| IntroductionDisorder of cell cycle and misbalance of proliferation and apoptosis give contribution to the carcinogenesis. Disordered regulatory apoptosis plays an important role in tumorigenesis. Imperishability is the characteristics of tumor and lose control of cell division is another characteristics. Survivin, a member of the inhibitor of apoptosis (IAP) family of proteins, is widely expressed in transformed cell lines and in different primary cancer cells. It is not expressed in many non - malignant adult tissues, but is essential for fetal development. The expression of Survivin gene directly relates to the histological classification of carcinoma, its relapse, metastasis, and growth index and inversely relates to ap-optotic index.Objective: In the present study, we generated a series of antisense compounds, with the strongest efficient antisense oligonucleotides and highly efficient vector Lipofectamine. Using immunohistochemistry, western blot, RT -PCR and flow cytometry to observe the Survivin gene expression following anti-sense oligonucleotides and its effects on cell apoptosis and growth, which has laid a foundation for further studies on the functions of Survivin gene and genetic therapy involved in human renal cell carcinoma.Methods(1) With the method of immunohistochemical staining, we studied the expression of Survivin in 66 cases of renal cell carcinoma and 20 cases of normalrenal stoma. Survivin protein and mRNA were measured by western blot and RT - PCR assay respectively in 6 cases of renal cell carcinoma, 6 cases of normal renal stoma and two renal cell carcinoma cell lines 786 - 0 and ACHN. (2) To investigate the effect of transfected Survivin antisense oligonucleotide ( ASODN) on proliferation and apoptosis of renal cell carcinoma, we designed ASODNs targeting Survivin mRNA. Cells exposed to the potent oligonucleotide were also examined for apoptosis induction by FCM and transmission electron microscopy. Semiquantitive RT - PCR and Western blot examinations were carried for expression of Survivin mRNA and protein. Cell growth was detected by MTT assay.Results(1) Positive staining for Survivin was located in cytoplasm of tumor cells in 66 cases of renal cell carcinoma . negative staining for Survivin was common in 20 cases of normal tissue. The optical density was 0. 031 0.002 and 0.875 0124 in renal cell carcinoma and normal tissue respectively, the statistic analysis was significant ( p < 0. 01 ). the optical density was 0. 873 ± 0. 091,0. 904 ± 0. 103and 0. 813 ±0. 126 in clear cell carcinoma, chromophilic cell carcinoma, and chromophobic cell carcinoma, respectively. The statistic analysis was insignificant, the optical density was 0. 693 ±0.092,0. 762 ±0. 113,0. 876 ±0.106 and 0.903 ±0.092 in Gj ,G2 ,G3 and G4 grade. The difference was significant (p < 0.05). the optical density was 0. 864 ± 0. 121,0. 884 ± 0. 093 and 0. 853 ± 0. 106 in I , II and HI ^ IV stage. The statistic analysis was insignificant, the expression of Survivin protein about 16. 5 kDa was negative in the normal tissue and were detected in the renal cell carcinoma and RCC cell lines. The difference was significant( p <0.01) The expressions of Survivin mRNA were detected in a significant proportion of renal cell carcinoma and RCC cell line 786 - 0 and ACHN. Survivin mRNA was not detected in normal tissue, the difference was significant p <0.01).(2)Cancer cells in non - transfection control group grew rapidly, with a regular polygon shape. After Ixansfection, some cells presented reduced size, irregular shape, and mostly round profile. Under electronic microscope, somecells had characteristic morphological changes of apoptosis such as nuclear shrinkage, chromatin congregation around nuclear membranes, reduction of cell volume and integrity of nuclear membranes. Expression of Survivin was observed primarily in cytoplasm. Expression of Survivin in tranfection group decreased compared with the control group. Specific Survivin gene band were observed in all group. The ratio of Survivin/f$ - actin in non - transfection group wasl. 35 ± 0. 13 and was greater than its inr transfection group with ASODN. Western blotting detection found obvious 16.5 KD protein bands in non -transfected group. Expression of Survivin protein decreased when transfection concentration increased. In flow flowcytometry, the histogram showed that Gl cells were obviously decreased after transfection, Sub - Gl peak was seen in the histogram. G2/M peak increased. The apoptotic rate of 786 -0 cells were 6.05 ±0.48% , 5.70 ±0.41% ,10. 54 ±0. 72% ,12. 80 ±0. 38% ,and 22. 30 ± 1. 23% in non - transfection group , SODN and ASODN group respectively. The increase was significant. The apoptic activities of 786 - 0 cells were reduced in a dose dependent manner. The inhibitory efficiencies of 800ng/L transfection increased after 24h ,48h and 72h. The inhibition activities of 786 - 0 cells were significantly increasing in a time dependent manner.Conclusion1. No Survivin expression in normal adjacent tissue but high expression in RCC and RCC cell line by using immunohistochemistry and RT - PCR method. This study showed that Survivin gave contribution to carcigenosis.2. This study implies that there is no significant difference in different pathologic type and clinical phase about Survivin. Expression of Survivin increased with the poorer differentiation. This result showed that Survivin is correlation with tumor progress and can serve as maker of prognosis.3. This study showed ASODN of Survivin can block the expression of Survivin in renal cell carcinoma cell line 786 -0. The expression of Survivin mR-NA and protein decreased after transfection. SODN represents a useful experimental approach for treatment of renal cell carcinoma.4. Inhibition activity of Survivin was in a time dependent manner.5. ASODN of Survivin block the cells in G2 / M phase to inhibit the proliferation, of renal cell carcinoma.6. ASODN of Survivin activates the Caspase - 3 to induce the apoptosis of renal cell carcinoma.
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