Gv102 vector

Human hepatocytes (L02) were purchased from the Cell Bank (Chinese Academy of Sciences, Shanghai, China), and hepatic tumor cell lines (Hep3B, Huh-7, SMMC7721) were purchased from ATCC (Manassas, VA, USA). The cell lines were all grown in a cell incubator at 37°C with a 5% CO₂/95% air atmosphere.
Three shRNAs targeting CMTM2 were constructed from a GV102 vector by Genechem (Shanghai, China). The target sequence for KD-1# was 5ʹ-GGGCACGCTGAGATCAAGATT-3ʹ, the target sequence for KD-2# was 5ʹ-GGCCAGCAAAGGGAAAGAAAT-3ʹ, and the target sequence for KD-3# was 5ʹ-GACCTCTTCAACGACCTGATT-3ʹ.

An MMP-1 shRNA was utilized to knock down the expression of MMP-1 in MCF-7 and MDA-MB-231 cells. Specifically, MMP-1 shRNA and control (mock) DNA sequences were cloned into multiple cloning sites of a GV102 vector (Shanghai Genechem Co., Ltd., Shanghai, China) to generate GV102-MMP1-shRNA (shRNA-MMP1#1, shRNA-MMP1#2 and shRNA-MMP1#3) and a GV102-control (shRNA-mock). The target sequences against MMP-1 were 5′-CACATGACTTTCCTGGAAT-3′, 5′-CTAGAACTGTGAAGCATAT-3′ and 5′-ACAATTTCAGAGAGTACAA-3′, respectively, and the negative control sequence was 5′-TTCTCCGAACGTGTCACGT-3′. Following DNA sequencing confirmation, the vectors were transfected into the breast cancer cells.
Breast cancer MCF-7 and MDA-MB-231 cells were plated into a 6-well plate, grown to reach 80% confluence and then transfected with these plasmids (2 µg each well)(shRNA-MMP1#1, shRNA-MMP1#2, shRNA-MMP1#3 or shRNA-mock) using Lipofectamine™ 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) for 48 h, according to the manufacturer's protocol.

For Gli1 overexpression, the full-length Gli1 sequence was synthesized by cloning the Gli1 gene into the GV146 vector (Shanghai GeneChem Co., Ltd.) via XhoI and EcoRI sites, and was referred to as pGli1-IRES-EGFP. Empty GV146 vector acted as a negative control (NC) of pGli1-IRES-EGFP and was referred to as pIRES-EGFP. For knockdown of Gli1, short hairpin RNA (shRNA) directed against Gli1 was ligated into the GV102 vector (Shanghai GeneChem Co., Ltd.) and was referred to as sh-Gli1; a non-targeting sequence was ligated into the GV102 vector as the NC of sh-Gli1, and was referred to as sh-NC. The target sequence was 5′-CCTCTGTCTACTCACCACA-3′ and the NC sequence was 5′-TTCTCCGAACGTGTCACGT-3′. Eca109 and Eca109R cells were seeded in 6-well plates at a density of 6×10⁵ cells/well one day before transfection. Briefly, 5 µl Lipofectamine^® 3000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.) and 2.5 µg plasmid were diluted into 125 µl Opti-MEM (Gibco; Thermo Fisher Scientific, Inc.) and mixed for 5 min at room temperature. Cells were then incubated with this solution, and complete medium was added to the final volume of 2 ml. Cell transfection was performed for 24 h following the manufacturer's protocols prior to further experiments.