文冠果XsSCL6基因的克隆及表达分析

GRAS家族HAM亚家族（Scarecrow-like 6，SCL6）是一类植物特有的转录因子，在植物生长发育中具有关键作用。文冠果（Xanthoceras sorbifolium Bunge）是我国特有的重要林木能源植物，雄花的雌蕊败育是造成产量低的主要原因。为解析XsSCL6基因在文冠果（Xanthoceras sorbifolium Bunge）雌蕊发育中的调控作用，采用RT-PCR方法克隆文冠果4个XsSCL6同源基因，并对关键XsSCL6a蛋白进行亚细胞定位；对XsSCL6基因进行生物信息学分析，利用qRT-PCR分析XsSCL6基因在文冠果雌蕊败育过程中的表达模式。结果表明：（1）获得文冠果XsSCL6a、XsSCL6b、XsSCL6c和XsSCL6d基因ORF全长，分别编码751、669、717和717个氨基酸，均属于亲水不稳定蛋白。（2）文冠果XsSCL6蛋白主要由无规则卷曲和α-螺旋构成，定位于细胞核。（3）XsSCL6a、XsSCL6c和XsSCL6d均包含GRAS保守结构域， 文冠果XsSCL6蛋白和克莱门柚及甜橙的亲缘关系最近。（4）文冠果XsSCL6基因启动子中包含多种激素响应元件、植物发育相关元件。（5）qRT-PCR结果显示XsSCL6基因各个时期在雄花中的表达量显著高于雌花，表达量的趋势和转录组FPKM值一致。（6）xso-miR171和XsSCL6基因均存在裂解方式的靶向关系，TPM值和FPKM值为负相关关系。XsSCL6基因可能受到xso-miR171调控，共同参与文冠果雌蕊发育过程，对文冠果花发育具有重要作用。本研究为今后研究进一步文冠果中XsSCL6基因的功能和雌蕊败育机制提供了参考，为采用分子育种方法提高种子产量提供了理论依据。     

Irreversible repolarization of tumour-associated macrophages by low-Pi stress inhibits the progression of hepatocellular carcinoma

Numerous studies have shown the positive correlation between high levels of Pi and tumour progression. A critical goal of macrophage-based cancer therapeutics is to reduce anti-inflammatory macrophages (M2) and increase proinflammatory antitumour macrophages (M1). This study aimed to investigate the relationship between macrophage polarization and low-Pi stress. First, the spatial populations of M2 and M1 macrophages in 22 HCC patient specimens were quantified and correlated with the local Pi concentration. The levels of M2 and M1 macrophage markers expressed in the peritumour area were higher than the intratumour levels, and the expression of M2 markers was positively correlated with Pi concentration. Next, monocytes differentiated from THP-1 cells were polarized against different Pi concentrations to investigate the activation or silencing of the expression of p65, IκB-α and STAT3 as well as their phosphorylation. Results showed that low-Pi stress irreversibly repolarizes tumour-associated macrophages (TAMs) towards the M1 phenotype by silencing stat6 and activating p65. Moreover, HepG-2 and SMCC-7721 cells were cultured in conditioned medium to investigate the innate anticancer immune effects on tumour progression. Both cancer cell lines showed reduced proliferation, migration and invasion, as epithelial–mesenchymal transition (EMT) was inactivated. In vivo therapeutic effect on the innate and adaptive immune processes was validated in a subcutaneous liver cancer model by the intratumoural injection of sevelamer. Tumour growth was significantly inhibited by the partial deprivation of intratumoural Pi as the tumour microenvironment under low-Pi stress is more immunostimulatory. The anticancer immune response, activated by low-Pi stress, suggests a new macrophage-based immunotherapeutic modality.