摘要
目的:明确垂体瘤转化基因(pituitary tumor transforming gene ,PTTG)的表达与胶质瘤临床病理级别和患者预后的相关性。构建PTTG RNA干扰载体,观察PTTG RNA干扰对细胞增殖、凋亡及对化疗敏感性的影响。
方法:选择80例胶质瘤手术标本,按WHO胶质瘤分级标准,包括Ⅰ级16例、Ⅱ级22例,Ⅲ级26例、Ⅳ级16例,患者生存时间<12月22例,12-36月41例,>36月17例,复发时间<12月18例,12- 36月39例,>36月23例。免疫组化检测PTTG表达,测定其积分光密度值(IOD)。
根据PTTG基因cDNA序列,设计针对PTTG基因的3组干扰序列,并设计1组阴性对照序列,克隆到真核表达载体pGenesil-1中,进行酶切鉴定和测序鉴定。将构建好的4组重组质粒分别命名为PTTG-1组,PTTG-2组,PTTG-3组、阴性对照NC(negative control)组。利用RNA干扰技术,将以上shRNA分别转染细胞中,逆转录聚合酶链式反应(RT-PCR)检测PTTGmRNA表达水平、Western Blot检测PTTG蛋白表达水平。
选取干扰效果最强的shRNA片段转染U251细胞,另用替莫唑胺(temozolomide,TMZ)作用于转染shRNA片段的U251细胞,四甲基偶氮唑盐( methyl thazoy terazolium, MTT)比色法检测细胞生长状况,流式细胞仪检测细胞凋亡。
结果:免疫组化结果显示80例胶质瘤标本中,其IOD值Ⅰ、Ⅱ、Ⅲ、Ⅳ级分别为0.69±0.06、0.76±0.08、1.32±0.12、1.67±0.15,随着肿瘤级别的增高而升高,各组间比较差异有统计学意义(P<0.05或0.01)。患者复发时间越长,PTTG蛋白表达越低,复发时间越短,PTTG蛋白表达越高,其IOD在复发时间<12月组、12-36月组、>36月组分别为1.60±0.20、0.98±0.10、0.61±0.08,各组间比较差异有统计学意义(P<0.01)。PTTG蛋白表达与生存时间的关系显示患者生存时间越长,PTTG蛋白表达越低,生存时间越短,PTTG蛋白表达越高,其IOD在生存时间<12月组、12-36月组、>36月组分别为1.55±0.14、1.01±0.13、0.65±0.08,各组间比较差异有统计学意义(P<0.01)。
构建的PTTG RNA干扰序列和阴性对照序列经酶切和基因测序证明插入序列正确,说明PTTG RNA干扰真核表达质粒和阴性对照表达质粒构建成功。
细胞在转染PTTG干扰载体后,提取细胞的总RNA,经RT-PCR电泳发现,在转染24h后,PTTG-2、PTTG-3即显示出抑制作用,PTTGmRNA表达水平降低,PTTG-1组对目的基因的抑制作用不明显;在转染48h后,各组均显示出抑制作用,但PTTG -3的抑制作用更明显;转染72h后,PTTG-1组有微弱的抑制作用,PTTG -2及PTTG -3组仍有显示明显的抑制作用,但PTTG -3抑制作用较PTTG -2更强烈。
Western blot检测不同时间PTTG蛋白的变化显示,在转染24h后,PTTG-1、PTTG-2、PTTG -3组对目的基因的抑制作用均不明显;在转染48h后,PTTG-1、PTTG-2、PTTG-3组均显示出抑制作用,但PTTG-3的抑制作用更明显;转染72h小时后,PTTG-1组没有抑制作用,PTTG-2及PTTG -3组仍有显示明显的抑制作用,但PTTG -3抑制作用较PTTG-2更强烈。
PTTG-3及PTTG-3联合TMZ作用细胞48h后,MTT实验结果显示,细胞生长和存活能力受到明显的影响,细胞增殖活性结果显示OD值在对照组、TMZ组、PTTG -3转染组、PTTG-3联合TMZ组分别为0.85±0.07、0.58±0.06、0.55±0.07、0.41±0.05,TMZ组、PTTG -3转染组、PTTG-3联合TMZ组细胞增殖抑制率分别为(31.56±5.51)%、(35.53±4.60)%、(51.49±6.74)%,PTTG-3组及TMZ组与对照组比较差异具有统计学意义(P<0.01),而PTTG-3联合TMZ组抑制细胞增殖更明显,与PTTG -3组及TMZ组比较差异有统计学意义(P<0.01)。流式细胞仪检测细胞凋亡显示48h对照组凋亡率为(6.29±0.78)%,TMZ组为(33.63±4.88)%,PTTG-3组为(39.61±4.95)%,PTTG -3联合TMZ组为(66.23±7.60)%,PTTG -3组及TMZ组与对照组比较差异差异有统计学意义(P<0.01),而PTTG -3联合TMZ组的凋亡率较PTTG-3组及TMZ组明显增高,组间比较与差异有统计学意义(P<0.01)。
结论:PTTG与胶质瘤病理级别和预后密切相关,可以作为肿瘤恶性程度和预后的指标, PTTG RNA干扰可以抑制胶质瘤细胞生长,诱导细胞凋亡,且可以增强胶质瘤细胞对化疗药物的敏感性,提高化疗效果。
Objective:To investigate the relationship between the expression of pituitary tumor transforming gene (PTTG) protein and pathological grade, prognosis in glioma。To constructed recombinant expression vector PTTG, and glioma U251 cell line was treated with recombinant expression vector PTTG and PTTG shRNA combined with temozolomide(TMZ), proliferation,apoptosis and chemotherapeutic sensitivity of glioma cells were observed.
Methods:The protein expression of PTTG was detected by immunostaining assay using Streptavidin-Peroxidase (S-P) method in 80 cases of glioma. Of the 80 cases,16 cases were with pathological gradeⅠ,22 with gradeⅡ,26 with gradeⅢ,16 with gradeⅣ.The survival time was <12 months in 22 cases,12-36 months in 41 cases and >36 months in 17 cases, recurencs time was<12 months in 18 cases,12-36 months in 39 cases and >36 months in 23 cases.
Three potential shRNA target sites were identified on the PTTG and recombined shRNA-PTTG plasmid. One negative control sequence was designed. The three recombine shRNA-PTTG plasmids and the negative control plasmid were detected by enzyme digestion and sequencing analysis,and named as PTTG-1,PTTG-2,PTTG-3,the negative control was named as NC(negative control),which were transiently transfected into U251 cell lines via lipofectamine 2000 respectively.
After the recombinant PTTG shRNA expression vector was transfected into cells,PTTG mRNA was dectected by reverse transcription polymerase chain reaction (RT-PCR) ,the protein expression of PTTG was dectected by western blot.
Select the validest shRNA , the glioma U251 cell line was treated with PTTG shRNA and PTTG shRNA combined with TMZ ,MTT assay was used to detected cell proliferation, the apoptosis of U251 cell line was evaluated by flow cytometry.
Results : Of the 80 cases, the IOD in gradeⅠ,Ⅱ,Ⅲ,Ⅳwere 0.69±0.06,0.76±0.08,1.32±0.12,1.67±0.15 respectively,the protein expression of PTTG increased with the increasing of pathological grade of glioma,there were signifcantly difference between the groups(P<0.05 or 0.01).The expression of PTTG protein in survival groups were 1.60±0.20,0.98±0.10,0.61±0.08 respectively,the expression of PTTG protein in recurrence groups were 1.55±0.14,1.01±0.13,0.65±0.08 respectively,there were significantly difference among the survival groups or recurrence groups (P< 0.01) .
Successful construction of vector PTTG was comfirmed by enzyme digestion and sequencing analysis.
Total RNA was eluted after the U251 cells were transfected, RT-PCR technique was used to measure the expression of PTTG mRNA. Compared with control group, PTTG -3 showed inhibiting effect after 24h, the inhibiting effect of PTTG-1,PTTG-2 were insidiously. PTTG-1,PTTG-2 and PTTG -3 showed inhibiting effect after 48,however, inhibiting effect of PTTG-3 was stronglyest. After 72h, PTTG-1 had no inhibiting effect, PTTG-2 had a weak inhibiting effect, PTTG -3 still had a strong inhibiting effect. Western blot was used to measure the expression of PTTG protein, compared with control group, PTTG -1,PTTG -2,PTTG-3 showed no inhibiting effect at 24h. PTTG -1,PTTG-2 and PTTG-3 showed inhibiting effect at 48h,however, inhibiting effect of PTTG -3 was stronglyest. PTTG -1 had no inhibiting effect, PTTG -2 had a weak inhibiting effect, PTTG still had a strong inhibiting effect at 72h.
The outcome of MTT showed that the growth and survive were influenced after PTTG -3 and PTTG-3 combined with TMZ, the OD of control group,TMZ group, PTTG -3 group and PTTG-3 combined with TMZ group were 0.85±0.07,0.58±0.06,0.55±0.07,0.41±0.05,inhibitory rate of TMZ group,PTTG -3 group and PTTG-3 combined with TMZ group were(31.56±5.51)%,(35.53±4.60)%,(51.49±6.74)% respectively, there was a statistically significance between control group and PTTG-3 group or TMZ group, and there was a statistically significance between group PTTG-3 combined with TMZ and PTTG -3 group and TMZ group.
The apoptosis rate at 48h of control group、TMZ、PTTG -3 group、PTTG-3 combined with TMZ group were(6.29±0.78)%,(33.63±4.88)%,(39.61±4.95)%,(66.23±7.60)% respectively in the flow cytometry analysis, there was a statistically significance between control group and PTTG-3 group or TMZ group, and there was a statistically significance between group PTTG-3 combined with TMZ and PTTG -3 group or TMZ group.
Conclsion:The expressions of PTTG protein were related to pathological grade and the prognosis of glioma, and can be considered as the indicator of the maligant degree and the prognosis, PTTG RNA interference may inhibit the cell proliferation and induce apoptosis in glioma cells ,which can upregulate chemotherapeutic sensitivity.
引文
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