Hk2 overexpression vector

The study on animal subjects was approved by the Ethics Committee of Affiliated Hospital of Inner Mongolia Medical University. In vivo xenograft mouse experiments were performed as previously described (14 (link), 15 (link)). Female mice (BALB/c nude, 4–6 weeks old) were purchased from Vital River Laboratory Animal Technology (Beijing, China). CC cells transfected with HK2 overexpression vector (OriGene, Rockville, MD) or HK2 shRNA (Santa Cruz Biotechnology; Santa Cruz, CA), were subcutaneously injected into the flank of nude mice under aseptic conditions. After a 7-day administration, mice were treated with CDDP (30 mg/kg) intraperitoneally. The tumors were measured in two dimensions by using manual calipers every 3 days. The tumor volume was calculated using the following formula: volume = 0.5 × length × width². After 30 days, all mice were sacrificed and tumor samples were weighted.

Transfection was conducted using the Lipofectamine 3000 reagent (Invitrogen) according to the manufacturer’s instructions. The control vector, HK2 overexpression vector, control shRNA, or HK2 shRNA was obtained from OriGene and Santa Cruz Biotechnology, respectively. Control siRNA, HK2 siRNA, control mimic, miR-145 mimic, miR-148a mimic, miR-497 mimic, control inhibitor, miR-145 inhibitor, miR-148a inhibitor, or miR-497 inhibitor, was purchased from Invitrogen. When cells achieved 70% confluence, the above plasmid, shRNA, siRNA, or miRNA mimic (or inhibitor) was transfected into CC cells for 48 h. Finally, stable HK2-overexpressing or HK2 knockdown CC cell lines were established by selecting cells using G418 (Sigma-Aldrich) or Puromycin (Sigma-Aldrich) for 4 weeks.