Analysis of microRNAs in Exosomes of Breast Cancer Patients in Search of Molecular Prognostic Factors in Brain Metastases

Brain metastases are the most severe tumorous spread during breast cancer disease. They are associated with a limited quality of life and a very poor overall survival. A subtype of extracellular vesicles, exosomes, are sequestered by all kinds of cells, including tumor cells, and play a role in cell-cell communication. Exosomes contain, among others, microRNAs (miRs). Exosomes can be taken up by other cells in the body, and their active molecules can affect the cellular process in target cells. Tumor-secreted exosomes can affect the integrity of the blood-brain barrier (BBB) and have an impact on brain metastases forming. Serum samples from healthy donors, breast cancer patients with primary tumors, or with brain, bone, or visceral metastases were used to isolate exosomes and exosomal miRs. Exosomes expressed exosomal markers CD63 and CD9, and their amount did not vary significantly between groups, as shown by Western blot and ELISA. The selected 48 miRs were detected using real-time PCR. Area under the receiver-operating characteristic curve (AUC) was used to evaluate the diagnostic accuracy. We identified two miRs with the potential to serve as prognostic markers for brain metastases. Hsa-miR-576-3p was significantly upregulated, and hsa-miR-130a-3p was significantly downregulated in exosomes from breast cancer patients with cerebral metastases with AUC: 0.705 and 0.699, respectively. Furthermore, correlation of miR levels with tumor markers revealed that hsa-miR-340-5p levels were significantly correlated with the percentage of Ki67-positive tumor cells, while hsa-miR-342-3p levels were inversely correlated with tumor staging. Analysis of the expression levels of miRs in serum exosomes from breast cancer patients has the potential to identify new, non-invasive, blood-borne prognostic molecular markers to predict the potential for brain metastasis in breast cancer. Additional functional analyzes and careful validation of the identified markers are required before their potential future diagnostic use.     

Identification and In Silico Characterization of a Novel COLGALT2 Gene Variant in a Child with Mucosal Rectal Prolapse

Rectal prolapse is influenced by many factors including connective tissue dysfunction. Currently, there is no data about genetic contribution in the etiology of this disorder. In this study, we performed trio whole-exome sequencing in an 11-year-old girl with mucosal rectal prolapse and her parents and sibling. Genetic testing revealed a novel heterozygous missense variant c.1406G>T; p.G469V in exon 11 of the COLGALT2 gene encoding the GLT25 D2 enzyme. Sanger sequencing confirmed this variant only in the patient while the mother, father and sister showed a wild-type sequence. The pathogenicity of the novel variant was predicted using 10 different in silico tools that classified it as pathogenic. Further, in silico prediction, according to Phyre2, Project HOPE, I-Mutant3.0 and MutPred2 showed that the missense variant can decrease protein stability and cause alterations in the physical properties of amino acids resulting in structural and functional changes of the GLT25D2 protein. In conclusion, the present study identifies a previously unknown missense mutation in the COLGALT2 gene that encodes the enzyme involved in collagen glycosylation. The clinical features observed in the patient and the results of in silico analysis suggest that the new genetic variant can be pathogenic.     

Microfluidic-Based Cationic Cholesterol Lipid siRNA Delivery Nanosystem: Highly Efficient In Vitro Gene Silencing and the Intracellular Behavior

Safe and efficient delivery of small interfering RNA (siRNA) is essential to gene therapy towards intervention of genetic diseases. Herein, we developed a novel cationic cholesterol lipid derivative (CEL) in which cholesterol hydrophobic skeleton was connected to L-lysine cationic headgroup via a hexanediol linker as the non-viral siRNA delivery carrier. Well-organized CEL/siRNA nanocomplexes (100–200 nm) were prepared by microfluidic-assisted assembly of CEL and siRNA at various N/P ratios. The CEL and CEL/siRNA nanocomplexes have lower cytotoxicity compared with bPEI25k. Delightfully, we disclosed that, in Hela–Luc and H1299–Luc cell lines, the micro-fluidic-based CEL/siRNA nanocomplexes exhibited high siRNA transfection efficiency under both serum-free condition (74–98%) and low-serum circumstances (80–87%), higher than that of lipofectamine 2000. These nanocomplexes also showed high cellular uptake through the caveolae/lipid-raft mediated endocytosis pathway, which may greatly contribute to transfection efficiency. Moreover, the time-dependent (0–12 h) dynamic intracellular imaging demonstrated the efficient delivery to cytoplasm after lysosomal co-localization. The results indicated that the microfluidic-based CEL/siRNA nanosystems possessed good stability, low cytotoxicity, high siRNA delivery efficiency, rapid cellular uptake and caveolae/lipid raft-dependent internalization. Additionally, this study provides a simple approach for preparing and applying a “helper lipid-free” cationic lipid siRNA delivery system as potential nanotherapeutics towards gene silencing treatment of (tumor) diseases.     

摄食低钠盐对三高病人局部基因网络模式的共同影响

应用局部基因网络模式(Local gene network pattern)研究摄食低钠盐在高血压、高脂血及高血糖三种疾病人群血液基因表达谱引起的若干共同差异.测试人群包含19个样本(8正常,11三高疾病),经过检测食用低钠盐前后的基因表达芯片,在监测的150个高血压候选基因中,有17个有明显表达,其中的14个发现可以通过明确的信号通路相互连通成包含约60个基因的局部基因网络.该局部基因网络相对于正常组在三高疾病组中的共同变化表现在4个基因上.结果表明,局部基因网络可在分子水平上研究营养因子对不同人群的营养效应.上述三高病人响应低钠盐的共同局部基因网络可以从方法学上作为进一步研发功能食品的基础,也为低钠盐作为针对三高人群的非药物保健因子在基因表达上提供了进一步研究所需的素材.     

Genome-Wide Identification,Expansion, and Evolution Analysis of Homeobox Gene Family Reveals TALE Genes Important for Secondary Cell Wall Biosynthesis in Moso Bamboo (Phyllostachys edulis)

The TALE gene family is a subfamily of the homeobox gene family and has been implicated in regulating plant secondary growth. However, reports about the evolutionary history and function of the TALE gene family in bamboo are limited. Here, the homeobox gene families of moso bamboo Olyra latifolia and Bonia amplexicaulis were identified and compared. Many duplication events and obvious expansions were found in the TALE family of woody bamboo. PhTALEs were found to have high syntenies with TALE genes in rice. Through gene co-expression analysis and quantitative real-time PCR analysis, the candidate PhTALEs were thought to be involved in regulating secondary cell wall development of moso bamboo during the fast-growing stage. Among these candidate PhTALEs, orthologs of OsKNAT7, OSH15, and SH5 in moso bamboo may regulate xylan synthesis by regulating the expression of IRX-like genes. These results suggested that PhTALEs may participate in the secondary cell wall deposition in internodes during the fast-growing stage of moso bamboo. The expansion of the TALE gene family may be implicated in the increased lignification of woody bamboo when divergent from herbaceous bamboos.     

Validation of Tuba1a as Appropriate Internal Control for Normalization of Gene Expression Analysis during Mouse Lung Development

The expression ratio between the analysed gene and an internal control gene is the most widely used normalization method for quantitative RT-PCR (qRT-PCR) expression analysis. The ideal reference gene for a specific experiment is the one whose expression is not affected by the different experimental conditions tested. In this study, we validate the applicability of five commonly used reference genes during different stages of mouse lung development. The stability of expression of five different reference genes (Tuba1a, Actb Gapdh, Rn18S and Hist4h4) was calculated within five experimental groups using the statistical algorithm of geNorm software. Overall, Tuba1a showed the least variability in expression among the different stages of lung development, while Hist4h4 and Rn18S showed the maximum variability in their expression. Expression analysis of two lung specific markers, surfactant protein C (SftpC) and Clara cell-specific 10 kDA protein (Scgb1a1), normalized to each of the five reference genes tested here, confirmed our results and showed that incorrect reference gene choice can lead to artefacts. Moreover, a combination of two internal controls for normalization of expression analysis during lung development will increase the accuracy and reliability of results.     

microRNA沉默卡氏肺孢子菌主要表面糖蛋白基因对大鼠卡氏肺孢子菌肺炎的治疗作用

目的探讨靶向卡氏肺孢子菌(Pneumocystis carinii,PC)主要表面糖蛋白基因(Major surface glycoprotein gene,MSG)上游保守序列(Upstream conserved sequence,UCS)的microRNA重组质粒对大鼠卡氏肺孢子菌肺炎(Pneumocystis cariniipneumonia,PCP)的治疗作用。方法将SD大鼠经腹股沟皮下注射地塞米松磷酸钠7周,制备大鼠PCP感染模型。将模型大鼠随机分为5组:microRNA重组质粒治疗组(M组)、磺胺药物治疗组(H组)、生理盐水对照组(C1组)、空载体质粒对照组(C2组)和模型对照组(C3组),其中M组和C1、C2组经尾静脉分别注射含microRNA重组质粒pPC-UCS、生理盐水和空载体,H组用磺胺药物灌胃治疗,C3组不做处理,每日1次,疗程为7 d。1周后吉姆萨染色,油镜下计数大鼠肺组织液印片中PC包囊数;HE染色,光镜观察肺组织病理改变;ELISA法检测大鼠血清中IL-10和IFNγ的表达水平;RT-PCR法检测肺组织中PC MSG-UCSmRNA的表达水平;电镜观察PC的超微结构。结果与C1、C2和C3组相比,M组和H组大鼠肺组织PC包囊数均明显减少(P<0.05),肺组织炎症较轻,大鼠血清IL-10的表达水平均显著降低(P<0.05),而IFNγ的表达水平显著升高(P<0.05),大鼠肺组织PC MSG-UCS mRNA的表达水平均显著降低(P<0.05);电镜观察显示,M组和H组PC包囊表面均出现破损,C1、C2、C3组未发现破损。结论 microRNA重组质粒pPC-UCS对大鼠PCP有明显的治疗作用,为治疗PCP提供了新的思路。     

进化驱动型产品创新设计方法研究

分析了市场竞争中产品"适者生存"的规律,提出新的产品创新设计方式--进化驱动模式.该模式强调:为适应市场竞争,在未发现潜在市场需求之前,产品应先行进化.基于该模式,构建了进化驱动型产品创新设计方法.该方法采用"结构→功能"的设计过程,以组件和变量作为结构遗传信息,构建产品基因及产品基因组.采用5种进化策略对产品基因组树进行进化操作;体系化并导向性地辅助设计人员进行产品创新设计.最后对该方法进行了实例演示.