Mus musculus Raw sequence reads. Mus musculus

Pancreatic cancer patient survival is the lowest of all common cancers. Given that pancreatic cancer therapies do little to improve survival, there is a significant need to identify additional potential therapeutic targets and treatment strategies. The ROCK1 locus on chromosome 18 is amplified in 15% of pancreatic patient tumors (Biankin et al. 2012), accompanied by concordant copy number/gene expression changes (Bailey et al. 2016). The ROCK1 and ROCK2 kinases promote actomyosin contractility through phosphorylation of substrates including the myosin regulatory light chain 2 (MLC2), myosin-binding subunit of the MLC phosphatase (MYPT1) and LIM kinases 1&2 (Rath and Olson 2012, Julian and Olson 2014). In addition to direct effects on the organization and dynamics of the actin cytoskeleton that impact cell morphology, ROCK-mediated cell contractility also affects gene transcription (Sanz-Moreno et al. 2011). How ROCK-mediated actomyosin contractility might contribute to pancreatic cancer by altering gene expression has not been established.In this study, mouse pancreatic ductal adenocarcinoma tumour cells were transduced with retrovirus encoding conditionally-activated estrogen-receptor hormone-binding domain (hbER) fusions with ROCK1 (ROCK1:ER) or ROCK2 (ROCK2:ER) kinase domains, or green fluorescent protein (GFP:ER). GFP:ER expressing cells were treated with ethanol vehicle or 1 micromolar 4-hydroxytamoxifen (4HT) to identify any effects of the estrogen analogue, while ROCK1:ER and ROCK2:ER cells were treated with 1 micromolar 4HT to activate the ER fusion proteins. RNA was isolated, and enriched for poly A+ transcripts prior to sequencing.Bailey, P., et al. (2016). Nature 531: 47-52.Biankin, A. V., et al. (2012). Nature 491: 399-405.Julian, L. and M. F. Olson (2014). Small GTPases 5: e29846.Rath, N. and M. F. Olson (2012). EMBO Rep 13: 900-908.Sanz-Moreno, V., et al. (2011). Cancer Cell 20: 229-245.

胰腺癌患者的生存率为所有常见癌症中最低。鉴于胰腺癌治疗手段对提升生存率收效甚微，学界亟需发掘更多潜在治疗靶点与治疗策略。18号染色体上的ROCK1基因座（ROCK1 locus）在15%的胰腺癌患者肿瘤中发生扩增（Biankin等，2012），并伴随一致的拷贝数/基因表达改变（Bailey等，2016）。ROCK1与ROCK2激酶可通过磷酸化包括肌球蛋白调节轻链2（MLC2）、MLC磷酸酶的肌球蛋白结合亚基（MYPT1）及LIM激酶1和2在内的底物，促进肌动蛋白-肌球蛋白收缩力（Rath与Olson，2012；Julian与Olson，2014）。除直接调控肌动蛋白细胞骨架的组织与动态变化以影响细胞形态外，ROCK介导的细胞收缩力还可参与调控基因转录（Sanz-Moreno等，2011）。目前，ROCK介导的肌动蛋白-肌球蛋白收缩力如何通过改变基因表达促进胰腺癌发生，尚未明确。

本研究中，研究人员将编码条件激活型雌激素受体激素结合结构域（hbER）分别与ROCK1激酶结构域、ROCK2激酶结构域融合的蛋白（ROCK1:ER与ROCK2:ER），以及编码绿色荧光蛋白与hbER融合蛋白（GFP:ER）的逆转录病毒，分别转导至小鼠胰腺导管腺癌细胞中。对于表达GFP:ER的细胞，分别以乙醇溶剂或1微摩尔浓度的4-羟基他莫昔芬（4HT）处理，以明确该雌激素类似物的潜在影响；对于表达ROCK1:ER与ROCK2:ER的细胞，则使用1微摩尔浓度的4HT处理以激活ER融合蛋白。随后提取RNA，并对聚腺苷酸化转录本进行富集后开展测序。

参考文献：
Bailey, P.等（2016）. 《Nature》531: 47-52.
Biankin, A. V.等（2012）. 《Nature》491: 399-405.
Julian, L.与M. F. Olson（2014）. 《Small GTPases》5: e29846.
Rath, N.与M. F. Olson（2012）. 《EMBO Rep》13: 900-908.
Sanz-Moreno, V.等（2011）. 《Cancer Cell》20: 229-245.