Supplementary Material for: miR-466 Contributes to the Enhanced Antitumor Effect of Bortezomib on Non-Small-Cell Lung Cancer by Inhibiting CCND1

Introduction: Changes in microRNAs (miRs) contribute to the alternative chemo-resistance of cancers. Bortezomib (BTZ) is a well-characterized anticancer agent that inhibits proteasome, and its effect is associated with the function of miRs. Based on the data of microarray assay and comprehensive bioinformatics analyses, in the current study, we explored the role of miR-466 and its downstream effector CCND1 in the BTZ-resistance of non-small-cell lung cancer (NSCLC) cells. Methods: miR expression profiles in NSCLC tissues and paratumor tissues were determined with microarray assay. The potential miR involved in the chemo-resistance of NSCLC cells was explored via a series of bioinformatics analyses, and miR-466 was selected. Afterward, levels of miR-466 and CCND1 were investigated in NSCLC samples and analyzed by clinicopathologic parameters, including age, sex, stage of NSCLC, tumor size, tumor differentiation status, and lymphocytic infiltration status. The expression of CCND1 and miR-466 was then modulated in vitro to explore the influence on cell phenotypes, which was then verified with mouse models. Results: Based on microarray detection, 287 miRs were dysexpressed between NSCLC tissues and paratumor tissues, including 90 upregulated members and 197 downregulated members. After bioinformatics analyses and reverse transcription quantitative PCR validation, miR-466 and CCND1 were selected. Following clinical investigations, miR-466 was downregulated, while CCND1 was upregulated in NSCLC samples, contributing to the advanced cancer progression. The overexpression of CCND1 increased cell viability, suppressed cell apoptosis, decreased p21 and induced N-cadherin, CCND2, and CDK4 under BTZ treatment. The induced expression of miR-466 re-sensitized NSCLC cells to BTZ treatment. In the animal model, the overexpression of CCND1 impaired the inhibitory effect of BTZ on the growth and metastasis of solid tumor, which was restored by miR-466 induction. Conclusion: The findings showed that the interaction between BTZ, miR-466, and CCND1 determined the antitumor effect of BTZ on NSCLC.

引言：微小RNA（microRNAs，miRs）的表达异常可参与癌症的化疗耐药调控。硼替佐米（Bortezomib，BTZ）是一种特征明确的蛋白酶体（proteasome）抑制剂类抗癌药物，其抗肿瘤活性与miRs的功能密切相关。本研究基于微阵列芯片检测（microarray assay）与综合生物信息学（bioinformatics）分析的数据，探讨了miR-466及其下游效应分子细胞周期蛋白D1（CCND1）在非小细胞肺癌（non-small-cell lung cancer，NSCLC）细胞硼替佐米耐药中的作用。

方法：采用微阵列芯片检测技术，测定非小细胞肺癌组织与癌旁组织（paratumor tissues）中的miR表达谱。通过一系列生物信息学分析，筛选与非小细胞肺癌细胞化疗耐药相关的潜在miR，并最终选定miR-466。随后，检测非小细胞肺癌样本中miR-466与CCND1的表达水平，并结合临床病理参数（clinicopathologic parameters）——包括年龄、性别、非小细胞肺癌分期、肿瘤大小、肿瘤分化程度及淋巴细胞浸润状态——进行相关性分析。在体外实验中调控CCND1与miR-466的表达，以探究其对细胞表型的影响，并通过小鼠模型开展体内验证。

结果：微阵列芯片检测结果显示，非小细胞肺癌组织与癌旁组织间共有287个miR存在异常表达，其中90个呈上调表达，197个呈下调表达。经生物信息学分析与逆转录定量聚合酶链反应（reverse transcription quantitative PCR，RT-qPCR）验证后，选定miR-466与CCND1作为研究靶点。临床样本检测结果表明，非小细胞肺癌样本中miR-466呈低表达，而CCND1呈高表达，且该表达异常与肿瘤进展密切相关。在硼替佐米处理条件下，CCND1过表达可提高细胞活力（cell viability）、抑制细胞凋亡（cell apoptosis），同时降低p21的表达水平，并诱导N-钙粘蛋白（N-cadherin）、细胞周期蛋白D2（CCND2）及细胞周期蛋白依赖性激酶4（CDK4）的表达。过表达miR-466可使非小细胞肺癌细胞重新对硼替佐米治疗产生敏感性。动物模型实验结果显示，CCND1过表达会削弱硼替佐米对实体瘤（solid tumor）生长与转移的抑制作用，而诱导miR-466表达可逆转这一效应。

结论：本研究结果表明，硼替佐米、miR-466与CCND1三者间的相互调控作用，决定了硼替佐米对非小细胞肺癌的抗肿瘤活性。