RT‑qPCR
Both tissues and cell lines were processed utilizing TRIzol reagent
(Invitrogen; Thermo Fisher Scientific, Inc.) to extract total RNA. The
PrimeScript RT reagent kit was employed for the synthesis of cDNA
following package guidelines (Takara Bio, Inc.). The SYBR Green
PrimeScript™ PLUS RT‑PCR kit (Takara Bio, Inc.) was then utilized to
carry out qPCR. The procedure included an initial denaturation phase at
95˚C for 30 seconds, followed by 40 cycles of 95˚C denaturation for 15
seconds, 60˚C annealing for 25 seconds, and 72˚C extension for 1 minute,
ending with a final extension step at 72˚C for 10 minutes. The internal
controls used were U6 for miRNA and β‑actin for mRNA, with the relative
gene expression being determined via the 2‑ΔΔCq method[24]. The
primers employed were as follows: 5’‑CTT GTC CTT CAT TCC ACC GGA‑3’ and
5’‑TGC CGC CTG AAC TTC ACT CC‑3’ for miR‑205‑3p; 5’‑CAC AGA GAA GA GTC
TGG CCC‑3’ and 5’‑AGG TTG CTT GTG ACA GGT CC‑3’ for HINT1; 5’‑GAT GGA
CTC TGG TGA TGG TGT GAC‑3’ and 5’‑TTT CTC TTT CGG CTG GTG GTG TG‑3’ for
β‑actin; and 5’‑CTC GCT TCG GCA GCA CA‑3’ and 5’‑AAC GCT TCA CGA ATT TGC
GT‑3’ for U6.