| Renal cell carcinoma accounts for approximately 3% of adult malignancies, the incidence of which has risen steadily in the past of 20 years.For the purpose of developing more effective drugs for renal carcinoma,researchers need to find some molecules more specific in the renal tumorigenesis and aggression, which could be a biomarker for early diagnosis of the renal cell carcinoma, and a drug target for the treatment. Besides, Clear cell renal cell carcinoma and papillary renal cell carcinoma make up about90% of all kidney malignant tumors. As one of the most commonly seen types of renal cell carcinoma, clear cell renal cell carcinoma calls for greater efforts in its further research. Micro RNA(mi RNA) is a short non-coding RNA that is able to regulate gene expression post-transcriptionally. Many mi RNAs are involved in a variety of biological processes, such as tumorgenesis, tumor metastasis. Since recent years, mi R-630 has become one of the most frequently studied mi RNAs in cancer biology, and up-regulation of mi R-630 has been found in various kinds of cancer. However, it is still not clear whether mi R-630 is expressed in the clear cell renal cell carcinoma, or whether expression of mi R-630 is correlated with clinical biological behavior of clear cell renal cell carcinoma, including clinical stage and prognosis.Part Ⅰ The expression of mi R-630 in the clear cell renal cell carcinoma and evaluated correlation between mi R-630 expression level and clinical-pathological variables.Objective: To analyze the expression level of mi R-630 in the clear cell renal cell carcinoma and clinical-pathological variables, including clinical stage and prognosis of patients.Methods: Detect mi R-630 expression in specimens of clear cell renal cell carcinoma by means of Real-time PCR, and investigate into therelationship between mi R-630 expression and clinicopathologic variables of patients.1 Inclusion criteria determined by HE staining: patients were included who were diagnosed as having renal cancer clinically. None of the patients had received adjuvant therapy before surgery. Renal tumor tissue and adjacent normal tissue were collected after radical nephrectomy, and stained by HE.With the histomorphology of all specimens examined under a microscope, it was confirmed that inclusion cases were clear cell renal cell carcinoma, and that the margins of adjacent normal tissues were negative.2 Determine mi R-630 expression level quantitatively by means of Real-time PCR: Specific primers for human mi R-630 are to be designed in order to determine the mi R-630 expression of all samples quantitatively by using Real-time PCR.3 Relation between mi R-630 expression and clinicopathologic variables of patients: The relation between mi R-630 expression and clinicopathologic variables of patients was assessed by Mann-Whitney U and Kruskal Wallis tests. The relation between mi R-630 expression and prognosis of patients was assessed by Kaplan-Meier method. Multivariate survival analysis was performed using the Cox proportional hazards model, which can be adopted in case there are more than two independent variables and in case there are continuous variables.Results:1 The total RNA was extracted from renal tumors and their matching adjacent normal tissues. And it was confirmed after a test that all the A260/280 ratios of RNA samples were around 2. Relative expression of mi R-630 in renal cancer was found after a real-time quantitative test, to be7.36±1.27, while that in the matching adjacent normal specimens was2.86±0.57. Statistical results showed that the expression of mi R-630 in renal cancer was significantly higher than that in adjacent normal specimens(P<0.05).2 The mi R-630 expression level in patients with high pathology grade(grade III-IV) was significantly higher than that in patients with low(grade I-II) pathology grade(P=0.001). Mi R-630 expression level in lymph node-positive patients was much higher than that in lymph node-negative patients(P=0.015), while mi R-630 expression level in patients with tumor distant metastasis was much higher than that in the patients without tumor distant metastasis(P=0.004). However, the results also showed that there was no difference in mi R-630 expression level between high clinical stage(T3-T4) and low(T1-T2) clinical stage(P=0.717). No significant difference in mi R-630 expression was observed with gender, age(P>0.05).3 Kaplan-Meier analysis demonstrated a significant impact of clinical pathological prognostic parameters, such as lymph node status(RR=5.108,P=0.015), tumor distant metastasis(RR=5.979, P=0.004) and histological grade(RR=3.264, P<0.001) on patient survival. Assessment of survival in patients revealed that higher expression of mi R-630 was correlated with adverse survival of patients(RR=3.378, P=0.008).4 The results also showed that the expression of mi R-630 was an independent prognostic factor for overall patient survival(relative risk: 3.021,CI: 2.074-5.726, P=0.016). With regard to other parameters, lymph node status(RR=3.681, CI=2.171-4.472, P=0.031), histological grade(RR=2.781,CI=1.876-4.638, P=0.006), and tumor distant metastasis status(RR=3.936,CI=2.074-5.726, P=0.016) were also shown to be an independent prognostic factors accounting for overall survival.Part Ⅱ The detection of expression of mi R-630 in the involved cell lines including renal cancer cell lines and normal human proximal tubule epithelial cell line.Objective: Compare the differences in mi R-630 expression between renal cancer cell lines and normal human proximal tubule epithelial cell line,and check to see whether there are differences in mi R-630 expression among different renal cancer cell lines.Methods: Detect the expression mi R-630 in the involved cell lines by Real-time PCR. Respectively culture human renal cancer cell lines 786-O,ACHN, Caki-1, Caki-2 and normal human proximal tubule epithelial cell line HK-2. The mi R-630 expression of these cells was detected by Real-time PCR.Results: Total RNA was extracted from cells(786-O, ACHN, Caki-1,Caki-2 and HK-2), and then converted to c DNA via reverse transcription. The Real-time PCR results showed that the expression level of mi R-630 in four renal cancer cell lines was significantly higher than that in normal human proximal tubule epithelial cell line(P<0.05). But there was no significant difference in mi R-630 expression level among different renal cancer cell lines(786-O, ACHN, Caki-1, and Caki-2)(P>0.05).Part Ⅲ The inhibition effects of mi R-630 on the growth, invasion and apoptosis of renal cancer cells(786-O).Objective: To investigate the effects of mi R-630 in renal cell cancer growth, invasion and apoptosis in vitro.Methods:1 Cell growth assay by MTT: 786-O cells transfected with Anti-mi R-630 or negative control were seeded in the dish respectively, and cultured for 96 hours. Then cell growth was investigated by MTT.2 Cell apoptosis assay by FACS Caliber: 786-O cells were cultured for48 hours after being transfected with Anti-mi R-630 or negative control respectively. Then cells were collected respectively, and stained with Annexin V-EGFP and Propidium Iodide. Then apoptotic cells were analyzed by using FACS Caliber.3 Wound healing assay: 786-O cells transfected with Anti-mi R-630 or negative control were seeded in plates, respectively, and grown to confluence.Wounds were made and then cell were cultured. The speed of wound closure was monitored, to evaluate the difference in cell migration between two groups.4 cell invasion assay: 786-O cells transfected with Anti-mi R-630 or negative control in each group were collected, and then cell suspensions were added into the upper Transwell chamber. After incubation for 48 hours, the number of invaded cells was counted under a microscope respectively, toinvestigate the effect of mi R-630 on the cell invasion ability.Results: When 786-O cells were transfected with Anti-mi R-630, the expression of mi R-630 was significantly decreased compared with the mi RNA inhibitor negative control group(P<0.05).1 Cell growth assay: The cell growth assay showed that down-regulation of mi R-630 significantly inhibited the growth of 786-O cell compared with the mi RNA inhibitor negative control group(P<0.05).2 Cell migration assay: Anti-mi R-630 decreased expression of mi R-630 in 786-O cells, and wound healing assay showed that the migratory rate of786-O cells transfected with Anti-mi R-630 significantly decreased compared with negative control transfection group, which indicated that mi R-630 could promote 786-O cell migration.3 Cell invasion assay: Transwell invasion assay indicated that the invasion of 786-O cells transfected with Anti-mi R-630 significantly decreased compared with negative control transfection group(P<0.05), which indicated that down-regulation of mi R-630 could resist the invasion ability of 786-O cells in vitro.4 Cell apoptosis assay: 786-O cells were stained with apoptosis-related protein. Using FACS Caliber, apoptosis assay showed a significantly increased rate of apoptosis in the 786-O cells transfected with Anti-mi R-630,as compared with negative transfection control group(P<0.05).Part Ⅳ The prediction and verification of target genes about renal cancer-related mi R-630Objective: To predict and verify the target genes about renal cancer-related mi R-630, and preliminarily probe into their functions.Methods:1 Use bioinformatics software to predict the target genes of mi R-630, the results of which will undergo KEGG pathway enrichment analysis. And the target genes will be screened according to the scores in the candidate target genes by bioinformatics software and its relation with renal cancer.2 Use Liposome transfection technique in 786-O cells to establish modelsfor mi R-630 over expression and inhibition.3 Use real-time quantitative PCR and Western-blot and FCM technique to detect the correlation of mi R-630 in the cell models with the expression level of target genes and functions of mi R-630.Results:1 The target genes were predicted by using common database and detected by real-time quantitative PCR. It was found that two of the theoretical target genes for mi R-630, i.e. RASGRF1 and CDC14 A have been significantly decreased(The difference was statistically significant.(P<0.01)after transfected with the mi R-630 mimics and increased(The difference was statistically significant(P<0.05) after the mi R-630 inhibited.2 Western-blot results show that the expression of m RNA and protein levels of RASGRF1 and CDC14 A decrease significantly(P<0.01), in the over expression group, while in the inhibition expression group, the expression of RASGRF1 and CDC14 A on m RNA and protein levels is significantly increased(P<0.05).3 FCM resluts show that the mi R-630 mimics group G1 phase was significantly shorter, and G2, S phase was significantly prolonged, while the mi R-630 mimics inhibitor group G1 phase was significantly prolonged, and G2, S phase was significantly shorter. It suggests that a retardation of the G1 cell cycle appear after transfection of the mi R-630 mimics inhibitor.Conclusions:1 The expression level of the mi R-630 was significantly higher in renal cancer in comparison to adjacent normal tissues, which indicated that mi R-630 could play a role in tumorigenesis and progression of clear cell renal cell carcinoma.2 The mi R-630 expression in clear cell renal cell carcinoma was significantly correlated with renal cancer histologic grade, lymphnode metastasis, distant metastasis, which indicated that the mi R-630 expression could be related with clear cell renal cell carcinoma malignancy, invasion and metastasis. Patients with high mi R-630 level in tumors had poor prognosis andshort survival, so the mi R-630 could play a role in clear cell renal cell carcinoma progression.3 The up-regulation of the mi R-630 could be a commonly seen event in renal cell carcinoma, which indicated that the mi R-630 might be one of valuable biomarkers for renal cell carcinoma. There was no difference in the mi R-630 level among different renal cancer cell lines. Therefore it can be presumed that the mi R-630 could play an important role in basic bio-behavior of renal cancer cells.4 The up-regulation of the mi R-630 expression could promote the growth of cells in(clear cell) renal cell carcinoma, inhibit its apoptosis, and help with its migration and metastasis. Therefore it is believed that the mi R-630 play an important role in the progression of(clear cell) renal cell carcinoma.Meanwhile, the Anti-mi R-630 could significantly down-regulate mi R-630 expression, which could in turn, lead to cell apoptosis, slow down the cell growth, and weaken its invasive power. Thus the mi R-630 could be a potential therapeutic target for renal cancer treatment.5 It is confirmed that RASGRF1 and CDC14 A are one of the theory target genes for mi R-630, for clear the pathogenesis of renal cancer,providing a new theoretical basis for and a new direction to its treatment.There exists a negative relation between the expression of mi R-630 and RASGRF1 CDC14 A protein expression levels, which may further affect the regulation of the cell cycles in renal cell carcinoma.To sum up, the mi R-630 plays an important role in the kidney tumorgenesis and cancer progression. So the mi R-630 might be a valuable biomarker for early diagnosis of renal cell carcinoma. Also the mi R-630 could be a potential target for renal cell carcinoma treatment. |
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