Alteration in the expression of microRNA-21 regulated target genes: Role in breast cancer

Breast cancer, also recognized as the principal cause of cancer-related deaths among women, is the second most familiar and prevalent form of cancer. New diagnostic and prognostic biomarkers that are highly specific are urgently needed for its early prognosis. MicroRNAs (miRNAs), a class of non-coding RNAs, are known to control the biological processes involving transcription, post-transcriptional and covalent modifications, splicing, translation, cell differentiation, proliferation, apoptosis, cancer progression, and invasion. Any dysregulation in miRNA expression, demonstrating their oncogenic and tumor-suppressive functions, contributes to cancer progression. MicroRNA-21 (miR-21), an ‘onco-miR’ in breast cancer, is involved in tumor progression and metastasis by suppressing the activity of the target gene via its interaction with the 3’UTR of the target gene. The upregulation of miR-21 is observed in many instances of breast cancer. Our review aims to summarize the current understanding of miR-21 in the regulation of important cellular functions via regulation of its target genes. We discuss its biosynthesis, oncogenic function in breast cancer, and different methods used for its detection. This will increase the current understanding of the role of miR-21 in breast cancer tumorigenesis, which will offer a perception of using miR-21 as an early detection molecular prognostic and diagnostic biomarker and as a therapeutic target in breast cancer care.

(miR-21), an 'onco-miR' in breast cancer, is involved in tumor progression and metastasis by suppressing the activity of the target gene via its interaction with the 3'UTR of the target gene. The upregulation of miR-21 is observed in many instances of breast cancer. Our review aims to summarize the current understanding of miR-21 in the regulation of important cellular functions via regulation of its target genes. We discuss its biosynthesis, oncogenic function in breast cancer, and different methods used for its detection. This will increase the current understanding of the role of miR-21 in breast cancer tumorigenesis, which will offer a perception of using miR-21 as an early detection molecular prognostic and diagnostic biomarker and as a therapeutic target in breast cancer care.

Introduction
The second most complex and heterogeneous cancer which brings mortality in 45-55 years older women across the world is breast cancer (Hemmatzadeh et al., 2016;Becker, 2015;Quan et al., 2020;Ataollahi et al., 2015). Aging is one of the most significant recognized risk factors associated with breast cancer. However, other risk factors such as deficiency of iodine, high estrogen level, obesity, intake of alcohol, menopause, family history, physically inactive and chest radiation exposure are also involved in breast cancer (Yager and Davidson, 2006;Steiner et al., 2008;Stoddard et al., 2008;Ataollahi et al., 2015). Breast cancer occurs in tissues that include mammary glands and ducts, resulting in lumps, swelling, redness, irritation in breast skin and nipples discharge (Ataollahi et al., 2015). The breast cancer cells are categorized into six major subtypes based on their phenotype and gene expression profile, including i) luminal A, ii) luminal B, iii) tumor enriched with human epidermal growth factor receptor 2 (also known as Her2), iv) basal-like (triple-negative), v) normal-like, and vi) claudin-low subtype (Singh and Mo, 2013;Sørlie et al., 2003;Eroles et al., 2012). It is believed that the mortality rate due to breast cancer can be reduced through the early diagnosis and detection of these six subtypes (Imani et al., 2017). The mammographic screening tool is the most operative tool used for the early diagnosis of tissue-based tumors in the breast (Taplin et al., 2008;Imani et al., 2017). But this tool has certain limitations such as high false-positive results, low reactivity, and low preciseness and, not adequate to diagnose the subtypes, so there is a need for new diagnostic and prognostic biomarkers that are highly specific (Adhami et al., 2018;Imani et al., 2017). Various non-coding RNAs (ncRNAs) that involve microRNAs (miRNAs), PIWIinteracting RNAs (piRNA), circular RNAs (circRNAs), and long non-coding RNAs (lncRNAs) are found to regulate biological processes like transcription, post-transcriptional modifications, covalent modifications, splicing, and translation in the metastasis phase of breast cancer cells (Klinge, 2018). Of all the ncRNAs studied in breast cancer, miRNAs are the most thoroughly studied and have diagnostic potential (Lo et al., 2016). miRNA in breast cancer miRNAs perform a crucial function in cancer research as predictive, diagnostic and, prognostic biomarkers because of their regulatory roles in vital processes of life like cell differentiation, proliferation, apoptosis, cancer progression and invasion (Kim, 2005;O'Day and Lal, 2010;Yu and Cheah, 2017). miRNAs are 15-27 nucleotides, short endogenous ncRNA molecules that are well known for posttranscriptionally regulating gene expression in eukaryotes by base pairing with protein-coding mRNA genes 3'-UTR (Feng and Tsao, 2016;Han et al., 2016;Wilczynska and Bushell, 2015;Adhami et al., 2018). Around half of the human miRNAs are placed on human cancer-related chromosomal regions (fragile sites), enabling them to alter the tumor suppressor or oncogenic pathways (Calin et al., 2004;Hemmatzadeh et al., 2016;Tang et al., 2012;Casalini and Iorio, 2009;Croce, 2009). Various studies have shown the miRNAs involvement as oncogenic/tumor suppressor in the pathogenesis of breast cancer as defined in Tab. 1 Si et al., 2007;Yan et al., 2008;Kong et al., 2012;Hassan et al., 2012;Fabian and Sonenberg, 2012).
Identifying miRNA in breast cancer cells miRNAs have crucial role in gene regulation, so it is becoming imperative to develop and improve methods that can detect miRNAs because their detection in breast cancer is difficult due to their small size, low abundance, high level of sequence similarities (Chandrasekaran et al., 2019;Ye et al., 2019). The most well-known traditional methods for the detection of miRNA are microarray, in situ hybridization, bead-based flow cytometry, next-generation sequencing (NGS), northern blotting, and quantitative reverse transcription polymerase chain reaction (qRT-PCR) (Ye et al., 2019;van Schooneveld et al., 2015). Out of these methods, northern blotting and in situ hybridization are low throughput methods, whereas microarray, bead-based flow cytometry, qRT-PCR and NGS are high throughput methods (van Schooneveld et al., 2015).
Northern blotting is the most standardized and highly specific method that can detect mature miRNA and their precursors (Ye et al., 2019;Várallyay et al., 2008). Besides these, the northern blotting method has a poor sensitivity and cannot detect the RNAs present in low amounts in few cells (Ye et al., 2019). qRT-PCR method is a highly sensitive and specific method used for evaluating miRNA and for the authentication of data prevailed from other detection platforms (Balcells et al., 2011;Yu and Cheah, 2017). The bead-based flow cytometry method is moderate in sensitivity and strong in specificity, whereas the in situ hybridization method is low in sensitivity and specificity (van Schooneveld et al., 2015). Microarray is a rapid method that can examine plenty of miRNAs from vast numbers of samples, with low sensitivity and specificity (Li and Ruan, 2009;Cissell and Deo, 2009). All these methods recognize only known miRNA structures, but NGS is the latest high sensitivity and specificity approach that enables new miRNAs to be recognized (van Schooneveld et al., 2015;Creighton et al., 2009). In the case of breast cancer, the upregulated levels of miR-21 can also be detected from tissues, blood, and serum (Yu and Cheah, 2017;Savad et al., 2012).

Programmed Cell Death 4 (PDCD4)
In both immune/non-immune cells, the programmed cell death 4 gene is a tumour suppressor . The PDCD4 protein contains three domains (two MA-3 domains and the N-terminal domain) that play a role in transcription and translation inhibition . Owing to the binding of its MA-3 domains to eIF4A, PDCD4 prevents the translation of mRNAs having structured 5'UTRs, hindering eIF4A binding to eIF4G, even blocking eIF4A's helicase activity, thus repressing the translation initiation mechanism of many genes, such as p53, Atg5, pro-caspase 3, and LXR-α (Kroczynska et al., 2012;Yang et al., 2004). By interacting with its N-terminal domain with A-myb & c-myb (RNA secondary structures), PDCD4 represses the elongation mechanism of translation (Singh et al., 2011). PDCD4 prevents tumor cell proliferation and invasion via inducing their apoptosis, leading to its upregulation in various tumors like breast, ovarian, gastric, esophageal, lung, hepatocellular, colon and glioma tumors (Powers et al., 2011;Wei et al., 2012;Jiang et al., 2017). The protein arginine methyltransferase 5 (PRMT5) deregulates the activity of the PDCD4 gene, resulting in breast cancer cell proliferation and invasiveness (Powers et al., 2011). miR-21 has been shown to target PDCD4 in tumour cells, thereby downregulating its expression (Sheedy et al., 2010). PDCD4 is a target of miR-21 for breast cancer (Frankel et al., 2008). The miR-21 binding sites in the PDCD4 3'UTR are shown in Fig. 3A, which shows how miR-21 interacts with the PDCD4 3'UTR. To assess the fact that miR-21 specifically targets PDCD4, the firefly luciferase reporter assay was carried out. Frankel and his colleagues first cloned PDCD4's 400-500 bp of 3'UTR into the pGL3 vector and then developed single (pGL3-PDCD4MUT1) and double (pGL3-PDCD4MUT2) mutations in the pGL3-PDCD4 seed region. PGL3-PDCD4, pGL3-PDCD4MUT1 & pGL3-PDCD4MUT2 were transfected into HEK293 cells and luciferase activity was tested after 24 h.
Results showed that miR-21 had no effect on pGL3-PDCD4MUT2 regulation but had little effect on pGL3-PDCD4MUT1 regulation (as shown in Fig. 3B), leading to the inference that there is a clear association between miR-21 and PDCD4 seed region 3'UTR. To verify the effects of miR-21 inhibition, they knocked down PDCD4 in MCF-7 cells via siRNA and found that PDCD4 siRNA mediated the antiproliferative effect on MCF-7 cells (see Figs. 3C, D). Thus, revealing the significance of PDCD4 by miR-21 as a putative target for breast cancer (Frankel et al., 2008).
Another research by Zhu et al. (2008) showed that miR-21 interacted with PDCD4's 3'UTR, resulting in its reduced expression in MDA-MB-231 cells. Anti-miR-21 also led to increased PDCD4 expression, indicating the role of miR-21 in breast cancer cell invasion by directly interacting with the PDCD4 target gene .
Tropomyosin-1 (TPM1) Tropomyosin-1, also known as TM1 and the actin-binding cytoskeletal protein, is a TPM family isoform that functions as a tumour suppressor gene by preventing cancer cell invasion by persuading apoptosis Pan et al., 2017;Qi et al., 2009;. Downregulation of TPM1 has been observed in breast cancer, glioma cancer, renal cell carcinoma, human oral squamous cell carcinoma (OSCC), and cholangiocarcinoma Dube et al., 2015;Pan et al., 2017;Bharadwaj and Prasad, 2002;Yang et al., 2013;Ku et al., 2010). The expression of TPM1 in the OSCC is regulated by miR-21 (Pan et al., 2017). In addition, Zhu et al. (2007) in their report, showed that TPM1 is targeted by miR-21 in breast cancer to inhibit the TPM1 mediated apoptosis of cancer cells. They conducted twodimensional differentiation in-gel electrophoresis, where they found the upregulated TPM1 expression in tumors procured from MCF-7 cells by antisense miR-21 oligonucleotides (anti-miR-21). miR-21 controls TPM1 expression by base-pairing with the V1 variant 3'UTR of TPM1, including miR-21's putative binding sites. For further evidence, the V1-3'UTR was cloned into a luciferase reporter. Luciferase assay results suggested that anti-miR-21 increased Luc-TPM1-V1-UTR activity, while miR-21 decreased its activity. Moreover, the effect of miR-21 inhibition on TPM1 was also tested at the level of translation. MTT assay revealed that TPM1-V1 repressed the growth of MCF-7 cells in vitro, and soft agar assay results also showed that TPM1-V1 reduced colony formation leading to anchorage-independent growth repression. Altogether, we can conclude that miR-21 uses TPM1 to control its expression in MCF-7 cells as a novel target.
Phosphatase and tensin homolog (PTEN) PTEN on chromosome 10 is recognized as a tumor suppressor gene coding 403 amino acids protein, which functions as a metabolic regulator of a number of cellular processes like glycogen synthesis, lipid and mitochondrial metabolism, glycolysis, and gluconeogenesis . It has dephosphorylation activities for both proteins and lipids due to which it is known as dual phosphatase enzyme (Chu and Tarnawski, 2004). PTEN has five domains: 1) N-terminal domain, responsible for the binding of phosphatidylinositol phosphate (PIP) substrates; 2) Phosphatase domain, carries acetylation sites and CX5R signature motif for the enzymatic actions; 3) C2 domain, a regulatory domain which contains a phospholipid-binding site and known for PTEN cell localization; 4) C-tail, which preserves phospho-sites and is responsible for the stability of protein; 5) PDZ domain, which is not well known (as shown in Fig. 4) (Chu and Tarnawski, 2004;Jerde, 2015;Chen et al., 2018). PTEN has regulatory functions in several processes, including cell migration, cell cycle arrest, MAP kinase signaling and angiogenesis (Chu and Tarnawski, 2004). Any mutations in PTEN gene lead to the progression of sporadic breast cancer, renal cell carcinoma, ovarian cancer, lung cancer, thyroid cancer, lymphoma cancer, hepatocellular carcinoma, lymphoma, head & neck cancer, prostate cancer, and glioblastoma (Chu and Tarnawski, 2004;Meng et al., 2007;Halachmi et al., 1998;Shao et al., 1998;Forgacs et al., 1998;FIGURE 1. Biosynthesis of miRNA. RNA polymerase II is responsible for the transcription of 100-120 nts pri-miRNA in the nucleus. The pri-miRNA is trimmed into 70 nts pre-miRNA by DROSHA and DGCR8, which is then exported by exportin 5/1 into the cytoplasm, where it is further processed by Dicer enzyme forming 20-30 nts duplex miRNA with the guide (5' to 3') and passenger (3' to 5') strands. The guide strand interacts with the PAZ domain of the RISC, causing Argonaute 2 (AGO2) to cause post-transcriptional gene silencing and cleavage of the target mRNA, while the RISC's PIWI domain degrades the passenger strand.  Represents that PDCD4 is a putative target of miR-21. A) shows the interaction of miR-21 with the 3'UTR of PDCD4. B) shows firefly luciferase reporter assay results, where co-transfection of HEK cells was done with pGL3-PDCD4, pGL3-PDCD4MUT1, pGL3-PDCD4MUT2 along with a Renilla luciferase transfection control plasmid, either alone or together with miR-21/lin-4 precursor miRNAs. Shown are relative luciferase values normalized to transfections without miRNA. Data are shown as the mean S.D. of four replicates and are representative of two independent experiments. ***, p_0.001 using a two-tailed t test. C) shows western blotting results where siRNA mediated knockdown of PDCD4 was shown in MCF-7 cells. D) shows the growth assay of PDCD4/control siRNA treated 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide in MCF-7 cells and transfection with inhibitors against miR-21/a scrambled control. Quantification of cell number was done after 5 days of transfection. Data are shown as the mean _ S.D. of three replicates and are representative of three independent experiments. The p value was calculated using a two-tailed t test (Frankel et al., 2008). Saito et al., 2000). In human hepatocellular cancer, miR-21 is known to regulate the PTEN expression and pathways which are mediated by PTEN (Meng et al., 2007).
A research conducted by Gong et al. (2014) showed that miR-21 decreases PTEN expression in tumors of breast phyllodes. Like PDCD4 and TPM1, PTEN also has miR-21 binding sequences at 3'UTR Frankel et al., 2008;Gong et al., 2014). PTEN protein levels were increased via miR-21 antisense oligonucleotides while, decreased via miR-21 mimics in stromal fibroblasts, demonstrating the role of miR-21 in targeting PTEN to increase the proliferation of phyllodes tumors cells (Gong et al., 2014). PTEN is known for the regulation of fibroblast activation protein (FAP) function (FAP is familiar as a serine protease and myofibroblast marker to increase the proliferation of phyllodes tumors) Gong et al., 2014). The PTEN knockdown raised the protein expression levels of FAP during the myofibroblast differentiation process, suggesting the role of PTEN in proliferation. Moreover, reintroduction of PTEN resulted in a decrease in both PTEN mRNA and protein levels in benign tumors, thus inhibiting the proliferation of tumor cells by miR-21. Together, these findings concluded that miR-21 downregulates the PTEN expression to induce cell proliferation by increasing FAP expression in breast phyllodes tumor (Gong et al., 2014).

Mammary Serine Protease Inhibitor (MASPIN)
Mammary serine protease inhibitor is related to the serpin family (serine protease inhibitor family) (Zhang, 2002). Due to its ability to prevent tumor invasion by inducing apoptosis in tumor cells found on the 18q21.3-q23 chromosome, it is also recognized as a tumor suppressor gene (Berardi et al., 2013;Zhang, 2002). It is also known to prevent the process of angiogenesis (Zhang, 2002). Owing to its nuclear/cytoplasmic location in cancer cells, its expression is downregulated in various cancers like breast, gastric, prostate, melanoma and upregulated in gallbladder, thyroid, colorectal, pancreatic cancers (Berardi et al., 2013).
Maspin decreased MDA-MB-231 tumour cell invasion in the case of breast cancer . With Flag-tagged maspin, they transfected MDA-MB-231 cells and confirmed the invasiveness of tumour cells through western blotting. However, by targeting it, the oncomir miR-21 downregulated maspin expression in MDA-MB-231 cells. Maspin also has 3'UTR binding sites for miR-21, so they cloned 3'UTR of maspin into pGL3 vector (luciferase gene) to check the Luc-Maspin-3'UTR luciferase activity. They found that miR-21 repression of luciferase activity was greater than 40%. Furthermore, anti-miR-21 increased the maspin expression, leading to the conclusion that by targeting its 3'UTR, miR-21 inhibits the maspin expression in MDA-MB-231 cells .
B-cell Lymphoma 2 gene (Bcl-2) B-cell lymphoma 2 is a 26 kDa protein present on human chromosome 18q21 with a hydrophobic carboxyl terminus positioned on the outer mitochondrial membrane and an anti-apoptotic gene as well (Lu et al., 1996). It suppresses  the caspase-mediated cell death and stimulates cell proliferation by restricting the function of pro-apoptotic proteins, Bak and Bax (Lu et al., 1996;Wickramasinghe et al., 2009). Upregulated levels of bcl-2 gene are found in many cancers like lymphoma, breast, colorectal, thyroid, and cervical (Flangea et al., 2008;Manne et al., 2000;Zhou and Wang, 2015). Research on human glioblastoma U87MG cells revealed that miR-21 overexpression resulted in decreased Bax expression and increased bcl-2 gene expression along with reduced caspase-3 activity (Shi et al., 2010). Similarly, miR-21 controls tumorigenesis by upregulating the expression of the bcl-2 gene in breast cancer as miR-21 was highly overexpressed in breast tumors compared to the matched normal breast tissues 157 human miRNAs analysed. . To investigate the function of miR-21, MCF-7 cells were transfected with anti-miR-21 oligonucleotide.
They showed that anti-miR-21 inhibits in vitro cell growth and tumor growth in the xenograft mouse model. Furthermore, to assess anti-miR-21 induced apoptosis, transfected cells were given Z-VAD-fmk, a caspase inhibitor, to counteract growth inhibition mediated by anti-miR-21. Apoptosis was caused by reduced Bcl-2 protein expression in anti-miR-21 transfected MCF-7 cells. These findings concluded that miR-21 should be used as a therapeutic target for breast cancer because of its essential function in controlling the Bcl-2 gene . In another study, breast cancer cell lines were developed, which are paclitaxel-resistant, and it was shown that down regulation of miR-21 via its mimic enhance the sensitivity against paclitaxel in the developed cell lines and increase the expression of Bcl-2 .
Leucine Zipper Transcription Factor-like 1 (LZTFL1) The 3p21.3 region of the chromosome contains the leucine zipper transcription factor-like 1 (LZTFL-1) gene expressed in epithelial cells of a diversity of normal cells (Wei et al., 2010). LZTFL-1, a cytoplasmic protein, regulates β-catenin nuclear signaling, ciliary protein trafficking, and the epithelial-mesenchymal transition (EMT) process (Wei et al., 2016). It represses EMT by inhibiting the mitogen-activated protein kinase (MAPK) signaling pathway, which is especially important in lung cancer (Wei et al., 2010;Wei et al., 2019). It functions as a tumor suppressor by stabilizing E-cadherin-mediated adherens junctions and inhibiting β-catenin relocation into the nucleus, preventing cell invasion and EMT-mediated breast cancer metastasis (Wang et al., 2014;.  discovered that LZTFL1 could be used as a new target in breast cancer cells via miR-21, facilitating cell invasion and metastasis. miR-21 was found to be upregulated in the plasma of three patient groups and various cell lines. The level of plasma miR-21 was significantly higher in the breast cancer patient group than the benign breast cancer patient group and healthy control group (see Fig. 5A), suggesting that plasma miR-21 level may be used as a potential diagnostic biomarker for breast cancer. The histopathological examination showed an upregulated level of miR-21 in breast cancer types Her-2+ and luminal B (as shown in Fig. 5B), and in vitro analysis results showed miR-21 upregulate in all cell lines of breast cancer (MDA-MB-231, Hs578T, MCF-7, and SK-BR3) compared to immortalized mammary epithelial (HBL-100) cell line (see Fig. 5C). As demonstrated by luciferase reporter assay, miR-21 directly targets LZTFL1, where miR-21 overexpression resulted in decreased luciferase activity in HEK293T cells transfected with luciferase gene containing either wild-type or mutant 3'UTR of LZTFL1 (see Fig. 6).
The miR-21/LZTFL1/-catenin axis induces the EMT process. The effect of miR-21 inhibition and LZTFL1 knockdown on various EMT markers such as N-cadherin, vimentin, E-cadherin and claudin-1. miR-21 inhibition decreased N-cadherin and vimentin expression while increasing E-cadherin and claudin-1 expression, whereas LZTFL1 knockdown reversed the expression of these markers (as shown in Fig. 7). Immunofluorescence assay  revealed that translocation of β-catenin in the nucleus was increased by miR-21 which contributed to the transcription of snail and slug transcription factors (EMT markers) (as shown in Fig. 7), while LZTFL1 repressed it. Therefore, miR-21 controls EMT by suppressing the LZTFL1, which increases β-catenin nuclear translocation, resulting in breast cancer proliferation and metastasis. This was also proved in BALB/c nude mice inoculated with Hs578T (human breast cancer) cells. miR-21 overexpression increased tumor size, weight, and volume (see Figs. 8A-8C) as well as lymph node invasion (see Figs. 8D and 8E). Furthermore, overexpression of miR-21 increased metastasis in liver and lung tissues (as shown in Figs. 8F and 8G) .

Summary
There is an extensive role of 'oncomiR' miR-21 in various stages breast cancer viz invasion, development, and metastasis. Different studies have demonstrated miR-21 upregulation in breast cancer cells by targeting multiple genes involved in breast cancer tumorigenesis. As mentioned above, miR-21 overexpression, through the downregulation of tumour suppressor genes, increases cell proliferation (Frankel et al., 2008;Zhu et al., 2007;Gong et al., 2014). miR-21 specifically interacts with PDCD4, TPM1, PTEN, and maspin 3'UTR, contributing to the suppression of apoptotic activities (Frankel et al., 2008;Zhu et al., 2007;Zhu et al., 2008;Gong et al., 2014). It also promotes breast cancer cell proliferation by increasing the bcl-2 gene expression. These studies indicate that alteration in the expression of miR-21 can be a beneficial intervention in the treatment of breast cancer. Various molecular biology-based techniques have been developed to detect the alteration in the expression of miR-21. Besides this, biosensor-based detection of miR-21 is a new area of research that could be more accessible and can help in point-of-care testing Meng et al., 2020;Sun et al., 2018). Another sensitive tool to detect miR-21 in breast cancer is DNA-Peptide dendrimer and mass spectrometric method. It would also be essential to understand the role of miRNA in connection with the changing microbiome, which will help in early diagnosis and be used as a therapeutic biomarker (Allegra et al., 2020;Rastogi et al., 2020;Kashyap et al., 2021). Acknowledgement: Authors acknowledge the support from Maharishi Markandeshwar (Deemed to be University) Mullana and Shoolini University, Solan.
Author Contribution: The authors confirm contribution to the paper as follows: study conception and design: AKS, RVS; data collection: PT, NKS, AC; draft manuscript preparation: PT, VKT, SSS. All authors compared different studies and approved the final version of the manuscript.
Funding Statement: Authors received no specific funding for this study.

Conflicts of Interest:
The authors declare that they have no conflicts of interest to report regarding the present study.