The relative mRNA levels of integrin β1, FAK in the BEL-7402 cells of each group was respectively determined by qRT-PCR assay. qRT-PCR was performed by a ABI StepOne Real-time PCR system (Applied Biosystems, foster, CA, USA). The total reactive volume is 20 μL, including 10 μL of 2 × SYBR Premix Ex TaqⅡ, 0.4 μL of 50 × ROX Reference Dye (TaKaRa, Dalian, China), 0.8 μL of 10 μM forward primer, 0.8 μL of 10 μΜ reverse primer, 2 μL of cDNA, double distilled water 6 μL. The reaction was performed as the manufacture’s instruction: incubation at 95 °C for 30 s followed by 40 cycles of 95 °C for 5 s and 60 °C for 30 s. Total RNA was extracted using Trizol Plus RNA purification kit (Tiangen, Beijing, China). Concentration of RNA was determined by absorption at 260 and 280 nm. The quantitative analysis was conducted by using the formula as follows: ΔCt = Ct1 − Ct2, where the Ct1 was Ct value of target gene and Ct2 was Ct value of GAPDH. The results were presented in 2−ΔΔCt. The experiment was repeated three times (n = 3). The sequences of primers were as follows:
GAPDH forward: 5′-AAGTTCAACGGCACAGTCAAGG-3′,
GAPDH reverse: 5′-CATACTCAGCACCAGCATCACC-3′;
Integrin β1forward: 5′-TTCGATGCCATCATGCAAGTTG-3′,
Integrin β1 reverse: 5′-CCATCTCCAGCAAAGTGAAACC-3′,
FAK-forward: 5′-ACTCATCGAGAGATCGAGATGG-3′,
FAK reverse: 5′-GCCCTAGCATTTTCAGTCTTGC-3′.