PinX1抑制胃癌细胞增殖、增强其对5-FU敏感性及机制研究
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摘要
目的:
端粒、端粒酶在瘤肿细胞的永生化和演进中扮演重要角色,端粒酶在人类肿瘤细胞中高表达而在人正常细胞中无表达或仅在生殖细胞中低表达,因此在治疗上常作为治疗靶标。端粒酶逆转录酶(hTERT)是端粒酶活性关键的限速酶。研究表明,恶性肿瘤端粒酶活性不仅受hTERT调节,而且受其它因子的调节。PinX1为2001年报导,以细胞周期蛋白Pin2为诱饵,应用酵母双杂交方法,在人类宫颈癌细胞中发现的一种新基因。研究发现,PinX1及其端粒酶抑制域TID域可以直接结合于人端粒酶,进而抑制端粒酶活性,是端粒酶活性的强力抑制剂,但其抑制端粒酶活性的确切机制仍不清楚。我们利用分子克隆技术、RNA干涉技术及脂质体转染技术,将PinX1基因转染入本身无该基因表达的胃癌MKN28细胞,观察转染前后MKN28细胞生物学行为及对化疗药物5-FU的敏感性变化;将针对PinX1编码区的干涉序列转染入高表达该基因的胃癌BGC823细胞。检测转染前后各组细胞中端粒酶活性及Mad1/c-Myc水平的改变。探讨PinX1抑制端粒酶活性可能的新机制,初步为该基因应用于临床胃癌的治疗提供实验依据。
方法:
1利用分子克隆技术,构建包含全长人PinX1序列的真核表达载体;同时根据RNA干涉序列的设计原则,在线设计3条针对PinX1编码区的干涉序列,并通过BLAST设计了阴性对照序列,构建PinX1的RNA干涉真核表达载体以及阴性对照载体。2 RT-PCR、Western Blotting分别检测3种分化程度不同的胃癌细胞系(高分化MKN28细胞、中分化SGC7901细胞、低分化BGC823细胞)中PinX1表达水平;将构建成功的真核表达载体及干涉载体利用脂质体分别转染胃癌MKN28及BGC823细胞,经G418抗性筛选获得稳定表达的细胞株。3半定量RT-PCR、Western Blotting检测转染前后各细胞系中PinX1表达变化。4、通过HE染色、透射电镜等观察转染前后细胞形态特征的变化。5、通过MTT法观察转染前后细胞的增殖能力的变化,绘制细胞生长曲线。6通过MTT法检测转染前后胃癌MKN28细胞对化疗药物5-FU的敏感性变化。7通过流式细胞仪检测细胞凋亡和细胞周期的改变。8通过半定量RT-PCR及Western Blotting检测转染前后各细胞系中Mad1/c-Myc水平的改变。9、采用荧光素标记的端粒酶重复序列扩增法(TRAP)法检测转染前后各细胞系端粒酶活性的变化。
结果:
1经酶切及测序证明,成功构建重组PinX1真核表达载体及针对PinX1编码区的的RNA干涉真核表达载体与阴性对照载体;2、检测表明:高分化的MKN28细胞中无PinX1表达,低分化的BGC823细胞PinX1高表达,中分化的SGC7901细胞PinX1中等表达;3成功地将构建好的重组真核表达载体及干涉载体通过脂质体分别转染胃癌MKN28与BGC823细胞,经G418抗性筛选获得稳定表达的细胞株;检测表明,PinX1基因成功地转染入MKN28细胞;有2条干扰序列对PinX1有干扰效果;4细胞形态特征:HE染色可见转染PinX1基因的MKN28细胞为多边形,但是大小不一更为明显,大而扁平的细胞更为常见,巨细胞比例明显增加,最大的细胞体积可以达到小细胞的5～10倍,多核细胞常见;未转染的MKN28细胞呈梭形、短梭形,大小不一。透射电镜特征:转染PinX1基因的MKN28细胞内可见凋亡小体,小体内可见半月形异染色质;而未转染或仅转染空载体的胃癌MKN28细胞内可见细胞器减少,幼稚,线粒体呈杆状,核浆比例增大,核仁突出;5 MTT检测表明:转染PinX1基因的MKN28细胞较之未转染或仅转染空载体的胃癌MKN28细胞生长明显减慢(P<0.05);6 MTT检测表明:转染PinX1基因的MKN28细胞对5-FU的半数抑制浓度(IC50)值为0.253±0.021mg/L,明显低于未转染或仅转染空载体的胃癌MKN28细胞的IC50值(分别为0.560±0.017及0.590±0.026 mg/L)(P<0.05);7流式细胞仪检测细胞周期和细胞凋亡表明:转染PinX1基因的MKN28细胞的G0/G1期细胞数(%)为84.76±3.92,明显高于未转染或仅转染空载体的胃癌MKN28细胞的G0/G1期细胞数(分别为68.74±6.96及65.91±3.00)(P<0.05);而转染PinX1基因的MKN28细胞的增殖指数(%)为15.24±0.61,明显低于未转染或仅转染空载体的胃癌MKN28细胞(分别为31.28±0.53及34.08±0.80)(P<0.05);转染PinX1基因的MKN28细胞的凋亡率(%)为11.41±2.85,明显高于未转染或仅转染空载体的胃癌MKN28细胞的凋亡率(分别为0.85±0.10及2.40±1.31)(P<0.05);8通过半定量RT-PCR及Western Blotting技术分别检测基因(Mad1及c-Myc)的mRNA及蛋白水平发现,转染PinX1基因后,Mad1的表达水平明显上升而c-Myc的表达水平明显下降;相反,通过iRNA技术抑制PinX1基因的表达后,Mad1的表达水平明显下降而c-Myc的表达水平明显上升;9利用TRAP对各组细胞进行端粒酶活性检测,结果表明:转染PinX1基因后,细胞端粒酶活性明显降低;相反,抑制PinX1的表达后,细胞端粒酶活性相应升高。
结论:
1.成功构建PinX1的真核和干涉表达载体,并获得它们稳定转染的细胞株。
2.筛选出2条特异性针对PinX1的干涉序列,其中干扰2序列的干扰效果更强。
3.转染入PinX1基因后,细胞生长速度明显减慢,细胞阻滞于G0/G1期,且出现早期凋亡改变。
4. PinX1基因转染能增强胃癌MKN28细胞对化疗药物5-FU治疗的敏感性。
5. PinX1基因可抑制胃癌细胞端粒酶活性,是端粒酶活性抑制因子。
6. PinX1通过Mad1/c-Myc途径抑制端粒酶活性。
Objective Since telomeres and telomerase play crucial roles in maintaining cell immortalization and cancer progression, they may be targets for anticancer treatment. The telomerase level is high in most human cancer while it is low or no in the somal cells. The telomerase reverse transcriptase (hTERT) is the key of telomerase activity regulation. PinX1 was reported in 2001 in human cervical cancer using the yeast two-hybrid method. The previous studies showed that PinX1 is a potent inhibitor of telomerase activity and its TID domain could suppress telomerase activity by coupling telomerase directly. However, the role of PinX1 in telomere regulation, as well as in cancer, is still poorly understood. We transfected PinX1 gene into the gastric carcinoma line MKN28 without expression of PinX1 and transfected PinX1 siRNA plasmids into the gastric carcinoma line BGC823 with a high level expression of PinX1. drug-resistant clones were screened with G418 in the study. Then we observed the biological behaviour and Mad1/c-Myc expressions in MKN28 cells before and after gene transfection, and explored the possible novel mechanism.
Methods 1.we constructed the eukaryotic expression vector pIRES2-EGFP contained PinX1 gene by molecular cloning technology. We also constructed PinX1 RNAi plasmids including interference sequence and negative control sequence. 2. we examined the expression of PinX1 in three different gastric carcinoma cell lines including MKN28,SGC7901 and BGC823 using RT-PCR and Western Blotting. Then, we transferred the recombinant plasmids into gastric carcinoma cell lines using lipofectamineTM 2000. drug-resistant clones were screened with G418. 3. We observed the change of PinX1 expression level before and after gene transfection by semi-quantitative RT-PCR and Western Blotting. 4. The Cell morphology was observed by HE staining and transmission electron microscopy. 5. MTT assay was used to observe cell growth and to test the sensitivety of gastric carcinoma cell lines to 5-FU. 6. Cell cycle and cell apoptosis were analyzed by FCM. 7. Mad1/c-Myc expressions were detected with semi-quantitative RT-PCR and Western Blotting. 8. Telomerase activity was detected by telomeric repeat amplification protocol (TRAP).
Results 1. Sequencing suggested that the recombinant eukaryotic expression vector and siRNA vectors targeting PinX1 was correct. 2. PINX1 expression was negative in MKN28 cells,weak positive in SGC7901 cells,but strong positive in BGC823 cells. 3. we transfected PinX1 into MKN28 cell and the void vector was transfected as control. we also transfected siRNA eukaryotic expression vectors into BGC823 and the negative sequence vector was transfected as control. Drug-resistant clones were screened with G418 and established their stable transfected cell line. The PinX1 expression was detected by semi-quantitative RT-PCR and western blotting. It was indicated that the PinX1 was transfected into MKN28 successfully. Two siRNA vectors could suppress the expressive level of PinX1 and the interference 2 was more effective. 4. PinX1-transfected cells became larger in size and the coenocyte was common. Apoptotic body with meniscus heterochromatin could be seen in the tranfected cells under transmission electron microscope. At the same time, the karyoplasm proportion was increased and the nucleolus was prominent before gene transfection. 5. PinX1-transfected MKN28 cells grew more slowly than the cells not transfected or transfected with void vectors by MTT examination (P<0.05). 6. The IC50 value(0.253±0.021mg/L) decreased markedly in PinX1-transfected cells than that in the PinX1-untransfected cells or transfected with void vectors(0.560±0.017\0.590±0.026 mg/L)by MTT examination (P<0.05). 7. The apoptosis rate was higher in PinX1-transfected MKN28 than that in PinX1-untransfected cells or transfected with void vectors only by FCM. The growth of PinX1-transfected cells was retarded, and was blocked into G0/G1 stage. 8. PinX1-transfected MKN28 showed up-regulation of Mad1 and, down-regulation of c-Myc, whereas RNAi led to down -regulation of Mad1 and up-regulation of c-Myc in BGC823 cells. 9. The telomerase activity reduced obviously after PinX1 transfection while it was elevated when PinX1 gene was suppressed by PinX1 RNAi.
Conclusions 1. we constructed the recombinant eukaryotic expression vector and siRNA vectors targeting PinX1 gene and which were tranfected in to gastric cancer cells successfully. 2. Two RNAi vectors were indentified to suppress the expression of PinX1 specifically and the interference 2 was more effective. 3. Flow cytometric analysis displayed that PinX1 transfanction inhibited gastric cancer MKN28 cells growth,and induced them apoptosis and an arrests of G0/G1 phase. 4. The IC50 value decreased markedly in PinX1-transfected cells,suggesting that PinX1 transfection enhances the sensitivity of MKN28 cells to 5-FU. 5. The PinX1 gene mayasuppressor of telomerase activity. 6. PinX1 may suppress telomerase activity through Mad1/c-Myc pathway.
端粒、端粒酶在瘤肿细胞的永生化和演进中扮演重要角色,端粒酶在人类肿瘤细胞中高表达而在人正常细胞中无表达或仅在生殖细胞中低表达,因此在治疗上常作为治疗靶标。端粒酶逆转录酶(hTERT)是端粒酶活性关键的限速酶。研究表明,恶性肿瘤端粒酶活性不仅受hTERT调节,而且受其它因子的调节。PinX1为2001年报导,以细胞周期蛋白Pin2为诱饵,应用酵母双杂交方法,在人类宫颈癌细胞中发现的一种新基因。研究发现,PinX1及其端粒酶抑制域TID域可以直接结合于人端粒酶,进而抑制端粒酶活性,是端粒酶活性的强力抑制剂,但其抑制端粒酶活性的确切机制仍不清楚。我们利用分子克隆技术、RNA干涉技术及脂质体转染技术,将PinX1基因转染入本身无该基因表达的胃癌MKN28细胞,观察转染前后MKN28细胞生物学行为及对化疗药物5-FU的敏感性变化;将针对PinX1编码区的干涉序列转染入高表达该基因的胃癌BGC823细胞。检测转染前后各组细胞中端粒酶活性及Mad1/c-Myc水平的改变。探讨PinX1抑制端粒酶活性可能的新机制,初步为该基因应用于临床胃癌的治疗提供实验依据。
方法:
1利用分子克隆技术,构建包含全长人PinX1序列的真核表达载体;同时根据RNA干涉序列的设计原则,在线设计3条针对PinX1编码区的干涉序列,并通过BLAST设计了阴性对照序列,构建PinX1的RNA干涉真核表达载体以及阴性对照载体。2 RT-PCR、Western Blotting分别检测3种分化程度不同的胃癌细胞系(高分化MKN28细胞、中分化SGC7901细胞、低分化BGC823细胞)中PinX1表达水平;将构建成功的真核表达载体及干涉载体利用脂质体分别转染胃癌MKN28及BGC823细胞,经G418抗性筛选获得稳定表达的细胞株。3半定量RT-PCR、Western Blotting检测转染前后各细胞系中PinX1表达变化。4、通过HE染色、透射电镜等观察转染前后细胞形态特征的变化。5、通过MTT法观察转染前后细胞的增殖能力的变化,绘制细胞生长曲线。6通过MTT法检测转染前后胃癌MKN28细胞对化疗药物5-FU的敏感性变化。7通过流式细胞仪检测细胞凋亡和细胞周期的改变。8通过半定量RT-PCR及Western Blotting检测转染前后各细胞系中Mad1/c-Myc水平的改变。9、采用荧光素标记的端粒酶重复序列扩增法(TRAP)法检测转染前后各细胞系端粒酶活性的变化。
结果:
1经酶切及测序证明,成功构建重组PinX1真核表达载体及针对PinX1编码区的的RNA干涉真核表达载体与阴性对照载体;2、检测表明:高分化的MKN28细胞中无PinX1表达,低分化的BGC823细胞PinX1高表达,中分化的SGC7901细胞PinX1中等表达;3成功地将构建好的重组真核表达载体及干涉载体通过脂质体分别转染胃癌MKN28与BGC823细胞,经G418抗性筛选获得稳定表达的细胞株;检测表明,PinX1基因成功地转染入MKN28细胞;有2条干扰序列对PinX1有干扰效果;4细胞形态特征:HE染色可见转染PinX1基因的MKN28细胞为多边形,但是大小不一更为明显,大而扁平的细胞更为常见,巨细胞比例明显增加,最大的细胞体积可以达到小细胞的5～10倍,多核细胞常见;未转染的MKN28细胞呈梭形、短梭形,大小不一。透射电镜特征:转染PinX1基因的MKN28细胞内可见凋亡小体,小体内可见半月形异染色质;而未转染或仅转染空载体的胃癌MKN28细胞内可见细胞器减少,幼稚,线粒体呈杆状,核浆比例增大,核仁突出;5 MTT检测表明:转染PinX1基因的MKN28细胞较之未转染或仅转染空载体的胃癌MKN28细胞生长明显减慢(P<0.05);6 MTT检测表明:转染PinX1基因的MKN28细胞对5-FU的半数抑制浓度(IC50)值为0.253±0.021mg/L,明显低于未转染或仅转染空载体的胃癌MKN28细胞的IC50值(分别为0.560±0.017及0.590±0.026 mg/L)(P<0.05);7流式细胞仪检测细胞周期和细胞凋亡表明:转染PinX1基因的MKN28细胞的G0/G1期细胞数(%)为84.76±3.92,明显高于未转染或仅转染空载体的胃癌MKN28细胞的G0/G1期细胞数(分别为68.74±6.96及65.91±3.00)(P<0.05);而转染PinX1基因的MKN28细胞的增殖指数(%)为15.24±0.61,明显低于未转染或仅转染空载体的胃癌MKN28细胞(分别为31.28±0.53及34.08±0.80)(P<0.05);转染PinX1基因的MKN28细胞的凋亡率(%)为11.41±2.85,明显高于未转染或仅转染空载体的胃癌MKN28细胞的凋亡率(分别为0.85±0.10及2.40±1.31)(P<0.05);8通过半定量RT-PCR及Western Blotting技术分别检测基因(Mad1及c-Myc)的mRNA及蛋白水平发现,转染PinX1基因后,Mad1的表达水平明显上升而c-Myc的表达水平明显下降;相反,通过iRNA技术抑制PinX1基因的表达后,Mad1的表达水平明显下降而c-Myc的表达水平明显上升;9利用TRAP对各组细胞进行端粒酶活性检测,结果表明:转染PinX1基因后,细胞端粒酶活性明显降低;相反,抑制PinX1的表达后,细胞端粒酶活性相应升高。
结论:
1.成功构建PinX1的真核和干涉表达载体,并获得它们稳定转染的细胞株。
2.筛选出2条特异性针对PinX1的干涉序列,其中干扰2序列的干扰效果更强。
3.转染入PinX1基因后,细胞生长速度明显减慢,细胞阻滞于G0/G1期,且出现早期凋亡改变。
4. PinX1基因转染能增强胃癌MKN28细胞对化疗药物5-FU治疗的敏感性。
5. PinX1基因可抑制胃癌细胞端粒酶活性,是端粒酶活性抑制因子。
6. PinX1通过Mad1/c-Myc途径抑制端粒酶活性。
Objective Since telomeres and telomerase play crucial roles in maintaining cell immortalization and cancer progression, they may be targets for anticancer treatment. The telomerase level is high in most human cancer while it is low or no in the somal cells. The telomerase reverse transcriptase (hTERT) is the key of telomerase activity regulation. PinX1 was reported in 2001 in human cervical cancer using the yeast two-hybrid method. The previous studies showed that PinX1 is a potent inhibitor of telomerase activity and its TID domain could suppress telomerase activity by coupling telomerase directly. However, the role of PinX1 in telomere regulation, as well as in cancer, is still poorly understood. We transfected PinX1 gene into the gastric carcinoma line MKN28 without expression of PinX1 and transfected PinX1 siRNA plasmids into the gastric carcinoma line BGC823 with a high level expression of PinX1. drug-resistant clones were screened with G418 in the study. Then we observed the biological behaviour and Mad1/c-Myc expressions in MKN28 cells before and after gene transfection, and explored the possible novel mechanism.
Methods 1.we constructed the eukaryotic expression vector pIRES2-EGFP contained PinX1 gene by molecular cloning technology. We also constructed PinX1 RNAi plasmids including interference sequence and negative control sequence. 2. we examined the expression of PinX1 in three different gastric carcinoma cell lines including MKN28,SGC7901 and BGC823 using RT-PCR and Western Blotting. Then, we transferred the recombinant plasmids into gastric carcinoma cell lines using lipofectamineTM 2000. drug-resistant clones were screened with G418. 3. We observed the change of PinX1 expression level before and after gene transfection by semi-quantitative RT-PCR and Western Blotting. 4. The Cell morphology was observed by HE staining and transmission electron microscopy. 5. MTT assay was used to observe cell growth and to test the sensitivety of gastric carcinoma cell lines to 5-FU. 6. Cell cycle and cell apoptosis were analyzed by FCM. 7. Mad1/c-Myc expressions were detected with semi-quantitative RT-PCR and Western Blotting. 8. Telomerase activity was detected by telomeric repeat amplification protocol (TRAP).
Results 1. Sequencing suggested that the recombinant eukaryotic expression vector and siRNA vectors targeting PinX1 was correct. 2. PINX1 expression was negative in MKN28 cells,weak positive in SGC7901 cells,but strong positive in BGC823 cells. 3. we transfected PinX1 into MKN28 cell and the void vector was transfected as control. we also transfected siRNA eukaryotic expression vectors into BGC823 and the negative sequence vector was transfected as control. Drug-resistant clones were screened with G418 and established their stable transfected cell line. The PinX1 expression was detected by semi-quantitative RT-PCR and western blotting. It was indicated that the PinX1 was transfected into MKN28 successfully. Two siRNA vectors could suppress the expressive level of PinX1 and the interference 2 was more effective. 4. PinX1-transfected cells became larger in size and the coenocyte was common. Apoptotic body with meniscus heterochromatin could be seen in the tranfected cells under transmission electron microscope. At the same time, the karyoplasm proportion was increased and the nucleolus was prominent before gene transfection. 5. PinX1-transfected MKN28 cells grew more slowly than the cells not transfected or transfected with void vectors by MTT examination (P<0.05). 6. The IC50 value(0.253±0.021mg/L) decreased markedly in PinX1-transfected cells than that in the PinX1-untransfected cells or transfected with void vectors(0.560±0.017\0.590±0.026 mg/L)by MTT examination (P<0.05). 7. The apoptosis rate was higher in PinX1-transfected MKN28 than that in PinX1-untransfected cells or transfected with void vectors only by FCM. The growth of PinX1-transfected cells was retarded, and was blocked into G0/G1 stage. 8. PinX1-transfected MKN28 showed up-regulation of Mad1 and, down-regulation of c-Myc, whereas RNAi led to down -regulation of Mad1 and up-regulation of c-Myc in BGC823 cells. 9. The telomerase activity reduced obviously after PinX1 transfection while it was elevated when PinX1 gene was suppressed by PinX1 RNAi.
Conclusions 1. we constructed the recombinant eukaryotic expression vector and siRNA vectors targeting PinX1 gene and which were tranfected in to gastric cancer cells successfully. 2. Two RNAi vectors were indentified to suppress the expression of PinX1 specifically and the interference 2 was more effective. 3. Flow cytometric analysis displayed that PinX1 transfanction inhibited gastric cancer MKN28 cells growth,and induced them apoptosis and an arrests of G0/G1 phase. 4. The IC50 value decreased markedly in PinX1-transfected cells,suggesting that PinX1 transfection enhances the sensitivity of MKN28 cells to 5-FU. 5. The PinX1 gene mayasuppressor of telomerase activity. 6. PinX1 may suppress telomerase activity through Mad1/c-Myc pathway.
引文
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