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Abstract：
To elucidate the currently unknown molecular mechanisms responsible for the similarity and difference during the acquirement of resistance against gemcitabine (GEM) and paclitaxel (PTX) in patients with pancreatic carcinoma, we examined two-dimensional (2D) and three-dimensional (3D) cultures of parent MIA PaCa-2 cells (MIA PaCa-2-PA) and their GEM resistance cell line (MIA PaCa-2-GR) and PTX resistance (MIA PaCa-2-PR). Using these cells, we examined 3D spheroid configurations and cellular metabolism, including mitochondrial and glycolytic functions, with a Seahorse bio-analyzer and RNA sequencing analysis. Compared to the MIA PaCa-2-PA, (1) the formation of the 3D spheroids of MIA PaCa-2-GR or -PR was much slower, and (2) their mitochondrial and glycolytic functions were greatly modulated in MIA PaCa-2-GR or -PR, and such metabolic changes were also different between their 2D and 3D culture conditions. RNA sequencing and bioinformatic analyses of the differentially expressed genes (DEGs) using an ingenuity pathway analysis (IPA) suggested that various modulatory factors related to epithelial -mesenchymal transition (EMT) including STAT3, GLI1, ZNF367, NKX3-2, ZIC2, IFIT2, HEY1 and FBLX, may be the possible upstream regulators and/or causal network master regulators responsible for the acquirement of drug resistance in MIA PaCa-2-GR and -PR. In addition, among the prominently altered DEGs (Log2 fold changes more than 6 or less than -6), FABP5, IQSEC3, and GASK1B were identified as unique genes associated with their antisense RNA or pseudogenes, and among these, FABP5 and GASK1B are known to function as modulators of cancerous EMT. Therefore, the observations reported herein suggest that modulations of cancerous EMT may be key molecular mechanisms that are responsible for inducing chemoresistance against GEM or PTX in MIA PaCa-2 cells.
摘要：
为了阐明目前未知的分子机制，导致胰腺癌患者在获得吉西他滨（GEM）和紫杉醇（PTX）耐药性过程中的相似性和差异，我们检查了亲本MIAPaCa-2细胞（MIAPaCa-2-PA）及其GEM抗性细胞系（MIAPaCa-2-GR）和PTX抗性（MIAPaCa-2-PR）的二维（2D）和三维（3D）培养物。使用这些细胞，我们检查了3D球体结构和细胞代谢，包括线粒体和糖酵解功能，海马生物分析仪和RNA测序分析。与MIAPaCa-2-PA相比，（1）MIAPaCa-2-GR或-PR的3D球体的形成要慢得多，（2）它们的线粒体和糖酵解功能在MIAPaCa-2-GR或-PR中受到极大的调节，并且这种代谢变化在它们的2D和3D培养条件之间也是不同的。RNA测序和差异表达基因（DEGs）的生物信息学分析，使用基因通路分析（IPA）表明，与上皮间质转化（EMT）相关的各种调节因子，包括STAT3，GLI1，ZNF367，NKX3-2，ZIC2，IFIT2，HEY1和FBLX，可能是负责获得MIAPaCa-2-GR和-PR耐药性的可能的上游调节因子和/或因果网络主调节因子。此外,在显著改变的DEG中(Log2倍改变大于6或小于-6)，FABP5，IQSEC3和GASK1B被鉴定为与其反义RNA或假基因相关的独特基因，其中，已知FABP5和GASK1B充当癌性EMT的调节剂。因此,本文报道的观察结果表明，癌性EMT的调节可能是在MIAPaCa-2细胞中诱导针对GEM或PTX的化学抗性的关键分子机制。
