Netherlands Cancer Institute, Division of Tumor Biology and Immunology, Amsterdam, The Netherlands.
VU University, Center for Neurogenomics and Cognitive Research, Amsterdam, The Netherlands.
PLoS One. 2018 May 22;13(5):e0196979. doi: 10.1371/journal.pone.0196979. eCollection 2018.
To identify coding and non-coding suppressor genes of anchorage-independent proliferation by efficient loss-of-function screening, we have developed a method for enzymatic production of low complexity shRNA libraries from subtracted transcriptomes. We produced and screened two LEGO (Low-complexity by Enrichment for Genes shut Off) shRNA libraries that were enriched for shRNA vectors targeting coding and non-coding polyadenylated transcripts that were reduced in transformed Mouse Embryonic Fibroblasts (MEFs). The LEGO shRNA libraries included ~25 shRNA vectors per transcript which limited off-target artifacts. Our method identified 79 coding and non-coding suppressor transcripts. We found that taurine-responsive GABAA receptor subunits, including GABRA5 and GABRB3, were induced during the arrest of non-transformed anchor-deprived MEFs and prevented anchorless proliferation. We show that taurine activates chloride currents through GABAA receptors on MEFs, causing seclusion of cell volume in large membrane protrusions. Volume seclusion from cells by taurine correlated with reduced proliferation and, conversely, suppression of this pathway allowed anchorage-independent proliferation. In human cholangiocarcinomas, we found that several proteins involved in taurine signaling via GABAA receptors were repressed. Low GABRA5 expression typified hyperproliferative tumors, and loss of taurine signaling correlated with reduced patient survival, suggesting this tumor suppressive mechanism operates in vivo.
为了通过有效的功能丧失筛选来鉴定锚定非依赖性增殖的编码和非编码抑制基因，我们开发了一种从消减转录组中酶促产生低复杂度 shRNA 文库的方法。我们生成并筛选了两个 LEGO（通过基因关闭富集的低复杂度）shRNA 文库，这些文库富集了针对编码和非编码多聚腺苷酸化转录物的 shRNA 载体，这些转录物在转化的小鼠胚胎成纤维细胞（MEFs）中减少。LEGO shRNA 文库每个转录物包含约 25 个 shRNA 载体，这限制了脱靶伪影。我们的方法鉴定了 79 个编码和非编码抑制转录物。我们发现，牛磺酸反应性 GABAA 受体亚基，包括 GABRA5 和 GABRB3，在非转化锚定剥夺的 MEFs 停滞期间被诱导，并阻止无锚增殖。我们表明，牛磺酸通过 MEFs 上的 GABAA 受体激活氯离子电流，导致细胞体积在大膜突起中隔离。牛磺酸引起的细胞体积隔离与增殖减少相关，相反，抑制该途径允许锚定非依赖性增殖。在人类胆管癌中，我们发现几种涉及 GABAA 受体的牛磺酸信号通路的蛋白质受到抑制。高增殖肿瘤的 GABRA5 表达水平较低，牛磺酸信号的丧失与患者生存时间的缩短相关，这表明这种肿瘤抑制机制在体内起作用。
