Studies On The Expression Of MiR-155 In The Renal Cell Carcinoma And The Effects On Biological Characteristics Of Renal Cell Carcinoma Cell Line ACHN

Background:Renal cell carcinoma (RCC) is one of the most commom seen malignant tumors which is secondary to bladder cancer in the urinary system carcinoma. The early detection of RCC is not easy, about 50% members of those patients are in their late stage when they were firstly diagnosed of this cancer and lost their best opportunity for treatment. The 5-year survival rate of advanced RCC is less than 10%. In our country, the morbidity of RCC is increasing gradually, but the mechanism of RCC remains unclear. Except surgery, the general conservative treatment including radiotherapy, chemiotherapy and immunotherapy has unsatisfactory results, so to find the tumorigenesis and seek perfect therapies of RCC are the goals of researcher.MicroRNAs (miRNAs) are a group of small noncoding functional RNAs that ara approximately 21?25 nucleotides in length. By binding to the 3'-untranslated regions of target mRNAs, miRNAs can either cleave RNA target or repress protein translation dependent upon its complementarity to the target mRNAs. Through their binding to target mRNAs sequences, miRNAs have a large number of biologically diverse functions. They have the capacity to control the expression of many downstream genes which can affect several cell regulatory pathways, such as cell growth, differentiation, mobility and apoptosis. Aberrant expression and metabolism of miRNAs are associated with human diseases including malignancy. Some miRNAs have been shown to possess oncogenic or tumor suppressor activity, influencing transformation, tumor progression or metastasis. Accumulating evidences suggest that miRNAs could potentially be widely used in the diagnosis, prognosis and therapy of human cancer.miR-155 is locates on human chromosome 21 q21, which is encoded by BIC. miR-155 is a typical multi-functional gene, involves in various physiological and pathological processes, such as tumorigeness, inflammation and immunoreaction. miR-155 is overexpressed in many types of cancer, including Non-small cell lung cancer, breast cancer, Pancreatic cancer, et al.Although the function and regulatory mechanisms of miR-155 are still unclear, however, over-expression in many types of cancer suggested its significance role in cancer diagnosis and treatment. In order to explore the relationship between miR-155 and clear cell renal carcinoma, we detected the expression of miR-155 in ccRCC and adjacent non-tumor tissue through real-time PCR, and to find out whether it is some relationships between its expression and clinical stage, histological grade of the patients. Furthermore, the expression of miR-155 was knockdown by using of has-mir-155 inhibitor, to explore the effects of miR-155 on biological behavior of ACHN cells, including proliferation, apoptosis and invasion ability. Last, we predicted miR-155 target genes and explore its possible role in renal cell carcinoma, we hope our study could provide convincing evidence on miR-155's association with renal tumorigenesis, and facilitate the search of suitable candidates for cancer targeting therapy. Part IThe expression of miR-155 and correlation with clinical stage, histological grade in ccRCCObjective To investigate the expressions of miR-155 in ccRCC with regard to the clinical significance.Methods Expressions of miR-155 was detected by real-time PCR in 16 cases of ccRCC and matched adjacent non-tumor tissue. The relevance between the expressions of miR-155 and the clinical stage and pathologic grade were also studied.Results Real-time PCR demonstrated that the expression of miR-155 was up-regulated in 15 samples, down-regulated in one sample compared to the matched adjacent non-tumor tissue. The relative expression content of miR-155 in ccRCC is 12.70±6.52, which was significantly higher than that in the matched adjacent non-tumor tissue with 2.82±1.25 (P< 0.05). According to the pathological grades, the relative expression content of miR-155 was 11.43±6.18 in well differentiated group, and 14.33±7.04 in low-moderately differentiated group, which had no significantly difference (P> 0.05). According to clinical stages, the relative expression content of miR-155 was 13.50±6.55 in I phase, and 10.93±6.79 in?-?phase, which had no significantly difference (P> 0.05). Conclusion1. The expression of miR-155 was up-regulated in ccRCC compared with matched adjacent non-tumor tissues, suggesting that up-regulation of miR-155 may be associated with the pathogenesis of ccRCC.2. The expression of miR-155 has no significant correlation with pathological grade and clinical stage in ccRCC. Part?The effects of miR-155 on biological characteristics in human renal carcinoma cell lineObjective To explore the effects of miR-155 on biological characteristics in human renal carcinoma cell line, including proliferation, apoptosis and invasion ability.Methods The expression of miR-155 in ACHN, KC and HK2 cells was detected by real-time PCR. Select the cell line which had the highest expression of miR-155 as model cell. Then the miR-155 expression of the model cell was knockdown by has-mir-155 inhibitor. The cell proliferative vitality, apoptosis and migration activity were determined by MTT assay, flow cytometry and transwell migration assay respectively.Results The relative expression content of miR-155 was 9.3,3.0 and 1.0, respectively. Select ACHN cell lines as cell model in vitro. Successfully transfected ACHN cells with hsa-mir-155 inhibitor which inhibited the expression of miR-155 compared with negative control group (P< 0.05).48 hours after transfection, the relative survival rate of inhibition group was 66.6±1.0%, while the negative control group was 97.1±1.5%, has significant difference (P< 0.05); The cell apoptosis rate of inhibition group was 13.76±0.86%, while the normal group and the negative control group, the apoptosis rates were 5.98±0.44% and 7.16±1.07%, which had significant difference (P< 0.05); Transwell invasion assay showed that the control group was 42.17±1.38, control group was 41.22±1.65, while the inhibition group was 14.74±1.06, which had significant difference compared with the normal group and control group (P< 0.05).Conclusion1. Hsa-mir-155 inhibitor could be effectively transfected to ACHN cells, leading the expression of miR-155 decreased.2. The down-regulated expression of miR-155 can inhibit the proliferation, invasion and make apoptosis increased in ACHN cells.3. miR-155 may play an oncogenic activity role in renal cell carcinoma, and was a potential tool in gene therapy of RCC. Part?Studies on the mechanism of miR-155 affecting the renal cell carcinomaObjective To explore the mechanism of microrna-155 affecting the renal cell carcinomaMethods Predicted target genes of miR-155 By bioinformatics methods (such as miRanda, Pictar, Targetscan), real-time PCR and Western blot were used to detecte the expression of miR-155 and target gene protein, study the mechanism of growth inhibition in renal cell carcinoma when microrna-155 was down-regulated.Results Select TargetScan, PicTar and miRanda as prediction software to predict the target genes of miR-155, all the three predicted that SOCS-1, BACH1, and RAB34 may be the potential target genes. The real-time PCR results showed that the relative expression content of miR-155 was 1.25±0.22 in inhibition group,7.03±0.18 in negative control group, which had significant difference (P< 0.05). Western blot showed the protein expression of SOCS-1 ratio (SOCS-1/GAPDH) in the negative control group and the inhibition group were 0.053±0.015 and 0.047±0.006, had no significant difference (P> 0.05). BACH1 protein expression ratio (BACH1/GAPDH) were 0.125±0.005 and 0.376±0.015 respectively, which had significant difference (P< 0.05). RAB34 protein expression ratio (RAB34/GAPDH) were 0.164±0.015 and 0.399±0.091 respectively, had significantly different (P< 0.05).Conclusion1. In ACHN cells, the expression of miR-155 had negatively correlation with the expression BACH1 and the RAB 34 protein, while had no correlation with the expression of SOCS1 protein.2. BACH1, RAB 34 may be the potential target genes of miR-155, while SOCS1 was not the target genes of miR-155.