Overexpressed PRR11-SKA2-miR301a/454 bidirectional transcription unit essentially and coordinately promotes PI3K-AKT pathway activation and lung cancer progression

Gene pairing is a highly conserved and special mode of eukaryotic gene organization, and critically implicated in development and diseases including cancer. We previously found that PRR11 and SKA2 constitute a classic head-to-head gene pair. Here, we further demonstrate that PRR11, SKA2, and its intronic miR301a and miR454 constitute a more exquisite bidirectional transcription unit that are overexpressed in various types of cancers. Functional studies using lung cancer as a model system reveal that co-overexpression of PRR11, SKA2, miR301a and miR454 together remarkably accelerates cell growth, cell cycle progression and cell motility in lung cancer cells, and promotes tumor growth in mouse models in vivo, whereas CRISPRi-mediated repression of the entire transcription unit inhibits these malignant phenotypes. Mechanistically, the four component genes do not display any additive or synergistic effect, but rather compensate for each other for robustly sustained activation of PI3K-AKT pathway, with PRR11 interacting with GRB2, and SKA2 with EGFR. Notably, miR301a and miR454 exert their oncogenic functions at least partially via repressing PTEN translation. Moreover, the transcription unit presents as a prominent prognostic meta-marker for lung cancer. Collectively, these findings demonstrate the essential and coordinated roles of PRR11-SKA2-miR301a/454 bidirectional transcription unit in lung cancer progression, highlighting its potential diagnostic and therapeutic values in cancers. Overall design: To investigate the molecular mechanisms by which the PRR11/SKA2/miR301a/miR454 transcriptional unit promotes lung cancer development, we conducted four sets of experiments in A549 cells. In the first set, we established stable cell lines with suppressed bidirectional promoter transcriptional activity of PRR11/SKA2 using the CRISPRi system. In the second set, we established stable cell lines with individual overexpression of PRR11, SKA2, as well as co-overexpression of PRR11 and SKA2, using the Lenti-pCDH-CMV system. In the third set, we established stable cell lines with individual overexpression of miR301a, miR454, as well as co-overexpression of miR301a and miR454, using the Lenti-pCDH-CMV system. In the fourth set, we established cell lines with knockout of pre-miR301a and pre-miR454, respectively, using the CRISPRd system. We subsequently conducted gene expression profiling analysis using RNA-seq data obtained from these four experimental groups. Comparative gene expression profiling analysis was conducted on the RNA-seq data. For the first group, we compared the stable cell lines with suppressed bidirectional promoter transcriptional activity of PRR11/SKA2 (CRISPRi_PRR11+SKA2 promoter-sgRNA#1) with their corresponding control cell lines (CRISPRi_Mock sgRNA). In the second group, we compared the stable cell lines with individual overexpression of PRR11, SKA2, and co-overexpression of PRR11 and SKA2 (LV-PRR11, LV-SKA2, LV-P+S) with their respective control cell lines (LV-vector-PS). For the third group, we compared the stable cell lines with individual overexpression of miR301a, miR454, and co-overexpression of miR301a and miR454 (LV-miR301a, LV-miR454, LV-3+4) with their corresponding control cell lines (LV-vector-34). Lastly, for the fourth group, we compared the cell lines with knockout of pre-miR301a and pre-miR454 (CRISPRd_miR301a-#2, CRISPRd_miR454-#1) with their respective control cell lines (CRISPRd_Control).

基因配对是真核生物基因组织的高度保守且独特的模式，在包括癌症在内的发育与疾病进程中发挥关键作用。我们前期研究发现，PRR11与SKA2构成经典的头对头基因对。本研究进一步证实，PRR11、SKA2及其内含子miR301a与miR454共同构成更为精巧的双向转录单元，该单元在多种癌症中呈过表达状态。以肺癌为模型系统开展的功能实验显示，PRR11、SKA2、miR301a与miR454的共过表达可显著促进肺癌细胞的增殖、细胞周期进程与运动能力，并在体内小鼠模型中加速肿瘤生长；而CRISPRi介导的该转录单元整体抑制则会削弱上述恶性表型。机制层面，这四个组分并未表现出相加或协同效应，而是通过相互代偿，持续稳健激活PI3K-AKT通路：PRR11与GRB2相互作用，SKA2与EGFR结合。值得注意的是，miR301a与miR454至少部分通过抑制PTEN的翻译过程发挥致癌功能。此外，该转录单元可作为肺癌的显著预后元标志物（meta-marker）。综上，本研究证实了PRR11-SKA2-miR301a/454双向转录单元在肺癌进展中的关键协同作用，凸显其在癌症诊疗中的潜在应用价值。整体实验设计：为探究PRR11/SKA2/miR301a/miR454转录单元促进肺癌发生的分子机制，我们在A549细胞中开展了四组独立实验。第一组：利用CRISPRi系统构建PRR11/SKA2双向启动子转录活性受抑制的稳定细胞系；第二组：采用Lenti-pCDH-CMV系统，分别构建PRR11、SKA2单基因过表达，以及PRR11与SKA2共过表达的稳定细胞系；第三组：同样采用Lenti-pCDH-CMV系统，分别构建miR301a、miR454单基因过表达，以及miR301a与miR454共过表达的稳定细胞系；第四组：利用CRISPRd系统分别构建pre-miR301a与pre-miR454基因敲除的细胞系。随后，我们对上述四组实验样本的RNA-seq数据进行基因表达谱分析，并开展组间对比：对于第一组实验，将PRR11/SKA2双向启动子转录活性受抑制的稳定细胞系（CRISPRi_PRR11+SKA2 promoter-sgRNA#1）与对应的对照细胞系（CRISPRi_Mock sgRNA）进行比较；对于第二组实验，将PRR11、SKA2单过表达及二者共过表达的稳定细胞系（LV-PRR11、LV-SKA2、LV-P+S）与各自的对照细胞系（LV-vector-PS）进行比较；对于第三组实验，将miR301a、miR454单过表达及二者共过表达的稳定细胞系（LV-miR301a、LV-miR454、LV-3+4）与对应的对照细胞系（LV-vector-34）进行比较；对于第四组实验，将pre-miR301a与pre-miR454分别敲除的细胞系（CRISPRd_miR301a-#2、CRISPRd_miR454-#1）与各自的对照细胞系（CRISPRd_Control）进行比较。