A total of 83 patients with CRC were enrolled in the present study at the Affiliated Hospital of Nantong University (Nantong, China) from March 2009 to February 2010. A total of 39 females and 44 males were included in the present study, ranging from 25–65 years, with a mean age of 57 years. Specimens of tumor tissues and adjacent paracancerous histological normal tissues (PCHNTs) were collected during surgery. The PCHNT specimens were extracted >3 cm from the tumor margin and assessed microscopically for the presence of normal cells and the absence of dysplastic cells. Following surgical removal, the fresh specimens were divided into two equal halves. One half was fixed in formalin, embedded in paraffin, and used for immunohistochemical staining. The other half was preserved at −80°C after freezing in liquid nitrogen for further analysis by western blotting and RT-qPCR. Clinicopathological data were obtained, and the tumor-node-metastasis (TNM) stages and lymph node status were determined. The stages and grades of tumors were determined according to the criteria listed in the fifth edition of TNM classification of the International Union Against Cancer (21). None of the patients with CRC had received preoperative radiotherapy or chemotherapy prior to surgery. Following surgery, all patients received standard treatments according to the National Comprehensive Cancer Network guidelines (22). The present study was approved by the Ethics Committee of the Affiliated Hospital of Nantong University. All experiments were performed in accordance with the approved guidelines of the Affiliated Hospital of Nantong University and the 1964 Helsinki Declaration and its later amendments. Written informed consent was obtained from all participants prior to undergoing surgery.

The paraffin-embedded tissue sections (~2 mm), were dewaxed in xylene, rehydrated in a series of ethanol (70% for 30 min, 80% for 60 min, 90% for 60 min, 95% for 60 min, 95% for 60 min, 100% for 60 min and 100% for 60 min) and immersed in 3% hydrogen peroxide at room temperature for 5 min to suppress the endogenous peroxidase activity. The tissues sections were heated at 100°C for 10 min in 0.01 mol/l sodium citrate buffer (pH 6.0) to facilitate antigen retrieval. After three washes with phosphate-buffered saline (PBS) for 5 min each, the sections were incubated at room temperature for 2 h with a polyclonal rabbit anti-human GRK6 primary antibody (cat. no. sc-566; 1:200; Abcam, Cambridge, UK) diluted in PBS. The anti-GRK6 monoclonal antibody was used to determine the expression of the GRK6 protein. The sections were washed three times in PBS for 5 min each, and incubated with a HRP-conjugated goat anti-rabbit secondary immunoglobulin G (cat. no. SA00004-6; 1:200; ProteinTech Group, Inc., Chicago, IL, USA) for 1 h at room temperature. After three additional washes with PBS, the antigen-antibody complexes were developed with diaminobenzidine (DAB, 20 µl + PBS, 1,000 µl + 3% H₂O₂, 5 µl) staining at room temperature for 10 min.

A total of three observers determined the immunohistochemical staining scores (ISS), which is based on staining frequency and intensity. Staining frequency was scored as follows: No staining was scored as 0; 1–25% of stained cells was scored as 1; 25–50%, as 2; 51–75%, as 3; and >75%, as 4. Staining intensity was rated on a scale of 0–3 as follows: 0, negative; 1, weak; 2, moderate; and 3, strong. The raw data were converted into ISS by multiplying the corresponding frequency scores and the staining intensity scores. Therefore, the ISS could range from 0 to 12. An ISS of 9–12 was designated as strong immunoreactivity (+++); 5–8, as moderate (++); 1–4, as weak (+); and 0, as negative (−) immunoreactivity. The sections with staining in the cell cytoplasm of >25% tumor cells were deemed as positive for GRK6. Staining was scored independently by three individuals. Based on the results of immunohistochemical staining for GRK6 expression, patients with CRC were divided into two groups, one with low GRK6 expression (− to +) and another with high GRK6 expression (++ to +++).

Total protein was extracted using a lysis buffer (Thermo Fisher Scientific, Inc., Waltham, MA, USA) that contains protease inhibitors. A total of 30 µg of protein were separated by 10% SDS-PAGE gel electrophoresis and transferred to polyvinylidene fluoride (PVDF) membranes. Non-specific binding was blocked by incubating the membranes with 5% non-fat milk in Tris-buffered saline containing 0.1% Tween-20 (TBST) for 2 h at room temperature. Following incubation with the polyclonal rabbit anti-human GRK6 antibody (dilution 1:1,000; cat. no. sc-566; Abcam), or rabbit anti-GAPDH antibody (dilution 1:2,000; cat. no. SAB2701826; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany), overnight at 4°C, the membranes were washed three times with TBST for 5 min. The membranes were then incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (dilution 1:1,000; Sigma-Aldrich; Merck KGaA) at room temperature for 2 h. The signals were detected using enhanced chemiluminescence (GE Healthcare, Chicago, IL, USA) followed by film development.

In addition, 19 other pairs of samples of cancerous tissues and matched non-cancerous fresh frozen tissues were collected for reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis. Similarly, western blot analysis was performed on 19 other pairs of cancerous tissues and matched non-cancerous fresh frozen tissues, by the aforementioned method.

GRK6 mRNA expression was analyzed by RT-qPCR (23). Total RNA was extracted using TRIzol^® reagent (Gibco; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions from the CRC tissues. Reverse transcription was performed at 42°C for 60 min followed by 70°C for 5 min using the Transcriptor First Strand cDNA Synthesis kit (Roche Diagnostics GmbH, Mannheim, Germany). RT-qPCR was performed using FastStart Universal SYBR-Green Master (Rox; Roche Diagnostics GmbH). According to the protocol, 2 µl cDNA was added to a 20 µl final reaction volume. The RT-qPCR analysis was performed in 96-well plates in a thermocycler (ABI Prism 7500; Applied Biosystems; Thermo Fisher Scientific, Inc.). The cycling parameters used were as follows: Hot start at 95°C for 10 min, 40 cycles of amplification, quantification at 95°C for 15 sec, and 57°C for 1 min, during which time fluorescence was measured, at 72°C for 30 sec. Continuous fluorescence acquisition was performed at 65–97°C in order to perform melting curve analysis. These cycling parameters generated a single amplification for the primer set used, according to the presence of a single melt peak. GAPDH was selected as the internal reference. All RT-qPCR reactions were repeated 3 times for each gene, and each sample was set up in triplicate. Primer sequences were designed based on published human gene sequences. The primer sequences were as follows: GRK6 forward primer, 5′-AAAACACCTTCAGGCAATACCG-3′; and reverse primer, 5′-AGGCCAAGCTCACTACAAACCTA-3′. GAPDH forward primer, 5′-CATGAGAAGTATGACAACAGCCT-3′; and GAPDH reverse primer, 5′-AGTCCTTCCACGATACCAAAGT-3′. The 2^−ΔΔCq method was used to calculate relative changes in gene expression (24).

SPSS statistical software (version 17.0; SPSS Inc., Chicago, IL, USA) was used for data analyses. The GRK6 mRNA levels in CRC and PCHNT specimens were compared using the paired Student's t-test. Student's t-test for independent samples was performed to compare the means of two groups. The χ² test was used to determine the significance of GRK6 expression and the clinicopathological variables of CRC. Kaplan-Meier analysis was used to compute survival rates, and statistical significance was assessed by using the log-rank test. A univariate analysis with the Cox regression model was used to determine the identified prognostic factors, and multivariate analysis with the Cox regression model was used to investigate the combined effects. Spearman's rank correlation analysis was used to analysis the association between GRK6 expression and TNM stage in colorectal carcinoma. The results are presented as mean ± standard deviation of a minimum of three independent experiments. P<0.05 was considered to indicate a statistically significant difference (25). Furthermore, the survival rates of patients based on GRK6 positivity were analyzed by separating the patients into two groups based on the stage of disease: I–II and III–IV.

The results of the immunohistochemical analysis demonstrated that GRK6 expression was significantly increased in patients with CRC compared with normal CRC tissues (Fig. 1). GRK6 expression was observed in 70 of 83 patients with CRC (84.3%); 33 patients demonstrated strong expression (33/70×100=47.1%; Fig. 1A), and 37 demonstrated weak expression (37/70×100=52.9%; Fig. 1B). In the majority of the PCHNT and non-cancerous specimens, negative or weakly positive immunostaining of GRK6 in the cytoplasmic region was observed (Fig. 1C and D). GRK6 expression was significantly higher in carcinoma tissues compared with PCHNT and non-cancerous tissues (P<0.05; Table I). However, there were no statistically significant differences in GRK6 expression between normal colorectal tissues and PCHNT specimens (P=0.289).

The expression of GRK6 protein was examined by western blotting in 19 pairs of randomly selected specimens of CRC tissues and their matched normal colorectal tissues. The representative results of western blotting in 8 cases are presented in Fig. 2A. The expression levels of GRK6 protein were markedly increased in tumor tissues compared with adjacent non-cancerous tissues (Fig. 2B). Therefore, GRK6 may be a potential candidate for the regulation of CRC initiation and progression.

The aforementioned results were further confirmed by RT-qPCR analysis. GRK6 mRNA expression was examined in 19 CRC tissues and corresponding PCHNT specimens, which were randomly selected. GRK6 expression was significantly upregulated in samples of CRC tissues compared with PCHNT specimens (P<0.05; Fig. 2C).

The association between GRK6 expression and clinicopathological factors in patients with CRC is summarized in Table II. The overexpression of GRK6 was significantly associated with histological differentiation, lymph node metastasis, venous invasion, depth of invasion, distant metastasis, and TNM stages (P=0.001, P=0.045, P=0.009, P=0.026, P<0.0001, P=0.020, respectively) in patients with CRC. However, no significant associations were observed between GRK6 expression and age, sex, tumor size and growth patterns in patients with CRC. In addition, no significant association was observed between the levels of preoperative carcinoembryonic antigen (CEA) (P=0.705), CA19-9 (P=0.443) and GRK6 expression. The results demonstrated that GRK6 expression in CRC specimens was positively associated with TNM stage, which indicated that advanced TNM stages corresponded to increased GRK6 expression in CRC (r_s=0.467, P<0.01; Table III).

The survival statuses of 83 patients with CRC were evaluated by Kaplan-Meier survival curves and the log-rank test at the end of clinical follow-up. Based on the results of immunohistochemical staining for GRK6 expression, patients with CRC were divided into two groups, namely, the patient group with low GRK6 expression (− to +) and the patient group with high GRK6 expression (++ to +++). There were 50 patients in the group with low levels of GRK6 expression, of which 11 succumbed to disease. The 5-year overall survival rate was 78%. There were 33 patients with high levels of GRK6 expression, of which 22 succumbed to disease. The 5-year overall survival rate was 33.3%. Therefore, the overall survival of patients with CRC and low GRK6 expression was significantly longer compared with patients with CRC with high GRK6 expression (Fig. 3A).

Furthermore, the survival rates of patients based on GRK6 positivity were analyzed by separating the patients into two groups based on the stage of disease: I–II and III–IV. In patients with stage I–II disease, there were 36 patients with low GRK6 expression (− to +), of which 6 succumbed to disease, and there were 17 patients with high GRK6 expression (++ to +++), of which 7 succumbed to disease. In patients with stage III–IV disease there were 14 patients with low GRK6 expression, of which 5 succumbed to disease. There were 16 patients in the high GRK6 expression group, of which 15 succumbed to disease. The overall survival of the group with high GRK6 expression was significantly shorter compared with the group with low GRK6 expression, irrespective of the differences in the stage of disease (Fig. 3B and C).

To evaluate the independent prognostic value of GRK6 expression on overall survival in patients with CRC, multivariate analysis was performed to evaluate the prognostic factors in the present study cohort using a Cox proportional hazard model (Table IV). In these patients, TNM stages, lymph node metastases, histological differentiation, tumor invasion, GRK6 expression, and the levels of CEA and CA19-9 were associated with poor prognosis (P<0.001, P=0.023, P<0.001, P=0.021, P=0.003, P=0.001, P=0.034, respectively).