Patients and blood samples collection
Ten biopsy-proven patients with invasive primary breast cancer treated with TA regimen at Chongqing University Cancer Hospital from April 2017 to October 2018 were enrolled in this study. The clinicopathological characteristics of the patients are summarized in Figure 1A. Whole blood was collected before any treatments, frozen in liquid nitrogen immediately, and preserved at -80℃ until used. The clinical response was assessed according to RECIST criteria. Thus, patients with complete remission (CR) or partial remission (PR) were regarded as clinical responders, while patients with stable disease (SD) or progressive disease (PD) were regarded as non-responders for further analysis. The present study was approved by the Ethics Committee of Chongqing University Cancer Hospital, and all patients signed informed consent.
Total RNA was quantified by the NanoDrop 2000 and RNA integrity was assessed by standard denaturing agarose gel electrophoresis. The sample preparation and microarray hybridization were performed based on the manufacturer’s standard protocols. The microarray data were selected by threshold values of >2 and < 2-fold change under FDR protection (P < 0.05).
The breast cancer cell lines MCF-7 and MCF-7/ADR (adriamycin-resistant) were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The cells were cultured in DMEM (Gibco, Suzhou, China) supplemented with 10% heat-inactivated fetal bovine serum (Zhejiang TianHang Biotechnology Co. LTD, Hangzhou, China) 100 units/ml penicillin and 100 μg/ml streptomycin (KeyGen Biotech, Jiangsu, China) in a humidified atmosphere at 37 °C under 5% CO2. The MCF-7/ADR cells were cultured in the above-mentioned media that were additionally supplemented with 1 mg/mL ADR (KeyGen Biotech, Nanjing, China) to maintain the drug-resistant phenotype.
LncRNA CTB-104H12.5 small interfering RNAs (si-122, si-1365. si-2484), negative control (si-control), LncRNA CTB-104H12.5 vector (CTB-104H12.5) and control vector (Control) were purchased from RiboBio (Guangzhou, China). The cells were transfected with Lipofectamine® 2000 (Invitrogen, USA) according to the manufacturer's instructions. Cells were harvested for further evaluation at 48 h after transfection. The sequence of siRNA as followed: si-control, 5’-GAGAAACAATTAAAAATACCC-3’, si-122, 5’-CTCAAAAAAAAGAAGTGGGACACT-3’, si-1365, 5’- TTGGCCCCAAGCATACAGCCCCCTA-3’, si-2484, 5’- TGAGCTATGATTGTACCACT-3’
The viability ability and ADR chemosensitivity were detected by CCK8 assay (Biotool, Shanghai, China) according to the manufacturer’s instructions. Briefly, MCF-7 and MCF-7/ADR cells were plated at the density of 1×104 cells per well in 96-well plates and incubated with 100 µl anticancer drugs adriamycin (KeyGen Biotech, Nanjing, China) at a final concentration of (0.02, 0.1, 0.5, 1, 2.5, 5, 10) ug/ml and (1, 2.5, 5, 10, 20, 40, 60) ug/ml for 48 h, respectively. Each well was added with a 10µl CCK-8 working solution and incubated for another one hour. The absorbance was tested at a wavelength of 450nm on the synergy2 microplate reader (BioTek, NV, USA). The IC50 values were calculated with these absorbance data. Each sample was performed in triplicate and repeated three independent experiments.
Colony formation assay
A colony formation assays were conducted to evaluate cell proliferation capability. Briefly, cells (500 cells/well) were seeded into 6-well plates and transfected with siRNA or CBT 104H12.5 vectors. Then, the plates were incubated for 8 days and the medium was refreshed every two days. Cells were fixed in 10% formaldehyde for 40min and stained with a 0.1% crystal viola solution for 20min. Finally, the clone number was counted under a microscope.
Apoptosis and cell cycle analysis
Twenty-four hours after the transfection as described above, ADR was added, with the final concentration of 10 mg/mL for MCF-7/ADR and 1 mg/mL for MCF-7. After incubation for 24 hours, cells were harvested and washed twice with cooled PBS and stained with FITC Annexin V and PI. Cell apoptosis was measured by a FITC Annexin V apoptosis detection kit (KeyGen Biotech, China) while cells were stained by PI for cell cycle analysis according to the manufacturer’s protocols. Analyses were conducted by FACS flow cytometer (BD Biosciences, San Jose, CA, USA).
RNA isolation and qRT-PCR assay
Total RNA was extracted from whole blood or cells using the Trizol reagent (TianGEN, Beijing, China) according to the manufacturer’s instructions. Reverse transcription was performed with PrimeScript RT-PCR Kit (Takara Bio companies, Beijing, China) following the manufacturer’s protocols. The qRT-PCR analysis was conducted with the SYBR Green PCR master mix (QIAGEN, Germany). The RNA amount of target genes was quantified by the 2-ΔΔCT method, and GAPDH used as an internal control. Primer sequences are listed as followed. Each PCR assay was performed on Applied Biosystems 7500 for triplicates. GAPDH: Forward 5’-TGTTCGTCATGGGTGTGAA-3’, Reverse 5’-ATGGCATGGACTGTGGTCAT-3’, RP11-53O19.: Forward, 5’-CATGGTGACTGACCCTTGAGC-3’, Reverse 5’-TCGGGACTACAGGAAGAGTGGAA-3’, RP11-264F23.3, Forward, 5’-CATGCAGCCATCCATTTGTG-3’, Reverse 5’-GGTCTGATAGCCCAGCCAAG-3’, RP11-414K1.3, Forward 5’-GGAAACGGCAAGCAGAAGAAT-3’, Reverse 5’-GGCTGGGACACTGGAAACATA-3’, CHEK1, Forward 5’-GGAAAGCATTTGTCTCCCACC-3’, Reverse 5’-TAAATCACAATCGCCACTCCC-3’, CTB-104H12.5, Forward 5’-CCTCCTTTGTCCCATAACCTG-3’, Reverse 5’-CGTCCCTTTGAATGTCTGTCC-3’, ARHGEF26, Forward 5’-ATGGAGATGAAGGATGGGAGAT-3’, Reverse 5’-AGCGGACAGTAGTTGGGACAT-3’, GAS7C, Forward 5’- CGAGCTACGTGCAGTTGCT-3’, 5’-CATGTGGGCAGTCTCTGGAG-3’
Western blot analysis.
Cells were harvested and suspended in RIPA buffer (TianGEN, Beijing, China) and quantified with a BCA protein assay kit (Boster, Wuhan, China). After denatured by boiling for 5 minutes, total protein samples (10μg each sample) were separated with 10% SDS-PAGE and subsequently transferred into the PVDF membrane (EMD Millipore, Billerica, MA, USA). Then the membranes were blocked by 5% non-fat milk for 1 h at 37℃, incubated overnight at 4°C with following primary antibodies: anti-CDK1, anti-Cyclin D1, anti-Caspase-3, anti-Bcl-2 (Cell Signaling Technology, USA), anti-GAS7C (Invitrogen, USA) and anti-GAPDH (Santa Cruz, USA). And Horseradish peroxidase-conjugated anti-mouse, anti-rabbit antibodies were used as secondary antibodies. The bands were visualized by LICOR C-digit (Biocompare, USA) and quantified by ImageJ. Each experiment was conducted at least three times.
All data are displayed as mean ± standard deviation (SD) and analyzed by using SPSS 17.0 software (SPSS, Chicago, IL, USA). The Student’s t-test was used to evaluate the significance of differences in multiple comparisons. The statistical significance was defined as *P < 0.05, **P< 0.01 or ***P< 0.001. All experiments were performed at least three times.