Ethical approval and Informed consent statements
Written informed consent was obtained from 104 participants before the study. The clinical protocol for this study was approved by the Medical Research Ethics Committee of Xiangya Hospital, Central South University (CSU) (#201403216). All mice experiments were approved by the Animal Ethics Committee and conducted by the official recommendations for the Care and Use Laboratory Animals of Xiangya Hospital, CSU (#201403217).
Tissue specimens
A total of 104 fresh NACLC tumor specimens were archived by radical resection from those NSCLC patients without any chemotherapy and radiotherapy intervention at the Department of Thoracic Surgery of Xiangya Hospital from January 2010 to December 2011. The tumor-node-metastasis was classified according to the criteria of the 8th AJCC/UICC (American Joint Committee on Cancer/International Union Against Cancer) TNM staging system. There were 29 cases in Stage I, 33 cases in Stage II and 42 cases in Stage III. All freshly collected NSCLC tumor tissues were frozen and stored in liquid nitrogen until required. All surgical resection samples were evaluated by a board-certified pathologist.
Cell culture
The human NSCLC cell lines A549 and H1299 (ATCC, Manassas, VA, USA) were cultured in RPMI 1640 (Gibco, Gaithersburg, VA, USA) supplemented with 10% fetal bovin serum (FBS) and penicillin/streptomycin and then incubated at 37 °C in a humidified 5% CO2 atmosphere.
RNA extraction and qRT-PCR analyses
Total RNA from cultured cells was extracted using the TRIzol reagent (Invitrogen, Thermo Fisher Scientific, USA) according to the manufacturer’s manual. The expression level was quantified using SYBR Green Assay Kit (Takara, Japan). Real-time PCR was performed in triplicate using the Applied Biosystems ViiA 7 Sequence Detection system (Life technologies, CA, USA). GAPDH was used to normalize mRNA and calculate the relative expression of each transcript. The following pairs of primers were synthesized as: C-Myc (F): 5′-CCTCCACTCGGAAGGACTATC-3′, (R): 5′-TTCGCCTCTTGACATTCTCC-3′; GAPDH (F): 5′-ACCCACTCCTCCACCTTTGACG-3′, (R): 5′- TCTCTTCCTCTT GTGCTCTTG-3′.
Immunohistochemistry (IHC)
IHC analysis was performed on 4 µm-thick formalin-fixed and paraffin-embedded NSCLC tissue sections from each tumor sample, using the PV-60001/PV-6002 Two-Step IHC Detection Reagent (ZhongShan Golden Bridge Bio, Beijing, China) according to the manufacturer’s protocol. In brief, antigen retrieval was performed in buffer for 10 min in a microwave oven. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide for 20 min at room temperature. Slides were incubated with
the following primary antibodies, anti-SH2B1 (1:100, ab196575, Abcam), anti-Akt (1:500, ab8805, Abcam), anti-Akt (phospho S473) (1:200, ab8932, Abcam), anti-mTOR (1:1000, ab2732, Abcam), anti-mTOR (phosphor S2448) (1:50, ab109268, Abcam), anti-PTEN (1:100, ab31392, Abcam, Cambridge, MA, USA), anti-Ki67 antibody (1:200, Cell signaling, Danvers, MA, USA) at 4 °C overnight. Following washing with PBS, slides were incubated with HRP-labeled secondary antibody for 50 min at room temperature. Immunoreactive products were visualized with 3,3′-diaminobenzidine tetrahydrochloride (DAB) and counterstained with Mayer’s hematoxylin. For negative controls, slides were treated with 0.01 mol/L PBS instead of primary antibodies under the same conditions.
The IHC staining scores were determined by combining the intensity of staining and the proportions of positively stained cells. The standard of staining intensity was graded that: 1, no staining; 2, weak staining (light yellow); 3, moderate staining (yellow brown); and 4, strong staining (brown). The positive cell proportions were scored according to the following standard: 0, < 5% positive cell; 1, 6–25% positive cells; 2, 26–50% positive cells; 3, 51–75% positive cells; 4, 76–100% positive cells. We evaluated protein expression using staining index (SI), which was calculated of the proportion of positive cells and the staining intensity score as possible total scores of 0, 1, 2, 3, 4, 5, 6, 7, and 8. Samples with SI ≤ 4 were defined as low (negative) expression and SI > 4 as high (positive) expression. The degree of SI was reviewed and scored separately by two independent pathologists.
Xenografted tumor model and H&E staining
Male BALB/c-nu mice (age of 4–5 weeks, 16–18 g) were purchased from the Hunan Slack King of Laboratory Animal Co., Ltd (Changsha, China). Mice were randomly divided into groups (n = 6 per group) before injection. A total of 1 × 107 cells were subcutaneously injected into the left dorsal flank per mouse. Measure tumors length and width and then calculate tumor volumes (L × W2/2) with calipers on alternate days. On day 30, the mice were euthanized and tumors were excised to paraffin embedded. Serial 4.0 μm sections were cut and subjected to haematoxylin and eosin (H&E) staining and IHC analyzed using an anti-Ki67 (1:200, Cell signaling, Danvers, MA, USA). The proportion of Ki67 positive cells were counted as proliferation index. All mice experiments were approved by the Animal Ethics Committee and conducted by the official recommendations for the Care and Use Laboratory Animals of Xiangya Hospital, CSU (#201403217).
Western blotting
Whole-cell lysates were prepared from human NSCLC cell lines after harvesting and centrifuging. Protein concentrations were quantified using BCA kit (Beyotime Bio, Shanghai, China). The proteins were denatured, electrophoresed (SDS-PAGE gel Kit, Beyotime Bio, Shanghai, China), and then transferred to PVDF membranes (Millipore Co., MA, USA). Subsequently, the PVDF membranes were blocked with non-fat milk powder, incubated with primary and secondary (HRP-labeled goat anti-rabbit IgG) antibodies, and visualized (Bio-Rad Image Lab Software).
Western blotting was performed using the primary antibodies, anti-SH2B1 (1:1000, ab196575, Abcam), anti-Akt (1:600, ab8805, Abcam), anti-Akt (phospho S473) (1:300, ab8932, Abcam), anti-mTOR (1:1500, ab2732, Abcam), anti-mTOR (phosphor S2448) (1:1000, ab109268, Abcam), anti-PTEN (1:600, ab31392, Abcam), anti-C-Myc (1:1000, ab32072, Abcam), antibodies (Abcam, Cambridge, MA, USA), and anti-GAPDH antiboty (1:3000. D110016, Sangon Biotech, Shanghai, China). Following the initial western blotting assay, the membranes were stripped and the band intensities were relative to GAPDH. The quantification of protein bands was performed using Image J software.
Cell proliferation assay
2 × 103 cells were seeded and cultured in triplicates in 96 well plates. Cell viability was measured every day over a 5-day period using cell count kit-8 (CCK8, #CK04, Dojindo Molecular Technologies, Rockville, MD 20850, USA). Cells were incubated with 10 μl CCK8 for 1 h at 37 °C, and absorbance was measured at 450 nm using the Elx808 Absorbance Microplate Reader (BioTek Instruments, USA).
Colony formation assays
Cells were suspended in 0.6% agarose and placed in 24-well plates at low density (2.5 × 102 cells per plate). After 2 weeks, the colonies were fixed with methanol and counted.
Cell cycle analysis
Cells were trypsinized, collected and rinsed twice with PBS and fixed with cold absolute alcohol at 4 °C overnight. After fixation, cells were incubated with 0.5 ml PBS, 5 μl propidium iodide (20×) and 10 μl RNase A (50×) for 30 min at 37 °C without light before analysis. The cell cycle was analyzed by a flow cytometry (BD, San Jose, CA, USA).
Flow cytometry detection
Cells were collected and stained using the FITC Annexin V Apoptosis Detection Kit I (BD Pharmingen, San Diego, CA, USA). Cell apoptosis were quantified using a flow cytometry (BD, San Jose, CA, USA).
Gene knockdown and gene overexpression
shRNA against SH2B1 and control shRNA were constructed into the lentiviral vector pHY-LV-KD5.1 (GFP-lentivirus, Hanyin Co. Shanghai, China). The recombinant SH2B1-shRNA lentivirus (target sequence: 5′-TAACAACCAGTACTCCTTCGTGTGAGC-3′) and the negative control lentivirus (target sequence: 5′-TTCTCCGAACGTGTCACGT -3′) were prepared and titered to 109 TU/ml (transfection unit). To obtained a stable SH2B1-knockdown cell line, 2 × 105 A549 cells seeded in per well of six-well dishes. The cells were then infected with the same titer virus with 8 μg/ml polybrene on the following day. Approximately 72 h after viral infection, the culture medium was replaced with selection medium containing 4 μg/ml puromycin after GFP expression was confirmed under a fluorescence microscope. The cells were cultured for at least 14 days. The puromycin-resistant cell clones were isolated, amplified in medium containing 2 μg/ml puromycin for 7–9 days, and then transferred to a medium without puromycin. The clones were designated shSH2B1 (knock down) or shCtrl (negative control) cells.
Lentiviral vectors for human SH2B1 expression sequence were constructed by Hanyin Co. (Shanghai, China). The cDNA of SH2B1 with a C-terminal Flag-tag were cloned into the lentiviral vector pHY-LV-OE1.6. Stable cell lines expressing SH2B1 were generated by retroviral infection using H1299 cells, and selected with 2 μg/ml puromycin for 7–9 days. Further details of methods were carried out as gene knockdown described. The clones were designated H1299-SH2B1 (overexpression) or H1299-vector (negative control) cells. Transfection was conducted using Lipofectamine 2000 (Invitrogen, Grand Island, NY, USA) according to the manufacturer’s instructions [10].
Statistical data analysis
All statistical analyses in this study were carried out using SPSS 22.0 statistical software. Data were shown as mean ± SD, and 2-tailed paired Student’s t-test was used for comparing the difference between groups unless otherwise stated. P < 0.05 was considered statistically significant.
