Lee Woo Joo, Shin Chang Hoon, Ji Haein, Jeong Seong Dong, Park Mi-So, Won Hong-Hee, Pandey Poonam R, Tsitsipatis Dimitrios, Gorospe Myriam, Kim Hyeon Ho
Department of Health Sciences and Technology, Samsung Advanced Institute for Health Sciences and Technology, Sungkyunkwan University, Seoul, 06351, Republic of Korea.
Department of Digital Health, Samsung Advanced Institute for Health Sciences and Technology, Sungkyunkwan University, Seoul, 06351, Republic of Korea.
Cell Death Dis. 2021 Mar 17;12(4):290. doi: 10.1038/s41419-021-03575-1.
Malignant characteristics of cancers, represented by rapid cell proliferation and high metastatic potential, are a major cause of high cancer-related mortality. As a multifunctional RNA-binding protein, heterogeneous nuclear ribonucleoprotein K (hnRNPK) is closely associated with cancer progression in various types of cancers. In this study, we sought to identify hnRNPK-regulated long intergenic non-coding RNAs (lincRNAs) that play a critical role in the regulation of cancer malignancy. We found that hnRNPK controlled malignant phenotypes including invasiveness, proliferation, and clonogenicity. RNA sequencing and functional studies revealed that LINC00263, a novel target of hnRNPK, is involved in the oncogenic functions of hnRNPK. Knockdown of LINC00263 mitigated the malignant capabilities. Conversely, increased malignant phenotypes were observed in LINC00263-overexpressing cells. Since LINC00263 was mainly localized in the cytosol and highly enriched in Argonaute 2-immunoprecipitation (Ago2-IP), we hypothesized that LINC00263 acts as a competitive endogenous RNA (ceRNA), and thus sought to identify LINC00263-associated microRNAs. Using small RNA sequencing followed by antisense oligonucleotide pull-down, miR-147a was selected for further study. We found that miR-147a negatively regulates LINC00263 via direct interaction, thus suppressing malignant capabilities. Moreover, knockdown of hnRNPK and LINC00263 upregulated miR-147a, indicating that LINC00263 serves as a ceRNA for miR-147a. By analyzing RNA sequencing data and miRNA target prediction, calpain 2 (CAPN2) was identified as a putative target of miR-147a. Ago2-IP and luciferase reporter assay revealed that miR-147a suppressed CAPN2 expression by directly binding to the 3'UTR of CAPN2 mRNA. In addition, we found that the weakened malignant capabilities following knockdown of hnRNPK or LINC00263 were restored by miR-147a inhibition or CAPN2 overexpression. Furthermore, our findings were validated in various other types of cancer cells including lung cancer, colorectal cancer, neuroblastoma, and melanoma. Collectively, we demonstrate that hnRNPK-regulated LINC00263 plays an important role in cancer malignancy by acting as a miR-147a decoy and thus upregulating CAPN2.
以快速的细胞增殖和高转移潜能为代表的癌症恶性特征，是癌症相关高死亡率的主要原因。作为一种多功能RNA结合蛋白，异质性细胞核核糖核蛋白K（hnRNPK）与多种类型癌症的进展密切相关。在本研究中，我们试图鉴定在癌症恶性调节中起关键作用的hnRNPK调控的长链基因间非编码RNA（lincRNA）。我们发现hnRNPK控制包括侵袭性、增殖和克隆形成能力在内的恶性表型。RNA测序和功能研究表明，LINC00263作为hnRNPK的一个新靶点，参与了hnRNPK的致癌功能。敲低LINC00263可减轻恶性能力。相反，在LINC00263过表达的细胞中观察到恶性表型增加。由于LINC00263主要定位于细胞质中且在Argonaute 2免疫沉淀（Ago2-IP）中高度富集，我们推测LINC00263作为一种竞争性内源RNA（ceRNA），因此试图鉴定与LINC00263相关的微小RNA。通过小RNA测序，随后进行反义寡核苷酸下拉实验，选择了miR-147a进行进一步研究。我们发现miR-147a通过直接相互作用负向调节LINC00263，从而抑制恶性能力。此外，敲低hnRNPK和LINC00263会上调miR-147a，表明LINC00263作为miR-147a的ceRNA发挥作用。通过分析RNA测序数据和miRNA靶标预测，钙蛋白酶2（CAPN2）被鉴定为miR-147a的一个假定靶点。Ago2-IP和荧光素酶报告基因实验表明，miR-147a通过直接结合CAPN2 mRNA的3'UTR抑制CAPN2表达。此外，我们发现敲低hnRNPK或LINC00263后减弱的恶性能力可通过抑制miR-147a或过表达CAPN2得以恢复。此外，我们的发现也在包括肺癌、结直肠癌、神经母细胞瘤和黑色素瘤等多种其他类型的癌细胞中得到了验证。总的来说，我们证明了hnRNPK调控的LINC00263通过作为miR-147a的诱饵从而上调CAPN2，在癌症恶性过程中发挥重要作用。
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