SUMO1 modification of KHSRP regulates tumorigenesis by preventing the TL-G-Rich miRNA biogenesis

Haihua Yuan, Rong Deng, Xian Zhao, Ran Chen, Guofang Hou, Hailong Zhang, Yanli Wang, Ming Xu, Bin Jiang, Jianxiu Yu

MicroRNAs (miRNAs) are important regulators involved in diverse physiological and pathological processes including cancer. SUMO (small ubiquitin-like modifier) is a reversible protein modifier. We recently found that SUMOylation of TARBP2 and DGCR8 is involved in the regulation of the miRNA pathway. KHSRP is a single stranded nucleic acid binding protein with roles in transcription and mRNA decay, and it is also a component of the Drosha-DGCR8 complex promoting the miRNA biogenesis. The in vivo SUMOylation assay using the Ni(2+)-NTA affinity pulldown or immunoprecipitation (IP) and the in vitro E.coli-based SUMOylation assay were used to analyze SUMOylation of KHSRP. Nuclear/Cytosol fractionation assay and immunofluorescent staining were used to observe the localization of KHSRP. High-throughput miRNA sequencing, quantantive RT-PCR and RNA immunoprecipitation assay (RIP) were employed to determine the effects of KHSRP SUMO1 modification on the miRNA biogenesis. The soft-agar colony formation, migration, vasculogenic mimicry (VM) and three-dimensional (3D) cell culture assays were performed to detect the phenotypes of tumor cells in vitro, and the xenograft tumor model in mice was conducted to verify that SUMO1 modification of KHSRP regulated tumorigenesis in vivo. KHSRP is modified by SUMO1 at the major site K87, and this modification can be increased upon the microenvironmental hypoxia while reduced by the treatment with growth factors. SUMO1 modification of KHSRP inhibits its interaction with the pri-miRNA/Drosha-DGCR8 complex and probably increases its translocation from the nucleus to the cytoplasm. Consequently, SUMO1 modification of KHSRP impairs the processing step of pre-miRNAs from pri-miRNAs which especially harbor short G-rich stretches in their terminal loops (TL), resulting in the downregulation of a subset of TL-G-Rich miRNAs such as let-7 family and consequential tumorigenesis. Our data demonstrate how the miRNA biogenesis pathway is connected to tumorigenesis and cancer progression through the reversible SUMO1 modification of KHSRP.