Cell transfection
HINT1-specific small interfering RNAs (50 nM; HINT1-siRNA1, 5’ -ATU TUC
CAT CAC GCA TAT GTT-3’; HINT1-siRNA2, 5’ -TTG CTT AAA TTA TTG TCA
GCT-3’) and a nonspecific control (50 nM; si-NC, 5’ -CAT CTG ATT TCA ATG
GTG CTT AT-3’) were produced by Shanghai GenePharma Co., Ltd.
Additionally, a miR‑205‑3p inhibitor (50 nM; 5’ -ATG TTC AGT GGG AGT GAG
TGT TC-3’) and its negative controls (miR‑NC; 50 nM; 5’ -CTA CAA CCT CCT
AGA AAG AGT AGA-3’) were procured from Changzhou Ruibo Bio‑Technology
Co., Ltd. In a 6-well plate, 3x105 cells were planted
per well and subjected to culture till they reached ≥70% confluence.
Lipofectamine 3000 reagent was employed to transfect the cells with the
miR205 inhibitor, HINT1 siRNA1, HINT1 siRNA2, and their matching NCs, as
per the package instructions. After 72 hours post-transfection, the
cells were harvested for additional examination. Both RT-qPCR and
Western blotting were conducted to verify the transfection efficiency.
The Lipofectamine 2000 reagent (Invitrogen; Thermo Fisher Scientific,
Inc.) was applied in all transfection procedures.