Next Generation Sequencing Facilitates Quantitative Analysis of HCC cells transfected with NCsiRNA or CDCA8siRNA

We hypothesized if targeting of CDCA8 with small interfering (si) RNA could inhibit HCC progression. We also investigated molecular mechanism to mediate HCC cell death caused by CDCA8 silencing. To accomplish this, Huh1 and Huh7 human HCC cells were transfected with CDCA8 siRNA and tested for growth inhibition and apoptotic induction using MTS, FACS and microscopic analysis. To obtain insights into the molecular changes in response to CDCA8 knockdown, global changes in gene expression were examined using RNA sequencing. As results, siRNA silencing of CDCA8 inhibited HCC cell growth by blocking cell-cycle progression and inducing apoptosis. RNA sequencing showed that, representatively, the anti-proliferative effects were driven by a subset of molecular alterations including the upregulation of tumor suppressive ATF3 and GADD34 genes, whereas a key regulator of cell growth and invasiveness BGLAP was repressed. Subsequent Western blotting also revealed that CDCA8 silencing decreases the levels of pro-caspase 3 and PARP-1, accelerating apoptotic signaling in HCC cells. In addition, targeting CDCA8 effectively suppressed HCC tumor growth growth in a murine xenograft model. Taken together, these findings suggest that CDCA8 could be a promising molecular target for systemic therapy of HCC. Examination of 2 different siRNA transfected cells in 2 cell lines.

本研究假设，使用小干扰RNA（small interfering RNA, siRNA）靶向CDCA8可抑制肝细胞癌（hepatocellular carcinoma, HCC）的进展，同时探究CDCA8沉默诱导肝癌细胞死亡的分子调控机制。为此，本研究将CDCA8 siRNA转染至Huh1和Huh7两株人肝癌细胞中，并通过MTS法、流式细胞术（fluorescence-activated cell sorting, FACS）以及显微镜观察，检测细胞生长抑制与凋亡诱导情况。为解析CDCA8敲低后的分子表达变化，本研究通过RNA测序分析了全基因表达谱的整体改变。实验结果显示，CDCA8的siRNA沉默通过阻滞细胞周期进程并诱导凋亡，抑制了肝癌细胞的生长。RNA测序结果表明，其抗增殖效应主要由一系列分子改变所介导：例如肿瘤抑制基因ATF3与GADD34的表达上调，而调控细胞生长与侵袭性的关键因子BGLAP的表达则被抑制。后续的蛋白质印迹（Western blotting）实验进一步证实，CDCA8沉默可降低procaspase-3与PARP-1的表达水平，加速肝癌细胞的凋亡信号通路激活。此外，在小鼠异种移植模型中，靶向CDCA8可有效抑制肝癌的肿瘤生长。综上，本研究结果表明，CDCA8有望成为肝细胞癌系统性治疗的潜在分子靶点。本研究对2株细胞系中的2种不同siRNA转染细胞样本进行了检测。