A total of 32 Chinese patients (mean age, 61.2 years) who had been diagnosed with locally advanced renal cell carcinoma by post surgery pathological examination at The People’s Hospital of Guangxi Zhuang Autonomous Region (Nanning, China) between 2010 and 2011 were included in this study. The TNM staging of cancer was performed according to the American Joint Committee on Cancer Cancer Staging Manual (10). The demographic and clinicopathological characteristics of the patients are listed in Table I. A routine physical examination was conducted and the immune function of the patients was detected prior to the surgery. No abnormalities were identified in the patients. All patients received a radical nephrectomy and post surgery adjuvant immunotherapy with IFN-α2b (Schering-Plough, Kenilworth, NJ, USA). The treatment protocol was performed according to the instructions in the guidelines of the Chinese Urological Association (11). Considering the toxic side-effects of IFN, the patients were advised to receive a low dose of IFN-α2b (3 MIU subcutaneously, three times weekly) treatment for 12 weeks. The patients were followed-up for 24 months, and the local relapse and metastasis rates were recorded.

The tumor samples from the 32 patients were collected as soon as they were removed from the body. Each tumor sample was divided into two sections. One section was ground and stimulated in RPMI-1640 medium (HyClone Laboratories, Inc., Logan, UT, USA) containing IFN-α2b (final concentration, 3 MIU/ml) for 4 h, and the other section was incubated in RPMI-1640 medium without IFN, which served as a control. The benign renal tissues were collected from 10 patients who underwent a nephrectomy as a result of nonmalignant disease. All samples were stored at −80°C. The present study was approved by the Ethics Committee of The People’s Hospital of Guangxi Zhuang Autonomous Region. Written consent was obtained from all patients.

The expression levels of JAK1, P-JAK1, STAT1, P-STAT1, STAT2 and P-STAT2 were detected by immunohistochemical staining of the renal cell carcinomas and benign renal tissues. The antibodies of P-JAK1, STAT2 and P-STAT2 were purchased from Abcam (Cambridge, UK). The antibodies of JAK1, STAT1 and P-STAT1 were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). The immunohistochemical staining and evaluation were performed as previously described (12). All sections were blindly analyzed by experienced pathologists. Briefly, the immunohistochemical analysis was performed after recoding the sections to create a blind set. The overall intensity and percentage were evaluated under a light microscope (Olympus, Tokyo, Japan). Briefly, the immunohistochemical results were categorized into four grades based on the percentage of positive cells and/or the staining intensity, which was identified by comparing the section with the positive controls in each experiment. The grades were as follows: (−), positive cells <5% of the cancer tissue and/or weakly stained; (+), <25% and/or weakly stained; (++), <50% and/or moderately stained; and (+++), >75% and/or strongly stained.

Total RNA was isolated from the tumor and benign tissues using TRIzol^® reagent (Invitrogen, Carlsbad, CA, USA). The RNA concentration was determined using a spectrophotometer (Bio-Rad Laboratories, Hercules, CA, USA). Complementary DNA was synthesized from 3 μg total RNA with a RevertAid First Strand cDNA Synthesis kit (Fermentas, Hanover, MD, USA) according to the manufacturer’s instructions. The qPCR assay was performed with 20 μl reaction mixture, and the PCR was conducted with 35 cycles of 94°C for 30 sec and 72°C for 1 min. β-actin served as an internal control. The PCR products underwent electrophoresis by 2% agarose gel analysis. The expression levels of the target genes were evaluated by calculating the average ratios of gray scale using a computerized gel imaging system (Bio-Rad Laboratories). The primers that were used for the amplification are listed in Table II.

The tumor samples, which had been stimulated with IFN, were lysed and homogenized (SanShon Machinery, Zhejiang, China) in radioimmunoprecipitation assay buffer and the control samples were treated in the same way. The protein concentration in the samples was subsequently determined. Equal amounts of protein were separated on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and transferred to a polyvinylidene difluoride membrane (Millipore, Billerica, MA, USA). The membrane was blocked with non-fat milk for 2 h and incubated with the primary antibodies overnight. Following washing with Tris-buffered saline and Tween 20 (TBST) for 30 min, the membrane was incubated for 1 h, washed with TBST and the proteins were detected with an enhanced chemiluminescence kit (Bio-Rad Laboratories). The protein expression levels were evaluated by calculating the average ratios of gray scale using a computerized gel imaging system; GAPDH served as the control.

The positive expression rates between the tumor and benign tissues were assessed with χ² test. The qPCR and western blot data were analyzed using Student’s t-test. P<0.05 was considered to indicate a statistically significant difference. The data were analyzed with SPSS software version 19.0 (IBM, Armonk, NY, USA).

Positive immunohistochemical staining was observed in the cytoplasm or cytomembranes of the renal tubules. The positive expression rates of JAK1, STAT1 and P-STAT1 in the renal cell carcinomas were significantly lower than those observed in the benign renal tissues (P<0.05). The expression rates of STAT2 in the renal cell carcinomas were elevated compared with those in the benign renal tissues (P<0.05). No differences in the expression rates of P-JAK1 and P-STAT2 between the renal cell carcinoma tissues and benign renal tissues were identified (Figs. 1–3; Table III).

The JAK1, STAT1 and STAT2 mRNA levels were detected in the renal cell carcinomas and benign renal tissues. The relative expression levels of JAK1 (0.696±0.102) and STAT1 (0.341±0.068) in the tumor tissues were lower than those in the benign tissues (0.957±0.103 and 0.547±0.082, respectively; P<0.05). No significant differences were identified in the relative mRNA expression levels of STAT2 (0.702±0.101 vs. 0.676±0.105) between the two types of tissue (Fig. 4).

To determine the active status of JAK-STAT in the tumor tissues following stimulation with IFN-α2b, the expression levels of JAK-STAT in the IFN stimulated and control tumor tissues were identified using western blot analysis. The expression levels of JAK1, P-JAK1, STAT1 and P-STAT1 were examined in the two types of tissues. The results showed that IFN stimulation significantly increased the expression levels of P-STAT1 (Figs. 5–7), however, not those of STAT1, JAK1 and P-JAK1 (P<0.05; Table IV). Enhanced expression levels of P-STAT1 were detected in 75% (24/32) of the samples, however, the levels varied markedly between the samples. Enhanced expression levels of P-STAT1 were not detected in 25% of the samples. The percentage of enhanced expression levels of P-STAT1 increased significantly when compared with those of the control (P<0.05).

The patients were followed up for 24 months after the immunotherapy with IFN. Clinical evaluation was performed by regular interviews, and relapse and metastasis of the tumors were diagnosed using enhanced computed tomography scanning and tissue biopsy analysis. The patients were divided into two groups (high and low expression) based on whether the enhancement of the P-STAT1 expression levels was above or below the mean value. The rates of relapse and metastasis were compared between the high and low expression level groups. All patients tolerated the immunotherapy well and completed their treatment courses. By the end of the follow-up period, nine patients suffered local relapse and lung metastasis was identified in one patient. The high expression levels group exhibited reduced relapse and metastatic rates compared with those of the low expression group (P<0.05; Table V).