客户采用我司磁珠进行RNA pull-down实验，SCI论文发表在《IUBMB LIFE》。

2.10 | RNA pull-down assay
The probe targeting the junction site of circ-ARL3 was synthesized and labeled with biotin, followed by addition into HCC cell lysates and incubation overnight at 4
C. The next day, above cell lysates were incubated with PuriMag G-streptavidin beads (Invitrogen) for 2 hr. After being washed five times, the purified RNA was collected and quantified by qRT-PCR to analyze the enrichment of circ-ARL3 and the indicated miRNAs.

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N6‐methyladenosine modification of circular RNA circ‐ARL3 facilitates Hepatitis B virus‐associated hepatocellular carcinoma via sponging miR‐1305

环状RNA circ-ARL3的N6-甲基腺苷修饰可通过海绵miR-130促进乙型肝炎病毒相关的肝细胞癌
Xi Rao  Lingling Lai  Xiaopeng Li  Liang Wang  Ai Li  Qian Yang
First published: 28 December 2020 https://doi.org/10.1002/iub.2438
IUBMB LIFE, Volume73, Issue2, February 2021, Pages 408-417

ABSTRACT: Hepatitis B virus (HBV) infection is a major risk factor for hepatocellular carcinoma (HCC), whether circular RNA (circRNA) is involved in this process remains unknown. In this study, we performed circRNA microarray profile and found an HBV‐related circRNA, circ‐ARL3 (hsa_circ_0092493). Stable knockdown of circ‐ARL3 inhibited the proliferation and invasion of HBV+ HCC cells. High circ‐ARL3 was positively correlated with malignant clinical features and poor prognosis. In terms of mechanism, HBx protein upregulated N6‐methyladenosine (m6A) methyltransferases METTL3 expression, increasing the m6A modification of circ‐ARL3; then, m6A reader YTHDC1 bound to m6A‐modified of circ‐ARL3 and favored its reverse splicing and biogenesis. Furthermore, circ‐ARL3 was able to sponge miR‐1305, antagonizing the inhibitory effects of miR‐1305 on a cohort of target oncogenes, thereby promoting HBV+ HCC progression. Importantly, depletion of circ‐ARL3 significantly retarded HBV+ HCC cell growth in vivo, whereas this effect was evidently blocked after silencing of miR‐1305. Collectively, our data suggest that circ‐ARL3 is a critical regulator in HBV‐related HCC, targeting the axis of circ‐ARL3/miR‐1305 may be a promising treatment for HBV+ HCC patients.

乙型肝炎病毒（HBV）感染是肝细胞癌（HCC）的主要危险因素，目前尚不清楚环状RNA（circRNA）是否参与该过程。在这项研究中，我们进行了circRNA微阵列分析，并发现了与HBV相关的circRNA，circ-ARL3（hsa_circ_0092493）。 circ-ARL3的稳定敲低抑制了HBV + HCC细胞的增殖和侵袭。 circ-ARL3高与恶性临床特征和预后不良呈正相关。在机制上，HBx蛋白上调N6-甲基腺苷（m6A）甲基转移酶METTL3的表达，从而增加circ-ARL3的m6A修饰。然后，m6A阅读器YTHDC1与circ-ARL3的m6A修饰结合，并支持其反向剪接和生物发生。此外，circ-ARL3能够抑制miR-1305，从而拮抗miR-1305对目标癌基因群的抑制作用，从而促进HBV + HCC的进展。重要的是，circ-ARL3的耗竭可显着延缓体内HBV + HCC细胞的生长，而在miR-1305沉默后，这种作用明显被阻止。总体而言，我们的数据表明，circ-ARL3是HBV相关HCC的关键调节剂，靶向circ-ARL3 / miR-1305的轴可能是HBV + HCC患者的有希望的治疗方法。