| 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 grade?,22 with grade?,26 with grade?,16 with grade?.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 grade?,?,?,?were 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. |
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