Cell Culture
The breast cancer cell line MCF-7, still maintaining mammary epithelium properties, including estrogen in the cell cytoplasm. MCF-7 cells are less aggressive and non-invasive, resulting in a versatile xenograft therapy response model for early and advanced ER + breast tumor development [13–15]. In the current investigation, the MCF-7 cell line has been provided from the Iran National Cell Bank (Pasteur institution, Tehran, Iran). This cell line was cultivated at 37 ° C in the 10% fetal bovine serum RPMI-1640 medium (Gibco), including streptomycin, and penicillin antibiotics (100 µg / mL and 100 IU / mL respectively), providing 5% CO2 and 95% humidity. The cells were isolated with Trypsin-EDTA (0.25 percent) and cultivated each 24–48 h with a 70–80 percent confluence of cell monolayers.
MicroRNA transfection
MicroRNA 34a was transfected into MCF-7 cells at different doses ( 40, 60, 80 and 100 pmol) using (Bio-Rad) Gene Pulser electroporation system, according to supplied protocols. Transfection was performed with a total volume of 1×106 MCF-7 cells per 500 mL electroporation buffer in voltage of 150 v for 12 ms. Then, 6×105 transfected MCF-7 cells were cultured into each well of 6-well plate, then followed up for 24, 48, and 72 hours. After harvesting the cells for evaluating optimized time and dose of drug and miRNA, RNA extraction was done, and the qRT-PCR was assessed the expression amount.
Extraction of RNA and quantitative real-time PCR
The Trizol RNA isolation package (GeneAll, Korea) was applied to separate the whole RNA following the producer's instructions. To measure the concentration and quantity of the extracted RNA, nano Drop spectrophotometer (Thermo Fisher Scientific Life Sciences) was used at A260/A280 nm. For cDNA synthesis, purified RNA (1µg) was used with RT Master Mix in the Bio-Rad T100 thermal cycler. To demonstrate the amounts of mRNA expression of miR-34a-5p, P-53, Bax, Caspase-3, MDR1, and Bcl-2 quantitative real-time PCR was utilized, and as a reference gene, GAPDH was used (Table 1). With the StepOnePlus Real-Time PCR Device and the BioFACT 2X RealTime PCR Master Mix, the reactions were performed triplicated. To assess the relevant quantitation presentation of genes levels, the 2 − ΔΔCt procedure was used.
Table 1
Genes Name | Primers | Sequence of primers |
miR-34a-5p | Target sequence | 5´ UGGCAGUGUCUUAGCUGGUUGU 3´ |
GAPDH | F R | 5’- CAAGATCATCACCAATGCCT − 3’ 5’- CCCATCACGCCACAGTTTCC-3’ |
P53 | F R | 5´ ACTTGTCATGGCGACTGTCC 3´ 5´ CACCCCTCAGACACACAGGT 3´ |
Bax | F R | 5′-GACTCCCCCCGAGAGGTCTT-3′ 5′-ACAGGGCCTTGAGCACCAGTT-3′ |
Bcl-2 | F R | 5´ CCTGTGGATGACTGAGTACC 3' 5´ GAGACAGCCAGGAGAAATCA 3´ |
Caspase-3 | F R | 5´ TGTCATCTCGCTCTGGTACG 3´ 5´ AAATGACCCCTTCATCACCA 3´ |
MDR1 | F R | 5′CCATCATTGCAATAGCAGG3′ 5′ GAGCATACATATGTTCAAACTTC 3′ |
Cell Viability Assay
The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) tetrazolium decrement assay has been used to detect cell survival with (Sigma-Aldrich, St. Louis, MO) kit [16]. First, the MTT assay was used to test the carboplatin IC50 value. After obtaining the IC50 amount for carboplatin, the cell transfection was performed subsequently with miR-34a and treated with varying amounts of carboplatin. Thus, various doses of carboplatin including 0.25 to 100 µM (Mylan, Saint-Priest, France) were applied to every electroporated well after 24 hours of transfection. Tests were carried out in triplicate.
Statistical analysis
All quantitative analyses have been carried out by Graph Pad Prism edition 6.00 (GraphPad, San Diego, CA), except for annexin V / PI assay. All concepts were stated as mean ± SD, and triple trials (n = 3) were conducted. Student's t test and variance analyzis were applied to assess the statistical significance of the discrepancies between groups. A p < .05 was seen as demonstrating a statically important discrepancy.