| Background Renal cell carcinoma(RCC) also called renal adenocarcinoma or renal cancer, it is a malignant tumor derived from renal tubular epithelial cells. According to statistics,renal cell carcinoma ranks second in all types of urological malignancies. Studies in recent years show that the incidence of renal cell carcinoma was significantly higher than in the past. Epidemiological studies have shown that adults with renal cell carcinoma each year approximately 3% of malignant tumors, and its incidence has been rising over the past two decades. clear cell renal cell carcinoma(cc RCC) is the most common histological type of renal cell carcinoma, the incidence rate of about 80 to 85% in all types of kidney cancer incidence rates. Stage clinical treatment of kidney cancer is still the main radical nephrectomy for renal cell carcinoma tumor confined perirenal fascia, to take the best curative effects of surgical resection, but surgery for advanced kidney cancer and renal cell carcinoma patients after recurrence and metastasis is still a lack of effective treatment. Although molecular targeted therapy has been used in clinical treatment of kidney cancer among, and the prognosis of patients also improved greatly, but the overall effect of the treatment of kidney cancer patients is still poor. Past studies have shown that the body as well as a variety of factors contributed to the occurrence of malignant process of renal cell carcinoma, and therefore the basis of experiments carried out in-depth study on the causes of cancer and renal cell carcinoma in the signal transduction pathways of change and cancer gene / changes in tumor suppressor genes, looking for a more targeted key targets and a more effective treatment of clinical significance. mi R-182 as a mi RNA. At present, studies have confirmed that the expression of mi R-182 in a variety of solid malignant tumors abnormal increase or decrease,suggesting its role in tumor occurrence and development of tumor promoters or tumor suppressor genes, but in renal clear cell carcinoma of the situation and the role of the mechanism is still not yet proven. IGF(insulin-like growth factors, IGFS) is an important member of the growth factor, which plays an important role in the human body bone and muscle growth and differentiation, whereas most of the biological activity of IGFS by IGF-1R mediated generation. IGF-1R is widely expressed in various cell types in living organisms, and its signal transduction pathway and the close links between the occurrence and development of malignant tumors. IGF-1R via a variety of signal transduction pathways mediated by the vicious proliferation,invasion and metastasis of tumor cells, but also mediates tumor angiogenesis and tumor cell anti-apoptotic effects. In addition, the occurrence and development of IGF-1R and clear cell carcinoma also has an important influence.Objective We examined the expression of mi R-182 in renal clear cell tumor cancer patients,study the relationship between mi R-182 in patients with clinical parameters, and analyze the impact of mi R-182 in renal cell cancer cell proliferation, invasion and metastasis explore mi R-182 signals activate or inhibit the expression of IGF-1R in renal cell cancer, study its effects in renal cell cancer cell invasion and metastasis,aimed at understanding the mechanisms of development and progression of renal cell carcinoma, while providing a reliable basis for renal cell carcinoma clinical diagnostics and targeted therapy.Method(1) We collected 57 cases of cancer tissue and peripheral blood cells in patients with renal cancer treatment in the Department of Urology, First Affiliated Hospital of Zhengzhou University, radical nephrectomy surgery- August 2015 period in April2010. 38 patients were male, female 19 patients, aged between 50 to 79 years old. Renal cell carcinoma histological grade according to Fuhrman grading system is divided: there are 15 cases of stage I, II period of 26 cases, III ~ IV grade 16 cases.Robson clinical stages according to the method divided: I of 22 cases, II 16 cases,III ~ IV 19 cases. According to whether the occurrence of lymph node metastasis were divided: lymph node metastasis and 19 patients without lymph node metastasis in 38 cases. Also selected 10 cases of physical examination in our hospital volunteers were served as healthy control group. Fluorescence quantitative PCR mi R-182 in normal tissue adjacent renal clear cell carcinoma and cancerous plasma of healthy volunteers and patients with the expression,analyze the correlation between mi R-182 and clinicopathological parameters in patients with renal cell carcinoma.(2) We designed and synthesized mi R-182 mimic, mi R-182 inhibitor and mi R-control,then the three were transfected into human renal clear cell carcinoma Caki-1 cells,in order to build mi R-182 overexpression or inhibit the expression of Caki-1 cells cell lines, and to verify the expression of the above-mentioned three groups of cells after transfection of mi R-182 detected by fluorescence quantitative PCR method. Then using the MTT assay after transfection proliferative activity of the above three groups of cells; clonogenic assay growth; migration and invasion chamber assay Transwe cells.(3) We use bioinformatics software screening target genes of mi R-182. We use the expression of real-time PCR and western blot to detect transfected with mi R-182 control, mi R-182 mimic and mi R-182 inhibitor of human clear cell carcinoma Caki-1 cells, IGF-1R m RNA and protein. We IGF-1R 3'UTR using site-directed mutagenesis was constructed IGF-1R 3'UTR Mut(mutant) plasmid vector containing the luciferase reporter gene, the key structure while IGF-1R 3'UTR WT(wild type) plasmid vector the two were with the mi R-182 mimic co-transfected Caki-1 cells. We used luciferase reporter system for detecting the expression of luciferase cells. We used to build si-IGF1 R inhibition of IGF-1R expression in Caki-1 cells, and cells expressing IGF-1Rm RNA and proteins by fluorescence quantitative PCR and western blot were used to detect. We human clear cell carcinoma Caki-1 cells were divided into 3 groups Transwe II cells were detected by Control group, mi R-182 inhibitor groups and si-IGF1 R + mi R-182 inhibitor invasion and migration of cells.Results(1) Fluorescence quantitative PCR results showed that renal cell carcinoma expression of mi R-182 expression was significantly lower than in the corresponding adjacent normal tissues(P <0.05); mi R-182 in the plasma of patients with renal clear cell carcinoma in It was significantly lower than in healthy volunteers(P <0.05). mi R-182 and clinicopathological parameters analysis showed that renal cell carcinoma, the expression of mi R-182 in patients with renal cell carcinoma histological grade, clinical stage and lymph node metastasis, regardless of gender and age of the patient.(2) Fluorescence quantitative PCR results showed that compared with the control group, mi R-182 mimic cells in the expression level of mi R-182 was significantly higher(P <0.05), mi R-182 inhibitor cells in the expression of mi R-182 was significantly reduced( P <0.05), showed that overexpression of mi R-182 / inhibit the expression of Caki-1 cell line was successfully constructed. MTT assay test results show that mi R-182 mimic transfection of Caki-1 cells at 48 h, 72 h, 96 h when the growth rate was significantly lower than the control group(P <0.05);transfected with mi R-182 inhibitor of Caki- 1 cells at 48 h, 72 h, 96 h when the growth rate was significantly higher(P <0.05). Clonogenic assay results show that the number of colony forming mi R-182 mimic group Caki-1 cells was significantly lower than the control group(P <0.05); cloning mi R-182 inhibitor group Caki-1 cells was significantly higher(P <0.05). Test results Transwe II cell method shows that migration mi R-182 mimic group Caki-1 cells and the invasive cells were significantly lower than the control group(P <0.05); and migration mi R-182 inhibitor group Caki-1 cells and the invasive cells number were significantly higher(P <0.05).(3) Bioinformatics prediction results show that, IGF-1R is a target gene of mi R-182.Respectively, after transfection of mi R-182 mimic, Caki-1 cells, the expression of IGF-1R m RNA and protein was significantly decreased(P <0.05); transfected mi R-182 inhibitor, Caki-1 cells, IGF-1R m RNA and protein the expression was significantly increased(P <0.05). Luciferase Assay results showed that compared with the control group, mi R-182 mimic IGF1 R 3'UTR WT co-transfected with luciferase expression of transfected group was significantly lower(P <0.05); and mi R-182 mimic and IGF1 R 3 ' UTR Mut co-transfected cells in the expression of the enzyme luciferase is no significant difference(P> 0.05) compared with the control group. After the expression of interference si-IGF1 R expression Caki-1cells IGF-1R m RNA and protein was significantly decreased. Test results Transwe II cell method shows that, compared with the control group, the migration mi R-182 inhibitor cells and invasion of cells was significantly increased(P <0.05),while migration si-IGF1 R + mi R-182 inhibitor cells and invasive cells number significantly decreased(P <0.05).Conclusion The expression of mi R-182 was significantly reduced in renal clear cell carcinoma and peripheral blood, and its expression level in patients with renal cell carcinoma histological grade, closely related to the clinical stage and lymph node metastasis. mi R-182 can directly inhibit the expression of IGF-1R, thereby regulating renal cell cancer cell proliferation, invasion and metastasis, and ultimately inhibit the development and progression of renal cell carcinoma. |
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