| BackgroundThe prevalence of renal cell carcinoma (RCC), a renal disorder accounting for approximately 2-3% of all adult malignancies and 90-95% of adult kidney neoplasm, is increasing in the recent years. The high mortality rate of such disease was due to the aggressive nature of the neoplasia, the lack of early detection, and the limited response to available treatments. Several histological subtypes are available with different genetics, biology and behaviors of RCC. Clear-cell RCC (ccRCC) as the most common type accounts for 70% of all RCC. In the past decades, studies on RCC have mainly focused on the genome mutations, expression of protein coding genes, as well as epigenetic changes. However, increasing evidence indicates that dys-regulation of a class of noncoding RNA genes, miRNAs, is also closely associated with RCC. Their expression profiles can be correlated with diseases pathogenesis and development, which possess important significance for early diagnosis and prognostic assessment.MicroRNAs (miRNAs) are a group of short non-coding RNAs with lengths of approximately 19-25 nucleotides that mediate post-transcriptional control of gene expression. It has been acknowledged that mature miRNA could load onto the RNA-induced silencing complex (RISC) which results in the silencing of target mRNA throughing mRNA cleavage or translational repression. Through their repressive action, these short RNA species play important roles in a wide range of biological functions, including cell growth, differentiation, apoptosis and cell cycle control. The dys-regulation of miRNA expression plays crucial roles in the pathogenesis and development of human malignancies. Accumulated evidences have revealed that miRNA are important regulator of all human malignancies and play key roles in tumour initiation, development and evasion of apoptosis, angiogensis, tissue invasion and metastasis. Besides, miRNA has been reported to show the function as either tumor-suppressors genes or oncogenes. The aberrant expression of miRNA in RCC has also been well described, which can induce cell proliferation, migratory activity and reduce cell apoptosis eventually contribute to the initiation and progression of RCC. In addition, apoptosis was induced in cancer cells subject to miRNA over-expressing knock-out tests or silencing tests. Taken together, it is reasonable to speculate that microRNA is a new target for cancer therapy.MiR-155, localized within a region known as B cell integration cluster (BIC), plays important roles in haematopoiesis, immune response, inflammation as well as pathogenesis and development of cancer. MiR-155 was first implicated in the oncogenesis of hematopoietic malignancies based on the finding that BIC/mir-155 expression is up-regulated in B-cell lymphomas and chronic lymphocytic leukemia. MiR-155 is also overexpressed in various solid tumors, including breast, lung, colon, pancreatic, and thyroid cancers. For instance, miR-155 was found to be up-regulated in RCC through a miRNA signature, which identified 35 miRNAs expressions were altered the most between normal and cancer. However, its exact function in the pathogenesis of ccRCC is still not well defined.The phosphoinositide 3-kinase (PI3K)/Protein kinase B (AKT) signaling pahway is a classic pathway which regulates many fundamental biological processes, such as cell proliferation, apoptosis, migration, gene transcription and ribosomal function and is associated with the initiation and development of various malignancies, including RCC. PI3K can be activated by a variety of cytokines, via phosphorylation of AKT consequently, resulting in cell proliferation, apoptosis suppression, and migration. Blockage of PI3K/AKT pathway induces programmed cellular apoptosis and growth inhibition of tumor cells. Recently, it was reported that miR-155 targets PI3K in RCC. As an up-stream regulatory factor, miR-155 could activate PI3K/AKT pathway.FOXO3a, a target gene of AKT, is a family member of forkhead transcriptional factor. It is reported to be distributed in nucleus, and plays crucial roles in the cellular apoptosis through up-regulating the BCL-2 interacting mediator of cell death (BIM) and Fas. Besides, it could activate the growth arrest and DNA damage-inducible genes 45A (GADD45A), based on which to induce cell arrest in G2/M phase. FOXO3a is an important tumor suppressor.Thus, it is reasonable to speculate that miR-155 is involved in the pathogenesis and development of RCC through regulating the PI3K/AKT/FOXO3a signaling pathway. In this study, we aimed to demonstrate that miR-155 is a determinant of cell proliferation and invasion by targeting PI3K/AKT/FOXO3a signaling pathway in RCC.Chapter 1 Significance of miR-155 expression in renal cell carcinomaObjective(1) To idendify the expression of miR-155 in renal cell carcinoma, matched adjacent non-tumor tissues and renal carcinoma cell lines.(2) To idendify the relationship between miR-155 expression and International Society of Urological Pathology (ISUP) grades or TNM stages of RCC.Materials and methods1. Tissue Specimens:A total of 20 RCC tissue specimens and patient-matched normal kidney tissues, were obtained from patients at Binzhou Medical University Hospital immediately after radical nephrectomy. And the clinical information, TNM stage, pathological grades, treatment and survival conditions of patients were recorded. None of these patients received antitumor treatment, such as radiotherapy or chemotherapy, before the operation. Tissue samples were immediately frozen in liquid nitrogen after resection and stored at -80℃ before RNA extraction. Written informed consent was obtained from each patient. The study protocols were approved by the Institutional Review Board.2. The histopathological diagnosis and TNM stage:The diagnosis as RCC was histologically confirmed. The pathological grades were reviewed by 2 qualified pathologists blinded according to 2012 ISUP renal carcinoma grades. The cases were staged according to the American Joint Committee on Cancer (AJCC) TNM Classification.3. Cell culture:The RCC cell lines ACHN、CAKI-1 and 786-0 were obtained from the Institute of Biochemistry and Cell Biology and normal human kidney tubular epithelial cell line (HK-2) were purchased from American Type Culture Collection. Cells were cultured in DMEM medium containing 10% fetal bovine serum (Gibco). All cells were maintained at 37℃ in a humidified incubator with 5% CO2.4. RNA extraction and real-time quantitative PCR:Total RNAs were extracted from frozen samples and cells using TRIzol reagent and reverse transcribed using the real-time PCR miRNA detection kit according to the protocol. PCR reactions were performed by SYBR Green on ABI 7500 Real-Time PCR System at the following conditions:95℃,10 min for 1 cycle; followed by 40 cycles of 95℃ for 15 s,60℃ for 1 min. All values were normalized to an endogenous U6 control.5. Statistical analyses:Each experiment was performed at least in triplicate. All data are presented as mean ± standard deviation. For comparison of differences, the statistical analysis was performed by analysis of variance (ANOVA) using SPSS 17.0. P<0.05 was considered to be statistically significant.Results1. RCC tissue specimens and matched normal kidney tissues were obtained from 20 patients including 12 male cases and 8 female cases with mean age of 66 years (34-73years). The diagnosis as ccRCC was histologically confirmed. There are 6 cases of grade 1; 7 cases of grade 2; 4 cases of grade 3; 3cases of grade 4 according to ISUP grades and 10 cases of stage Ⅰ; 7 cases of stage Ⅱ; 3 cases of stageⅢ; 0 case of stageIV according to TNM stages. No metastasis was found in all patients confirmed by imaging examination. None of these patients received antitumor treatment, such as radiotherapy or chemotherapy, before the operation.2. miR-155 expression was significantly up-regulated in RCC compared with adjacent normal kidney tissues, with an average 5.6-fold increase (P<0.05).3. The up-regulation of miR-155 was also found significantly in the RCC human cell lines ACHN、CAKI-1 and 786-0 compared with the human kidney tubular epithelial cell HK-2 (P<0.05).4. The expression of miR-155 was significantly increased in cases of grade 3-4 (0.102 ±0.034) compared with the cases of grade 1～2 (0.044±0.024, P<0.05).5. The expression of miR-155 was significantly increased in cases of stage Ⅱ～Ⅲ (0.085±0.039) compared with the cases of stage I (0.043±0.026, P<0.05).Conclusions1. miR-155 expression was significantly up-regulated in RCC compared with adjacent normal kidney tissues and this pattern was recapitulated by RCC cell lines. These results suggested that miR-155 may function as an oncogene in RCC.2. The expression of miR-155 was different in different ISUP grades and the higher the grade, the higher the expression.3. The expression of miR-155 was different in different TNM stages and the higher the stage, the higher the expression.Chapter 2 Biological function of miR155/PI3K/AKT/FOXO3a signaling pathway in renal cell carcinoma cellsObjective(1) To investigate the effects of miR-155 on the proliferation, apoptosis and migratory activity of renal carcinoma cells.(2) To investigate the biological function of miR155/PI3K/AKT/FOXO3a signaling pathway in renal carcinoma cells.Materials and methods1. miR-155 inhibitor cell transfection:ACHN cell lines were randomly divided into three groups:(1) miR-155 inhibitor group:miR-155 hairpin inhibitor was transfected using Lipofectamine 2000 according to the manufacturer’s instructions. (2) Negative control group:miR-CON was transfected using the same method. (3) Untreated gourp: the normal ACHN cell with untreated. The level of miR-155 in transfected cells was examined by real-time PCR.2. Cell proliferation analysis:ACHN cells (3×104 cells/well) were seeded onto 96-well plates overnight, and then transfected with miR-155 inhibitor or miR-CON. About 24 h,48 h and 72 h after transfection, cell proliferation were determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyl-tetrazolium bromide (MTT) assay according to the manufacturer’s instructions. The absorbance rate was measured at 490 nm in a microplate spectrophotometer.3. Colony formation assay:ACHN cells were transfected with miR-155 inhibitor or negative control as previously described and plated into 6-well plates at a density of 1,000 cells/well. After incubation at 37℃ for 10 days, the colonies were washed and then fixed with formalin and stained with crystal violet. The number of colonies containing more than 50 cells was counted under a microscope for three independent replicates.4. Flow cytometric analysis of cell cycle:ACHN cells were cultured in 6-well plates overnight, then transfected with miR-155 inhibitor or control as previously described. After 48 h incubation, cell cycle distribution was determined using flow cytometry. Briefly, the cells were collected, washed with ice-cold PBS twice and fixed with 70% cold ethanol at 4℃ overnight. After incubation in 100μg/mL RNase A at 37℃ for 30 min, the cells were stained with 50μg/mL propidium iodide. The DNA contents analyses of cells were performed using a FACSCalibur flow cytometer. All the tests were performed at least in triplicate.5. Apoptosis assays:ACHN cells were collected and transferred to 6-well plates overnight, then transfected with miR-155 inhibitor or control. After 48 h incubation, the cells were harvested and washed twice with cold PBS. A total of 1.0×105 cells were resuspended in 100μL binding buffer, and mixed with 5μL FITC-labelled Annexin V and 5μL of propidium iodide (PI) at room temperature for 15 min in the dark. After incubation,400μL binding buffer was added. Apoptosis was analyzed by flow cytometry.6. Wound-healing assay:ACHN cells were seeded on 6-well plates overnight, then transfected with miR-155 inhibitor or negative control. After 48 h incubation, the cells were scratched with a 200μL pipette tip and washed 3 times with PBS buffer subsequently to remove cell debris. Fresh medium was added to each well, and the cells were allowed to close the wound for 48 h under normal conditions. Photographs were taken at the same position of the wound with a computer-assisted microscope.7. Cell invasion assay:Cell invasion assays were evaluated using a Boyden chamber containing 24-well Transwell plates with 8-μm-pore on the membrane. ACHN cells were transfected with miR-155 inhibitor or miR-CON. After 48 h incubation, approximately 5x 104cells in 200 μL culture medium supplemented with 5% FBS were placed in the upper chamber. The complete medium containing 10% FBS was added to the lower chamber as a chemoattractant. After 12 hours of incubation at 37℃ in a 5% CO2 atmosphere, cells on the lower chamber were fixed with paraformaldehyde and stained with crystal violet. The lower surfaces of the membranes were photographed in five random fields at x 100 magnifications for each chamber to determine the migration.8. Western blot:The expressions of PI3K (P110α), p-AKT (Ser473) and FOXO3a were measured by Western blot. Cells were lysed with RIPA Buffer. Then, the extracts were resolved on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The membranes were blocked with 5% non-fat milk and incubated with primary antibodies followed by horseradish peroxidase-conjugated secondary antibody. The bands were detected by enhanced chemiluminescence (ECL) kit. Membranes loaded with GAPDH served as control.9. Statistical analyses:All data are presented as mean ± standard deviation. For comparison of differences, the statistical analysis was performed by analysis of variance (ANOVA) using SPSS 17.0. P<0.05 was considered to be statistically significant.Results1. Altered miR-155 expression inhibited cell proliferation in ACHN cells. miR-155 inhibition significantly decreased the growth rate of ACHN cells according to MTT assay(P<0.05). Colony formation analysis also revealed that miR-155 inhibition significantly decreased the colony sphere formation after the cells was cultured in medium for 10 days(P<0.05).2. Altered miR-155 expression induced apoptosis and cell cycle arrest of RCC cells. Flow cytometric analysis revealed that the rate of apoptosis was dramatically increased in ACHN cells of miR-155 inhibitor group. These results suggested that inhibition of miR-155 could induce enhanced apoptosis (P<0.05). Inhibition of miR-155 dramatically decreased the percentage of cells in S phase and significantly pushed the accumulation of cells in Gl/GO phase. Collectively, these results indicate that downregulation of miR-155 resulted in the G1/G0 arrest and suppressed RCC cell proliferation in vitro.3. The effects of miR-155 on cell migration and invasion of RCC cells. Wound-healing assay demonstrated that inhibition of miR-155 reduced the migration capacity of the ANCH cells. Transwell assay showed that miR-155 inhibition resulted in reduction of ANCH cells invasion compared with the miR-CON transfected cells. (P<0.05). Therefore, these data indicated that miR-155 enhanced migration and invasion of RCC cells.4. PI3K/AKT signaling pathway was down-regulated. Western bolt results indicated that the expression of PI3K (P110α) and p-AKT(Ser473) were reduced effectively after miR-155 inhibition (P<0.05).These suggest that miR-155 promote renal carcinoma by activating PI3K/AKT signaling pathway.5. The expression of FOXO3a was significantly up-regulated. The expression of FOXO3a was increased effectively after miR-155 inhibition (P<0.05).These suggest that miR-155 promote renal carcinoma by suppressing FOXO3a.Conclusions1. The proliferation, invasion and migration of RCC cells were decreased after miR-155 inhibition. Moreover, miR-155 inhibition induced apoptosis and cell cycle arrest of RCC cells.2. miR-155 promoted the RCC through activating PI3K/AKT signaling pathway and targeting FOXO3a. |
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