Involvement of miR-141-3p dysregulation and its targets genes in trastuzumab resistance of HER2-positive breast cancer cells
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Mina Makki,1 Valentina Gambacorta,2 Kazem Dastjerdi,3,*
1. 1. Department of medical biotechnology, Faculty of Medicine, Birjand University of Medical Sciences.
2. 3. Unit of Senescence in Stem Cell Aging, Differentiation and Cancer, San Raffaele Telethon Institute for Gene Therapy, IRCCS San Raffaele Scientific Institute,
3. 2. Cellular and molecular research center, Birjand University of medical sciences,
- Introduction: One of the most prevalent malignancy in the world is breast cancer which has led to high mortality rate in women. Trastuzumab is one of the most effective drugs for targeted therapy of breast cancer (BC). The response of HER2-positive BC to trastuzumab in early stages is approximately up to 50%. After one year of treatment, several patients show resistance to trastuzumab. In order to increase the therapeutic effectiveness of patients, it is also important to study the molecular mechanisms causing trastuzumab resistance. Studying MicroRNAs (miRNAs) are regarded in cancer prognosis and treatment. Based on the targets, miRNAs can act as either an oncogene or a tumor suppressor. miRNAs dysregulation have been reported in a variety of cancers. Numerous studies have shown that dysregulation of microRNA expression is associated with cancer invasion, metastases, and chemotherapy resistance. According to previous studies, the mechanism of resistance to treatment may be regulated by changing in miRNA expression. Latest experiments have shown that microRNA-141 is concerned in breast tumor cell proliferation and metastases. In current study, we performed the prediction and verification of miR-141-3p target genes including IGF1R and PTEN and the expression of these genes in trastuzumab-resistant cell line compared with sensitive cell line as control sample.
- Methods: Cell culture and generation of trastuzumab-resistant cells:
BT-474 cells were cultured in supplemented media using a humidified incubator at 37 °C. Generation of trastuzumab-resistant cells (BT-474-R) was performed by exposing culture of BT-474 cells to trastuzumab for 6 months. Then, trastuzumab-resistant and sensitive BT-474 cells were cultured with and without trastuzumab, respectively.
Cell survival assay:
Cell survival was measured by MTT assay. Cells were seeded in each well of a 96-well plate and incubated for 24 h. Then, trastuzumab was added and incubation continued for 72 h. After removing the medium MTT solution was added and incubated for 4 h at 37 °C. After addition of
DMSO, agitation was done for 45 min at room temperature and the absorbance values were
read at 570 nm. Cell survival was measured by following formula: survival percentage= (absorbance of drug-treated wells – blank wells)/(absorbance of untreated wells – blank wells)· 100. IC50 values was calculated by the ED50 Plus v1.0 online software.
RNA extraction and quantitative real-time PCR:
Total RNA was extracted from BT-474 and BT-474-R cells. The isolated RNA samples were reverse-transcribed into double-stranded cDNA. The samples were subjected to quantitative PCR (qPCR) in the StepOne Real-Time PCR System. GAPDH was used as internal control. Relative abundance of detected transcripts was calculated using the 2−ΔΔCt method. The experiments were performed in triplicate.
Prediction of target genes:
TargetScan and miRDB were used for prediction of target genes. Related pathway to target genes was predicted Using KEGG pathway analysis.
- Results: Trastuzumab-resistant breast cancer cells display
survival over parental cells:
The parental cells showed a significant reduction in cell proliferation in comparison to resistant cells (p < 0.01). At the highest dose of trastuzumab the percentage of cell viability for resistant cells was about 80%, whereas it was about 44% for sensitive cells. The results suggest that trastuzumab-resistant HER2-positive breast cancer cells exhibit proliferation advantages over parental cells.
Expression status of the tow selected genes
detected by qRT-PCR:
Using qRT-PCR the expression level of target genes including IGF1R and PTEN were evaluated in resistant and sensitive BT-474 cells. The expression of both genes were significantly downregulated in BT-474-R cells compare with sensitive cells.
- Conclusion: In this study, we found that the development of resistance to trastuzumab may be related to the downregulation of PTEN that is related with AKT/PI3K pathway. The results suggest that downregulation of PTEN induces senescence and consequently secretion of SASP factors. It causes progression and invasion of cancer cells. Increasing the activation of AKT/PI3K pathway as a results of PTEN depletion causes resistant to trastuzumab in BT-474 cell line. Our results emphasize on the importance to identify proper biomarkers and molecular targets to improve personalized medicine.
- Keywords: miR-141-3p, AKT/PI3K pathway, HER2-positive breast cancer, trastuzumab resistance
