| Renal cell carcinoma (RCC) is one of the malignant tumors which issecondary to carcinoma of urinary bladder in the urinary carcinoma, and renal cell carcinomas (RCCs) of the clear-cell type is the majority tissular type of RCC. It's estimated that the morbility of RCC is increased 2% annumly and about 100 000 patients died from this kind of carcinomas every year in the world. About half members of those patients are in their late stage when they were firstly diagnosed of this cancer, about 40% will be recurrenced after and metabasised before radical renal resection, the survival rate is lower than 3% within 3 years who had not received any treatment. RCC has more malignant and insensitivity to radiotherapy and chemotherapy, and immunotherapy is also has little effect on it. Although radical renal resection is a reliable treatment, but many patients have lost their opportunity for it, this urge us to seek new methods for treatment. Today the gene therapy to RCC has been the hotspot in tumor therapy.Survivin gene is a new member of inhibitors of apoptosis protein (IAP) family. It is observed to express mainly in the developing embryonic tissues and the common human malignant tumor tissues, but not in the adult differentiated tissues. It has been found in previous studies that Survivin possesses a double regulatory activity on cell apoptosis and cell division cycle by exerting a direct inhibitory effect on the activities of caspase, especially caspase-3 and caspase-7 or caspase-9, to block the corporate pathway of the down-stream cell apoptosis induced by various stimulants. Besides, Survivinhas a specific expression in the cells in G2/M stage of cell division circle, and is involved in the regulation of cell gene's transcription by combing with the microtubule of cell mitosis spindle. And Survivin can directly inhibit the activity of caspase-3 to reduce tumor cells' sensitivity to chemotherapeutic agents. Survivin gene's selective expression in tissues and its special activity on the inhibition of cell apoptosis suggest that it is likely to be identified as a special index in diagnosis and a potential therapeutic target in RCC.The advantage of the target-oriented therapy with tumor apoptosis-inhibiting gene as its target is that it can increase tumor cells' apoptosis and inhibit their proliferation, while it has no bad effect on normal tissues. Using antisense oligonucleotides to inhibit the expression of survivin and to investigate whether survivin antisense oligonucleotide (ASODN) down-regulate survivin protein and mRNA, induce apoptosis, inhibit proliferation and enhance the sensitivity of RCC to chemotherapy is the major part of our work.Aims:To investigate the expression of survivin gene in renal cell carcinoma of clear-cell type and its correlation with clinicobiological behavior. To investigate whether survivin antisense oligonucleotide (ASODN) down-regulate survivin protein and mRNA, induce apoptosis. , inhibit proliferation and enhance the sensitivity of RCC to chemotherapy, and to provide a basis for gene therapy or affiliate gene therapy to RCC of clear-cell type.Methods:1. The expressions of survivin , PCNA in 48 cases of clear-cell RCC tissues, 11 adjacent noncancerous tissues of clear-cell RCC and 5 normal renal tissues were detected by immunohistochemistry of SABC. Apoptosis cell was detected by TUNEL. It's correlation with clinicobiological behavior were also investigated.2. Human clear-cell renal carcinoma cells line (786-O) was transfected with different concentration of survivin ASODN through lipofection to construct RCC cell model. And then SABC immunohistochemical staining and Western blot were utilized to assay the expression of survivin protein in 786-O cell; RT-PCR was utilized to assay the expression of survivin mRNA in 786-Ocell; and the cell relative viability was examined using 3(4,5-dimethylethiazoly 1-2-) 2,5-diphonyl tetrazolium bromide (MTT) assays.3. 786-O cells were transfected with different concentration of survivin ASODN through lipofection, electron microscopy was used to demonstrate apoptotic morphological changes in treated cells. The apoptosis rates and cell cycle were detected by flow cytometry. DNA gel electrophoresis were utilized to detect the debris of DNA in treated cells.4 Human renal carcinoma 786-0 cells were implanted subcutaneously in BALB/c-nu mice to develop the implanted tumor model of human renal carcinoma.In ASODNs treated group, ASODNs (10 nmol/100 μL) were directly injected into tumors in nude mouse every day for 4 days. In control group nude mice were treated with the same volume of RPMI 1640 or the same concentration of SODN (10 nmol/100 uL). Tumor sizes were measured and tumor volumes were calculated every three days. Three nude mice were killed respectively at 6th, 12th day in each group, survivin expresion of transplanted tumor was detected by immunohistochemical technique and the apoptosis of tumor was measured by TUNEL assay.5 .The expression of survivin protein and mRNA were assayed by immunohistochemical staining of SABC and RT-PCR respectively in 786-0 cell line after different concentration of epirubicin treated. Using untreated cells as control group, ASODN and SODN were transfected into 786-0 cells by lipofection, then the cytotoxicity of epirubicin was determined by MTT, and apoptosis rate were detected with the use of flow cytometry (FCM).Results:1. An up-regulation expression of Survivin gene and PCNA gene were observed in clear-cell RCC tissues. The positive rate of survivin and PCNA is 87.5%, 89.4%, 92.3% and 87.5%, 89.4%, 92.3% in Fuhrman grade I, II and III respectively; The positive rate of survivin and PCNA is 83.3%, 91.7%, 100%, 90% and 66.7%, 66.7%, 62.5%, 60% in Robson stage I, II, III and IV respectively; there were no correlation between these expression and the age and gender of the patients, the size of the tumors, and pathological grade and clinical stages respectively (P>0.05) . Instead, the apoptosis index (AI) is (1.03±0.42) in survivin highly expressed group and in the lower group is (1.35±0.71), the PI/AI index closely related with Fuhrman grade and Robsonstage.2. The adhesive rate of SODN group, 200nmol/L ASODN group, control group is (95.3±3.4) %,(92.6±8.1) %, (96.5±4.4) % respectively, there is no remarkable significancebetween each other (P >0.05), but the adhesive rate of 400 nmol/L ASODN group and 600 nmol/L ASODN group is (87.5±7.8)% and (51.5±4.5)%, there are remarkable significance compared with each control group(P<0.05). Cell viability was inhibited in a dose-dependent and time-dependent manner, and IC50 is 400 nmol/L at the time of 24h. Survivin protein expression and its mRNA expression were down regulated in 786-0 cells transfected by survivin ASODN, and have a time and dose dependence.3. The cell cycle of survivin ASODN treated cells were blocked at G2/M stage, The percentage of G2/M is 10.1%, 11.8%, 11.4%, 25.1%, 34.6% and 61.1% for control group, SODN group, 200nmol/L ASODN group, 400nmol/L ASODN group, 600nmol/L ASODN group respectively. There are hypodiploid peaks in front of G1 peak. The Apoptosis rate is (13.5±0.9) %,(19.7±2.3) %, (31.4±5.1) % for 200 nmol/L ASODN group, 400 nmol/L ASODN group, 600 nmol/L ASODN group respectively. Some typical manifestations of cell apoptosis were detected by transmission electron microscope, and DNA gel electrophoresis show "DNA ladder", which is typical for apoptosis.4. The tumor volume in ASODNs group was significantly less than that in the control group and SODN group (P < 0.01). The survivin positive expression rate in ASODNs group was significantly lower than that in the control group and SODN group (P < 0.01). The apoptosis rate in control group , SODN group and ASODNs group is (10.6±2.1) %, (9.8±1.6) %,(23.4±2.7) % respectively on the sixth day after the treatment started, there is a significance between ASODNs group and control group (P < 0.01).5. Epirubicin could reduce the proliferation and induce the apoptosis of 786-0 cells significantly. The inhibition ratio is (51±4)% after 24h treatment at the concentration of 10mmol/L .The expression of survivin induced by epirubicin has time and dose dependence manner. The survival rate of 786-0 cell is significantly decreased, which are transfected by survivin ASODNs 24hours late and then treated with epirubicin. The apoptosis rate in the 786-O cells transfected by survivin ASODNs 24 hours late and then treated with epirubicin is (35.7±1.67)%, which is significantly higher than that of the control group(18.1±1.7)%and SODN group(19.3±2.7)%(P<0.05). Conclusions:1. The results of the detect of Survivin , PCNA protein and apoptosis index in the clear-cell RCC tissues demonstrate that survivin gene contributes to the occurrence of clear-cell RCC by enhancing the clear-cell RCC cells' proliferation and inhibiting their apoptosis. The findings suggest that detecting PI/AI index in the clear-cell RCC tissues may be to some extent valuable to diagnose and treat clear-cell RCC in the clinical application.2. Survivin protein expression and its mRNA expression were down regulated in 786-0 cells transfected by survivin ASODN. The proliferation activity of 786-O cells was also decreased obviously, and has a time and dose dependence. The possible mechanism is that survivin ASODN could directly represse the translation of survivin mRNA, and also active the RNaseH, then degradate survivin mRNA in 786-0 cells.3. It very important for survivin gene to maintain the normal cell cycle and inhibit apoptosis in carcinoma. The cell cycle of survivin ASODN treated cells were blocked at G2/M stage, and inducing 786-0 apoptosis have a time and dose dependent.4. The results of the transfection by survivin ASODN in vitro and the tumorigenic experiment in nude mice in vivo confirm that survivin ASODN can reduce the proliferation of clear-cell RCC cells and induce their apoptosis both in vivo and in vitro, which provides new evidence to prove that survivin gene is involved in the occurrence and development of clear-cell RCC. The results of the transfection by survivin ASODN in vitro and tumorigenic experiment in nude mice in vivo also prove that survivin ASODN both in vivo and in vitro can specifically down regulate the Survivin expression in clear-cell RCC cells, promote their apoptosis, and inhibit their proliferation, which may serve as a scientific evidence for survivin gene as the target gene in the gene therapies for clear-cell RCC.5. The increased survivin protein in 786-0 cells treated with different concentration of epirubicin reveal that survivin gene maybe one of the...
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