TRIzol reagent (Invitrogen, Carlsbad, CA, USA) was used to extract the RNA from LUSC clinical specimens and cell lines. miR-665 expression and TRIM8 mRNA expression were examined through using PrimeScript RT Reagent Kits and SYBR Premix Ex Taq II Kit (Takara Biotechnology, Takara, Dalian, China). The correlative primers of miR-665 and TRIM8 were purchased from Takara Biotechnology (Takara, Dalian, China). All reactions were performed in triplicate by using the iCycler iQ Multicolor Real-Time PCR System (Bio-Rad, CA, USA). The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was regarded as a control for TRIM8 mRNA. RNU6B (U6) was as a control for miR-665. The primers were as follows: miR-665 reverse-transcription primer (5′-GTCGTATCCAGTGCGTGTCGTGGAGTCGGCAATTGCACTGGATACGAC GGCCCCT-3′), miR-665 (F: 5′-ATCCAGTGCGTGTCGTG-3′; R: 5′-TGCT ACCAGGAGGCTGA-3′); U6 reverse-transcription primer (5′-CGCTTCACGAATTTGCGTGTCAT-3′), U6 (F: 5′-GCTTCGGCAGCACATATACTAAAAT-3′; R: 5′-CGCTTCACGAATTTGCGTGTCAT-3′); TRIM8 (F: 5′- CGTGGAGATCCGAAGGAATGA-3′; R: 5′-CAGGCGCTTGTCTGACTCG-3′), GAPDH (F: 5′-GCCACATCGCTCAGACAC-3′; R: 5′-GCCCAATACGACCAAATCC-3′). miR-665 reactions conditions: 95 °C for 15 min, followed by 40 cycles at 95 °C for 5 s, 58 °C for 30 s, and 72 °C for 30 s. TRIM8 reactions conditions: 95 °C for 10 min, followed by 40 cycles at 95 °C for 10 s, 60 °C for 20 s, and 72 °C for 30 s. The 2−ΔΔCt method was applied in the qRT-PCR analysis.

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