Cell transfection and Lentivirus infection

The small/short interfering RNA (siRNA) designed for MCM2 interference was synthesized by Hanbio (https://www.hanbio.net/cn) and the sequences used are shown in Table S1. On the contrary, the vector carrying the wild-type full-length human MCM2 cDNA was transfected into HCC cells for MCM2 upregulation. The siRNAs and vectors were both transfected into the cells using a Lipofectamine 3000 system (Invitrogen, USA) according to the manufacturer’s instructions.

To achieve stable and durable MCM2 expression interference in the HCC cells, the sh-MCM2 lentivirus was constructed and packaged. The functional sequences of sh-MCM2 lentivirus refer to hs-MCM2-si1 and hs-MCM2-si3 in Table S1. The cells were infected with the lentivirus in the culture medium along with 10% serum according to the manufacturer’s instructions (Hanbio, Shanghai, China). After 24 h of infection, the HCC cells were cultured in a medium containing 5 μg/ml puromycin for 7 days.