MiR‐548 K regulatory effect on the ABCG2 gene expression in MDR breast cancer cells

Abstract Background multidrug resistance (MDR) is One of the foremost challenges in overcoming breast cancer. Various molecular processes are involved in the development of MDR in breast cancer cells, including over expression of ABC transporters such as ABCG2 (BCRP), increase breast cancer stem cells drug resistance, and epithelial mesenchymal transition. Aims In the present study, we used bioinformatics and experimental analysis to investigate the role of miR‐548 K, in the modulating of ABCG2, in MDR breast cancer cells. Methods and Results In silico inspections introduce 14 microRNAs targeting 3′‐UTR region of ABCG2 transcripts, which are probably involved in breast cancer drug resistance. An association was highlighted between miR‐548 k with ABC transporter family. The expression level of ABCG2 gene in MCF7‐MX cell lines was significantly more than MCF7 cell lines. On the other hand, we increased the expression of miR‐548 K in MCF7‐MX and MCF7 cell lines through its transfection, which dramatically coincided with decreasion in the ABCG2 transcripts level. Additional studies on patient samples revealed that the expression of ABCG2 showed an increase in ABCG2 level in neoadjuvant chemotherapy drugs resistance (NCDR) patients compared to primary pre‐operative chemotherapy drugs response (PCDR) patients. Also, a reduction in the expression of miR‐548 K in NCDR patients was revealed. Conclusion The results of our study suggest that miR‐548 K may be involved in modulating the expression of ABCG2 in MDR breast cancer cells.


| INTRODUCTION
Breast cancer is known as the most common cancer among women. This condition is a multifactorial disease which various factors involved in its development. 1 Genetic, epigenetic, environmental, and lifestyle major factors involved in cancer development. Genomic mutations, chromosomal instabilities, genomic imprinting, and non-coding RNAs are the most significant genetic and epigenetic factors in tumorigenesis. 2,3 Cancer cells acquire various properties that cause cell growth and proliferation as well as resistance to stresses in later stages. Some principal problems in cancer treatment are the transformation of cancer cells and adaptation to the conditions and stresses imposed on them. Multi-drug resistance (MDR) is a challenge in overcoming cancer cells. In breast cancer, chemotherapy is a treatment strategy employed before surgery to limit the tumor cells (neoadjuvant therapy) or after surgery to prevent the recurring growth of cancer cells (adjuvant therapy). Drug resistance may be observed at any of these steps. Various mechanisms are involved in the development of drug resistance; the most well-known is the overexpression of drug transporters like the ATP-binding cassette (ABC), which are located on the cytoplasmic membrane. 4 ABC transporters have similar transmembrane domains that can pump out the chemotherapy drugs in an energy-dependent ATP manner from cancer cells. ATP-binding cassette, subfamily G, member 2 (ABCG2), also known as breast cancer resistance protein (BCRP), is a member of the drug transporter family whose role in MDR is known. 5 It was shown that in the later stages of breast cancer, which is associated with drug resistance, ABCG2 gene expression increases, which leads to a more frequent appearance of this protein on the cell membrane. This rise in the level of ABCG2 protein helps pump out the chemotherapy drugs from the cancer cells thereby causing drug resistance. Although ABCG2 role in the development of drug resistance in cancer cells is known, the regulatory mechanisms that enhance the expression of this protein in cancer cells and create a growth advantage have not yet been identified. [6][7][8][9][10] MicroRNAs are small non-coding RNAs that have been shown to play a role in various cellular and pathogenic processes. Changes in the miRNAs expression patterns in different diseases, including cancer, are tracable; this feature introduces the miRNAs as a potential biomarker. 11,12 Recent studies have shown the role of microRNAs in cancer drug resistance (CDR). So far, many miRNAs have been reported to be involved in regulating the expression of the ABC transporter family genes. 13,14 MiR-548 k is one of the most effective miRNAs in regulating the expression of genes involved in breast cancer and has been suggested to be possibly involved in ABC transporters -dependent MDR. 4 In the present study, we aimed to investigate the role of miR-548 k on ABCG2 gene expression in breast cancer cells.    For miR-548 K cDNA synthesis, miRNAs polyadenylation was done and subjected to reverse transcription using BON-miR 1st-strand cDNA synthesis kit and then QRT-PCR was performed using BON-miR QPCR (Stem Cell Technology, Iran), according to the manufacturer's protocol. Small Nucleolar RNA, C/D Box 47 (SNORD47) was used as an internal control for data normalization. The Pfaffl method was used to report fold changes. 15

| Statistical analysis
The REST 2009 software (Qiagen, Hilden, Germany) was used for relative expression analysis.The quantities of mRNA and miRNA in the tissues or cell lines were standardized to the GAPDH mRNA and F I G U R E 1 MicroRNAs and breast cancer. MicroRNAs that are probably involved in breast cancer drugs resistance mediated by ABCG2 and miR-548 k connection with other members of the ABC transporter family. The Cytoscape software was used to make the visualization of the network (A). The has-mi-R548k expression in breast tissue according to the GTEX database (B). (www.gtexportal.org); the microRNAs that did not have expression in the breast tissue were excluded from the study. In the next step, we checked the filtered microRNAs in terms of their expression in breast tissue, in the published documents, and in terms of their role in drug resistance in breast cancer. Fourteen microRNAs were determined, which may probably be involved in breast cancer drug resistance mediated by ABCG2 (Fig 1A). One of these microRNAs is miR-548 k which has three targets site in positions 494-500, 2736-2742, and 5197-5203 of ABCG2 3'-UTR region ( Figure 1B) (Table 1). An association was deciphered between miR-548 k and some other members of ABC transporter family that involved in drug resistance ( Figure 1A).

| Increased miR -548 k expression is associated with decreased ABCG2 gene expression
The statistical analyses revealed that the expression of ABCG2 gene in MCF7-MX was 34.876 (P < .0001) fold greater than in MCF7 cell lines. Also, it was highlighted that the expressions of ABCG2 gene in treatment mimic MCF7 cell lines was 0.407 (P = 0.003) fold and in MCF7-MX was 0.573 (P = 0.001) fold fewer in comparison with treatment scrambles respectively (Fig 2A).
On the other hand, a miR-548 k expression in treatment mimic MCF7 cell line was 7.359 fold (P = 0.002) and in MCF7-MX cell lines was 3.046 (P = 0.003) times more than treatment scrambles respectively ( Fig 2B). on patient samples showed that the expression of ABCG2 elevated in F I G U R E 3 ABCG2 gene expression in NCDR and PCDR patients. ABCG2 gene expression was more (P = 0.002; ** pronouncedly significant) in tumor tissue in comparison with adjacent normal tissue in NCDR patients (n = 10)(A). The changes of ABCG2 gene expression in tumor tissue was not significant than adjacent normal tissue in PCDR patients (n = 9)(B).

F I G U R E 4
MiR-548 k expression in NCDR and PCDR patients. miR-548 k expression was lower (P = 0.002; ** pronouncedly significant) in tumor tissue in comparison with adjacent normal tissue in NCDR patients (n = 10)(A). miR-548 k expression in tumor tissue was more (P = 0.019; * significant) than adjacent normal tissue in PCDR patients (n = 9)(B).
NCDR patients, compared with PCDR patients. Also, a decline in the expression of miR-548 K in NCDR patients was observed.
Previous studies confirmed the elevation in ABCG2 gene and pro-  20 and on the other hand, reducing its expression can lead to the occurrence of drug resistance. 4 Considering the results of enhanced expression of miR-548 K due to its transfection in drug-resistant cancer cells, which led to a decline in the expression of ABCG2 gene in these cells, as well as the results of miR-548 K and ABCG2 transcript alignment revealing 3 binding site for miR-548 K on ABCG2 mRNA 3 0 UTR, the role of miR-548 k in regulating ABCG2 gene expression was highlighted. One of the probable functions of miR-548 k is binding to the ABCG2 gene transcript and directing it to the cleavage pathway, similar to what happens with miR-519c. But this possibility requires investigation on more samples by more specific techniques to investigate the possible interaction of miR-548 k, but unfortunately, in this study, we were limited in terms of conducting complementary studies and more functional assays. Overall, miR-548 k may has the potential to be investigated as a therapeutic and pharmaceutical target for ABCG2-related MDRs, and future studies with a large study population and functional assay technics will be beneficial.

| CONCLUSION
The results of our study suggested that miR-548 K may be involved in