Introduction
MicroRNAs (miRNAs) can function as post-transcriptional regulators. It has been reported that the tissue-specific deregulation of miRNAs is correlated with several human diseases, such as cancer [1, 19]. Numerous studies have investigated the potential of miRNA expression profiles as biomarkers of cancer response to treatment, diagnosis, and prognosis [6]. Many studies have shown that miR-17-5p and miR-93-5p are important regulatory molecules for some biological behaviors, including cell proliferation processes that are important in cancer development [2, 12]. 
Nowadays, researchers investigate the change in the expression of miRNAs in the tissues and fluids of cancer patients compared to the control group.
 In this research, we focus on evaluating miR-17-5p and miR-93-5p expression levels in tissues and plasma of patients with breast ductal carcinoma compared with the control group.
Materials and Methods
In total, 180 samples consisting of 80 plasma and 100 breast tissues were examined in this project. The samples were taken from the patients referred to Asia Hospital, Tehran City, Iran (2017-2018). The expression levels of miR-17-5p and miR-93-5p were measured by SYBR Green-based real-time RT-PCR assay in plasma and breast tissues of cancerous patients compared with control. 
The total RNA (including miRNA) was extracted from breast tumor tissue samples and plasma. BON-miR cDNA kits were used for microRNA’s reverse transcription. The expression levels of miRNAs were quantified using SYBR Green-based real-time RT-PCR.
The expression level was calculated by the 2-∆∆CT method. The data were presented as the fold change in gene expression normalized to an endogenous reference gene relative to the controls.
Results
As shown in Figure 1, the expression of miR-17-5p increased in breast tumors and plasma of breast cancer patients compared to the normal control group. In contrast, the expression of miR-93-5p in breast cancer tumors and plasma decreased (Figure 2). A positive correlation between the expression levels of miR-17-5p and miR-93-5p in plasma and tumor samples was confirmed by the Spearman test.
As summarized in Table 1, the data revealed a statistically significant association between the expression level of miR-17-5p in the tissue and plasma of breast cancer patients with lymph node metastasis, advanced stages of the tumor, and the status of ER (estrogen receptor) and PR (progesterone receptor).


Also, the expression level of miR-93-5p in the tissue and plasma of breast cancer patients was significantly reduced in lymph nodal involvement and advanced stages of breast cancer (P<0.001).
Discussion
 Breast cancer is the leading cause of cancer mortality in women worldwide [20, 21]. In recent years, many studies have reported that miRNAs have key roles in providing sensitive mechanisms for precise regulation of gene expression [22, 23, 24].
In many studies, abnormal expression of the miR-17-92 family, especially miR-17-5p, has been reported to be associated with the spread of tumor growth in many cancers, including colorectal cancer, esophageal squamous cell carcinoma, hepatocellular carcinoma, hematopoietic malignancies, breast cancer, lung cancer, glioblastoma, and neuroblastoma by targeting cell cycle control [9, 25].
It was reported that miR-93-5p (miR-93) significantly inhibited cell proliferation and induced G1/S cell cycle arrest. Moreover, two well-established oncogenes, E2F1 and CCND1 were identified as dual targets of miR-93 [34].
In the present study, miR-17-5p showed significantly higher expression in tissues and plasma of the cancer group compared with the control group (P<0.0001), which was significantly associated with tumor stage and lymph node, ER, and PR status (P<0.0001). In contrast, decreased expression of miR-93-5p in plasma and tumor tissues was shown to be significantly associated with tumor stage and lymph node involvement (P<0.0001).
Our data revealed that upregulation of miR-17-5p and down-regulation of miR-93-5p in both plasma and breast tumor might be associated with poor prognosis in breast cancer. However, miR-17-5p, due to the greater difference in expression and ease of plasma detection, may serve as a possible non-invasive biomarker for breast cancer’s poor prognosis. Further follow-up studies are required to confirm this finding.

Ethical Considerations
Compliance with ethical guidelines
This study was approved by the ethics committee of institute, (IRAN 52d/4922, 6.10.2016). All individuals included in the study signed a consent form to use their clinical samples and personal data under the supervision of their physician.

Funding
This project was supported by the National Institute of Genetic Engineering and Biotechnology (NIGEB).

Authors' contributions
Implementation and collection of data and writing the draft of the article: Andia Sidi Moghadam; Designing the idea of the project, supervising the implementation, analyzing the results and finalizing the article: Mahdieh Salimi; Scientific advice: Najmeh Ranji and Hossein Mazdarani; Assistance in providing clinical samples: Hossein Mazdarani; Design, sample collection and research writing: all authors.

Conflicts of interest
The authors declared no conflict of interest.

Acknowledgements
We sincerely thank Dr. Parisa Aziminejadan for her kind support and all the individuals who participated in this study. 


References

1. O’Brien J, Hayder H, Zayed Y, Peng C. Overview of microRNA biogenesis, mechanisms of actions, and circulation. Front Endocrinol (Lausanne). 2018; 9:402.[DOI:10.3389/fendo.2018.00402] [PMID] [PMCID]
2. Peng Y, Croce CM. The role of microRNAs in human cancer. Signal Transduct Target Ther. 2016; 1:15004. [DOI:10.1038/sigtrans.2015.4] [PMID] [PMCID]
3. Shamsizadeh S, Goliaei S, Razaghi Moghadam Z. CAMIRADA: Cancer microRNA association discovery algorithm, a case study on breast cancer. J Biomed Inform. 2019; 94:103180.[DOI:10.1016/j.jbi.2019.103180] [PMID]
4. Zhang B, Pan X, Cobb GP, Anderson TA. MicroRNAs as oncogenes and tumor suppressors. Dev Biol. 2007; 302(1):1-12. [DOI:10.1016/j.ydbio.2006.08.028] [PMID]
5. Svoronos AA, Engelman DM, Slack FJ. OncomiR or Tumor Suppressor? The duplicity of microRNAs in cancer. Cancer Res. 2016; 76(13):3666-70. [DOI:10.1158/0008-5472.CAN-16-0359] [PMID] [PMCID]
6. Mohammadi E, Aminorroaya A, Fattahi N, Azadnajafabad S, Rezaei N, Farzi N, et al. Epidemiologic pattern of cancers in Iran; current knowledge and future perspective. J Diabetes Metab Disord. 2020; 20(1):825-9. [DOI:10.1007/s40200-020-00654-6] [PMID] [PMCID]
7. Filipów S, Łaczmański Ł. Blood circulating miRNAs as cancer biomarkers for diagnosis and surgical treatment response. Front Genet. 2019; 10:169. [DOI:10.3389/fgene.2019.00169] [PMID] [PMCID]
8. Bai X, Hua S, Zhang J, Xu S. The microRNA family both in normal development and in different diseases: The miR-17-92 Cluster. Biomed Res Int. 2019; 2019:9450240. [DOI:10.1155/2019/9450240] [PMID] [PMCID]
9. Liu F, Zhang F, Li X, Liu Q, Liu W, Song P, et al. Prognostic role of miR-17-92 family in human cancers: Evaluation of multiple prognostic outcomes. Oncotarget. 2017; 8(40):69125-38. [DOI:10.18632/oncotarget.19096] [PMID] [PMCID]
10. Chen Ch, Lu Z, Yang J, Hao W, Qin Y, Wang H, et al. MiR-17-5p promotes cancer cell proliferation and tumorigenesis in nasopharyngeal carcinoma by targeting p21. Cancer Med. 2016; 5(12):3489-99. [DOI:10.1002/cam4.863] [PMID] [PMCID]
11. Li Zh, Peng Zh, Gu S, Zheng J, Feng D, Qin Q, et al. Global analysis of miRNA-mRNA interaction network in breast cancer with brain metastasis. Anticancer Res. 2017; 37(8):4455-68. [DOI:10.21873/anticanres.11841]
12. Bobbili MR, Mader RM, Grillari J, Dellago H. OncomiR-17-5p: Alarm signal in cancer? Oncotarget. 2017; 8(41):71206-22. [DOI:10.18632/oncotarget.19331] [PMID] [PMCID]
13. Li Y, Liang M, Zhang Y, Yuan B, Gao W, Shi Zh, et al. miR-93, miR-373, and miR-17-5p negatively regulate the expression of TBP2 in lung cancer. Front Oncol. 2020; 10:526. [DOI:10.3389/fonc.2020.00526] [PMID] [PMCID]
14. Cloonan N, Brown M K, Steptoe A L, Wani Sh, Chan Ling, Forrest A RR, et al. The miR-17-5p microRNA is a key regulator of the G1/S phase cell cycle transition. Genome Biol. 2008; 9(8):R127. [DOI:10.1186/gb-2008-9-8-r127] [PMID] [PMCID]
15. Fang L, Wang X, Sun B, Zhang X, Zhu X, Yu Z, et al. Expression, regulation and mechanism of action of the miR-17-92 cluster in tumor cells (Review). Int J Mol Med. 2017; 40(6):1624-30. [DOI:10.3892/ijmm.2017.3164] [PMID] [PMCID]
16. Liu MX, Liao J, Xie M, Gao ZK, Wang XH, Zhang Y, et al. miR-93-5p transferred by exosomes promotes the proliferation of esophageal cancer cells via intercellular communication by targeting PTEN. Biomed Environ Sci. 2018; 31(3):171-85. [DOI:10.3967/bes2018.023] [PMID]
17. Hao J, Jin X, Shi Y, Zhang H. miR-93-5p enhance lacrimal gland adenoid cystic carcinoma cell tumorigenesis by targeting BRMS1L. Cancer Cell Int. 2018; 18:72. [DOI:10.1186/s12935-018-0552-9] [PMID] [PMCID]
18. Yang W, Bai J, Liu D, Wang Sh, Zhao N, Che R, et al. MiR-93-5p up-regulation is involved in non-small cell lung cancer cells proliferation and migration and poor prognosis. Gene. 2018; 647:13-20. [DOI:10.1016/j.gene.2018.01.024] [PMID]
19. Shan X, Zhang H, Zhang L, Zhou X, Wang T, Zhang J, et al. Identification of four plasma microRNAs as potential biomarkers in the diagnosis of male lung squamous cell carcinoma patients in China. Cancer Med. 2018; 7(6):2370-81. [DOI:10.1002/cam4.1490] [PMID] [PMCID]
20. Harbeck N, Penault-Llorca F, Cortes J, Gnant M, Houssami N, Poortmans P, et al. Breast cancer. Nat Rev Dis Primers. 2019; 5(1):66. [DOI:10.1038/s41572-019-0111-2] [PMID]
21. Sun Y, Zhao Z, Yang Z, Xu F, Lu H, Zhu Z, et al. Risk factors and preventions of breast cancer. Int J Biol Sci. 2017; 13(11):1387-97. [DOI:10.7150/ijbs.21635] [PMID] [PMCID]
22. Lewis BP, Burge CB, Bartel DP. Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets. Cell. 2005; 120(1):15-20. [DOI:10.1016/j.cell.2004.12.035] [PMID]
23. Xu W, Yang M, Gao M. MiR-21-3p and miR-21-5p in tumor tissue as diagnostic biomarkers for gastric cancer. Int J Clin Exp Pathol. 2016; 9(7):7195-201. [Link]
24. Mattie MD, Benz CC, Bowers J, Sensinger K, Wong L, Scot G K, et al. Optimized high-throughput microRNA expression profiling provides novel biomarker assessment of clinical prostate and breast cancer biopsies. Mol Cancer. 2006; 5:24. [DOI:10.1186/1476-4598-5-24] [PMID] [PMCID]
25. Lu S, Wang S, Geng S, Ma S, Liang Z, Jiao B. Increased expression of microRNA-17 predicts poor prognosis in human glioma. J Biomed Biotechnol. 2012; 2012:970761. [DOI:10.1155/2012/970761] [PMID] [PMCID]
26. Wang J X, Jia X J, Liu Y, Dong J H, Ren X M, Xu O, et al. Silencing of miR-17-5p suppresses cell proliferation and promotes cell apoptosis by directly targeting PIK3R1 in laryngeal squamous cell carcinoma. Cancer Cell Int. 2020; 20:14. [DOI:10.1186/s12935-020-1096-3] [PMID] [PMCID]
27. Chen YZ, Liu ZP, Zhou KY, Li B. [Value of serum miR-17-92 cluster in diagnosis of retinoblastoma (Chinease)]. Zhongguo Dang Dai Er Ke Za Zhi. 2017; 19(7):776-80. [PMID] [PMCID]
28. Fu F, Jiang W, Zhou L, Chen Z. Circulating exosomal miR-17-5p and miR-92a-3p predict pathologic stage and grade of colorectal cancer. Transl Oncol. 2018; 11(2):221-32. [DOI:10.1016/j.tranon.2017.12.012] [PMID] [PMCID]
29. Jiang H, Bu Q, Zeng M, Xia D, Wu A. MicroRNA-93 promotes bladder cancer proliferation and invasion by targeting PEDF. Urol Oncol. 2019; 37(2):150-7. [DOI:10.1016/j.urolonc.2018.08.001] [PMID]
30. Liang L, Zhao L, Zan Y, Zhu Q, Ren J, Zhao X. MiR-93-5p enhances growth and angiogenesis capacity of HUVECs by down-regulating EPLIN. Oncotarget. 2017; 8(63):107033-43. [DOI:10.18632/oncotarget.22300] [PMID] [PMCID]
31. Fang S, Gao M, Xiong S, Chen Q, Zhang H. Expression of serum Hsa-miR-93 in uterine cancer and its clinical significance. Oncol Lett. 2018; 15(6):9896-900. [DOI:10.3892/ol.2018.8553] [PMID] [PCMID]
32. Fang L, Du WW, Yang W, Rutnam ZJ, Peng C, Li H, et al. MiR-93 enhances angiogenesis and metastasis by targeting LATS2. Cell Cycle. 2012; 11:4352-65. [DOI:10.4161/cc.22670] [PMID] [PMCID]
33. Fabbri E, Brognara E, Montagner G, Ghimenton C, Eccher A, Cantùet CM et al. Regulation of IL-8 gene expression in gliomas by microRNA miR-93. BMC Cancer. 2015; 15:661. [DOI:10.1186/s12885-015-1659-1] [PMID] [PMCID]
34. Bao C, Chen J, Chen D, Lu Y, Lou W, Ding B, et al. MiR-93 suppresses tumorigenesis and enhances chemosensitivity of breast cancer via dual targeting E2F1 and CCND1. Cell Death Dis. 2020; 11(8):618. [DOI:10.1038/s41419-020-02855-6] [PMID] [PMCID]