Total RNA from the human tissues and cultured cells were extracted using TRIzol solution (Takara Biotechnology Co., Ltd., Dalian, China) and quantified using a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, Inc.). A 1 µg sample of mRNA was reverse transcribed using PrimeScript RT Master Mix (Perfect Real Time) kit (Takara Biotechnology Co., Ltd.) and qPCR was performed in an ABI PRISM 7900 Real Time system (Applied Biosystems; Thermo Fisher Scientific, Inc.) using the SYBR Premix Ex Taq kit (Takara Biotechnology Co., Ltd.). The primers used were as follows: Tim-3, forward 5′-GCTACTACTTACAAGGTCCTCAG-3′ and reverse 5′-ATTCACATCCCTTTCATCAGTC-3′; GAPDH, forward 5′-GTGGACATCCGCAAAGAC-3′ and reverse 5′-AAAGGGTGTAACGCAACTA-3′. U Initial denaturation was performed at 95°C for 30 sec, and PCR by 40 cycles of 95°C for 5 sec and 60°C for 35 sec. All experiments were performed in triplicate at least three times. Values were calculated used the 2−ΔΔCq method (25 (link))