Lentivirus containing short hairpin rna shrna

The lentivirus-containing short hairpin RNA (shRNA) targeting LINC01133 was purchased from GenePharma (Shanghai, China), and the pLenti-GIII-CMV vector for LINC01133 overexpression was purchased from Applied Biological Materials (Richmond, Canada); both shRNAs were transfected into the GC cell lines. At 48 h post-transfection, the cells were selected with puromycin (2 μg/mL) for 2 weeks to construct cell lines with stable LINC01133 knockdown or overexpression. The transfection efficiency was verified by qRT-PCR. The has-miR-106a-3p mimic, hsa-miR-106a-3p inhibitor, and negative control (NC) oligonucleotides were obtained from Ribobio (Guangzhou, China). The pCMV-Neo-Bam-APC vector for upregulation of the APC gene was obtained from Addgene (Cambridge, MA, USA). GC cells were transfected with the abovementioned oligonucleotides and plasmids using Lipofectamine 3000 (Invitrogen) according to the manufacturer’s instructions.

Three different small interfering (si)RNAs against PLOD2 were designed and synthesized by Ribobio(Guangzhou, China), and transfected into MG63 and 143B cells using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). Cells were collected 48h after transfection, and the knockdown efficiency was detected by reverse transcription-quantitative PCR (RT-qPCR). The lentivirus-containing short hairpin RNA(shRNA) targeting PLOD2 was purchased from GenePharma(Shanghai, China), shRNA was transfected into 143B cell line, 48h after transfection, cells were selected with puromycin(2μg/mL) for 2 weeks to construct stable PLOD2 knockdown cell line. The sequences of PLOD2 siRNA are listed in Table S1.