Our previous work shows that increased matrix stiffness not only alters malignant characteristics of hepatocellular carcinoma (HCC) cells, but also attenuates metformin efficacy in treating HCC cells. Here, we identified differential membrane proteins related to matrix stiffness-mediated metformin resistance for better understand therapeutic resistance of metformin in HCC. Differential membrane proteins in HCC cells grown on different stiffness substrates before and after metformin intervention were screened and identified using isobaric tags for relative and absolute quantification (iTRAQ) labeling coupled with the liquid chromatography-tandem mass spectrometry (LC–MS/MS), then bioinformatic analysis were applied to determine candidate membrane protein and their possible signaling pathway. A total of 5159 proteins were identified and 354 differential membrane proteins and membrane associated proteins, which might be associated with matrix stiffness-mediated metformin resistance were discovered. Then 94 candidate membrane proteins including 21 up-regulated protein molecules and 73 down-regulated protein molecules were further obtained. Some of them such as Annexin A2 (ANXA2), Filamin-A (FLNA), Moesin (MSN), Myosin-9 (MYH9), Elongation factor 2 (eEF2), and Tax1 binding Protein 3 (TAX1BP3) were selected for further validation. Their expressions were all downregulated in HCC cells grown on different stiffness substrates after metformin intervention. More importantly, the degree of decrease was obviously weakened on the higher stiffness substrate compared with that on the lower stiffness substrate, indicating that these candidate membrane proteins might contribute to matrix stiffness-mediated metformin resistance in HCC. There was an obvious change in membrane proteins in matrix stiffness-mediated metformin resistance in HCC cells. Six candidate membrane proteins may reflect the response of HCC cells under high stiffness stimulation to metformin intervention, which deserve to be investigated in the future.

中文翻译：

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肝细胞癌细胞基质刚度介导的二甲双胍耐药性相关差异膜蛋白分析

我们之前的工作表明，基质硬度的增加不仅改变了肝细胞癌（HCC）细胞的恶性特征，而且削弱了二甲双胍治疗 HCC 细胞的功效。在这里，我们鉴定了与基质刚度介导的二甲双胍耐药性相关的差异膜蛋白，以更好地了解 HCC 中二甲双胍的治疗耐药性。使用用于相对和绝对定量 (iTRAQ) 标记的同量异位标签结合液相色谱-串联质谱 (LC-MS/MS) 筛选和鉴定二甲双胍干预前后在不同刚度基质上生长的 HCC 细胞中的差异膜蛋白，然后应用生物信息分析来确定候选膜蛋白及其可能的信号通路。总共鉴定了 5159 个蛋白质，并发现了 354 个差异膜蛋白和膜相关蛋白，这些蛋白可能与基质刚度介导的二甲双胍耐药性有关。进一步获得94个候选膜蛋白，其中21个上调蛋白分子和73个下调蛋白分子。其中一些如膜联蛋白 A2 (ANXA2)、Filamin-A (FLNA)、Moesin (MSN)、Myosin-9 (MYH9)、伸长因子 2 (eEF2) 和 Tax1 结合蛋白 3 (TAX1BP3) 被选择进行进一步验证。在二甲双胍干预后，在不同硬度基质上生长的 HCC 细胞中，它们的表达均下调。更重要的是，与较低硬度的基质上相比，较高硬度基质上的降低程度明显减弱，表明这些候选膜蛋白可能有助于 HCC 中基质硬度介导的二甲双胍耐药。HCC细胞中基质硬度介导的二甲双胍耐药性中膜蛋白发生明显变化。六种候选膜蛋白可能反映了HCC细胞在高硬度刺激下对二甲双胍干预的反应，值得未来研究。