照组(SKOV-3) (P<0.05)。阴性对照组(SKOV-3/neg)和空白对照组(SKOV-3)的粘附率差异无统计学意义(P=0.587)。
6间皮素表达变化对细胞侵袭能力的影响
细胞侵袭实验显示,干扰组(SKOV-3/shRNA)、阴性对照组(SKOV-3/neg)以及空白对照组(SKOV-3)转入底层膜的细胞数分别为(7.5±1.78)个、(27.54±3.39)个、(29±2.29)个,结果分析差异有统计学意义(F=208.96,P<0.05)。干扰组细胞数少于空白对照组和阴性对照组,差异有统计学意义(P<0.05),空白对照组(SKOV-3)和阴性对照组(SKOV-3/neg)差别无统计学意义(P=0.227)。
迁移实验结果显示,各组迁移到滤膜下面的细胞数,干扰组为(18.7±2.11)个,阴性对照组为(44.7±4.57)个,空白对照组为(47±4.14)个,结果分析差异有统计学意义(F=174.51,P<0.05)。干扰组细胞数少于空白对照组和阴性对照组,差异有统计学意义(P<0.05),而空白对照组(SKOV-3)和阴性对照组(SKOV-3/neg)差别无统计学意义(P=0.183)。
结论:1病毒载体系统能有效的把目的基因整合至靶细胞基因组,实现稳定表达,并有较高的转染效率。
2通过Western blotting检测证实MSLN-RNAi能有效抑制MSLN蛋白的表达,同时干扰后细胞生长特性发生改变,说明MSLN影响细胞增殖能力。
3通过降调MSLN基因,发现细胞的粘附、侵袭以及迁移功能明显降低。说明MSLN影响卵巢癌细胞和基底膜之间的粘附以及肿瘤细胞的侵袭迁移功能。
以上结果为进一步研究MSLN的功能以及通过阻断MSLN基因表达治疗恶性肿瘤奠定了基础。
Objective:Mesothelin is a tumour differentiation antigen that is normally present on the mesothelial cells lining the pleura,peritoneum and pericardium.It is,however,highly expressed in several human cancers including malignant mesothelima,pancreatic,and epithelial ovarian carcinoma(EOC). The MSLN precursor protein is cleaved by furin to yield the 40-kDa fragment ,mesothelin, attached to the cell membrane by a glycosyphosphatidy inositol linkage and a smaller 32-kDa fragment named megakaryocyte-potentiating factor, that is released from the cell. Analysis the gene expression profiles between EOC and normal ovarian tissue by the cDNA microarray, the result showes that the mesothelial which has potential biological markers up-regulated the expression significantly. Recent studies have shown that CA125 binds to mesothelin in a specific manner with the two-mediated interaction between the cell adhesion,which may play a role in the peritoneal spread of EOC suggesting that mesothelin may be a promising tumor markers and therapeutic targets. But, so far, the function study of the mesothelial is still limited to cell adhesion,and the knowledge about the other mesothelial-function is still very poor.
RNAi can silence gene expression in long-term, that can be used for the study of gene function and gene therapy, and so on. Using lentiviral pRNAT-U6.2/Lenti vector was constructed five recombinant plasmid on preliminary studies, preparation of the recombinant plasmid cutting and sequencing proved successful reorganization. And packaging the virus particles,the virus particles were conformed to the following experiment.
To test whether this RNAi strategy can silence MSLN gene expressed in many tumors, following investigations were conducted in this study.
Methods:1 Observe the infection efficiency by the laser scanning confocal microscope after the SKOV-3 cells was infected with the virus which respectively carry pMSLN-negative, pMSLN-shRNA1, pMSLN-shRNA2, pMSLN-shRNA3, pMSLN-shRNA4 plasmid for 96 hours.
2 Using Western blotting methods to detect the expression of mesothelin protein,and choosing the high-efficiency cells carried out the following experiment.
3 Using indirect immunofluorescence staining to detect recombinant lentivirus-mediated RNAi of mesothelin expression.
4 The proliferation of cells changes was evaluated by proliferation experiments and clone formation methods.
5 Cell adhesion ability were examined by EDTA-induced cell detachment assay and cell adhesion test.
6 Cell invasion and migration assay to detect the invasion ability .
Results:1 Using the laser scanning confocal microscope to observe the efficiency of infection,found that the infection rate was 80% -90%.
2 Western blotting results showed that, compared with OVCAR-3 , OVCAR-3/neg, OVCAR-3/(shRNA1 ~ shRNA4) protein expression were reduced by 10%, 9%, 53%, 84%, 90%, that means the maximum interference efficiency is OVCAR-3/shRNA4, it is 90%.
3 It can be seen that MSLN marked as red fluorescence, mainly distributed in the membrane, SKOV-3/shRNA group MSLN protein fluorescence intensity was significantly lower than SKOV-3, SKOV-3/neg groups.
4 Cell proliferation results: The amount of cells were (9.89±2.0)×10~5,(18.9±2.24)×10~5,(19.81±2.5)×10~5,respectively, the results had a significant difference(F=29.76,P<0.05).The proliferating ability of SKOV-3/shRNA cells was significantly lower than the latter two groups (P<0.05), and the negative control group and the blank control group were no significant difference in the proliferation rate (P=0.531).
Cloning experiment results:The colony forming rate were (15.85±2.5) %in interference group, (28.3±2.7)% in the negative control group , (29.13±1.98)% in the blank control group , the results had a significant difference (F=47.62 ,P<0.05).SKOV-3/shRNA colony formation was significantly lower than the negative control group (SKOV-3/neg) or the blank control group (SKOV3) (P<0.05). Negative control group (SKOV-3/neg) and the blank control group (SKOV3) between cloning was no significant difference (P=0.612).
5 Adhesion test
The detachment rate of interference group (SKOV-3-shRNA) in time points was (13.86±1.05)%,(34.44±2.72)%,(56.71±3.4)% ,(83.76±4.37)%,respectively.They were significantly higher than that of the negative control group(SKOV-3/neg)and the blank control group(SKOV)(P<0.05), the negative control group and the blank control group were no significant difference in the cell detachment rate(P>0.05).
Cell adhesion test shown that the adhesion rate were (12.12±2.21)% in interference group, (49.78±3.2)% in the negative control group(50.74±2.65)% in the blank control group , the results had a significant difference( F=327.87,P<0.05).SKOV-3/shRNA cell adhesion rate was significantly lower than that of the negative control group(SKOV-3/neg)and the blank control group(SKOV3)( P<0.05), the negative control group and the blank control group were no significant difference in the cell adhesion rate(P=0.587).
6 Cell invasion and migration assay to detect the invasive ability of cells
The cell counts in assay of invasive ability were(7.5±1.78), (27.54±3.39), (29±2.29),respectively. The results had a significant difference( F=208.96,P<0.05).There were significant differences compared the interference group with the latter two groups(P<0.05),while there was no difference between the latter two(P=0. 227).
The cell counts of migratory test were (18.7±2.11), (44.7±4.57) and (47±4.14), respectively.The results had a significant difference( F=174.51,P<0.05).There were significant differences compared the interference group with the latter two group( P<0.05),while there was no difference between the latter two(P=0. 183).
Conclusion:1 Virus vector systems can integrate the target cell genome effectively, and achieve stable expression, and have high transfection efficiency.
2 Confirmed by Western blotting detection MSLN RNAi can effectively inhibit protein expression,and chang the characteristis of cell growth.
3 MSLN RNAi can decrease cell adhesion, invasion and migration ability significantly. Mesothelin impact of the ovarian cancer cells and the basement membrane of tumor cell adhesion and migration function of the invasion.
The above results are very helpful for the further study of MSLN function and treating the malignant tumor by blocking the mesothelin gene expression.
引文
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