Sirnas

The tRF‐1‐Ser overexpression model was constructed by transfecting synthetic tRF‐1‐Ser mimic, while the scrambled RNA was transfected as a negative control. The tRF‐1‐Ser knockdown model was accomplished by transfecting shtRF‐1‐Ser plasmid packed into lentivirus, and the negative control lentivirus was transfected as the control. Small interfering RNAs (siRNAs) against MBNL1 were used to construct the MBNL1 knockdown model. The MBNL1 overexpression plasmid was purchased from GeneChem. All mimic, siRNAs and lentiviruses were ordered from RIBOBIO. Lipofectamine 3000 reagent (Invitrogen) was used for cell transfection. Lentivirus transfection was performed in accordance with the manufacturer's protocol.

The transfection method was introduced in our previous study 25 (link). The agents in our study were as follows: small interfering RNAs (siRNAs) targeted to GNA14, DNMT1, DNMT3A, and negative control, which were purchased from RiboBio Co., Ltd. (Guangzhou, China). siRNA targeted to HBx and negative control were purchased from Jima Biotechnology Co., Ltd. (Shanghai, China). HBx overexpressed plasmid, HBVpg3 plasmid (GV146-HBx), were purchased from JiKai Gene Co., Ltd (Shanghai, China). Lentivirus containing the complete open reading frame of GNA14 and GFP (Lentivirus -GFP -GNA14) and plasmid containing GNA14 promoter and luciferase reporter (pGL3-GNA14 Promoter wt-LUC, pGL3-GNA14 Promoter mut-LUC) for the dual luciferase reporter system were purchased from AoQian Co., Ltd (Hangzhou, China). The siRNA sequences are listed in Table S1.

Normal epithelial colon cells (NCM460 cells) and human CRC cells, including Lovo, HCT116, SW620, DLD-1 and HT-29 cells, were obtained from American Type Culture Collection. RPMI-1640 medium and McCoy’5A medium (HyClone, Logan, UT, United States) were mixed with 10% fetal bovine serum to culture the DLD-1 and HCT116 cells, respectively. The cells were cultured in a humid incubator, which always maintained a temperature of 37°C and an atmosphere of 5% CO₂. The siRNAs targeting SCRIB and the corresponding negative controls (si-NC) were synthesized by RiboBio (Guangzhou, China). The siRNA target sequence (GAAGCAGCTATCCATCCTA) with the highest efficiency for knocking down the expression of SCRIB was cloned into the lentivirus vector pGLV3 to by GeneChem (Shanghai, China). GeneChem Company designed and constructed the SCRIB overexpression plasmid for our study. Lipofectamine 3000 (Invitrogen) was used for the transfection of cells.

SiRNAs obtained from Ribobio (Guangzhou, China) were designed to interfere with the two mRNAs encoding HSP70 (GenBank accession number AB170713 for HSPA1A and XM_001115060 for HSPA1B). MARC-145 cells were seeded into 6-well plates nearly 24 hours before tansfection. The SiRNAs were transfected into MARC-145 cells with lipofectamine 2000 (Invitrogen) according to the manufacturer’s instruction.

A total of 43 paired freshly frozen HCC and corresponding noncancerous tissues were obtained from patients who underwent liver resection at the Hepatic Surgery Center of Tongji Hospital and were used for qRT-PCR. This study was approved by the Medical Ethics Committee of Tongji Hospital and conducted in accordance with the Declaration of Helsinki. The HCC cell line MHCC-97H [97H] was purchased from the China Center for Type Culture Collection (CCTCC, Wuhan, China). The cell lines were cultured in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY) and incubated in a humidified atmosphere comprising 5% CO₂ at 37 °C. The small interfering RNAs (siRNAs) and si-control were purchased from RiboBio (Guangzhou, China). Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) was used for transient transfection according to the manufacturer's protocol. To conduct animal experiments, MHCC-97H cells were transfected with the specific short hairpin RNAs to BBOX1-AS1 (sh-BBOX1-AS1). SiRNA target sequences and sh-BBOX1-AS1 sequence are listed in Additional file 1: Table S1.