延边黄牛背最长肌差异表达基因的筛选、克隆及序列分析

应用引物复性控制技术筛选肌内脂肪含量差异极显著的延边黄牛背最长肌组织差异表达基因,寻找与肌内脂肪沉积的相关候选基因。文章选取30头28月龄延边黄牛阉牛的背最长肌组织测定肌内脂肪含量,选取肌内脂肪含量差异极显著的最高和最低各3头组成RNA池,采用引物复性控制技术,分析了两组个体背最长肌组织差异表达基因。利用20对随机引物差异显示扩增下,共获得12条ESTs(片段大小为200～890 bp),其中8个为已知的ESTs分别与细胞骨架形成、细胞因子信号转导、蛋白质合成、能量代谢和其他功能的差异基因,4个未知的ESTs。结果表明,应用引物复性控制技术筛选得到了12个可能参与了肌内脂肪沉积调控的ESTs,为进一步筛选肌内脂肪沉积相关的基因奠定了基础。     

家兔前体脂肪细胞分化不同时期基因表达谱分析

脂肪的过度积累严重危害人类健康。前体脂肪细胞分化是脂肪发育的关键过程,研究前体脂肪细胞分化相关基因的表达有助于认识脂肪沉积的机理。尽管家兔是一种理想的研究脂肪发育的动物模型,但是针对其前体脂肪细胞分化不同时期基因表达谱的研究鲜见报道。本研究通过诱导家兔前体脂肪细胞分化,在分化第0 d、3 d和9 d收集脂肪细胞,利用转录组测序(RNA-seq),在分化第3 d样本与第0 d样本的比较中筛选出1352个差异表达基因(differentially expressed genes, DEGs),在分化第9 d样本与第3 d样本的比较中筛选出888个DEGs。GO (gene ontology)功能富集和KEGG (kyoto encyclopedia of genes and genomes)通路分析发现,0~3 d分化期上调的DEGs显著富集在PPAR信号通路和PI3K-Akt信号通路上,3~9d分化期上调的DEGs显著富集到与细胞周期调控有关的GO条目和KEGG信号通路,0~3d和3~9d阶段特异上调的DEGs可能分别作用于细胞质和细胞核。通过DEGs的蛋白-蛋白互作(protein-protein interaction, PPI)网络分析发现,筛选出的核心节点(hub node)基因可能通过调控细胞周期而影响家兔前体脂肪细胞分化。     

Verification of suitable and reliable reference genes for quantitative real-time PCR during adipogenic differentiation in porcine intramuscular stromal-vascular cells

Intramuscular fat (IMF) is an important trait influencing meat quality, and intramuscular stromal-vascular cell (MSVC) differentiation is a key factor affecting IMF deposition. Quantitative real-time PCR (qPCR) is often used to screen the differentially expressed genes during differentiation of MSVCs, where proper reference genes are essential. In this study, we assessed 31 of previously reported reference genes for their expression suitability in porcine MSVCs derived form longissimus dorsi with qPCR. The expression stability of these genes was evaluated using NormFinder, geNorm and BestKeeper algorithms. NormFinder and geNorm uncovered ACTB, ALDOA and RPS18 as the most three stable genes. BestKeeper identified RPL13A, SSU72 and DAK as the most three stable genes. GAPDH was found to be the least stable gene by all of the three software packages, indicating it is not an appropriate reference gene in qPCR assay. These results might be helpful for further studies in pigs that explore the molecular mechanism underlying IMF deposition.     

Cloning and Expression of SFRP5 in Tibetan Chicken and its Relationship with IMF Deposition

Secreted frizzled related protein 5 (SFRP5), an anti-inflammatory adipokine, is relevant to the adipocyte differentiation. In order to clarify its role in regulating intramuscular fat (IMF) deposition in Tibetan chicken, the full-length sequence of the Tibetan chicken SFRP5 gene was cloned. The relative expression of SFRP5 gene was detected using quantitative RT-PCR in various tissues of 154 days old Tibetan chicken, as well as in breast muscle, thigh muscle, and adipose tissue at different growth stages. The results showed that SFRP5 gene was expressed in all examined tissues but highly enriched in adipose tissue. Temporal expression profile showed that the expression of SFRP5 was gradually decreased in breast muscle, but was fluctuated in thigh muscle and adipose tissue with the growth of Tibetan chicken. Furthermore, correlation analysis demonstrated that the expression of SFRP5 in breast muscle, thigh muscle and adipose tissue was correlated with IMF content at different levels. The results indicated that Tibetan chicken SFRP5 is involved in IMF deposition.     

Transcriptional alteration of p53 related processes as a key factor for skeletal muscle characteristics in <Emphasis Type="Italic">Sus scrofa</Emphasis>

The pig could be a useful model to characterize molecular aspects determining several delicate phenotypes because they have been bred for those characteristics. The Korean native pig (KNP) is a regional breed in Korea that was characterized by relatively high intramuscular fat content and reddish meat color compared to other western breeds such as Yorkshire (YS). YS grew faster and contained more lean muscle than KNP. We compared the KNP to Yorksire to find molecular clues determining muscle characteristics. The comparison of skeletal gene expression profiles between these two breeds showed molecular differences in muscle. We found 82 differentially expressed genes (DEGs) defined by fold change (more than 1.5 fold difference) and statistical significance (within 5% of false discovery rate). Functional analyses of these DEGs indicated up-regulation of most genes involved in cell cycle arrest, down-regulation of most genes involved in cellular differentiation and its inhibition, down-regulation of most genes encoding component of muscular-structural system, and up-regulation of most genes involved in diverse metabolism in KNP. Especially, DEGs in above-mentioned categories included a large number of genes encoding proteins directly or indirectly involved in p53 pathway. Our results indicated a possible role of p53 to determine muscle characteristics between these two breeds.     

Knockdown of CTRP6 reduces the deposition of intramuscular and subcutaneous fat in pigs via different signaling pathways

The regulation of porcine subcutaneous (SC) and intramuscular (IM) fat deposition significantly affects pork quality and the lean meat percentage of the carcass, respectively. The adipokine C1q/tumor necrosis factor-related protein 6 (CTRP6), plays a significant role in regulating animal fat deposition. The purpose of this study was to understand the effects of CTRP6 gene knockdown in IM and SC adipocytes by RNA-seq analysis. A total of 1830 and 2936 differentially expressed genes (DEGs) were identified in SC and IM adipocytes, respectively. 844 were down- and 2092 were upregulated in SC adipocytes, while 648 were down- and 1182 were upregulated in IM adipocytes. Furthermore, 1778 DEGs were detected only in SC adipocytes, 672 DEGs only in IM adipocytes, and 1158 DEGs in both types of adipocytes. GO analysis indicated that DEGs involved in adipocyte differentiation were significantly enriched in both SC and IM adipocytes following treatment with CTRP6-siRNA. Moreover, KEGG pathway enrichment analysis revealed differences of metabolic regulation between IM and SC adipocytes. With CTRP6-silencing, the signaling pathways related to Ras and arachidonic acid metabolism were significantly enriched in IM adipocytes, while four other signaling pathways, encompassing the TNF, MAPK, p53 and adipokine pathway were specifically enriched in SC adipocytes. Interestingly, the effect of CTRP6-siRNA treatment was attenuated by the specific Ras activator ML-097 in IM adipocytes, while the specific p53 activator SJ-172550 had the corresponding effect in SC adipocytes. Altogether, we suggest that CTRP6 may be a differential regulator of the development and metabolism of IM and SC adipose tissues.     

Bioinformatics analysis of the biological changes involved in the osteogenic differentiation of human mesenchymal stem cells

The mechanisms underlying the osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs) remain unclear. In the present study, we aimed to identify the key biological processes during osteogenic differentiation. To this end, we downloaded three microarray data sets from the Gene Expression Omnibus (GEO) database: GSE12266, GSE18043 and GSE37558. Differentially expressed genes (DEGs) were screened using the limma package, and enrichment analysis was performed. Protein‐protein interaction network (PPI) analysis and visualization analysis were performed with STRING and Cytoscape. A total of 240 DEGs were identified, including 147 up‐regulated genes and 93 down‐regulated genes. Functional enrichment and pathways of the present DEGs include extracellular matrix organization, ossification, cell division, spindle and microtubule. Functional enrichment analysis of 10 hub genes showed that these genes are mainly enriched in microtubule‐related biological changes, that is sister chromatid segregation, microtubule cytoskeleton organization involved in mitosis, and spindle microtubule. Moreover, immunofluorescence and Western blotting revealed dramatic quantitative and morphological changes in the microtubules during the osteogenic differentiation of human adipose‐derived stem cells. In summary, the present results provide novel insights into the microtubule‐ and cytoskeleton‐related biological process changes, identifying candidates for the further study of osteogenic differentiation of the mesenchymal stem cells.     

Identification of Gene Expression Changes from Colitis to CRC in the Mouse CAC Model

A connection between colorectal carcinogenesis and inflammation is well known, but the underlying molecular mechanisms have not been elucidated. Chemically induced colitis-associated cancer (CAC) is an outstanding mouse model for studying the link between inflammation and cancer. Additionally, the CAC model is used for examining novel diagnostic, prognostic, and predictive markers for use in clinical practice. Here, a CAC model was established in less than 100 days using azoxymethane (AOM) with dextran sulfate sodium salt (DSS) in BALB/c mice. We examined the mRNA expression profiles of three groups: control untreated mice (K), DSS-induced chronic colitis mice (D), and AOM/DSS-induced CAC (AD) mice. We identified 6301 differentially expressed genes (DEGs) among the three groups, including 93 persistently upregulated genes and 139 persistently downregulated genes. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses revealed that the most persistent DEGs were significantly enriched in metabolic or inflammatory components in the tumor microenvironment. Furthermore, several associated DEGs were identified as potential DEGs by protein-protein interaction (PPI) network analysis. We selected 14 key genes from the DEGs and potential DEGs for further quantitative real-time PCR (qPCR) verification. Six persistently upregulated, 3 persistently downregulated DEGs, and the other 3 genes showed results consistent with the microarray data. We demonstrated the regulation of 12 key genes specifically involved in Wnt signaling, cytokine and cytokine receptor interactions, homeostasis, and tumor-associated metabolism during colitis-associated CRC. Our results suggest that a close relationship between metabolic and inflammatory mediators of the tumor microenvironment is present in CAC.