Total RNA was extracted from the clinical specimens using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Subsequently, the RNAs were reversed transcribed into cDNA using a reverse transcription reagent kit (Takara Biotechnology Co., Ltd., Dalian China). Subsequently, cDNA was amplified using an SYBR Green mix kit and the ABI 7900 Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.) following the manufacturer's instructions. PCR was performed with the following thermocycling conditions: Initial denaturation at 94°C for 4 min, 40 cycles of 94°C for 30 sec, 56°C for 30 sec and 72°C for 25 sec, using the ABI 7900 Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific Inc.). The primer sequences are shown in Table II. Relative levels of miR-539 and mRNA expression levels of HMGA2 were normalized to that of small nuclear RNA U6 (for miRNAs) or GAPDH (for mRNAs) respectively. The relative expression of miRNA or mRNA was calculated using the 2−ΔΔCt method (15 (link)).