Twenty-one patients with malignant disease admitted to Zhongshan Hospital of Fudan University between August 2011 and December 2013 were enrolled in the present study. Diagnoses of malignant disease were confirmed by postoperative pathological examinations. Twenty-three patients undergoing surgery for benign abdominal diseases served as the control group. Exclusion criteria were patients with acute or chronic renal and liver failure, acute and chronic hepatitis, diabetes, metabolic acidosis, sepsis, AIDS, inflammatory bowel disease, autoimmune disorders, chronic heart failure, hyperthyroidism and chronic obstructive pulmonary disease. Written informed consent was obtained from all patients, and permission for the present study was obtained from the Ethics Committee of the Zhongshan Hospital, Fudan University.

The nutritional assessments of patients before surgery included height, actual body weight, body mass index (BMI), total lymphocyte count, serum albumin and prealbumin. A biopsy specimen was obtained from the rectus abdominis muscles according to the methods described by Bossola et al (21). The biopsy specimen was immediately frozen in liquid nitrogen and then stored at −70°C until further analysis. No complications occurred during the biopsy procedure.

BALB/c male mice (6–8 week of age) weighing 16–20 g were purchased from the Shanghai Laboratory Animal Center, Chinese Academy of Sciences. Mice were housed at 22±1°C, with a 12-h light/dark cycle, free access to water and conventional diet. Mice were acclimated to the environment for one week before the study started. All animal manipulations were carried out according to the guidelines of regulations for the use of experimental animals of the Chinese Academy of Science. All efforts were made to minimize animal suffering and to use only the number of animals necessary to produce reliable scientific data.

Colon 26/clone 20 cells, which have been reported to induce severe cachexia in BALB/c mice by subcutaneous inoculation, were purchased from the Shanghai Institute of Pharmaceutical Industry. The cells were cultured in RPMI-1640 supplemented with 5% fetal bovine serum and 1% penicillin-streptomycin at 37°C in 5% CO₂. On study day 0, 1.0×10⁶ cells suspended in 100 ml phosphate-buffered saline (PBS) were injected subcutaneously into the armpits of the mice in the tumor group. An equal volume of PBS without tumor cells was injected into the control group mice. The body weight of each mouse was recorded every two days after tumor cell inoculation. On day 16, the mice were sacrificed via cervical dislocation. Non-tumor body weights and gastrocnemius muscle weights were recorded. Muscle specimens were frozen at −70°C for further analysis.

Mouse skeletal muscle C2C12 cells were propagated and maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with antibiotics and 10% fetal bovine serum at 37°C in the presence of 5% CO₂. To induce differentiation, C2C12 cells were propagated in DMEM supplemented with antibiotics and 2% horse serum for the indicated period. After 3 days of differentiation, according to a previous report (22), C2C12 cells were treated with 10 ng/ml recombinant mouse TNF-α (R&D Systems, USA) for 96 h to extract proteins, and C2C12 cells were treated with 6 ng/ml recombinant mouse TNF-α for 2 h to extract RNAs.

Five pairs of siRNAs targeting the human atrogin-1 gene (Table I) were designed using Ambion siRNA Target Finder and were synthesized by Invitrogen Biotechnology Co., Ltd. (Shanghai, China). In addition, negative control siRNA was also synthesized for monitoring the influence of the exogenous genes. These siRNAs were cloned into vector pBS-hU6-I and then into vector FG12. The length and accuracy of the sequences were confirmed by DNA sequencing. The recombinant FG12 vectors were cotransfected with pRSVREV, pMDLg/pRRE and pHCMV-G into 293T cells to package lentivirus particles, with which C2C12 cells were infected. The infected C2C12 cells were cultured and differentiated to form myotubes before TNF-α was added to induce atrophy. Atrogin-1 mRNA and protein were identified by real-time PCR (RT-qPCR) and western blotting, respectively. Myotube morphology was observed and photographed directly in the culture plate without fixation.

Total proteins were extracted from cells or muscle tissues and quantified by the BCA method. Western blotting was performed to determine the protein expression of atrogin-1. The protocol used for western blotting was previously described (23). Briefly, 50 mg total proteins was separated by SDS-PAGE, transferred to PVDF membranes, and analyzed by western blotting with a goat polyclonal anti-human atrogin-1 antibody (Santa Cruz Biotechnology, USA). An antibody against α-tubulin (Santa Cruz Biotechnology) served as an endogenous control. Finally, the membrane was visualized by an imaging system in the dark to quantify the protein level.

Total RNAs were isolated from the cells or tissues with TRIzol (Invitrogen, Carlsbad, CA, USA). RNA integrity was detected by an agarose gel method, and the RNA concentration was quantified by measuring A260 and A280 absorbance in a NanoDrop spectrophotometer (Thermo Scientific, USA). cDNA synthesis was carried out with 2 μg of total RNAs by reverse transcription (RT) (Promega, Madison, WI, USA). RT-qPCR was performed using 7900HT Fast Real-Time PCR System (Applied Biosystems, Inc., Foster City, CA, USA). Real-time Master Mix (Toyobo, Osaka, Japan) was used to detect and quantify the expression levels of the target genes. The PCR reaction consisted of an initial denaturation at 95°C for 10 min followed by 40 cycles of 30 sec at 95°C, 10 sec at 60°C and 30 sec at 72°C, and finally 10 min at 72°C. Amplification of the target cDNA was normalized to β-actin expression. Relative levels of target mRNA expression were calculated using the 2^−ΔΔCt method. The primers used are listed in Table II.

Immunohistochemical staining (IHC) of the tissues was performed as previously described (23). Briefly, muscle tissues were fixed in 4% formalin for 24 h and paraffin-embedded using a conventional method. Sections (3-μm) were dehydrated in xylene and graded alcohols. Antigen retrieval was performed with 0.01 M citrate buffer at pH 6.0 at 95°C for 20 min. The slides were then incubated with atrogin-1 antibody at 4°C overnight and then with the fluorescein isothiocyanate (FITC)-conjugated secondary antibodies for 60 min at room temperature in the dark.

Statistical analysis was performed using GraphPad Prism 5.0 software (GraphPad Software, USA). Continuous variables were compared using the Student’s t-test and are expressed as the mean and standard deviation (SD). P<0.05 was considered to indicate a statistically significant result.

The clinical characteristics of the patients with malignant disease and benign disease are shown in Table III. Nutrition statuses such as BMI, total lymphocyte count, serum albumin and pre-albumin were significantly different in the two groups (P<0.05), while other characteristics showed no significant differences.

The mRNA levels of atrogin-1 and MuRF-1 in the rectus abdominis muscles were analyzed by RT-qPCR. Considering the mean ΔCt of the benign disease group as control, we determined the ΔCt of atrogin-1 and MuRF-1 in the malignant disease group. As shown in Fig. 1A and B, atrogin-1 and MuRF-1 mRNA levels were increased in 66.7% (14/21) and 76.2% (16/21) of the malignant disease patients, respectively. Univariate analysis indicated that the atrogin-1 and MuRF-1 mRNA levels were significantly highly expressed in the malignant disease patients (P<0.05, data not shown). In addition, to determine whether the expression of atrogin-1 and MuRF-1 mRNA increased before weight loss, we compared the mRNA levels of atrogin-1 and MuRF-1 in 16 malignant disease patients and 23 benign disease patients without weight loss. The results showed that both atrogin-1 and MuRF-1 mRNA levels tended to increase in the malignant disease patients but without statistical difference (P=0.099, P=0.132, respectively, Fig. 1C and D).

Subcutaneous tumors could be touched by hand four days after colon-26 adenocarcinoma cell inoculation and reached ~8 cm³ when the mice were sacrificed. As shown in Fig. 2A, the whole body weight of the tumor-bearing mice increased slowly at the beginning yet had a rapid growth afterwards due to the rapid growth of the subcutaneous tumors. Non-tumor body weight and gastrocnemius muscle weight of the tumor-bearing mice were significantly lower than the control group (Fig. 2B and C), indicating that cancer cachexia was effectively induced in this model.

We determined the expression of four ubiquitin proteasome pathway-associated genes (atrogin-1, MuRF-1, ubiquitin and E2-14K) in the muscles of colon-26-induced cachexia mice. Compared to the non-tumor mice, atrogin-1, MuRF-1, ubiquitin and E2-14K mRNA levels were significantly increased in the cachexia mice (P<0.05, Fig. 3A). Western blotting and IHC confirmed that the atrogin-1 protein was overexpressed in the cachexia mice (Fig. 3B and C).

The accuracy of the recombinant plasmids was confirmed by DNA sequencing. Two correct recombinant plasmids PBSU6I-atrogin-1 siRNA-1 and -2 were chosen for further analysis, while three incorrect recombinant plasmids PBSU6I-atrogin-1 siRNA-3, -4 and -5 were discarded. After transfection with the recombinant lentivirus vector expressing atrogin-1 siRNA-1 and -2, atrogin-1 was downregulated in the C2C12 cells. However, only atrogin-1 siRNA-1 was identified to be the most effective siRNA in inhibiting atrogin-1 expression. As shown in Fig. 4, atrogin-1 was downregulated in the C2C12 cells transfected with atrogin-1 siRNA-1 and -2 compared to the non-specific siRNA control, yet only siRNA-1 significantly downregulated the expression of atrogin-1 (P<0.05). Thus, we chose siRNA-1 for subsequent study.

After treatment with 10 ng/ml TNF-α, the C2C12 cells grew slowly and myotubes atrophied with a significantly decreased diameter (Fig. 5A and B). Atrogin-1 was significantly increased at the mRNA and protein levels after treatment with 6 ng/ml TNF-α for 2 h and 10 ng/ml TNF-α for 96 h, respectively (Fig. 5C and D).

To investigate whether inhibition of atrogin-l affects myotube atrophy induced by TNF-α, we compared the myotube morphology and myotube diameter in the C2C12 cells in the atrogin-1 siRNA-1 group and the non-specific siRNA control group. No differences were observed in the two groups before incubation with TNF-α. However, after treatment with TNF-α, the myotubes underwent atrophy with a significant decrease in myotube diameter in the control group, while no obvious changes were observed in the atrogin-1 siRNA-1 group (Fig. 6).