TCGA database analysis
The clinical information of each patients and the transcriptome expression datasets were downloaded from The Cancer Genome Atlas (http://cancergenome.nih.gov). 1. 1104 cancer and 113 normal tissues were used for analyzing the HINT3 expression in BRCA tissues and normal tissues. 2. 114 normal, 183 stage I, 615 stage II, 247 stage III and 20 stage IV BRCA tissues were used for analyzing the correlation between HINT3 expression and BRCA stage. 3. 114 normal, 516 N0, 362 N1, 120 N2 and 77 N3 BRCA tissues were used for analyzing the relationship between HINT3 expression and nodal metastasis status.
Human breast cancer cell lines MCF7 and MDA-MB-231 were purchased from ATCC. These cells were cultured in (Hyclone) medium, which was supplied with 10% fetal bovine serum (Gbico) and 1% penicillin and streptomycin solution (Hyclone). The cell culture was maintained at 37℃ with 5% CO2.
Lentivirus-mediated HINT3 knockdown and overexpression
The lentivirus vector was used to knock down HINT3 in MCF7 and MDA-MB-231 cells. This system composes three vectors: pGCSIL-GFP (inserted with the targeted shRNA), pHelper1.0 (gag/pol) and Helper2.0 (VSVG). The sequences of the shRNA were as follow: negative control, 5’-TTCTCCGAACGTGTCACGT-3’; HINT3#1, 5’-GCGAGAATGAGGACCTAATTT-3’ HINT3#2, 5’-GAGTCAATTCCTATTGGTTTA-3’. After cloning the shRNA into the pGCSIL-GFP vectors, the vectors were co-transfected with pHelper1.0 and Helper2.0 into 293FT cells using Lipofectamine 2000 (Invitrogen). Viral supernatants were harvested, filtered and centrifuged for subsequent infection. Knockdown efficiency was detected by qRT-PCR and Western blot assays.
The protein from shCtrl, shHINT3#1 and shHINT3#2, Ctrl and HINT3 overexpressed MCF7 and MDA-MB-231 cells were lysed using lysis buffer (Beyotime). BCA protein assay kit (Beyotime) was performed to detect the protein amount. The proteins were loaded on SDS-PAGE gels for separation and transferring onto the PVDF membranes. Subsequently, the membranes were blocked by 5% non-fat milk for 60 minutes at room temperature and incubated with indicated primary antibodies at 4℃ overnight. Antibody against HINT3 was from Abcam. Antibodies against β-actin and the secondary antibodies were from Proteintech.
shCtrl, shHINT3#1 and shHINT3#2, Ctrl and HINT3 overexpressed MCF7 and MDA-MB-231 cells were lysed using Trizol (Invitrogen). Total RNA was extracted using Ultrapure RNA Kit from CWBIO, according to the manufacturer’s protocols. After reversely transcribing the RNA to cDNA, qPCR reaction was conducted on the Biorad machine with SYBR master mixture. The sequences of qPCR primers are as follows: HINT3 forward, 5’-CTGGTTGAGAACATGGTAACT-3’, and reverse, 5’-TGATCAGCTGTGATAAACCAAT-3’; and GAPDH forward, 5’-TGACTTCAACAGCGACACCCA-3’, and reverse, 5’- CACCCTGTTGCTGTAGCCAAA-3. GAPDH is the internal control.
Equal number of shCtrl, shHINT3#1, shHINT3#2, Ctrl and HINT3 overexpressed MCF7 and MDA-MB-231 cells were seed into 96-well plates, which contained 200ul culture medium. 1, 2, 3 and 4 days later, 20ul of CCK regent was added into each well and incubated at 37oC for 3 hours. The optical density (OD) was detected at 450 nm on the micro-plate reader.
Equal number of shCtrl, shHINT3#1, shHINT3#2, Ctrl and HINT3 overexpressed MCF7 and MDA-MB-231 cells were seed into the 6-well plates. After cultured for 10 days, the colonies were fixed by methanol and stained by Giemsa solution. The images of the colonies were taken with the camera and the numbers of the colonies were counted using Photoshop CS5.
8.0-μm filter migration chambers (BD Biosciences, USA) was applied to detect the cell migration of shCtrl, shHINT3#1 and shHINT3#2 MCF7 and MDA-MB-231 cells. In brief, equal number of the cells were seeded onto the upper surface of the chambers. After incubating at 37°C for 24 hours, the cells on the upper surface were removed by cotton tips. The cells on the lower surface were fixed by methanol and stained by crystal violet. The stained cells were photographed using the microscope.
Dual luciferase activity reporter assay
The promoter sequence of PTEN was inserted into pGL3.basic reporter vectors. The coding sequence of HINT3 was inserted into pCDNA3.1 vectors. MCF7 cells were transfected with pGL3.basic, TK and siRNAs or pCDNA3.1 vectors. Luciferase activity was measured using Dual luciferase reporter kit (Promega), following manufacturer’s protocols.
In vivo xenograft assay
Balb/c nude mice (14-16 g per mice) were purchased from Animal Center of Beilun District People's Hospital and were fed with standard diet and water in a specific pathogen-free room. The temperature was maintained at 25°C and the room light was kept with 12-h light/12-h dark cycles. Ten mice were randomly divided into two groups (n=5 in each group). A total of 3*107 MDA-MB-231 cells were subcutaneously implanted into the right armpit of 5-week old female balb/c nude mice. The volume of the xenogrfted tumors was calculated using the following formula: v=ab2/2 (a, the long diameter, b, the short diameter). The mice were sacrificed by peritoneal injecting with 0.4 mg/g chloral hydrate per mice at 45 days after implantation. Animal experiments were approved by the Ethics Committee of and were performed according to the animal guideline of Beilun District People's Hospital.
The statistical analysis was conducted using GraphPad prism. The data were shown as mean ± standard error of mean (SEM). Students’t tests, one-way or two-way analysis of variance (ANOVA) were used to determine the statistical significance. Statistical difference was considered significant when P<0.05.