Cell Infection

Lentivirus was constructed for cell infection. Cells were assigned into short hairpin RNA (sh)-negative control (NC) group (cells infected with lentivirus carrying sh-NC), sh-SMAD5-AS1 group (cells infected with lentivirus carrying sh-SMAD5-AS1), mimic NC group (cells infected with lentivirus carrying mimic NC), miR-195 mimic group (cells infected with lentivirus carrying miR-195 mimic), sh-SMAD5 group (cells infected with lentivirus carrying sh-SMAD5), miR-195 mimic + overexpressed (oe)-NC group (cells infected with lentivirus carrying miR-195 mimic and oe-NC) and miR-195 mimic + oe-SMAD5 group (cells infected with lentivirus carrying miR-195 mimic and oe-SMAD5 plasmid). After cleavage with two endonucleases EcoRI and BamHI, fragments containing overexpressed and shRNAs and target vectors were inserted into pLVX-IRES-ZsGreenl vector and transformed into DH5α cells. Lentiviral supernatants were produced by infection into 293T cells with the helper plasmids pSPAX2 and pMD2G, and the supernatants were collected 48–72 h post-infection. The lentiviruses with a concentration higher than 10⁷ TU/mL were packaged and stored at −80°C for subsequent experiments. The supernatant containing different lentiviruses was dripped into CNE-2 cells upon 70–80% confluence. The medium was changed after 24 h, and the infection efficiency was tested after 48 h for subsequent experiments.