RBM15 promotes hepatocellular carcinoma progression by regulating N6-methyladenosine modification of YES1 mRNA in an IGF2BP1-dependent manner

The function of the N6-methyladenosine (m6A) methyltransferase RNA-binding motif protein 15 (RBM15) in hepatocellular carcinoma (HCC) has not been thoroughly investigated. Here we determined the clinical value, biological functions, and potential mechanisms of RBM15 in HCC. Expression of RBM15 was identified using tissue microarrays and online databases. A risk-prediction model based on RBM15 was developed and validated. We determined the biological role of RBM15 on HCC cells in vitro and in vivo. RNA sequencing was used to screen candidate targets of RBM15. Subsequently, the m6A dot blot assay, methylated RNA immunoprecipitation qPCR, dual-luciferase reporter assays, RNA decay assay, and RNA immunoprecipitation qPCR were employed to explore the mechanisms of RBM15. Our study showed that RBM15 was highly expressed in HCC, and overexpression of RBM15 indicated a worse outcome. A new nomogram combining RBM15 with age and TNM stage was developed and validated to predict the outcome of HCC patients; our nomogram increased the prediction accuracy of the TNM system. Functionally, RBM15 facilitates the proliferation and invasiveness of HCC. RBM15-mediated m6A modification contributed to a post-transcriptional activation of YES proto-oncogene 1 (YES1) in an insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1)-dependent manner. In addition, YES1 was confirmed as an oncogene in HCC cells by activating the mitogen-activated protein kinase (MAPK) pathway. In conclusion, RBM15-mediated m6A modification might facilitate the progression of HCC via the IGF2BP1–YES1–MAPK axis. RBM15 may be a promising biomarker in the outcome prediction of HCC.

Imaging system (Bio-Rad, USA) and enhanced chemiluminescence detection kit (Servicebio, Wuhan, China) were used for Western blotting. GAPDH was chosen as loading control. All the antibodies used in the study were listed in Supplementary Table 2.

Immunohistochemistry (IHC)
The IHC staining of TMA cohort was applied to determine expression of RBM15 protein and the connection between RBM15 and prognosis of HCC patients. IHC intensity scores and percentage of positive cells was defined as we described in our previous study 10 . All score was assessed by two pathologists who were unaware of the prognosis of patients independently. Besides, subcutaneous tumor specimens from mice were fixed with formalin and embedded in paraffin. Then IHC staining of RBM15, YES1 and PCNA were performed on these tumors.
Transient transfection was performed according to the manufacturer's protocol by jetPRIME Polyplus Kit (France). All the sequences were summarized in Supplementary   Table 3.
Huh7 and HCC-LM3 cells were applied to establish stable RBM15 knockdown models.
Infected cells were selected by 3μg/ml puromycin for two weeks before subsequent assays. All the targeted sequences were listed in Supplementary Table 3.

Cell proliferation assay, colony formation and EdU incorporation assay.
Cell proliferation ability was determined by Cell Counting Kit-8 according to the manufacturer's protocol. In colony formation assay, 1.5× 103 cells were plated in 6well cell culture plate with three repetitions. After incubation of two weeks, the plates were fixed by paraformaldehyde and stained with 1% crystal violet for 10min. In Edu assay, A 5-ethynyl-20-deoxyuridine (EdU) assay kit (Ribobio, Guangzhou, China) was used to evaluate the proliferation ability of HCC cells according to the manufacturer's protocol. The results were visualized by a fluorescence microscope.

Migration and invasion assays
In migration or invasion assays, a 24-well plate was used with a transwell filter insert (Corning, NY, USA) with a pore size of 8 μm. In the invasion experiment, diluted matrix was added to the transwell filter insert in advance. 8 × 10 4 HCC cells in serumfree medium were placed in the upper cavity, and then the medium containing 10% fetal bovine serum was added to the lower cavity. After 48 h (migration) or 72 h (invasion) culture at 37 ° C, the submembranous cells were fixed and stained with crystal violet.
Then cell counts in five random domains were then performed under the microscope.

Subcutaneous xenograft experiments
Four-week-old male Balb/c nude mice were obtained from Shanghai Experimental Animal Center of Chinese Academic of Sciences (Shanghai,China). 5 × 10 6 Huh7 and HCC-LM3 cells resuspended in 100μl PBS were subcutaneously injected to the left flank of the mice (randomly selected, five mice per group for Huh7 cells in the first time and ten mice per group for HCC-LM3 cells in the second time. No blinding was performed). Tumor sizes were measured regularly. After feeding of more than 3 weeks, mice were sacrificed and tumors were surgically dissected for histology analyses. The tumor volume was calculated with the equation: (length×width 2 )/2. The animal experiments were approved by the Ethics Committee for Laboratory Animals of the First Affiliated Hospital, Zhejiang University.

RNA immunoprecipitation (RIP)
Magna RIP kit (Millipore, Germany) was applied to conduct RIP assay in accordance with manufacturer's recommendation. In Brief, magnetic beads were mixed with anti-IGF2BP1 (Abclonal, China) and anti-rabbit IgG (Millipore, Germany) and added to sufficient cell lysates. Then, target RNA-protein complexes were eluted and purified for qPCR.

Luciferase reporter assay
cDNAs containing 3' UTR sequence of YES1 were cloned into luciferase reporter vectors (pcDNA3.1 vector including firefly and renilla luciferase). For mutant report plasmids, two adenosine (A) in m6A sties were replaced with cytosine (C). RBM15knockdown HCC cells were transfected with wild-type or mutated YES1 reporter plasmids. After 24h, the luciferase activity was tested using Dual Luciferase Reporter Assay Kit (Vazyme Biotech Co.,Ltd, China) The inserted sequences were listed Supplementary Table 4 RNA decay assay RNA decay assay was performed to evaluate RNA stability. HCC cells were cultured in 6-well plates followed by treatment of RBM15 knock-down. Actinomycin D (MCE, HY-17559) was added into each well. After 0, 12 and 24h, we collected cells to quantify the relative abundance of YES1 mRNA (relative to 0h).