【作者】 张冰；

【导师】 史本康；

【作者基本信息】 山东大学 ， 临床医学（专业学位）， 2015， 博士

【摘要】 研究背景肾细胞癌(renal cell carcinoma, RCC)的发病率在肾脏肿瘤中占90-95%,约占成人恶性肿瘤的2-3%,且发病率和死亡率逐年增加。RCC起病隐匿,患者早期常无明显临床症状,容易被忽视。除此之外,RCC具有侵袭性,且治疗相对单一,以手术切除为主,放疗、化疗、免疫治疗等其他辅助治疗效果欠佳。这些都是造成晚期肾癌预后差、死亡率高的原因。RCC有多种不同的病理分型,不同分型肿瘤的遗传学、生物学行为也不尽相同。其中,肾透明细胞癌(clear cell renal cell carcinoma, ccRCC)约占70%,是最常见的病理类型。在最近的几年中,对RCC发生、发展的生物学机制的研究主要集中在基因突变,蛋白质编码的基因表达异常和表观遗传变异等。然而,随着研究的深入,人们发现,一种非蛋白编码的RNA, microRNA的异常调节与RCC密切相关。microRNA表达的研究对明确RCC的发病机理,早期诊断,提高预后具有重大意义。microRNA (miRNA)是一种只有19-25个核苷酸的短链非编码RNA,其介导转录后基因表达的调节。成熟的miRNA与目标信使RNA (messenger ribonucleic acid, mRNA)结合,形成沉默复合体,从而沉默mRNA,导致其降解或抑制翻译。通过这种负向调节作用,这种小RNA,实现了包括细胞生长、分化、增殖、凋亡和细胞周期调控等多种广泛的生物学功能调节。此外,miRNA表达与肿瘤的关系亦非常密切。大量研究表明,miRNA在多种人类恶性肿瘤中表达异常,是恶性肿瘤的重要调节因子,在肿瘤的发生、发展、逃避凋亡,血管生成及组织浸润和转移中起非常重要的作用。miRNA在不同的肿瘤中可起到癌基因或抑癌基因样作用。此前的文献已报道了RCC中异常miRNA的表达谱,这些异常表达的miRNA可以促进细胞增殖,影响细胞侵袭,抑制细胞凋亡,最终导致RCC的发生和发展。除此之外,敲除过表达的miRNA或者重新表达在癌细胞中沉默的miRNA可导致肿瘤细胞的死亡。因此,我们可预计,miRNA会成为肿瘤治疗的新靶点。miR-155由B细胞整合簇(B-cell integration cluster, BIC)基因编码。研究表明,miR-155在造血细胞生成、炎症反应、免疫反应及肿瘤的发生和发展中发挥重要效应。miR-155的癌基因样作用在造血系统恶性肿瘤中首次被发现,miR-155在B细胞淋巴瘤和慢性淋巴细胞白血病表达升高。miR-155在许多实体恶性肿瘤中也高表达,如乳腺癌,肺癌,结肠癌,胰腺癌和甲状腺癌等。利用基因芯片对RCC及正常肾组织分析表明,共有35种miRNA在RCC中表达显著异常,其中miR-155在RCC中表达显著升高,但其详细机制尚不明确。磷脂酰肌醇三激酶(phosphoinositide 3-kinase, PI3K)/蛋白激酶B (protein kinase B, AKT)信号通路是调节众多生物学进程的经典通路。如：细胞增殖,凋亡,转移,基因转录,核糖体功能等等。PI3K/AKT与各种恶性肿瘤,包括RCC的发生、发展关系密切。P13K可被多种细胞因子激活,随后通过过AKT的磷酸化,而发挥功能,从而引起细胞增殖,凋亡抑制和细胞迁徙。抑制PI3K/AKT通路可诱导肿瘤细胞的程序性凋亡和生长抑制。最近,有文献表明,在RCC中,miR-155有可能作为上游调节因子,作用于PI3K,从而激活PI3K/AKT信号通路。但其具体机制尚不清楚。FOXO3a是AKT的下游靶基因之一,它是具有叉头样结构的转录因子家族的成员之一。FOXO3a在正常情况下分布于细胞核内,其可以上调细胞凋亡调节蛋白(B cell lymphoma/leukemia-2 interacting mediator of cell death, BIM)和Fas,从而在细胞凋亡过程中起非常关键的作用。除此之外,FOXO3s还可通过激活下游组件,如生长抑制及DNA损伤诱导基因(growth arrest and DNA damage-inducible genes, GADD45A),引起细胞G2/M期生长阻滞。FOXO3a是一种非常重要的肿瘤抑制因子。综上所述,我们有理由推断miR-155在RCC的发生、发展中发挥重要的作用。其调节机制是通过激活PI3K/AKT路径,引起FOXO3a表达异常而实现。在本次研究中,我们将证实miR-155在RCC中,是引起细胞增殖和侵袭的重要决定因素,且它的作用是通过介导PI3K/AKT/FOX03a信号通路实现的。第一章miR-155在人肾细胞癌中表达的临床意义研究目的(1)研究miR-155在人肾细胞癌组织中及肾癌细胞系中的表达情况。(2)观察miR-155与RCC的国际泌尿病理学会(ISUP)分级、TNM分期之间的关系。材料与方法1.组织样本：收集滨州医学院附属医院收治的20例行肾癌根治术的RCC患者新鲜肿瘤组织及周边正常肾组织,-80℃低温保存。并且记录患者就诊时临床资料、TNM临床分期、病理标本检查资料、治疗情况及其存活状况。所有患者术前均未行任何放疗、化疗及其他治疗。本次研究的病人均签署知情同意书,所有研究内容均经过滨州医学附属医院伦理委员会审批。2.组织病理学诊断和TNM分期：所有病例均经过病理诊断确诊。采用双盲法,请2位高年资病理学医师对所有病例按照2012年国际泌尿病理学会(ISUP)肾脏肿瘤组织学分级进行形态学复习并分级。根据病例资料,按照美国癌症联合委员会(AJCC)标准进行TNM分期。3.细胞培养：人肾细胞癌细胞系ACHN、CAKI-1、786-0购自中国科学院上海生命科学研究院细胞资源中心。人肾小管上皮细胞系HK-2,购自由美国ATC C公司。在37℃恒温,5%CO2含量的培养箱中,用包含10%胎牛血清的DMEM培养基培养。4.RNA提取及实时定量RT-PCR：应用实时定量RT-PCR技术检测新鲜组织及RCC细胞系中miR-155的表达。应用TRIzol试剂盒提取组织和细胞中总RNA,用miRNA检测试剂盒进行逆转录。PCR反应在ABI 7500 Real-Time PCR系统中反应完成。具体步骤为：95℃,10分钟1周期；95℃,15秒；然后60℃,1分钟,40周期。每一样本进行一式三份实验,计算取平均值,应用U6作为内参。5.统计学分析：数据以x±SD表示,使用SPSS17.0软件对数据进行方差分析,|根据统计学要求,P<0.05,认为差异有统计学意义。实验结果1.本实验共收集20例RCC患者新鲜肿瘤组织及配对非肿瘤组织标本。其中男性12例,女性8例。年龄34～73岁,平均66岁。病理类型均为肾透明细胞癌。按照ISUP病理分级：1级6例,2级7例,3级4例,4级3例。TNM分期：Ⅰ期：10例,Ⅱ期：7例,Ⅲ期3例,Ⅳ期0例。所有患者术前均通过影像学检查未发现远处转移。术前均未行任何放疗、化疗及其他治疗。2.miR-155在RCC组织中表达显著上调,较癌旁正常肾组织相比平均增加5.6倍。结果有显著统计学意义(P<0.05)。3.miR-155在RCC各细胞系中表达显著上调。与人类肾小管上皮细胞株HK-2相比,miR-155在ACHN、CAKI-1与786-0细胞系中表达均上调,结果有显著统计学意义(P<0.05)。4.miR-155在RCC不同病理分级的表达情况：根据病理分级进行分组,ISUP分级1～2级中miR-155的相对表达量为0.044±0.024,ISUP分级3-4级为0.102±0.034,有显著统计学差异(P<0.05)。5.在不同TNM分期组合样本中,Ⅰ期miR-155的相对含量为0.043±0.026,Ⅱ-Ⅲ期为0.085±0.039,有显著统计学差异(P<0.05)。实验结论1.在RCC新鲜组织及细胞系中,miR-155表达较癌旁正常肾组织或HK-2细胞系明显上调,提示miR-155可能在RCC的发病原因中起原癌基因样作用。2.不同ISUP分级的RCC中,miR-155表达存在差异。ISUP分级升高,miR-155表达也相应升高。3.不同TNM分期的RCC中,miR-155表达存在差异。TNM分期越高,miR-155的表达也相应升高。第二章miR-155及PI3K/AKT/FOXO3a通路与肾癌细胞生物学行为的关系研究目的(1)探讨miR-155表达的变化对肾癌细胞增值、凋亡、侵袭能力等生物学行为的影响。(2)探讨miR-155及PI3K/AKT/FOXO3a通路在调节肾癌细胞生物学行为中的作用。材料与方法1.转染miR-155抑制剂于肾癌细胞系：选取ACHN细胞系作为模型,随机分为3组,(1)miR-155 inhibitor组：使用Lipofectamine 2000方法将miR-155hairpin inhibitor转染至ACHN细胞系；(2)Negative control组：将miR-CON转染至细胞系；(3) Untreated组：正常未经转染的ACHN细胞。用实时定量RT-PCR检测转染效果。2.细胞增殖分析：ACHN细胞(3×104 cells/well),在96孔板中培养过夜,用上述方法进行转染,分别在转染后24h、48h和72h,用MTT法检测细胞的增值。样本的吸光度用微孔板分光光度计在490nnm检测,该实验重复3次。3.克隆形成实验：ACHN细胞被转染后,置入6孔板中,细胞密度为1000cells/well。在37℃培养10天后,将细胞洗涤,福尔马林固定,然后用结晶紫进行染色。用显微镜进行观察,超过50个细胞的克隆数为阳性,每组样本一式三份。4.细胞周期分析：将ACHN细胞置入6孔板中,过夜,用上述方法转染后,孵化48h,用流式细胞仪确定细胞周期分布。该实验重复3次。5.凋亡分析：将ACHN细胞置入6孔板中,过夜,转染后,孵化48h,收集细胞,用冰PBS洗2次,总量1.0×105细胞在100μLPBS中重悬,然后在常温避光下,与5μL FITC标记的膜联蛋白和5μL碘化丙啶混合15分钟,然后加入400μL缓冲液,应用流式细胞仪进行凋亡分析。6.细胞划痕实验(Wound-healing assay):将ACHN细胞置入6孔板中,过夜,转染后,孵化48h,用200μL枪头做横线划痕,用PBS洗细胞3次,去除划下的细胞,加入无血清培养基。在常规条件下培养48h,用显微镜在划痕的相同部位拍照。7.细胞侵袭实验(Transwell assay):ACHN细胞转染,孵化48h后,取200μL约含5×104细胞的培养液种于上室,下室用培养基填满,孵育12h后,将包含细胞的膜用多聚甲醛固定,结晶紫染色,将膜的底面放大100倍拍照,每个小室随机选取5个区域拍照。8.Western bolt实验：应用Western bolt检测ACHN细胞系中PI3K(P110α)、p-AKT(Ser473)、FOX03a蛋白的表达。细胞裂解以后,进行凝胶电泳,转膜,封闭,分别孵一抗和二抗,然后显影。应用GAPDH作内参。9.统计学分析：数据以x±SD表示,使用SPSS17.0软件对数据进行方差分析,根据统计学要求,P<0.05,认为差异有统计学意义。实验结果1.转染miR-155抑制剂于ACHN细胞株后,MTT法检测细胞增殖明显减弱(P<0.05),克隆形成实验也表明,培养10天后,克隆形成较Negative control组和Untreated组明显减少(P<0.05)。2.下调miR-155表达后,ACHN细胞的凋亡增加,并出现细胞周期阻滞。流式细胞仪分析显示：转染miR-155抑制剂使细胞凋亡率显著增加(P<0.05),这一结果表明,抑制miR-155表达,可以促进细胞凋亡。而且,抑制miR-155的表达,减少了处于S期的细胞比率,增加了处于G1/G0期细胞比率,使得细胞发生G1/G0期阻滞,减弱了细胞增殖。3.抑制miR-155的表达使得细胞迁徙和侵袭能力下降。Wound-healing assay实验和T ranswell实验均表明：抑制miR-155的表达使得细胞迁徙和侵袭能力下降(P<0.05)。4.抑制miR-155的表达使得PI3K(P110α)和p-AKT (Ser473)的表达降低。Western bolt实验表明,抑制miR-155的表达引起了PI3K(P110α)和p-AKT(Ser473)表达的降低(P<0.05)。这表明,miR-155通过上调PI3K-AKT路径而实现其癌基因样的作用。5.抑制miR-155的表达使得FOXO3a的表达升高。Western bolt实验表明,抑制miR-155的表达引起了约2倍的FOX03a表达的升高(P<0.05)。这表明,miR-155通过PI3K-AKT通路负向调节FOXO3a而实现其癌基因样的作用。实验结论1.通过转染,敲低miR-155表达可以使得ACHN细胞增殖减少,凋亡增加,侵袭能力下降,并且出现细胞周期阻滞。提示miR-155可能在RCC的进展,转移中起促进作用。2.抑制miR-155表达可以使得PI3K (P110α)和p-AKT(Ser473)表达的降低,FOXO3a的表达升高。表明：miR-155通过PI3K-AKT通路负向调节FOXO3a的表达,从而影响RCC的进展。更多还原

【Abstract】 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.更多还原