Dissertation
Dissertation > Medicine, health > Oncology > Genitourinary tumors > Urinary tumors > Kidney,renal pelvis tumor

RNA Interferencing Krüppel Like Factor 8 Inhibits the Renal Carcinoma 786-0 Cells Growth in Vitro and in Vivo

Author FuWeiJin
Tutor DingQiang;XiaGuoWei;FangZuJun
School Fudan University
Course Urology
Keywords KLF8 Kidney cancer RC - PCR Immunohistochemistry RNA interference Gene Protein Cell Cycle Apoptosis Attack RNAi Nude Growth curve Tumor weight
CLC R737.11
Type PhD thesis
Year 2009
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Renal cell cancer (renal cell carcinoma, RCC) is the most common cancer of the urinary system, accounting for 2% of all adult cancer incidence. Radical nephrectomy (radical nephrectomy, RN) is the treatment of localized renal cell carcinoma is the most effective method, due to the lack of early diagnosis and effective treatment for patients with advanced, these patients still maintain a high mortality. 25% of patients with renal cell carcinoma diagnosed in locally advanced or metastasis. Radiotherapy and chemotherapy for advanced renal cell carcinoma treatment results are poor, 5-year survival rate of only 5% -10%. Interleukin (interleukin) and interferon (interferon) single or combined immunotherapy because of toxicity, clinical use is limited, and the the immunotherapy overall response rate is not high. Therefore, understanding the molecular mechanisms of renal cell carcinoma metastasis and progress is very important for improving the early diagnosis and treatment of patient survival. Krüppel-like transcription factor 8 (the Krüppel--like Factor 8, KLF8) is one of the Krüppel-like transcription factor family. KLF8 was highly expressed in malignant tumors, low expression in normal tissues. Research indicates that KLF8 promoter by activating cyclin D1 (cyclin D1), focal adhesion kinase (focal adhesionkinase FAK) regulation of cell cycle, cell carcinogenic transformation, invasiveness of epithelial-mesenchymal transition (epithelial tomesenchymal transition, EMT ) play an important role. But the KLF8 the role in the occurrence of renal cell carcinoma, development, invasion is not clear. First, the subject by reverse transcription - polymerase chain reaction (reverse transcription-polymerasechain reaction, RT-PCR) and immunohistochemistry detected 42 cases of sporadic renal cell carcinoma patients KLF8 gene and protein expression, and explore the KLF8 gene occur with renal cell carcinoma, the possibility of development. Next will build chemically modified small interfering RNA (small interference RNA, siRNA) was transfected into 786-0 cells express kidney cancer of KLF8 genes. The clear KLF8-siRNA in gene and protein levels can effectively inhibit the expression of KLF8. And research interfere KLF8 gene on cell growth, cell invasion force, apoptosis and cell cycle, FAK, cyclinD1, cadherin (E-cadherin) gene and protein expression. Then inoculated 786-0 cells to form a nude mouse model to explore the RNA interference KLF8 gene after nude mice tumor and renal transplantation intratumoral KLF8 of gene and protein expression. The subject of the use of RNA interference method research KLF8 gene 786-0 cell proliferation and tumor growth, experimental evidence for the gene therapy of renal cell carcinoma. The first part KLF8 in renal cell carcinoma Expression and Clinical Significance Objective: To investigate Krüppel-like transcription factor 8 (KLF8) in renal clear cell carcinoma and its clinical significance. Method: by RT-PCR and immunohistochemistry detected from the gene and protein levels of 42 patients with primary distribution of patients with renal cell carcinoma and adjacent normal kidney tissue of KLF8 mRNA and protein expression. Results: in KLF8mRNA renal cell carcinoma relative expression (0.869 ± 0.049) and adjacent normal kidney tissue KLF8mRNA relative expression levels (0.416 ± 0.018), the difference between groups was statistically significant (P <0.001). 42 cases of renal cell carcinoma 36 cases expression KLF8 of protein expression rate was 85.7% (36/42); 42 cases of adjacent normal kidney tissue 16 cases of expression KLF8 of protein expression rate was 38.1% (16 / 42), renal cell carcinoma KLF8 protein levels were significantly higher than in adjacent normal kidney tissue, the difference between groups was statistically significant (P <0.001). KLF8 mRNA and protein expression with tumor size (P <0.001) and clinical stage (P <0.001) correlation has nothing to do with age, gender, degree of cell differentiation (P> 0.05). Conclusion: renal cell carcinoma KLF8mRNA and the increased protein expression may be associated with kidney cancer progression, may become the target of kidney cancer gene therapy. The second part of the RNA interference KLF8 effective gene sequence to build and screening purposes: To investigate the the RNA interference KLF8 gene Construction and screening of valid serial. Methods: Expression of the design and synthesis can inhibit KLF8, after chemical modification of the siRNA, and transfected into the cells 786-0. RT PCR and Western blot detection KLF8mRNA and protein expression. An inhibition efficiency filter out the highest KLF8-siRNA into further research. Results: Compared with the control group (0.977 ± 0.003) and negative control group (0.917 ± 0.014), each interference group KLF8 mRNA relative expression of siRNA1 (0.276 ± 0.017), siRNA2 (0.084 ± 0.005), siRNA3 (0.316 ± 0.014), differences between the groups was statistically significant (P <0.001). Compared with blank control group (0.822 ± 0.016) and negative control group (0.807 ± 0.019) the interference group KLF8 protein to express respectively siRNA1 (0.491 ± 0.012), siRNA2 ( 0.264 ± 0.029), siRNA3 (0.401 ± 0.013), difference between the groups was statistically significant (P <0.001). The blank group compared with the negative control group of KLF8 mRNA and protein expression, the difference between groups was not statistically significant (P> 0.05). Consistent with RT-PCR results, siRNA2 inhibit the highest efficiency of KLF8 protein. Conclusion: building a successful KLF8-siRNA sequence can effectively inhibit the KLF8 mRNA and protein expression in renal cell carcinoma 7860 cells, KLF8 siRNA2 sequence inhibition efficiency, select it to go to the next step study. The third part the RNA interference KLF8 gene 786-0 cells in vitro growth of purpose: To investigate the growth of 786-0 cells in vitro the RNA interference KLF8 gene. Methods: KLF8-siRNA2 sequence transfected into 786-0 cells. Flow cytometry renal cancer cell cycle by RT-PCR and Western blot method for detection of cyclinD1, FAK, E-cadherin mRNA and protein expression; MTT assay of renal cancer cell growth; Matrigel Invasion Assay to detect renal cancer cell invasion ability; and cell apoptosis circumstances. Results: compared with the control group (0.693 ± 0.008), (0.487 ± 0.020) and negative control group (0.715 ± 0.010), (0.490 ± 0.021) KLF8-siRNA2 group cyclinD1 mRNA (0.217 ± 0.018), FAK mRNA (0.1 13 ± 0.012) expression were inhibited (P <0.001); with the control group (0.780 ± 0. 017), (0.810 ± 0.021) and negative control group (0.607 ± 0.024), (0.593 ± 0.008) compared to, KLF8-siRNA2 group cyclin D1 protein (0.323 ± 0 .061 1), the expression of FAK protein (0.380 ± 0.021) was inhibited (P <0.001); cyclinD1, FAK mRNA and protein expression of blank group and negative control group, the difference between the groups was not statistically significance (P> 0.05). E-cadherin mRNA and protein expression. Compared with the control group and negative control group, MTT Show KLF8-siRNA2 renal cancer cell growth rate (P <0.05); Matrigel Invasion Assay method the group KLF8-siRNA2 cell invasion decreased (P <0.001) ; flow cytometry analysis showed that compared with the control group and the control group, KLF8-siRNA2 group the proportion of cells in G0/G1 phase increased (P <0.001), S phase was decreased (P <0.001), apoptotic cells The increase in the proportion (P <0.001). Conclusion: the RNA interference KLF8 gene can down-regulate the of cyclinD1, FAK mRNA and protein expression, downward KLF8 also up-regulated the expression of E-cadherin, and slow down the growth of kidney cancer cells; reduce cell invasion force; affect the cell cycle and increased apoptosis. KLF8 in promoting renal cancer cell growth, cell cycle regulation, inhibition of apoptosis, to maintain cell invasion force and other aspects may play an important role. May be KLF8 FAK regulation of renal cell cycle key factor exists between the two-way adjustment. RNA interference down KLF8 genes may reverse renal cell carcinoma in EMT. After the fourth part the RNA interference KLF8 gene growth of 786-0 cells in vivo Objective: To investigate interference KLF8 gene vivo human kidney cancer cell tumorigenicity impact. Methods: 18 nude mice were randomly divided into blank control group and negative control group, KLF8-siRNA2 groups of six. 786-0 cell suspension subcutaneously inoculated with 3.5 × 10 to 6/0.2ml (RP1640 and PBS solution 0.1ml) method produced a nude mouse model. Tumor of the kidney transplant successfully, were given the same amount of PBS solution 50μgscramble RNA solution and 50μgKLF8-siRNA2, liquid tumor injection therapy, and 3 days for 6 weeks. After 6 weeks of treatment, the mice were sacrificed calculate tumor weight and tumor volume growth curve; detected by RT-PCR and Western blot method the KLF8 expression at the mRNA and protein levels. Results: After 6 weeks of treatment KLF8-siRNA2 tumors in nude mice tumor weight (2.073 ± 0.067) g less than the blank control group (3.317 ± 0.144) g and negative control group (3.333 ± 0. 185) g, the difference between the groups was statistically significant (P <0.001), while the blank control group and negative control group between the two groups was no significant difference (P> 0.05). Compared with the control group (0.488 ± 0.021) and negative control group (0.483 ± 0.017), the of KLF8-siRNA2 KLF8mRNA (0.269 ± 0.028) significantly decreased the expression difference between the groups was statistically significant (P <0.001); compared with the control group (0.785 ± 0.016) and negative control group (0.731 ± 0.023), of KLF8-siRNA2 KLF8 decreased expression (0 .278 ± 0.044), the difference between the groups was statistically significant (P <0.001). The blank control group and negative control group KLF8mRNA and protein compared to the difference between the groups was not statistically significant (P> 0.05). Conclusion: the transfection KLF8-siRNA2 can effectively inhibit the nude mouse model of tumor growth and reduced tumor the body KLF8 gene and protein expression in kidney cancer, the development process prompt KLF8 gene may play an important role. This study provide a reliable theoretical and experimental basis for the target KLF8 tumor gene silencing therapy.

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