BACKGROUND Vasculogenic mimicry (VM), defined as a capability of aggressive tumor Cells to mimic embryonic vasculogenic networks, caused poor prognosis in hepatocellular carcinoma (HCC). Rho kinases (ROCK), p21-activated kinase (PAK), hypoxia or epithelial-mesenchymal transition (EMT) contributed to the VM potential. However, the details underlying these biological behaviors have not been completely elucidated. METHODS Kaplan-Meier analysis was conducted to predict relationship with hypoxia Inducible factor (HIF-1α), EMT related markers: Vimentin and patient prognosis. CD34/periodic acid-Schiff (PAS) double staining was examined to differentiate VM-positive (VM+) and VM-negative (VM-) samples. Cells were cultured under controlled hypoxic environments (1% O2) or normoxic conditions. The effect of hypoxia on RhoA/ROCK, Rac1/PAK and EMT were evaluated by real time-qPCR and western blot. HIF-1α small interfering RNA (siRNA), overexpressed or short hairpin RNA (shRNA) of ROCK and kinase inhibitors were used to explore the effect of HIF-1α, RhoA/ROCK, Rac1/PAK and Vimentin on VM. RESULTS HIF-1α or Vimentin was upregulated in VM+ HCC tissues, compared to non-cancerous tissues (P < 0.01), and patients with high expression of HIF-1α or Vimentin had worse prognosis (P < 0.001). We showed hypoxia induced RhoA/ROCK and Rac1/PAK signaling transduction, and EMT could be repressed by HIF-1α siRNA. Notably, RhoA/ROCK or Rac1/PAK stabilized HIF-1α in hypoxia, whereas HIF-1α did not significantly altered RhoA/ROCK or Rac1/PAK signaling in hypoxia. Moreover, we found distinct roles of ROCK1, ROCK2 and PAK in regulating Vimentin phosphorylation. CONCLUSIONS RhoA/ROCK and Rac/PAK signaling played crucial roles in hypoxia-induced VM via Ser72 and Ser56 Vimentin phosphorylation in HCC.

中文翻译：

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低氧诱导肝细胞癌血管生成拟态：HIF-1α，RhoA / ROCK和Rac1 / PAK信号传导的作用。

背景技术血管生成模拟（VM）定义为侵袭性肿瘤细胞模仿胚胎血管生成网络的能力，导致肝细胞癌（HCC）的不良预后。Rho激酶（ROCK），p21活化激酶（PAK），缺氧或上皮-间质转化（EMT）有助于VM的发展。但是，尚未完全阐明这些生物学行为的基础。方法采用Kaplan-Meier分析来预测与缺氧诱导因子（HIF-1α），EMT相关指标：波形蛋白和患者预后的关系。检查CD34 /高碘酸席夫（PAS）双重染色以区分VM阳性（VM +）和VM阴性（VM-）样品。在受控的低氧环境（1％O2）或常氧条件下培养细胞。缺氧对RhoA / ROCK的影响 Rac1 / PAK和EMT通过实时定量PCR和Western blot进行评估。使用HIF-1α小干扰RNA（siRNA），ROCK的过表达或短发夹RNA（shRNA）和激酶抑制剂来研究HIF-1α，RhoA / ROCK，Rac1 / PAK和波形蛋白对VM的作用。结果与非癌组织相比，VM + HCC组织中的HIF-1α或波形蛋白升高（P <0.01），HIF-1α或波形蛋白表达高的患者预后较差（P <0.001）。我们发现缺氧诱导的RhoA / ROCK和Rac1 / PAK信号转导，并且HIF-1αsiRNA可以抑制EMT。值得注意的是，在低氧条件下，RhoA / ROCK或Rac1 / PAK稳定了HIF-1α，而在低氧条件下，HIF-1α并未显着改变RhoA / ROCK或Rac1 / PAK信号。此外，我们发现了ROCK1，ROCK2和PAK在调节波形蛋白磷酸化中的独特作用。