*4.5. RNA Isolation and Reverse Transcription and Polymerase Chain Reaction (RT-PCR) for miR-143-Target Gene Expression*

Total cellular RNA was extracted from 1 × 106 cells using TrizolTM reagent (Invitrogen Inc., Carlsbad, California, MD, USA) according to the manufacturer's protocol. The concentration and purity of RNA were determined spectrophotometrically. Then, the synthesis of cDNA was performed according to a cDNA cycle kit K1621 (Fermentas Inc., Burlington Canada). To determine the expression of the target gene of miR-143, IL-13Rα1, we performed fluorescent quantitative real time RT-PCR assay. The sequences of the primers (TaKaRa Inc., Dalian, China) specific for IL-13Rα1 were performed with sense (ACCCGAGGGAGCCAGCTCAA) and antisense (GGTGCTACACTGGGACCCCACT) primers, wherein the expected size of the amplified sequence was 111 bp. 18sRNA was used as control. Then, the incubation of cDNA and primer was performed at 95 °C for 15 s, and the PCR reaction proceeded for 45 cycles as per the following conditions: 95 °C for 15 s and 60 °C for 30 s, in a programmable thermal cycler (Eppendorf realplex.2s, Eppendorf Co., Ltd., Hamburger, Germany) using a thermostable Taq DNA polymerase (SYBR premix ex taq, TaKaRa Inc., Dalian, China). Experiments were done in triplicate. For each sample, the amounts of the target and an endogenous control (18sRNA) were determined. To obtain a normalized target value, the amount of the target molecule was divided by the amount of the endogenous reference.

#### *4.6. Western Blotting Analysis*

Forty-eight hours after transfection, HMC-1 cells were harvested and centrifuged, and total protein was extracted. Protein concentrations were determined using the BCA protein assay. After heated for 10 min at 100 °C, 20 g of denatured protein was subjected to 10% SDS-PAGE. Then proteins were transferred electrophoretically for 1 h at 200 mA at 4 °C onto PVDF membranes. Membranes were blocked for 1 h at room temperature in TBS containing 5% non-fat dry milk. Blots were washed 3 times for 10 min each with 0.1% TBS-T and subsequently treated overnight at 4 °C with primary antibodies against IL-13Rα1 and actin (1:500). After washing 3 times for 10 min each with 0.1% TBS-T, the blots were incubated with anti-mouse antibody (1:5000) conjugated with horseradish peroxidase for 1 h at room temperature. Bands were visualized by using EZ-ECL detection reagents. The scanned images were quantified using Quantity One software. Actin used as an endogenous protein for normalization. Experiments were done in duplicate. The ratio of IL-13Rα1/actin was used for semiquantification and comparison between different groups.

### *4.7. Statistical Analysis*

All data were analyzed by SPSS10.0 software (SPSS, Chicago, IL, USA, 2000). Microarray data were analyzed by Cluster Analysis. All data were represented by mean values ± standard deviation. P values less than 0.05 were considered statistically significant. Independent simple T test was used to compare the results of different groups.
