Objective: To investigate the role of myosin heavy chain 9 (MYH9) in regulation of cell proliferation, apoptosis, and cisplatin sensitivity of non-small cell lung cancer (NSCLC).
Methods: Six NSCLC cell lines (A549, H1299, H1975, SPCA1, H322, and H460) and a normal bronchial epithelial cell line (16HBE) were examined for MYH9 expression using Western blotting. Immunohistochemical staining was used to detect MYH9 expression in a tissue microarray containing 49 NSCLC and 43 adjacent tissue specimens. MYH9 knockout cell models were established in H1299 and H1975 cells using CRISPR/Cas9 technology, and the changes in cell proliferation cell were assessed using cell counting kit-8 (CCK8) and clone formation assays; Western blotting and flow cytometry were used to detect apoptosis of the cell models, and cisplatin sensitivity of the cells was evaluated using IC50 assay. The growth of tumor xenografts derived from NSCLC with or without MYH9 knockout was observed in nude mice.
Results: MYH9 expression was significantly upregulated in NSCLC (P < 0.001), and the patients with high MYH9 expression had a significantly shorter survival time (P=0.023). In cultured NSCLC cells, MYH9 knockout obviously inhibited cell proliferation (P < 0.001), promoted cell apoptosis (P < 0.05), and increased their chemosensitivity of cisplatin. In the tumor-bearing mouse models, the NSCLC cells with MYH9 knockout showed a significantly lower growth rate (P < 0.05). Western blotting showed that MYH9 knockout inactivated the AKT/c- Myc axis (P < 0.05) to inhibit the expression of BCL2- like protein 1 (P < 0.05), promoted the expression of BH3- interacting domain death agonist and the apoptosis regulator BAX (P < 0.05), and activated apoptosis-related proteins caspase-3 and caspase-9 (P < 0.05).
Conclusion: High expression of MYH9 contributes to NSCLC progression by inhibiting cell apoptosis via activating the AKT/c-Myc axis.
目的: 探讨肌球蛋白重链9（MYH9）对非小细胞肺癌（NSCLC）细胞增殖、凋亡和对顺铂敏感性的影响并探讨其作用机制。
方法: 常规培养6株NSCLC细胞系（A549、H1299、H1975、SPCA1、H322、H460）和1株正常支气管上皮细胞系（16HBE）采用Western blot法检测MYH9的表达情况；采用免疫组织化学染色法检测NSCLC患者组织MYH9的表达，实验分为癌组织（49例）和癌旁组织（43例）；常规培养H1299和H1975细胞系，使用CRISPR/Cas9技术构建MYH9敲除的NSCLC细胞系，实验分为sgNC组，sgMYH9组，采用细胞计数试剂盒-8（CCK-8）、克隆形成实验检测敲除MYH9对肺癌细胞增殖的影响；Western blot实验、细胞凋亡流式细胞术检测MYH9对细胞凋亡的影响；通过IC50法检测敲除MYH9的肺癌细胞对顺铂敏感性的影响，实验分为sgNC组、sgMYH9组；培育小鼠肿瘤异种移植物模型验证MYH9体内生物学功能，实验分为sgNC组、sgMYH9组。
结果: 与癌旁组织相比，MYH9在癌组织中上调（P < 0.001），高表达患者具有更短的生存（P=0.023）。敲除MYH9抑制细胞增殖（P < 0.001），促进细胞凋亡（P < 0.05），增加对顺铂的化学敏感性。此外，MYH9的耗竭显著抑制了体内肿瘤生长（P < 0.001）。分子机制表明，敲除MYH9使AKT/c-Myc轴失活（P < 0.05），从而抑制BCL-2样蛋白1的表达（P < 0.05），促进BH3相互作用结构域死亡激动蛋白和凋亡调节剂BAX的表达（P < 0.05），并激活胱天蛋白酶3和胱天蛋白酶9（P < 0.05）。
结论: MYH9通过AKT/c-Myc轴调节细胞凋亡，从而促进NSCLC进展。
Keywords: Akt/c-Myc signaling; CRISPR-Cas9; MYH9; cell apoptosis; cisplatin resistance; non-small cell lung cancer.