Liposomal Delivery of MicroRNA-7-Expressing Plasmid Overcomes Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor-Resistance in Lung Cancer Cells
Liposomal Delivery of MicroRNA-7-Expressing Plasmid Overcomes Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor-Resistance in Lung Cancer Cells
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DOI：
10.1158/1535-7163.mct-11-0220
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发表时间：
2011-09-01
影响因子：
5.7
通讯作者：
Kiura, Katsuyuki
中科院分区：
文献类型：
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作者：
Rai, Kammei;Takigawa, Nagio;Kiura, Katsuyuki
表皮生长因子受体（EGFR）酪氨酸激酶抑制剂（TKI）在携带激活EGFR突变的肺癌中具有显著的疗效。不幸的是，癌细胞最终获得对EGFR-TKI的抗性。大约50%的获得性耐药性涉及二次T790 M突变。为了克服耐药性，我们专注于使用microRNA-7（miR-7）抑制EGFR，靶向EGFR mRNA的30个非翻译区中的多个位点。使用了两种EGFR-TKI敏感细胞系（PC-9和H3255）和两种携带T790 M的EGFR-TKI抗性细胞系（RPC-9和H1975）。我们构建了含miR-7 - 2的miR-7表达质粒。转染miR-7表达质粒后，使用阳离子脂质体，进行定量PCR和双荧光素酶测定以检查功效。使用细胞计数测定和异种移植模型评价抗增殖作用。Western blotting检测蛋白表达。转染子的miR-7表达水平高约30倍，荧光素酶活性降低92%。miR-7不仅在PC-9和H3255中显著抑制细胞生长，而且在RPC-9和H1975中也显著抑制细胞生长。胰岛素受体底物-1（IRS-1）、RAF-1和EGFR的表达在4种细胞系中均受到抑制。注射表达miR-7的质粒显示在使用RPC-9和H1975的小鼠异种移植模型中显著的肿瘤消退。EGFR、RAF-1和IRS-1在残留肿瘤中被抑制。这些发现表明miR-7表达质粒通过脂质体递送对EGFR癌基因成瘾性肺癌（包括T790M抗性）的有希望的治疗应用。Mol Cancer Ther; 10（9）; 1720 - 7.（c）2011年《非洲标准化评论》。
Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKI) have been strikingly effective in lung cancers harboring activating EGFR mutations. Unfortunately, the cancer cells eventually acquire resistance to EGFR-TKI. Approximately 50% of the acquired resistance involves a secondary T790M mutation. To overcome the resistance, we focused on EGFR suppression using microRNA-7 (miR-7), targeting multiple sites in the 30-untranslated region of EGFR mRNA. Two EGFR-TKI-sensitive cell lines (PC-9 and H3255) and two EGFR-TKI-resistant cell lines harboring T790M (RPC-9 and H1975) were used. We constructed miR-7-2 containing miR-7-expressing plasmid. After transfection of the miR-7-expressing plasmid, using cationic liposomes, a quantitative PCR and dual luciferase assay were conducted to examine the efficacy. The antiproliferative effect was evaluated using a cell count assay and xenograft model. Protein expression was examined by Western blotting. The miR-7 expression level of the transfectants was approximately 30-fold higher, and the luciferase activity was ablated by 92%. miR-7 significantly inhibited cell growth not only in PC-9 and H3255 but also in RPC-9 and H1975. Expression of insulin receptor substrate-1 (IRS-1), RAF-1, and EGFR was suppressed in the four cell lines. Injection of the miR-7-expressing plasmid revealed marked tumor regression in a mouse xenograft model using RPC-9 and H1975. EGFR, RAF-1, and IRS-1 were suppressed in the residual tumors. These findings indicate promising therapeutic applications of miR-7-expressing plasmids against EGFR oncogene-addicted lung cancers including T790M resistance by liposomal delivery. Mol Cancer Ther; 10(9); 1720-7. (C)2011 AACR.