Department of Microbiology & Molecular Cell Biology, Leroy T. Canoles Jr. Cancer Research Center, Eastern Virginia Medical School.
Department of Microbiology & Molecular Cell Biology, Leroy T. Canoles Jr. Cancer Research Center, Eastern Virginia Medical School;
J Vis Exp. 2022 Apr 25(182). doi: 10.3791/63704.
MicroRNAs (miRNAs) have emerged as important cellular regulators (tumor suppressors, pro-oncogenic factors) of cancer and metastasis. Most published studies focus on a single miRNA when characterizing the role of small RNAs in cancer. However, ~30% of human miRNA genes are organized in clustered units that are often co-expressed, indicating a complex and coordinated system of noncoding RNA regulation. A clearer understating of how clustered miRNA networks function cooperatively to regulate tumor growth, cancer aggressiveness, and drug resistance is required before translating noncoding small RNAs to the clinic. The use of a high-throughput clustered regularly interspaced short palindromic repeats (CRISPR)-mediated gene editing procedure has been employed to study the oncogenic role of a genomic cluster of seven miRNA genes located within a locus spanning ~35,000 bp in length in the context of prostate cancer. For this approach, human cancer cell lines were infected with a lentivirus vector for doxycycline (DOX)-inducible Cas9 nuclease grown in DOX-containing medium for 48 h. The cells were subsequently co-transfected with synthetic trans-activating CRISPR RNA (tracrRNA) complexed with genomic site-specific CRISPR RNA (crRNA) oligonucleotides to allow the rapid generation of cancer cell lines carrying the entire miRNA cluster deletion and individual or combination miRNA gene cluster deletions within a single experiment. The advantages of this high-throughput gene editing system are the ability to avoid time-consuming DNA vector subcloning, the flexibility in transfecting cells with unique guide RNA combinations in a 24-well format, and the lower-cost PCR genotyping using crude cell lysates. Studies using this streamlined approach promise to uncover functional redundancies and synergistic/antagonistic interactions between miRNA cluster members, which will aid in characterizing the complex small noncoding RNA networks involved in human disease and better inform future therapeutic design.
微小 RNA（miRNAs）已成为癌症和转移中重要的细胞调控因子（肿瘤抑制因子、致癌因子）。大多数已发表的研究在描述小 RNA 在癌症中的作用时，都集中在单个 miRNA 上。然而，约 30%的人类 miRNA 基因组织在簇单元中，这些单元通常是共表达的，这表明非编码 RNA 调控存在一个复杂而协调的系统。在将非编码小 RNA 转化为临床应用之前，需要更清楚地了解簇状 miRNA 网络如何协同作用来调节肿瘤生长、癌症侵袭性和耐药性。高吞吐量的簇状规律间隔的短回文重复（CRISPR）介导的基因编辑程序已被用于研究位于前列腺癌中约 35000bp 长的基因座内的七个 miRNA 基因的基因组簇的致癌作用。对于这种方法，用人源癌细胞系感染了含有 DOX 的培养基中培养 48 小时的 DOX 诱导型 Cas9 核酸酶的慢病毒载体。随后，将合成的反式激活 CRISPR RNA（tracrRNA）与基因组位点特异性 CRISPR RNA（crRNA）寡核苷酸复合物共转染到细胞中，以允许在单个实验中快速生成携带整个 miRNA 簇缺失和单个或组合 miRNA 基因簇缺失的癌细胞系。这种高通量基因编辑系统的优点是能够避免耗时的 DNA 载体亚克隆，能够以 24 孔格式灵活地转染具有独特引导 RNA 组合的细胞，以及使用粗细胞裂解物进行低成本的 PCR 基因分型。使用这种简化方法的研究有望揭示 miRNA 簇成员之间的功能冗余和协同/拮抗相互作用，这将有助于描述涉及人类疾病的复杂小非编码 RNA 网络，并更好地为未来的治疗设计提供信息。
