CDC6靶向RNA干扰质粒载体的构建及生物活性鉴定

目的:构建小鼠CDC6基因的RNAi真核表达载体PGCsilencer TM u6/Neo/GFP/RNAi,观察其转染小鼠肝细胞前后CDC6的表达变化。方法:根据GenBank中CDC6的序列,设计特异性siRNA序列,将模板序列克隆至PGCsilencer U6/Neo/GFP质粒中,通过测序鉴定后,用脂质体将重组子转染至正常小鼠肝细胞中,用RT-PCR检测CDC6的mRNA的表达及用Western blot方法检测CDC6蛋白水平的表达,并比较转染前后其表达水平的变化。结果:经测序,模板序列与设计序列完全正确,经过RT-PCR及Western blot方法检测,转染干扰质粒后,小鼠肝细胞中CDC6表达在mRNA及蛋白水平都有明显的下降。结论:成功构建了CDC6基因的RNAi真核表达载体并转染至小鼠肝细胞中,为下一步探讨CDC6在肝再生的作用奠定了基础。     

Chronic ethanol feeding enhances miR-21 induction during liver regeneration while inhibiting proliferation in rats

Liver regeneration is an important repair response to liver injury. Chronic ethanol consumption inhibits and delays liver regeneration in experimental animals. We studied the effects of chronic ethanol treatment on messenger RNA (mRNA) and microRNA (miRNA) expression profiles during the first 24 h after two-thirds partial hepatectomy (PHx) and found an increase in hepatic miR-21 expression in both ethanol-fed and pair-fed control rats after PHx. We demonstrate that the increase of miR-21 expression during liver regeneration is more robust in ethanol-fed rats. Peak miR-21 expression occurs at 24 h after PHx in both ethanol-fed and control rats, corresponding to the peak of hepatocyte S phase in control rats, but not in ethanol-exposed livers in which cell cycle is delayed. The induction of miR-21 24 h after PHx in control rats is not greater than the increase in expression of miR-21 due to sham surgery. However, in the ethanol-fed rat, miR-21 is induced to a greater extent by PHx than by sham surgery. To elucidate the implications of increased miR-21 expression during liver regeneration, we employed unbiased global target analysis using gene expression data compiled by our group. Our analyses suggest that miR-21 may play a greater role in regulating gene expression during regeneration in the ethanol-fed rat than in the control rat. Our analysis of potential targets of miR-21 suggests that miR-21 affects a broad range of target processes and may have a widespread regulatory role under conditions of suppressed liver regeneration in ethanol-treated animals.     

MicroRNA-99a-5p suppresses breast cancer progression and cell-cycle pathway through downregulating CDC25A

In this study, we aimed to explore the association between miR-99a-5p and CDC25A in breast cancer and the regulatory mechanisms of miR-99a-5p on breast cancer. The expressions of messenger RNA and microRNAs in breast cancer tissues and adjacent tissues were analyzed by the Cancer Genome Atlas microarray analysis. Quantitative real-time polymerase chain reaction was conducted to find out the expression levels of miR-99a-5p and CDC25A. The expression levels of proteins (CDC25A, ki67, cyclin D1, p21, BAX, BCL-2, BCL-XL, MMP2, and MMP9) were determined by Western blot analysis. The relationship between miR-99a-5p and CDC25A was predicted and verified by bioinformatics analysis and dual luciferase assay. After transfection, cell proliferation, invasion, and apoptosis of breast cancer tissues were, respectively, observed by cell counting kit-8 assay, transwell assay, and flow cytometry (FCM). Furthermore, the relationship among miR-99a-5p, CDC25A, and cell-cycle progression was determined by FCM assay. The nude mouse transplantation tumor experiment was performed to verify the influence of miR-99a-5p on breast cancer cell in vivo. The expression of miR-99a-5p in breast cancer tissues and cells was significantly downregulated, whereas CDC25A expression was upregulated. MiR-99a-5p targeted CDC25A and suppressed its expression in breast cancer cells. Overexpression of miR-99a-5p and decreased expression of CDC25A could suppress breast cancer cell proliferation and invasion and facilitate apoptosis. Cell-cycle progression was significantly activated by downregulated miR-99a-5p and upregulated CDC25A. Moreover, miR-99a-5p overexpression repressed the expressions of CDC25A, marker ki67, and Cyclin D1 proteins, whereas it upregulated the expression of p21 protein. MicroRNA-99a-5p suppresses breast cancer progression and cell-cycle pathway through downregulating CDC25A.     

LncRNA–CDC6 promotes breast cancer progression and function as ceRNA to target CDC6 by sponging microRNA-215

Rapid proliferation and metastasis of breast cancers resulted in poor prognosis in clinic. Recent studies have proved that long noncoding RNAs (lncRNAs) were involved in tumor progression. In this study, we aimed to determine the roles and mechanisms of lncRNA–cell division cycle 6 (CDC6) in regulating proliferation and metastasis of breast cancer. Clinically, lncRNA–CDC6 was highly expressed in tumor tissues and was positively correlated with clinical stages of breast cancers. Functionally, the ectopic expression of lncRNA–CDC6 promoted proliferation via regulation of G1 phase checkpoint, and further promoting the migration capability. Moreover, lncRNA–CDC6 could function as competitive endogenous RNA (ceRNA) via directly sponging of microRNA-215 (miR-215), which further regulating the expression of CDC6. Taken together, our results proved that lncRNA–CDC6 could function as ceRNA and promote the proliferation and metastasis of breast cancer cells, which provided a novel prognostic marker for breast cancers in clinic.     

NPAS2 regulates proliferation of acute myeloid leukemia cells via CDC25A-mediated cell cycle progression and apoptosis

Promoted proliferation and associated suppression of apoptosis at various stages of myeloid differentiation are well-known features of acute myeloid leukemia (AML), but understanding of the molecular processes involved remains limited. As a crucial circadian agent, neuronal PAS domain protein 2 (NPAS2) is widely recognized as a promising predictor of clinical outcome in various malignancies. Nevertheless, the understanding of its influence on AML is insufficient. Using KD cells and expression assays, we carried out detailed investigation of the role of NPAS2 in AML in vivo and in vitro. Firstly, we found that NPAS2 expression was elevated in AML cells both in vivo and in vitro. NPAS2 knockdown via lentiviral infection clearly suppressed proliferation of MV4-11 and MOLM-14 cells. Additionally, NPAS2 knockdown caused G1/S cell cycle arrest (CCA), which inhibited CDC25A expression. Moreover, NPAS2 knockdown promoted cell death, as evidenced by increased caspase-3 cleavage, and change in Bcl2/Bax production. Excessive CDC25A expression eliminated G1/S CCA triggered by NPAS2 knockdown and death of NPAS2 knocked down MOLM and MV4-11 cells. The expression of CDC25A was stabilized by NPAS2, which induced cell cycle progression and participated in suppression of cell death by modulating caspase-3 cleavage, and expression of Bcl2/Bax. We therefore indicated NPAS2 to be a crucial modulator of survival as well as proliferation. Our research sheds light on the etiology of the proliferation of promyelocytes modulated via NPAS2 with regard to AML.     

Eps15同源结构域包含蛋白2通过调控Cyclin D1-CDK4-pRb信号轴影响食管鳞癌细胞的增殖

微丝结合蛋白是微丝细胞骨架的重要组成成分,它们通过促进微丝的聚合和解聚来影响微丝的动力学。大量研究已经表明,微丝和微丝结合蛋白参与细胞癌变的所有阶段。我们通过对食管癌蛋白质组数据挖掘结果显示,微丝结合蛋白Eps15同源结构域包含蛋白2(EHD2)在食管癌组织中低表达,且EHD2低表达的食管癌患者预后不良。以往的研究已经证明,EHD2参与调控糖代谢、自噬和肿瘤迁移。然而,EHD2在食管癌进展中的作用和机制仍不清楚。本研究旨在探究EHD2在食管鳞癌细胞中的影响及其作用机制。免疫荧光和细胞组分分离结果显示,EHD2 不仅定位于细胞膜和细胞质,还存在于细胞核中。使用克隆形成实验、EdU细胞增殖实验和细胞流式术检测EHD2对食管鳞癌细胞增殖能力的影响。结果显示,过表达EHD2 和EHD2-3×NLS(核定位信号)抑制食管鳞癌细胞增殖和细胞周期G₁/S转换;同时,双荧光素报告基因结果显示,过表达EHD2 和EHD2-3×NLS抑制Wnt 信号通路活性。而siRNA敲降则获得相反的结果。免疫共沉淀和Duolink-PLA实验证明,EHD2与Wnt信号通路关键分子β-连环蛋白(β-catenin)和T细胞因子3(T-cell factor 3,TCF3)相互作用。蛋白质印迹和荧光定量PCR结果证实,过表达EHD2 和EHD2-3×NLS抑制TCF3下游与增殖和细胞周期相关的靶基因的转录,以及细胞周期蛋白D1(cyclin D1)、细胞周期蛋白激酶4(CDK4)和pRb的蛋白质表达。以上结果表明,核EHD2与β-catenin和TCF3 复合体相互作用,通过Cyclin D1-CDK4-pRb信号轴来调控食管鳞癌细胞的增殖和细胞周期进程。     

CDC20 regulates the cell proliferation and radiosensitivity of P53 mutant HCC cells through the Bcl-2/Bax pathway

Purpose: The incidence of hepatocellular carcinoma (HCC) is extremely high, and China accounts for approximately 50% of global liver cancer cases. Previous studies reported that CDC20 is involved in the occurrence and progression of a variety of malignant tumors. So, whether CDC20 will affect the development of HCC, we have conducted in-depth research on this.Methods: We selected Hep3B and HepG2 for cell culture, and performed siRNA transfection, lentiviral infection, western blot, MTS determination, cell cycle determination, apoptosis test, immunodeficiency test, clone survival test and subcutaneous parthenogenesis in nude mice.Results: Knockdown of CDC20 greatly enhanced the radiation efficacy on the growth retardation in HepG2, and protein level of CDC20 was decreased for the activation of P53 by radiation. Downregulation of CDC20 combined with radiation can inhibit proliferation, aggravate DNA damage, increase G2/M arrest, and promote apoptosis of HCC cells to a greater extent, and the relative survival fraction of HCC cells was gradually reduced with radiation dose increased in P53 mutated Hep3B cells. After knocking down CDC20 in HCC, Bcl-2 was down-regulated and Bax expression increased. Down-regulation of CDC20 can inhibit further invasion by promoting the radiosensitivity of HCC.Conclusion: In this study, we found that that CDC20 was highly expressed in HCC and participated in radio resistance of HCC cells with P53 mutation Bcl-2/Bax via signaling pathway. This study is the first to present evidence that CDC20 may play a role in improving the efficacy of radiotherapy in HCC.     

Regulatory crosstalk between KLF5, miR-29a and Fbw7/CDC4 cooperatively promotes atherosclerotic development

Atherogenesis is a chronic inflammatory process that involves complex interactions between endothelial dysfunction, lipid deposition and vascular smooth-muscle cell (VSMC) proliferation. However, the molecular mechanism is still unclear. We found that a pro-atherosclerotic factor (oxLDL) induced the expression of Krüppel-like factor 5 (KLF5), which in turn increased miR-29a expression levels. The increased miR-29a was retained within HASMCs and down-regulated Fbw7/CDC4 expression by targeting the 3´UTR of Fbw7/CDC4, subsequently increasing KLF5 stability by reducing the Fbw7/CDC4-dependent ubiquitination of KLF5, forming a positive feedback loop to enhance VSMC proliferation and promote atherogenesis. These results indicate a potentially important role for the oxLDL-activated feedback mechanism in VSMC proliferation and atherogenesis. Suppression of miR-29a may be an effective way to attenuate atherosclerosis. In conclusion, our data are the first to reveal that the regulatory crosstalk between KLF5, miR-29a, and Fbw7/CDC4 cooperatively promotes atherosclerotic development.     

过表达miR-155抑制C2C12成肌分化

为明确miR-155在C2C12成肌分化中的作用及分子机制,本研究构建了miR-155过表达腺病毒载体,运用过表达miR-155的腺病毒感染C2C12,并诱导其成肌分化。通过形态学观察,成肌标志基因mRNA和蛋白表达水平的检测,以及双荧光素酶报告基因系统对预测的miR-155靶基因(TCF4)的验证,结果表明,C2C12细胞分化中,过表达miR-155明显降低了肌管的形成,成肌标志基因MyoG和MyHC的mRNA表达量极显著地下降(P0.01),而MyoD差异不显著(P0.05),成肌标志基因蛋白检测结果与mRNA检测结果一致;进一步研究显示miR-155与预测的TCF4基因的3'UTR 3个靶点(1487-1493,1516-1522,4532-4583)中的1个(4532-4538)结合,并发现过表达miR-155显著降低了TCF4的mRNA水平(P0.05)。表明miR-155可能通过靶向TCF4抑制C2C12成肌分化。     

CDC20 promotes bone formation via APC/C dependent ubiquitination and degradation of p65

The E3 ubiquitin ligase complex CDC20‐activated anaphase‐promoting complex/Cyclosome (APC/C^CDC20) plays a critical role in governing mitotic progression by targeting key cell cycle regulators for degradation. Cell division cycle protein 20 homolog (CDC20), the co‐activator of APC/C, is required for full ubiquitin ligase activity. In addition to its well‐known cell cycle‐related functions, we demonstrate that CDC20 plays an essential role in osteogenic commitment of bone marrow mesenchymal stromal/stem cells (BMSCs). Cdc20 conditional knockout mice exhibit decreased bone formation and impaired bone regeneration after injury. Mechanistically, we discovered a functional interaction between the WD40 domain of CDC20 and the DNA‐binding domain of p65. Moreover, CDC20 promotes the ubiquitination and degradation of p65 in an APC11‐dependent manner. More importantly, knockdown of p65 rescues the bone loss in Cdc20 conditional knockout mice. Our current work reveals a cell cycle‐independent function of CDC20, establishes APC11^CDC20 as a pivotal regulator for bone formation by governing the ubiquitination and degradation of p65, and may pave the way for treatment of bone‐related diseases.     

胃癌中CDC4和cyclin E的表达及临床意义

检测胃癌中CDC4/Fbxw7、cyclin E的表达,分析其与胃癌临床病理特征的关系和临床意义.采用逆转录多聚酶链反应(RT-PCR)检测部分胃癌和胃正常组织中CDC4、cyclin E mRNA的表达;免疫组化(SP法)检测60例胃癌组织及对应正常组织中CDC4/FBXW7、cyclin E蛋白的表达,探讨两者的临床病理特征之间的关系.胃癌中CDC4蛋白表达显著低于正常组织(P<0.05),而cyclin E蛋白的表达在胃癌组织中显著增高,阳性率与癌旁正常组织比较,差异有统计学意义(P<0.01).CDC4、cyclin E蛋白和mRNA表达均与胃癌的分化程度、TNM分期、淋巴结转移及浸润深度有关.CDC4、cyclin E在mRNA表达水平上呈负相关.CDC4的表达缺失可能导致cyclin E的过表达.CDC4的低表达可能是胃癌诊疗及预后判断的重要生物学指标.