2.1. Clinical tissue samples
37 cases of BDM patients at the Third Hospital of Nanchang City (Nanchang, China) were enrolled in the study. The control group included 50 age-matched breast cancer patients without type 2 diabetes mellitus(NBDM)at the same period, based on the oncologist confirmation and negative familial history of diabetes. All patients with primary breast cancer who underwent surgical resection from December 2012 to October 2016.The fresh tumor tissue specimens were collected and frozen in liquid nitrogen immediately and stored at -80˚C until needed. All the patients were female, and the postoperative histologically diagnosed with invasive breast cancer. After surgery, all enrolled patients were followed up every 3 months and the survival time was recorded. The cut-off for the follow-up period was December 31, 2019.
The baseline tumor characteristics are listed and compared between BDM group and NBDM group (Supplementary data, Table S1). This study was conducted in accordance with the Helsinki Declaration and was approved by the Ethics Review Committee of Third hospital of Nanchang. Written informed consent was obtained from all breast cancer patients in our study.
2.2. Cell culture
The MDA-MB-231 breast cancer-derived cells were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA) and maintained in Dulbecco’s Modified Eagle’s medium (DMEM, Gibco BRL, Grand Island, NY, USA) containing normal glucose (5.6 mM/L), supplemented with 10% fetal bovine serum (FBS; Gibco), 100 U/ml penicillin and 100 mg/ml streptomycin (Invitrogen, USA) at 37˚C in a humidified incubator under 5% CO2-95% air.
2.3. Plasmids, lentivirus production and transduction
miRZip-29a, an shRNA-miR-29a structure cloned in a SBI’s pGreenPur™ lentivector, was purchased from System Biosciences (Cat No, MZIP29a-PA-1; Palo Alto, CA), lentivirus used to stably overexpress miR-29a in breast cancer cells were purhased from Genechem Company (Shanghai, China),and the Scr control plasmid was a generous gift from Professor F-C Zhang at Ruijin Hospital of Shanghai Jiaotong University.
MiRZip-29a plasmid or Scr control plasmid was transfected into 293T cells by Lipofectin Reagent (Invitrogen) according to the manufacturer’s protocol. Pseudoviral particles were applied on MDA-MB-231cells. 24 hours later, Virus-infected cells were subjected to drug selection (1 µg/ml puromycin) for 7 days to achieve stable cell subclones. They were labeled as blank group, anti-miR29a and vector group.SIRT1 plasmid (5’-CCGGATTGAAGAATGTTGG-3’, 5’-ATCTGCTCCTTTGCCACTC T-3’) cloned into pcDNA3.1 vector to increase expression of SIRT1 in MDA-MB-231 cells(named as pc-SIRT1 and pcDNA3.1).After drug-selection, cells of normal conditions (5.6 mM/L glucose + 5nM/L insulin) or BDM (25 mM/L glucose + 25 nM/L insulin) conditions were cultured in vitro in different experiments.
2.4. TaqMan miRNA analysis
The RNA extracted from the tissues and cells using Trizol Reagent (Invitrogen, Carlsbad, CA, USA) in accordance with the manufacturer’s protocol. MiRNAs were detected by real-time reverse transcriptase polymerase chain reaction (qRT-PCR). The cDNAs were produced by reverse transcription kits (Takara, dalian, China) and miRNA specific bulg-loop ™ miRNA RT primers (Ribobio Co, guangzhou, China) (Supplementary data,Table S2). Real-time PCR was performed on a StepOnePlus Real-Time PCR System (Applied Biosystems, Carlsbad, CA, USA) using an SYBR Green I Real-Time PCR kit (TaKara, Dalian,China). Using the comparative delta CT (2−ΔΔCt) method to estimates the relative expression of miR-29a-3p. U6 small nuclear 2 (RNU6) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were used as endogenous control in this study.
2.5. Western blot
Proteins collected from the relevant cells are electrophoretic on a polyacrylamide gel and transferred to a PVDF membrane. Then, 5% fat free dry milk was used to seal the membrane in TBS-T for 1 hour. The primary antibodies were applied to the cell membrane at 4˚C overnight and then washed off with TBS-T. The membrane was then cleaned with TBS-Tween and incubated with a secondary antibody at 1:10,000. The Clarity Western ECL substrate (Bio-Rad, Hercules, CA, USA) was used for protein detection. The relevant data were analyzed using GraphPad Prism 5 software (GraphPad software, San Diego, CA, USA). Repeat Western blots at least three times.
For immunohistochemistry, paraffin sections were treated with primary antibody against SIRT1 (1:50 dilution, DF6033, Affinity Biosciences) at 4°C overnight. After incubating with secondary antibody and DAB substrate for visualization, the sections were subjected to fluorescence microscopy (CKX53, Olympus) for imaging.
2.7. Luciferase reporter assay
The wild-type 3’UTR of SIRT1 mRNA and the mutant type 3’UTR of SIRT1 mRNA (UGGUGCU to ACCACGA) were amplified and inserted into pmiRGLO vectors (Promega Corporation, Madison, WI, USA). The HEK-293T cells were co-transfected with the reporter plasmids and miR-29a-3p mimics or miR-NC using Lipofectamine 3000 (Invitrogen). After cells transfection at 48 h, the cells were collected and Renilla luciferase activity was detected by using the Dual Luciferase Reporter Assay System (Promega Corporation, Madison, WI, USA).
2.8. CCK-8 assays
The cells of different groups were seeded in 96-well plates and and cultured for 12, 24, 48 or 72 h .Cell proliferation was detected with Cell Counting Kit-8 (CCK-8) Kit (Dojindo Molecular Technologies, Inc., Kumamoto, Japan). reference the manufacturer' instructions.The absorbance at 450 nm was examined using a microplate reader (Bio-Rad, Berkeley, CA, USA).Triplicate independent experiments were performed.
2.9. Flow cytometry analysis
The cells were cultured in the DMEM medium with insulin (25 nmol/L) plus high glucose (25 mmol/L) for 48 h, then the cells were harvested and washed twice with PBS and fixed overnight at -20 ℃ with 70% ethanol. After washed with PBS, the cells were stained with 5 ul of propidium iodide solution (10 ug/ml) and 100 ul of RNase (100 ug/ml) in PBS and incubated for 30 min at room temperature. The analysis was performed using FACS Calibur (Becton Dickinson, Franklin Lakes, NJ) and CellQuest Pro software (Becton Dickenson).
2.10. Wound healing assay
In order to determine MDA-MB-231 cells migration, the different groups of those cells were seeded onto 6-well plates, and cultured in BDM condition per well overnight. Wounds were made by gently scraping with a disposable pipette tip, and those MDA-MB-231 cells were photographed using a phase contrast microscope (Olympus, Japan) at 0 and 48 hours. Distance migration was measured by calculating the area of the wound using Image J software and represented in the form of bar graph. All experiments in this study were repeated independently three times with similar results.
2.11. Invasion assay
Cell migration assays were performed in 24-well transwell chambers (8.0 µm; Corning Incorporated, Corning, NY, USA) to detect the migration and invasion of transfected cells. Every 200 µl of cell suspensions were pre-coated with 50 ml of 1.0 mg/ml Matrigel (Millipore, Billerica, MA,USA), which obtain from serum‑free media with the density of 2x104 cells/ml and seeded into the upper chamber of transwells. After, the chemo-attractant (600 µl of medium containing 10% serum) was added to the lower chamber with 24 hours of incubation. The cells on the upper side of the transwell were removed with a cotton swab which were fixed with 100% methanol (30 min), than staining using Crystal Violet Staining Solution (Beyotime Institute of Biotechnology)
2.12. Xenograft mouse model for breast cancer with type 2 Diabetes Mellitus
All female BALB/c mouse with 4-week-old which were purchased from the Animal Research Center of Shanghai Jiaotong University.The mouse were fed ad libitum a high-fat diet (HFD) containing 60 kcal % fat for 10 weeks and acclimated to it for at least 7 days before the experiment. After 2 weeks of HFD treatment, the mouse was intraperitoneally injected for 5 consecutive day with small dose of 40 mg/kg streptozotocin (STZ) (Sigma-Aldrich, Beijing, China) dissolved in 100 µl of 0.05 M citrate buffer (pH 4.5). The STZ solution is prepared a few minutes before use. 3 days after STZ administration,the random blood glucose of mice was detected.The model of T2DM mice was established successfully if the blood glucose was more than 16.7mmol/l for two days.
MDA-MB-231 cells with Scr control plasmid or MiRZip-29a plasmid were washed with DPBS and resuspended in Matrigel (10 mg/mL). All mouse received s.c. injections at the fourth mammary fat pad with 0.1 mL of cell suspension (5×105 cells/mL). The mouse were sacrificed 8 weeks after inoculation. As the Tumor volumes formula: volume = 0.5LS2, which were assessed by measuring the longest (L) and shortest (S) diameter of the tumor under a microscope. The number of tumor nodules in lung was also counted after 8 weeks. Each tissue was excised and embedded in paraffin for histopathological examination. The schematic diagram of animal model construction process was shown in Fig. 1. This animal study was approved by Ethics Committee for animal experimental research of Third hospital of Nanchang.
2.13. Statistical analyses
T test, mann-whitney U test, bidirectional analysis of variance and correlation analysis were used to compare the differences between variables in this study. The Kaplan–Meier analysis was performed to compare Disease-specific Survival (DSS) and Recurrence-free Survival (RFS) between the groups. P < 0.05 was considered statistically significant. All analyses were performed using SPSS. The mean ± SD is displayed in the figures.