microRNA-7-5p inhibits melanoma cell proliferation and metastasis by suppressing RelA/NF-κB

microRNA-7-5p (miR-7-5p) is a tumor suppressor in multiple cancer types and inhibits growth and invasion by suppressing expression and activity of the epidermal growth factor receptor (EGFR) signaling pathway. While melanoma is not typically EGFR-driven, expression of miR-7-5p is reduced in metastatic tumors compared to primary melanoma. Here, we investigated the biological and clinical significance of miR-7-5p in melanoma. We found that augmenting miR-7-5p expression in vitro markedly reduced tumor cell viability, colony formation and induced cell cycle arrest. Furthermore, ectopic expression of miR-7-5p reduced migration and invasion of melanoma cells in vitro and reduced metastasis in vivo. We used cDNA microarray analysis to identify a subset of putative miR-7-5p target genes associated with melanoma and metastasis. Of these, we confirmed nuclear factor kappa B (NF-κB) subunit RelA, as a novel direct target of miR-7-5p in melanoma cells, such that miR-7-5p suppresses NF-κB activity to decrease expression of canonical NF-κB target genes, including IL-1β, IL-6 and IL-8. Importantly, the effects of miR-7-5p on melanoma cell growth, cell cycle, migration and invasion were recapitulated by RelA knockdown. Finally, analysis of gene array datasets from multiple melanoma patient cohorts revealed an association between elevated RelA expression and poor survival, further emphasizing the clinical significance of RelA and its downstream signaling effectors. Taken together, our data show that miR-7-5p is a potent inhibitor of melanoma growth and metastasis, in part through its inactivation of RelA/NF-κB signaling. Furthermore, miR-7-5p replacement therapy could have a role in the treatment of this disease.

IntroductIon
Melanoma is the most aggressive form of skin cancer and its incidence continues to increase globally [1]. Through early detection, primary melanoma may be cured by surgery, however metastatic disease is typically refractory to chemotherapy and radiation therapy, and these patients generally have a poor prognosis. Increased understanding of the key molecular events that drive melanoma development and progression has led to the development of targeted therapies to treat metastatic melanoma, such as small molecule inhibitors that block the mutant BRAF V600 kinase found in 40-50% of tumors [2,3]. However to date, the impact of targeted therapy in melanoma has been limited by the rapid and almost inevitable onset of therapeutic resistance via many different molecular mechanisms [4], the paucity of actionable mutations identified in NRAS mutant and BRAF/NRAS wild-type melanomas [5,6], and the considerable inter-and intra-tumoral heterogeneity of the disease [7]. In parallel, advances in immunotherapy, particularly from understanding immune checkpoint pathways, have improved melanoma patient survival [8]. However, despite a number of durable clinical responses, many patients do not benefit from these therapies and toxicity is a significant concern, particularly with combined immune checkpoint blockade [8]. Thus, there remains an urgent need to develop novel approaches to treat melanoma, especially those able to target the inherent metastatic potential of the disease.
MicroRNAs (miRNAs) are a class of small noncoding RNAs that regulate gene expression at the posttranscriptional level by binding to the 3ʹ-untranslated region (3ʹ-UTR) of specific mRNAs, either targeting those transcripts for degradation and/or repressing their translation. Altered miRNA expression has been widely reported in cancer, and models in which high levels of oncogenic miRNAs downregulate tumor suppressor genes and under-expressed tumor suppressor miRNAs result in elevated oncogene expression have been proposed [9]. Aberrant miRNA expression has been linked to various stages of melanomagenesis, including transformation initiation, cell proliferation, apoptosis resistance, metastasis, and therapy resistance [10][11][12][13]. In addition to their prognostic or predictive utility as biomarkers, miRNAs have emerged as novel molecules for cancer treatment [14][15][16], where modulating expression and activity of a single cancer-associated miRNA has the potential to coordinately alter the function of an entire biological pathway within a given tumor type. miRNA replacement therapy is emerging as a potential therapeutic strategy in several cancers, and is being actively evaluated in clinical trials (eg. NCT01829971, clinicaltrials.gov). This emphasizes the emerging potential of using miRNAs to treat patients with metastatic melanoma by coordinate inhibition of key signaling pathways. microRNA-7-5p (miR-7-5p) has diverse roles in development and disease and its expression is often aberrant in the latter [17]. In humans, miR-7 is transcribed from three genes, miR-7-1, miR-7-2 and miR-7-3, and these transcripts are processed to yield the same mature miRNA sequence, of which the 5 strand is the most commonly studied. In the context of cancer, microRNA-7-5p (miR-7-5p) is a well characterized tumor suppressor that is frequently downregulated in a variety of cancers [18][19][20][21]. miR-7-5p has been reported to decrease tumor cell proliferation, anchorage-independent growth, migration and invasion, and to promote apoptosis and chemosensitivity by repressing expression of specific oncogenic target genes [22][23][24][25][26][27][28], suggesting that it has potential utility in the treatment of certain cancers. We previously characterized the role of miR-7-5p as a tumor suppressor and inhibitor of epidermal growth factor receptor (EGFR) expression and signaling in lung, glioblastoma, breast and head and neck cancers [27,29]. miR-7-5p has been reported previously to be downregulated in several cases of metastatic versus primary melanoma, consistent with a tumor suppressor role, but, crucially, no studies have systematically investigated the role of miR-7-5p in metastatic melanoma development or its clinical significance [30][31][32]. We reported previously that miR-7-5p overexpression significantly inhibits the migration and invasion of melanoma cells in vitro, in part through regulation of insulin receptor substrate 2 (IRS-2) expression [30], but there are no reports investigating these effects and their mechanisms in vivo, or addressing the role of miR-7-5p in regulating melanoma cell growth.
Here, we demonstrate that miR-7-5p is a potent suppressor of melanoma cell growth and viability, and inhibits metastasis of melanoma cells to the lung. We also show that the anti-metastatic function of miR-7-5p is mediated, in part, via its direct suppression of RelA expression and NF-κB signaling. Importantly, high RelA expression is associated with a worse clinical outcome in melanoma patients.

mir-7-5p inhibits melanoma cell proliferation
To investigate the functional role of miR-7-5p in melanoma we first assessed its impact on proliferation in vitro. Our results show a significant reduction in cell viability 5 days after transfection of the metastatic melanoma cell lines WM266-4, A2058, 1205Lu (all BRAF V600-mutant) and SK-MEL-2 (BRAF WT, NRAS Q61K ) with miR-7-5p ( Figure 1A). In parallel, we transfected these cells with an antagomiR to inhibit miR-7-5p function (anti-miR-7-5p), and observed no significant change in cell viability ( Figure S1A), most likely due to the very low endogenous levels of miR-7-5p in melanoma cells. To further investigate the effect of ectopic miR-7-5p expression on melanoma cell proliferation, we assessed 2D and 3D colony formation, and observed fewer and smaller colonies in miR-7-5p-overexpressing WM266-4 cells in both assays ( Figure 1B). Similar results were obtained with 1205Lu cells ( Figure S2A). To investigate the mechanism underlying these effects, we assessed cell cycle distribution following propidium iodide (PI) staining and flow cytometry analysis. These experiments revealed that miR-7-5p induces G0/G1 cell cycle arrest in WM266-4 and 1205Lu cells ( Figure 1C and Figure S2B). However, the reduction in cell number following miR-7-5p transfection does not appear to be due to apoptosis, evidenced by the lack of pre-G1 peak in cell cycle analysis, absence of PARP cleavage or caspase 3 and 7 activation, and lack of an apoptotic fraction following annexin V and PI staining and flow cytometry ( Figure S3A-C). Together, these results indicate that miR-7-5p inhibits melanoma cell proliferation, but does not induce apoptosis.

mir-7-5p inhibits melanoma cell migration and invasion in vitro and metastasis in vivo
To further elucidate the functional role of miR-7-5p in melanoma, we assessed the impact of its overexpression on metastatic processes. We found that miR-7-5p abrogated migration and invasion of 1205Lu cells in vitro using real-time monitoring by xCELLigence ( Figure 2A). In parallel, we measured cell proliferation in real-time and observed no difference within the first 24 h, therefore the early repression of cell migration and invasion cannot be due to accounted for by cell death or altered proliferation in this time period ( Figure S4A). Next, we utilized a 3D spheroid invasion assay [33] as a more representative model of in vivo invasion, and found that miR-7-5p overexpression markedly inhibited the ability of 1205Lu cells to invade into the surrounding collagen ( Figure  2B). Similar results were observed in WM266-4 cells ( Figure S4B). Additionally, fewer cells migrated across a scratched wound following miR-7-5p augmentation, as assessed by wound healing assay of 1205Lu cells ( Figure  2C). However, transfection of 1205Lu with anti-miR-7-5p to inhibit miR-7-5p function resulted in no significant difference in wound closure ( Figure S4C), which is most likely due to the very low endogenous miR-7-5p expression in melanoma cells. To assess the impact of miR-7-5p on melanoma metastasis in vivo, 1205Lu cells with transient overexpression of miR-7-5p or miR-NC were injected into the tail vein of NSG mice and the lungs examined for metastatic deposits after 14 days. We found that the lungs from mice injected with 1205Lu cells that overexpressed miR-7-5p were significantly lighter and had markedly fewer tumors visible on the surface of the lungs ( Figure 2D). Histological examination confirmed the significant reduction in lung tumor burden, with both fewer and smaller 1205Lu metastatic nodules being present in lungs from the miR-7-5p group ( Figure 2D). 1205Lu cells formed tumors exclusively in the lungs of mice, as no nodules were found in the liver, heart or brain (data not shown), consistent with previous studies [34].   Collectively, these in vitro and in vivo data demonstrate the capacity for miR-7-5p to potently suppress melanoma metastasis in vitro and in vivo, and provide a foundation for subsequent studies to define the mechanisms responsible for these effects.

mir-7-5p represses a pro-metastatic gene expression program in melanoma
To better understand the mechanisms through which miR-7-5p regulates melanoma growth and metastasis, we Volcano plot representing fold change and significance of altered microarray probes in WM266-4 cells 24 h post-transfection with miR-7-5p or miR-NC precursor molecules (30 nM). b. Cluster analysis of genes downregulated (green) or upregulated (red) following miR-7-5p transfection. c. Signature of putative miR-7-5p target genes that contain a miR-7-5p seed sequence, are downregulated in the microarray and are associated with melanoma and metastasis. FC = Fold change. d. IPA network analysis showing growth-and metastasis-associated functions of putative miR-7-5p target genes in melanoma.
performed gene expression array analysis using RNA isolated from WM266-4 cells that were transfected with miR-7-5p or miR-NC for 24 h. After data normalization, differentially expressed genes were assigned based on a cut-off of a fold-change of at least 1.5 and p-value < 0.05 ( Figure 3A). Cluster analysis identified a total of 817 genes that were significantly upregulated or downregulated by miR-7-5p transfection, relative to miR-NC ( Figure 3B and Supplementary Tables S1 and S2).
To identify direct putative targets of miR-7-5p in melanoma that might account for its anti-proliferative and anti-metastatic effects, we focused on those genes that were significantly downregulated in WM266-4 cells following transfection of miR-7-5p, which refined the list to 575 genes, of which 129 were identified by Ingenuity Pathway Analysis (IPA) software as being predicted (by TargetScan) or proven miR-7-5p targets (Supplementary Table S3). Biofunctional analysis of this gene list with IPA showed enrichment for genes involved in cell movement, cell cycle, colony formation, proliferation and cancer (Supplementary Table S4). Further analysis of this putative target list identified 18 genes that are associated with growth and/or metastatic processes (Supplementary  Table S5), and of these 10 (CTSK, IRS2, PAK1, POLE4, RAF1, RELA, SMO, SP1, STMN3, TGFA) have been previously implicated in melanoma, thus representing a pro-metastatic gene signature that is repressed by miR-7-5p ( Figure 3C). RT-qPCR analysis confirmed significant downregulation of mRNA expression of each of these genes by miR-7-5p in WM266-4, 1205Lu and A2058 cells ( Figure S5). Additional bioinformatics analyses using this 10-gene signature identified an integrated network of interactions between the miR-7-5p-downregulated genes related to metastasis and invasion in melanoma ( Figure  3D), that potentially confer the in vitro and in vivo antimetastatic and anti-proliferative effects.

Impact of pro-metastatic gene signature on melanoma patient survival
To assess the clinical significance of increased expression of the above miR-7-5p target genes in melanoma, we analyzed publically available microarray data from The Cancer Genome Atlas melanoma study (TCGA; http://cancergenome.nih.gov). Data on survival and mRNA expression for 463 melanoma patients was analyzed by Cox regression, and after adjustment for age and sex, high expression of RelA (hazard ratio (HR) = 1.55, p-value = 0.007; Figure 4B), POLE4 (HR = 1.45, p-value = 0.023) and SP1 (HR = 1.40, p-value = 0.032) was predictive of poorer overall survival (Table 1). To validate this finding, we performed meta-analyses of these genes across three additional melanoma cohorts [35][36][37], in addition to the TCGA study, to increase the sample size and statistical power. Comparisons across these four datasets showed high expression of either RelA (consensus HR = 1.47), POLE4 (consensus HR = 1.39) or SP1 (consensus HR = 1.48) was consistently associated with an increased risk of death in melanoma ( Figure 4A and 4B). Furthermore, low expression of miR-7-1, which is the predominant isoform that gives rise to mature miR-7-5p, was associated with an increased risk of death in melanoma patients, with HR = 0.558, p = 0.05 ( Figure 4C) [37]. Additionally, TCGA melanoma data was analyzed for an inverse correlation between expression of miR-7 isoforms and putative target gene expression, and revealed a negative association between the miR-7-1 isoform and RelA with R = -0.12, p = 0.013, but no significant negative correlations with the other genes that were also associated with survival ( Figure 4D and Supplementary  Table S6). Taken together, these data suggest that RelA overexpression and low miR-7-1 levels are indicators of a poor prognosis in melanoma. In addition, the data identified a negative correlation between levels of miR-7-5p and RelA in these tumors, supporting a model whereby miR-7-5p loss may contribute to increased expression of RelA.

miR-7-5p reduces RelA expression by directly targeting its 3ʹ-UTR
We elected to focus on the NF-κB subunit RelA (p65) for further functional studies, given its prognostic significance in melanoma and its inverse correlation with miR-7-5p levels. To confirm the direct regulation of RelA expression by miR-7-5p in melanoma, we used quantitative real-time PCR (RT-qPCR) and western blotting and found that miR-7-5p reduces RelA mRNA ( Figure 5A) and protein expression ( Figure  5B) in WM266-4, A2058 and 1205Lu melanoma cells. TargetScan analysis identified two putative miR-7-5p binding sites within the RelA 3ʹ-UTR at positions 2030-2036 nt (site A, poorly conserved) and at 2301-2308 nt (site B, highly conserved, Figure 5C). To test the direct binding of miR-7-5p to these sites, wild type or mutant full length RelA 3ʹ-UTR luciferase reporter constructs were generated that included either: (i) wild type miR-7-5p sites A. Luciferase reporter assay with WM266-4, A2058 and 1205Lu cells that were transfected with NF-κB cignal luciferase reporter or negative control luciferase reporter, and either miR-7-5p or miR-NC precursor molecules (10 nM), RelA siRNA (si-RelA7, positive control for assay) or negative control siRNA (si-NC) at 5 nM. *, p-value < 0.01. b. ELISA analysis of secreted and intracellular expression of IL-1β, IL-6 and IL-8 in 1205Lu cells 3 d post-transfection with either miR-7-5p or miR-NC precursor molecules, or si-RelA7 or si-NC. *, p-value < 0.01.

miR-7-5p inhibits NF-κB transcriptional activity and downstream signaling
As RelA is a NF-κB subunit responsible for activating the transcription of many genes, we assessed the impact of miR-7-5p on NF-κB transcriptional activity in melanoma cells. Cells were co-transfected with a luciferase reporter construct containing multiple canonical NF-κB (green) or upregulated (red) by miR-7-5p in melanoma, as a summary of data from the NF-κB PCR array and further validation by RT-qPCR and ELISA. IPA software was used to map common miR-7-5p-downregulated genes (shown in green) in WM266-4 and 1205Lu melanoma cells onto the canonical NF-κB pathway. The density of shading represents the degree of gene regulation by miR-7-5p. transcriptional regulatory elements (TREs) or a negative control luciferase plasmid lacking TREs (for background measurement), plus either miR-7-5p, miR-NC, RelA siRNA (si-RelA, positive control) or non-targeting siRNA (si-NC). Cells transfected with miR-7-5p or si-RelA had significantly reduced NF-κB reporter activity, compared to their respective controls ( Figure 6A). In view of the reduced NF-κB transcriptional activity in melanoma cells, we next hypothesized that expression of key NF-κB target genes would also be reduced. To test this, we screened for miR-7-5p-regulated genes using a NF-κB pathway PCR array with WM266-4 cells transfected with miR-NC or miR-7-5p for 3 d. Multiple NF-κB target genes were found to be deregulated in response to miR-7-5p overexpression, and these changes were subsequently verified by RT-qPCR in WM266-4 and 1205Lu cells (Supplementary Table S7). We further investigated IL-1β, a cytokine associated with melanoma metastasis [38] as a miR-7-5p-regulated NF-κB target gene. Transfection of 1205Lu cells with either miR-7-5p or si-RelA resulted in reduced secretion and intracellular expression of IL-1β protein ( Figure 6B), as measured by ELISA. As IL-1β has also been linked to the expression of other cancer-associated cytokines [38], we tested the impact of miR-7-5p or si-RelA on expression of IL-6 and IL-8, and found that both miR-7-5p and si-RelA reduced the levels of secreted and intracellular IL-6 and IL-8 ( Figure 6B and Supplementary Table S7). These data indicate that the direct targeting of RelA by miR-7-5p reduces NF-κB activity and consequently expression of many of the NF-κB downstream target genes associated with tumor metastasis.

RelA is a functional target of miR-7-5p
Next, we sought to establish a functional role for RelA as a miR-7-5p target in melanoma, such that repression of RelA and its downstream signaling might in part explain the capacity of miR-7-5p to inhibit proliferation, migration and invasion of melanoma cells. WM266-4 and 1205Lu cells were transfected with five different RelA siRNAs, each of which reduced RelA mRNA and protein expression by approximately 70-95% in both cell lines ( Figure S6B-C). Silencing of RelA expression in WM266-4 cells significantly reduced their viability ( Figure 7A), colony forming potential in both 2D and 3D models ( Figure 7B), and also resulted in G0/G1 phase cell cycle arrest ( Figure 7C). Similar results were obtained with 1205Lu cells (Figure S7A-C). Furthermore, transfection of 1205Lu cells with RelA siRNAs significantly reduced the rate of cell migration and invasion, impaired wound closure in a wound healing assay, and suppressed invasion of melanoma spheroids ( Figure 7D-F). Thus, RelA knockdown was able to recapitulate the phenotypic effects of ectopic miR-7-5p overexpression in melanoma cells. Collectively, these data suggest that miR-7-5p is a potent inhibitor of melanoma growth, migration and invasion, at least in part through the targeted reduction of RelA expression/activity and its downstream targets. dIscussIon miR-7-5p suppresses the growth and metastasis of several tumor types, including hepatocellular carcinoma, breast cancer, glioma and gastric cancer, by inhibiting the expression of specific target genes, including EGFR and its signaling pathways [21,39,40]. Given that melanoma is typically not driven by aberrant EGFR expression and/ or signaling, this study aimed to clarify the functional and clinical relevance of miR-7-5p in melanoma, and to identify critical pathways through which miR-7-5p acts in this disease. We found that overexpression of miR-7-5p not only suppresses melanoma cell growth and cell cycle progression, but also tumor cell migration, invasion and metastasis in vivo. Gene expression analyses revealed a signature of putative miR-7-5p target genes associated with melanoma and metastasis. Further, aberrant expression of some of these genes including RelA, POLE4 and SP1 had prognostic implications in terms of melanoma patient survival. We focused on RelA, due to its negative association with miR-7-5p expression and emerging evidence establishing it as a potential target for metastatic melanoma treatment [41][42][43]. Our study shows that RelA is a direct target of miR-7-5p, via two independent binding sites in its 3'-UTR, which reduces its expression, activity and downstream signaling, and our functional experiments validate RelA as a critical mediator of the anti-metastatic effects of miR-7-5p in melanoma.
Analysis of four independent melanoma clinical cohorts showed that RelA overexpression is associated with worse overall survival, supporting its tumorpromoting role in this system [44][45][46]. Further analyses of these data revealed an inverse correlation between miR-7-1 and RelA levels in melanoma and that miR-7-1 levels were associated with survival, consistent with a model in which reduced expression of miR-7-5p promotes RelA overexpression and facilitates melanoma progression. While this manuscript was in preparation, Hanniford and coworkers reported that reduced miR-7-5p expression was associated with melanoma recurrence and reduced invasion in vitro [47]. Another recent report suggested that serum miR-7-5p levels might be a useful biomarker for tumors of the esophagus [48]. Certainly, additional studies are required to more clearly define the prognostic significance of miR-7-5p expression in large cohorts of primary and metastatic melanoma.
Our expression array data indicate that miR-7-5p functions to regulate large networks of genes in melanoma, and our network analysis supports the concept of regulation via "hub genes", which includes RelA, a transcriptional regulator that itself regulates expression of many other genes, and hence altering miR-7-5p expression produces a significant "flow on" effect in terms of broadly altering gene expression [62]. We found that downregulation of RelA by miR-7-5p resulted in reduced expression of various canonical targets of NF-κB, including IL-1β, thus providing additional evidence for the indirect inhibition of broad transcriptional networks. Many of these canonical NF-κB target genes, including IL-1β, IL-6 and IL-8, have been associated with melanoma tumorigenicity, metastasis and angiogenesis, and represent potential targets for therapy [38,49,50]. Importantly, using siRNAs against RelA, we were able to recapitulate the functional effects of miR-7-5p on melanoma cell proliferation, migration and invasion, thus validating RelA as a direct, functional target of miR-7-5p in melanoma.
Interesting links between miR-7-5p and the NF-κB pathway have recently emerged. A study by Ning et al. identified loss of expression of the transcription factor Hepatocyte Nuclear Factor 4α (HNF4α) as mediating reduced miR-7-5p levels in metastatic hepatocellular carcinoma, and showed that ectopic expression of HNF4α reduced NF-κB activity and inhibited tumor cell migration, invasion and lung metastasis [39]. It is unclear whether the reduced miR-7-5p levels observed in some melanomas can be explained by loss of HNF4α expression or activity. Recently, a report by Zhao et al. showed that miR-7-5p reduced gastric carcinoma cell proliferation, induced G0/G1 cell cycle arrest, promoted apoptosis, suppressed tumor growth in vivo, and suggested that RelA and FOS could mediate these effects [51]. Of particular interest, they demonstrated that RelA/NF-κB could repress miR-7-5p transcription. Thus, it seems possible that miR-7-5p can directly target RelA in melanoma, and that, in turn, NF-κB can act in a reciprocal feedback loop to downregulate expression of miR-7-5p [51].
Our study identified several other putative miR-7-5p target genes relevant to melanoma. Most notably, we report that high expression of specific protein 1 (SP1) and polymerase (DNA-directed), epsilon 4, accessory subunit (POLE4) was associated with significantly worse overall survival in the TCGA melanoma cohort. SP1 is a ubiquitous transcription factor that has been implicated in driving constitutive expression of cell adhesion molecules in metastatic melanoma [52], and Li et al. recently showed that miR-7a/b overexpression in rat cardiac fibroblasts (CFs) suppresses SP1 expression and CF cell growth and migration [53]. Interestingly, miR-22 has been shown to form an autoregulatory loop with SP1 in breast cancer, whereby miR-22 binds to the 3'-UTR of SP1 and represses its expression, SP1 binding to the miR-22 promoter suppresses miR-22 transcription, and miR-22 overexpression suppresses breast cancer cell growth, invasion, and metastasis [54]. In this regard, it would be interesting to examine whether SP1 binding to the miR-7-5p promoter can directly inhibit miR-7-5p expression, suggesting a similar feedback loop may also exist in melanoma. Intriguingly, there are SP1 binding sites in the RelA promoter and SP1 has been shown to interact with and form a complex with RelA in vitro [55,56]. As miR-7-5p can co-ordinately downregulate RelA and SP1, these observations may explain the altered expression of many genes by miR-7-5p that do not contain classic miR-7-5p seed regions. Elevated levels of POLE4 and other DNA repair genes have been associated with shorter time to melanoma relapse [57], and in previous studies we identified POLE4 as a putative miR-7-5p target gene in lung and head and neck cancer cell lines [27,29]. Together, our findings suggest that SP1 and POLE4 could represent additional functional targets of miR-7-5p in melanoma, emphasizing the coordinate manner in which miRNAs suppress expression of multiple target genes to exert their biological effects.
Therapy for metastatic melanoma has undergone dramatic change in the recent years with the development of molecularly-targeted therapies and immune checkpoint inhibitors. miR-7-5p is known to target several of the molecules, such as IGF1R, EGFR and IRS1/2, that promote resistance to BRAF inhibitors [58][59][60]. Thus, patients who have developed resistance to BRAF inhibitor therapy could potentially benefit from miR-7-5p replacement therapy, which might resensitize tumors to these treatments. Further, we and others have shown that miR-7-5p is capable of sensitizing other resistant tumor types to targeted therapies, such as the EGFR tyrosine kinase inhibitor erlotinib [61]. In support of this concept, Stark et al. have shown that miR-514a modulates BRAF inhibitor sensitivity in melanoma through its regulation of the tumor suppressor NF1 [62]. In addition, miR-7-5p could potentially augment the anti-melanoma immune response, by blocking expression of immunosuppressive cytokines, such as IL-6 [63]. Ultimately, this could lead to studies investigating the efficacy of combining miR-7-5p with immune checkpoint blockade therapy. As treatment of metastatic melanoma increasingly involves combination therapies, it will be of great interest to define which combinations of miR-7-5p and targeted therapies (BRAF/ MEK inhibitors) or immunotherapies are most effective or yield synergistic anti-melanoma effects.
In summary, we found miR-7-5p markedly suppresses the growth, migration and invasion of melanoma cells, and inhibits melanoma lung metastasis. Our data indicate miR-7-5p directly inhibits RelA expression, resulting in reduced NF-κB/RelA transcriptional activity. In turn, this leads to reduced expression and secretion of NF-κB downstream target molecules, including IL-1β, IL-6 and IL-8, which ultimately suppresses melanoma growth and metastasis ( Figure 8). Thus, loss of miR-7-5p in melanoma promotes RelA overexpression, facilitating melanoma growth and metastasis and ultimately resulting in a worse clinical outcome. miRNA-based cancer therapies have entered the clinic, suggesting that they may represent a viable strategy to inhibit tumor growth and metastasis and extend survival in some patients. This study provides novel insight into the anti-melanoma activity of miR-7-5p, and support further preclinical development of miR-7-5p replacement therapy for patients with metastatic melanoma.

cell proliferation and colony formation assays
Cell viability and 2D colony formation assays were performed as described [61], using miRNA precursors and anti-miRs at 5 nM for viability or 30 nM for clonogenicity or with siRNAs at 5 nM (final concentrations). For 3D colony formation assays, cells were transfected with miRNA precursors (30 nM) or siRNAs (5 nM) for 48 h, resuspended in a 1:1 mixture of RPMI 1640 and Matrigel Matrix (BD Biosciences) and added to 12-well plates, precoated with Matrigel. Cells formed colonies over a 14 d period, before staining with crystal violet for visualization.

cell cycle analysis
Cells were transfected with miRNA precursors (30 nM) or siRNAs (5 nM) for 72 h, trypsinized and fixed with ethanol, before propidium iodide (PI) staining and analysis by Flow cytometry on a BD Accuri C6 with Flowjo software (version 7.6.5).

Migration and invasion assays
Cells were transfected with miRNA precursors (30 nM) or siRNAs (5 nM) for 48 h. Cell migration and invasion was monitored with a Real-Time Cell Analyzer dual-plate (RTCA-DP) xCELLigence instrument as described previously [30]. For invasion assays, CIMplates were coated with Matrigel Matrix (diluted 1:40 in serum-free medium) 4 h prior to seeding cells and media with 20% FBS was used as a chemoattractant.
For wound healing assays, culture inserts (Ibidi) in 35mm μ-Dishes were used. Following transfection for 48 h with miRNA precursors and anti-miRs (30 nM) or siRNAs (5 nM), 70 μl of cell suspension (3 × 10 5 cells/ ml) was seeded into each well. After 24 h the inserts were removed, leaving a gap of 500 μm and wound closure was determined using ImageJ 1.48v imaging software (NIH).
The 3D spheroid invasion assay was performed as described previously [33,64]. Briefly, cells were transfected with miRNA precursors (30 nM) or siRNAs (5 nM) for 24 h, seeded into 96-well plates coated with 1.5% agarose and incubated for 72 h to form single spheroids. The spheroids were then harvested, embedded into bovine collagen I (Cultrex) matrix and invasion monitored by microscopy.

Animals
The metastasis study was conducted in male, 10-11 week old, NOD.CB17-Prkdcscid Il2rgtm1Wjl/ SzJ (NSG) mice (Jackson Laboratory). Use of animals was in accordance with the guidelines of the University of Western Australia Animal Ethics Committee under approval numbers RA/3/100/1216.

In vivo metastasis study
1205Lu cells were transfected with miRNA precursors (30 nM) for 48 h, at which time a total of 20 mice (randomized groups of 9 each for miR-NC or miR-7-5p, and 2 with non-transfected cells) each received tail vein injections of 1 x 10 6 cells in 0.1 ml of PBS. Mice were monitored for 14 d, sacrificed and lungs excised, weighed and examined for visible tumor burden. Lungs were next stained with Bouin's solution for 24 h (lung parenchyma stains yellow and tumor nodules are white) and then paraffin-embedded, sectioned and stained with H&E. Five sections at various depths were imaged by microscopy and the 1205Lu lung tumor nodules manually quantified by three independent researchers under blind conditions using ImageJ software. The liver, heart and brain were also analyzed for presence of tumor nodules.

cDNA microarray expression profiling and analysis
Total RNA was extracted from WM266-4 cells transfected with miR-NC or miR-7-5p precursor molecules (30 nM) for 24 h, using Qiazol reagent (Qiagen). The quantity and integrity of RNA samples was assessed using a 2100 Bioanalyzer (Agilent Technologies). Gene expression profiling by microarray hybridization was performed with three experimental replicates for each condition by the Australian Genome Research Facility (AGRF; Victoria, Australia) using a HumanHT-12 v4 array chip (Illumina). Signal intensity values underwent normalization, variance stabilization transformation and log 2 transformation using the lumiR package of R Bioconductor (www.Bioconductor.org). ANOVA analysis of normalized probe intensities values was performed in Partek® Genomic SuiteTM software (version 6.6 build 6.12.0420). Probes with an unadjusted p-value of 0.05 or less and an absolute fold change of 1.5 or more between miR-NC and miR-7-5p samples were classified as being differentially expressed. Clustering and volcano plots showing the distributions and correlations of gene expression between miR-NC and miR-7-5p samples were produced using normalized data and the R 'graphics' package. A heat map comparing significant differential gene expressions between the miR-NC and miR-7-5p microarrays was produced with the R 'gplots' package. Ingenuity Pathway Analysis (Ingenuity Systems, Inc) was used to identify canonical pathways and cellular functions affected by miR-7-5p, some of which were presented figuratively using Ingenuity's Pathway Designer. TargetScan (Version 6.2: June 2012) was used to identify putative miR-7-5p seed sites within the set of differentially expressed mRNAs.

Microarray data
The microarray data for WM266-4 cells transfected with miR-7-5p or miR-NC have been deposited in the Gene Expression Omnibus under Accession Number GSE77196.  [35]). The Kabbarah cohort consisted of 81 melanoma patients, 54 men and 27 women, with 50 primary and 31 metastatic melanomas, and a mean age of 58 years (GEO accession number GSE46517 [36]). The Jayawardana cohort consisted of 74 melanoma patients, with 29 stage I, 29 stage II, and 20 stage III tumors, and 1 melanoma of unknown staging (GEO accession numbers GSE54467 and GSE59334 [37]).

Reverse
transcription and quantitative polymerase chain reaction (rt-qPcr) Cells were transfected with miRNA precursors (30 nM) or siRNAs (5 nM) for 24 h and RNA isolated using Qiazol. For cDNA synthesis, 0.5 µg of total RNA was reverse transcribed using QuantiTect Reverse Transcription Kit (Qiagen). Quantitative PCR was performed with a Rotor-Gene 6000 thermocycler (Qiagen) or ViiA7 Real-Time PCR system (Life Technologies), using SensiMixPlus SYBR (Bioline). Primer sequences are listed in Supplementary Table S8. Validated primer sequences from PrimerBank were used where available [65]. Relative expression for each sample was calculated, taking into account PCR efficiency [66] with GAPDH or HPRT1 used as reference genes. A NF-κB pathway PCR array (RT 2 Profiler PCR Array, SABiosciences, Qiagen) was performed with WM266-4 cells transfected with miR-NC or miR-7-5p for 72 h, according to manufacturer's instructions and analyzed with the PCR Array Data Analysis Software provided from the company.

Luciferase plasmid construction
Construction of the miR-7-5p perfect target reporter has been described previously [68]. Full-length wild-type (WT) and mutant (MT) RelA 3ʹ-UTR reporter plasmids were synthesized by GenScript Inc. (Piscataway, NJ) to include the full length RelA 3ʹ-UTR (nt 1919-2700 of GenBankTM accession number NM_021975) within the pmiR-REPORT firefly luciferase reporter vector (Ambion). Modifications were made to the RelA 3ʹ-UTR at predicted miR-7-5p binding regions by replacing nt 2030-2036 (site A) and/or nt 2301-2308 (site B) with the sequence 5ʹ-CAG AAG GU-3ʹ. All constructs were verified by DNA sequencing.

Luciferase reporter assays
RelA 3ʹ-UTR luciferase reporter assays were performed as described [67], and using miRNA precursor molecules at 2.5 nM final concentration. NF-κB transcriptional activity was determined by co-transfection of a NF-κB-driven luciferase reporter or a negative control reporter lacking transcriptional response elements (TREs) (Cignal Reporter Assay, SABiosciences, Qiagen), with miRNA precursor molecules (10 nM) or siRNAs (5 nM). Transfection efficiency was assessed using a green fluorescent protein (GFP)-expressing positive control plasmid. Lysates were assayed for firefly and Renilla luciferase activities 48 h post-transfection with the Dual Luciferase Reporter Assay System (Promega). Firefly luciferase activity was first normalized to Renilla, then each condition expressed relative to the negative control reporter.

statistical and bioinformatic analyses
Gene expression data were analyzed using the R programming platform. Kaplan-Meier survival curves were produced using the R "survival" package. Cox regressions were calculated using the R "coxph" function from the "survival" package with ties addressed by Efron's method. Forest plots were generated using the R "forestplot" package. For Forest plots, Cox regressions and Kaplan-Meier survival curves, deaths were used as an endpoint. Analyses were adjusted for age and gender. The validity of proportional hazards assumptions were assessed by scaled Schoenfeld residuals [69]. Correlation coefficients were calculated using the R "cor.test" function. Unless otherwise indicated, all experiments are presented as a representative of three independent experiments and quantitative data shows mean ± SD. Student's t-test was used to compare test (miR-7-5p or si-RelA) to negative control and detect statistical significance. P-values < 0.05 were considered as statistically significant.

Annexin V-FItc and PI apoptosis assay
WM266-4 cells were transfected with miR-7-5p or miR-NC at 30 nM for 3 d, then both floating and adherent cells (harvested by trypsinization) were pooled and stained with Annexin V-FITC and PI using the Annexin V FITC Apoptosis Detection Kit I (BD Biosciences, 556547), as per manufacturer's instructions and analyzed on a BD Accuri C6 .

xcelligence proliferation assay
1205Lu cells were transfected with miR-7-5p or miR-NC at 30 nM for 48 h. Cells were seeded in CIMplates to measure migration and invasion (80,000 cells/ well) and E-plates (5000 cells/well) for proliferation and loaded into the xCELLigence instrument. Cell Index was normalized to 5 h for proliferation to account for time for cells to adhere.


Hypoglycemic effect of poly-herbal combination in streptozotocin-induced diabetic rats

The present study evaluates the combinatorial effect of a poly-herbal combination with multiple therapeutic effects as a hypoglycemic and hypolipidemic agent. Herbal combination of Salacia oblonga, Tinospora cordifolia, Emblica officinalis, Curcuma longa and Gymnema sylvestre was prepared and administered to streptozotocin (55 mg/kg, i.p.)-induced diabetic rats, at a dose of 100 mg/kg, for four weeks. The poly-herbal combination significantly elevated the plasma insulin (90%) and reduced serum glucose (42%), cholesterol (49%), triglycerides (43%), LDL cholesterol (62.5%) and plasma apo B/apo A in the diabetic rats. The liver lipogenic enzymes like HMG CoA reductase and glucose-6-phosphatase significantly declined (p<0.05) in their activity while glucose-6-phosphate dehydrogenase and malic enzymes showed higher activity compared to the diabetic control. The results suggest a significant hypoglycemic and hypolipidemic property for the poly-herbal combination.

Introduction expressed as %dry weight: 30% S. oblonga leaves, 10% T. cordifolia, 10% E. officinalis, 10% C. longa and 40% G. sylvestre prepared by Kerala Ayurvedic Pharmaceutical Company, Kerala, India. The herbal formulation of the anti-diabetic plants were prepared and named as G-400 based on the guidelines prescribed by ayurvedic antidiabetic formulations (Modak et al., 2007).

Induction of experimental diabetes
The animals were fasted overnight, and diabetes was induced by a single intraperitoneal injection of a freshly prepared solution of streptozotocin (55 mg/kg) in 0.1M citrate buffer (pH 4.5) (Rakieten et al., 1963). Control rats were injected with citrate buffer alone. On the third day of streptozotocin injection, the rats were fasted for 6 hours and blood was taken from the tail vein. Rats with moderate diabetes, having hyperglycemia (blood glucose range 250-400 mg/dL) were taken for further experiment. The rats were monitored for 15 days to stabilize the diabetic condition.
In the experiment, a total of 24 rats (18 diabetic surviving rats and 6 normal rats) were used. The rats were divided among 4 groups consisting of 6 animals within each group as follows: Group I (normal rats); Group II (diabetic untreated rats); Group III (diabetic treated G-400 (100 mg/kg) daily using an intragastric tube for 4 weeks); Group IV (diabetic treated with glibenclamide (0.025 mg/kg) daily using an intragastric tube for 4 weeks).
At the end of 4 weeks, the animals were fasted overnight and sacrificed. Blood samples were collected in 2 different tubes, one with anticoagulant, potassium oxalate and sodium fluoride for plasma and another without anticoagulant for serum separation. The blood was then centrifuge at 3,000 rpm for 20 min using refrigerated centrifuge at 4°C to separate the plasma and serum, used for various biochemical studies.

Biochemical analysis
Blood biochemical parameters were estimated by using diagnostic kits. Fasting blood glucose was estimated by the oxidase/peroxidise method (Trinder, 1969) and glycosylated hemoglobin by ion exchange method as per the standard protocol described in the kit from BioSystems Diagnostics Pvt. Ltd, India. Plasma insulin level was assayed by the Radioimmunoassay (RIA) kit (Diasorin, Saluggia, Italy), using human insulin as standard. Serum high density lipoproteins, low density lipoproteins + very low density lipoproteins were separated by the standard protocol described by Wranick and Alber (1978) and Falholt et al., (1973) procedure was used to extract and estimate serum fatty acids by utilizing standard diagnostic kits supplied by Dr. Reddy's Diagnostic Lab, Hyderabad, India. Apolipoprotein A1 and apolipoprotein B100 were estimated by immune-turbidometric assay kit.

Hepatic cholesterol
Cholesterol in the liver tissues were extracted by the method of Folch et al., (1957), and estimated by the method of Allain et al., (1974). By using chloroform/ methanol mixture lipids were extracted from 100 mg of liver. Cholesterol was then determined using a colorimetric assay kit performed according to the manufacturer's instruction.
Hepatic triglycerides Van Handel's method (1961) was used in the estimation of triglycerides after removing phospholipids with flurosil. Briefly, the hepatic tissue samples were homogenized in 2:1 chloroform-methanol solution and after separating the different phases, triglycerides were measured by using a commercially available colorimetric reagent.

Protein estimation
Lowry's methods (Lowry et al., 1951) was used to measure protein concentration in the hepatic tissue by using Folins reagents (Biorad, CA,USA) and measure the absorbance at 660 nm.

HMG CoA reductase
Glutaryl co-enzyme reductase activity was estimated by measuring the ratio of 3-hydroxy-3-methyl glutaryl coenzymes to mevalonate as described (Kurian et al., 2005). Briefly, the hepatic HMG-CoA react with hydroxylamine reagent at alkaline pH resulting hydroxamic acid by formation of complexes with ferric salts measured at 540 nm. The hepatic mevalonate was estimated by reaction with the same reagent but at pH 2.1. At this pH, the lactone form of mevalonate readily reacts with hydroxylamine to form the hydroxamate.
Glucose-6-phosphatase Activities of glucose-6-phosphatase was measured by previously described standard protocol (Garland et al., 1974). The reaction mixture consist of 0.1M phosphatebuffered saline (pH 7.4), 0.1M glucose-6-phosphate solution and the sample were incubated in a 37°C water bath for 15 min. The reaction was stopped by the addition of 1 mL of 10% trichloroacetic acid (TCA), followed by chilling in ice and centrifuging at 3000 × g for 10 min.
Glucose-6-phosphate dehydrogenase and malic enzyme The NADPH-linked activities of glucose-6-phosphate dehydrogenase (G6PDH) (Schroeder et al., 1984) and malic enzyme (ME) (Pongratz et al., 2007) were investigated after the increase in absorption at 340 nm. For the determination of G6PDH activity, a buffer containing 50 mM Tris-HCl, pH 8, 1 mM MgCl2, and 5 mM NADP was mixed with 60 µg of cytosolic protein/ mL, and the reaction was started with glucose-6-phosphate acid (8 mM final concentration) at 37°C. ME activity was measured in a buffer containing 50 mM Tris-HCl, pH 7.4, 1 mM MnCl2, 5 mM NADP, and 180 µg protein/mL. The reaction was started with L-malic acid (8 mM final concentration) at room temperature.

Lipoprotein lipase
Lipoprotein lipase (Mayes and Fells, 1968) was assayed by standard protocol. The standard reaction mixture contained 0.2 mM DMPTB, 0.8 mM DTNB, 1 mM EDTA, 0.05% Triton X-100, and 50 mM Tris-Cl2, pH 7.5. Microplate wells were filled with 180 µL of this mixture and 20 µL of the enzyme sample was added to each well. The microplate was immediately transferred to a 37°C incubator to start the reaction. After the indicated period of incubation, the absorbance of each well at 405 nm was measured.

Statistics
All data were expressed as the mean ± SD. Statistical analysis was performed using one-way analysis of variance (ANOVA) for multiple comparisons among the groups.

Effect on fasting blood glucose and plasma insulin level in diabetic rats
The effect of G-400 on fasting blood glucose in control and experimental diabetic rat are shown in Figure 1. A normal fasting blood glucose level was observed in the rats treated with the drug as compared to the diabetic animals (p<0.01). In correspondence to fasting blood glucose, plasma insulin level was significantly increased after drug treatment compared to diabetic control (p<0.01).

Effect on lipid profile and apoproteins in the blood samples of diabetic rats
Diabetes induced by streptozotocin resulted in a significant elevation in the levels of total cholesterol (TC), LDL + VLDL cholesterol (LDL + VLDL ©, triglycerides (TG) and free fatty acids (FFA) in comparison to the normal control group. After treatment of diabetic group with G-400, a significant reduction (p<0.05 in TC, LDL + VLDL©, TG and FFA was observed compared to the untreated diabetic group (Figure 2). In fact, the effect of G-400 on the lipid profile was as good as to the standard drug, glibenclamide.
The Apo A1 level in blood was significantly lower and Apo B100 and Apo B/Apo A were significantly higher in the diabetic rat than those of the normal control animals ( Figure 3). Apo A1, Apo B100 and its ratio were normalized to the control level by the administration of G-400 and the protection was even effective than the animals administrated with standard drug.
The effects of G-400 on cholesterol, triglycerides levels and HMG CoA reductase and lipoprotein lipase activities were measured in rat liver ( Figure 4). Rats administered with streptozotocin showed a significant elevation in the concentration of cholesterol and triglycerides with a corresponding increased HMG CoA reductase activity and lower lipoprotein lipase activity as compared to those in the control animals. Treatment of G-400 and standard drug to the animals reduced the TC and TG levels and normalized the respective enzymes significantly (p<0.05) to the control animals.
The activities of carbohydrate metabolic enzymes and level of glycogen in the liver of normal, diabetic and drug treated rats are given in Figure 5. The decreased activities of glucose-6-phosphate dehydrogenase and malic enzyme and the concentration of glycogen observed in the liver of diabetic rats as compared with normal control rats were reversed by treatment with G-400 and glibenclamide. Similarly, increased glucose-6phosphatase activity was improved to the near normal level by the treatment of G-400 and glibenclamide. Streptozotocin-treated diabetic rats were treated with G-400 (100 mg/kg b.wt.) daily using an intragastric tube for 4 weeks. The results were compared with diabetic control rats (group 2) and normal control (group 1), where no STZ was administered. The positive control in the study comprises of rats administered with glibenclamide (0.025 mg/kg b.wt.) daily using an intragastric tube for 4 weeks. Results are expressed as mean ± SD of n = 4-6 independent assays. b p<0.05, statistically different from the control group, a p<0.05, statistically different from diabetic control

Discussion
The present study demonstrated that oral administration of G-400 could improve not only the glucose level in streptozotocin-treated rats and thereby act as anti-diabetic agent, but also reduce the hyperlipidemia. We used streptozotocin to induce diabetes in rat. According to Szkudelski (2001), streptozotocin enters the B cell via a glucose transporter (GLUT2) and causes alkylation of DNA and induces increased supply of substrate for xanthine oxidase resulting into the formation of superoxide radicals, hydrogen peroxide, hydroxyl radicals and nitric oxide that inhibits aconitase activity and participates in DNA damage, ultimately leads to β cell's destruction (Szkudelski, 2001). When animals being treated with G-400, significant improvement in the plasma insulin suggests secretagogue/β cells regeneration capacity of the drug or even the reduction of free radicals. Evidence from the early report suggests that G. sylvestrae, one of the constituents of G-400, is having insulin secretagogues action by increasing cell permeability rather than exocytosis Streptozotocin-treated diabetic rats were treated with G-400 (100 mg/kg b.wt.) daily using an intragastric tube for 4 weeks. The results were compared with diabetic control rats (group 2) and normal control (group 1), where no streptozotocin was administered. The positive control in the study comprises of rats administered with glibenclamide (0.025 mg/kg b.wt.) daily using an intragastric tube for 4 weeks. Results are expressed as mean ± SD of n = 4-6 independent assays. bp<0.05, statistically different from the control group, ap<0.05, statistically different from diabetic control  (Persaud et al., 1999). In addition, E. officinalis can stimulate basal insulin output from the remaining beta cells within the pancreas (Nainet al., 2012). In an another study, the methanol extract of G. sylvestre leaf, and callus showed anti-diabetic activities through regenerating β cells (Ahmed and Rao, 2010). Furthermore, constituents of G-400 were already reported to have high anti-oxidant potential.
Growing evidences showed a positive correlation of fasting insulin levels with obesity, systolic and diastolic blood pressure, triglyceride, β-lipoprotein cholesterol and pre-β-lipoprotein cholesterol levels (Burke et al., 1986). Hence, we measured the lipid profile in the serum (total, LDL, non-HDL, and HDL cholesterol and triglycerides) and free fatty acid along with the LDL to HDL ratio and our results suggest prominent hypolipidemic effect for G-400. This effect was the consequence of the synergic effect of G-400 constituents as evident from early study of S. oblonga (Bhat et al., 2012), G. sylvestre (Daisy et al., 2009), T. cordifolia (Stanely Mainzen Prince and Menon, 2003), E. officinalis (Augusti et al., 2001) and C. longa (Babu and Srinivasan, 1997).
The elevated apoprotein A, an integral protein of HDL and declined apoprotein B, an integral apoprotein of LDL in G-400 treated rat blood signify the antiatherogenic effect of the drug as previous study showed that apoB/apoA-I ratio represents the balance of proatherogenic and antiatherogenic lipoproteins (Lima et al., 2007). Current reports suggest that cholesterol balance, determined as the apoB/apoA-I ratio has repeatedly been shown as a better marker than lipids, lipoproteins and lipid ratios (Lima et al., 2007). As shown in Figure 3, G-400 treatment significantly reduced apo B/apo A ratio, indicates the efficacy of the drug as a hypolipidemic agent as well.
In order to check, whether the alterations in blood lipid levels correlated with tissue lipid content, we measured lipid profiles in hepatic tissues and the results found to be agreeing with blood lipid profile results. In addition, in order to reconfirm the resultant alteration in the metabolic changes following diabetes and therapy in blood and tissues, we analyzed corresponding enzyme activities in hepatic tissues of rats to affirm our results. Hypoglycemic effect of G-400 was affirmed by the low HMG-CoA reductase, the key regulatory enzyme in cholesterol biosynthesis, indicating reduced endogenous synthesis of cholesterol in liver, resulting in  Figure 3: Effect of G-400 on apo A1, apo B100 and apo A1/apo B100 levels in blood samples Streptozotocin-treated diabetic rats were treated with G-400 (100 mg/kg b.wt.) daily using an intragastric tube for 4 weeks. The results were compared with diabetic control rats (group 2) and normal control (group 1), where no streptozotocin was administered. The positive control in the study comprises of rats administered with glibenclamide (0.025 mg/kg b.wt.) daily using an intragastric tube for 4 weeks. Results are expressed as mean ± SD of n = 4-6 independent assays. bp<0.05, statistically different from the control group, ap<0.05, statistically different from diabetic control Importantly, glibenclamide treatment also reduced HMG-CoA reductase activity without significant decline in total concentration of cholesterol in liver, pointing out the effect is indirect and is associated with anti-diabetic action of the drug. The difference in G-400 action may be due to the higher cholesterol catabolism mediated by C. longa (Babu and Srinivasan, 1997) and E. officinalis (Augusti et al., 2001), the constituents of G-400.
It has been proposed that inhibition of HMG-CoA reductase leads to a reduction in the concentration of hepatic cholesterol and therefore, up regulation of LDL receptors resulted in internalization of not only LDL but also VLDL remnants (Sato and Takano, 1995), resulting a reduced plasma triglyceride concentration. We found a similar observation agreeing the consequential positive correlation between hepatic triglyceride and plasma triglyceride concentrations, reported by previous investigators (Nikkila et al., 1977).
The action of lipoprotein lipase on triglyceride-rich lipoproteins, generates free fatty acids that are either transported into the tissues or mixed with circulating free fatty acids. Accumulated evidences suggested that higher triglyceride concentration, and lower LPL activities are the determinants of small LDL size, the predictor of ischemic heart disease risk in diabetic patients. G-400 treatment not only reduced triglycerides and free fatty acids in blood and hepatic tissues, it also elevated LPL to near normal level as compared to diabetic control (Xu et al., 2008).
A decreased G6PD/malic enzyme activity leads to decreased NADPH and makes cells very sensitive to oxidant damage, one of the major reasons for pathophysiology of diabetes mellitus was improved with G-400 treatment for 4 weeks. Recovered malic enzyme activities, an important player for that regulate insulin secretion (Xu et al., 2008) substantiate the improved insulin secretion in the drug treated group and this effect was reflected in plasma insulin level as well.
In addition, elevated glucose 6 phosphatase, one of the key enzymes that determine the endogenous glucose production is a consistent feature of type 2 diabetes is Streptozotocin-treated diabetic rats were treated with G-400 (100 mg/kg) daily using an intragastric tube for 4 weeks. The results were compared with diabetic control rats (Group 2) and normal control (Group 1), where no streptozotocin was administered. The positive control in the study comprises of rats administered with glibenclamide (0.025 mg/kg) daily using an intragastric tube for 4 weeks. Results are expressed as mean ± SD of n = 4-6 independent assays. b p<0.05, statistically different from the control group, a p<0.05, statistically different from diabetic control the consequence of increased differences in insulin sensitivity, resulted from increased fatty acid oxidation and hyperglycaemia. Therefore, reduced glucose-6phosphatase activity in our study especially in those animals treated with G-400 and glibenclamide suggested better recovery of animal from diabetic insult. Growing evidence showed that insulin can stimulates glycogen synthesis in the liver and skeletal muscle and thereby increase hepatic glycogen level and the improved hepatic glycogen content of G-400 treated animal support the improve hepatic function and insulin action. Furthermore, our clinical trial with this drug in small populations showed significant recovery in diabetic patients (Kurian et al., 2014).

Conclusion
The poly-herbal drug, G-400 has potential hypoglycemic and anti-diabetic activity beside it reduced the hyperlipidemia. Hypolipidemic and anti-diabetic effect may be due to the effect of active constituents in the combination which may be responsible not only for the scavenging free radicals liberated by streptozotocin in diabetic rats but also by stimulating insulin secretion.

Ethical Issue
The experimental protocol has been approved by the institutional animal ethics committee and by the regulatory body of the government (Reg. No. 817/ac/ CPCSEA, dated 06/08/04).  Figure 5: Effect of G-400 on glycogen level and the activities of glucose-6-phosphatase, glucose-6-phosphate dehydrogenase and malic enzyme in hepatic tissue Streptozotocin-treated diabetic rats were treated with G-400 (100 mg/kg) daily using an intragastric tube for 4 weeks. The results were compared with diabetic control rats (Group 2) and normal control (Group 1), where no streptozotocin was administered. The positive control in the study comprises of rats administered with glibenclamide (0.025 mg/kg b.wt.) daily using an intragastric tube for 4 weeks. Results are expressed as mean ± SD of n = 4-6 independent assays. b p<0.05, statistically different from the control group, a p<0.05, statistically different from diabetic control Glucose 6 phosphate dehy -drogenase (unit/g protein)


extfieldsofstudy: Medicine Biology

text:
Export of microRNAs: A Bridge between Breast Carcinoma and Their Neighboring Cells

Breast cancer is a leading type of cancer among women in India as well as worldwide. According to the WHO 2015 report, it has been anticipated that there would be a twofold rise in the death due to breast cancer among women. The heterogeneous property of breast carcinoma has been suggested to be linked with dedicated set of communication and signaling pathway with their surroundings, which culminate into progression and development of the cancer. Among the plethora of communication tools in the hand of breast carcinoma cells is the recently appreciated exocytosis of the tightly packed short non-coding RNA molecules, predominantly the microRNAs (miRNAs). Recent studies suggest that miRNAs may work as courier messengers to participate in endocrine and paracrine signaling to facilitate information transfer between breast carcinoma and their neighboring cells. Evidence suggests that breast tumor cells communicate via packaged miRNAs in the tumor-released microvesicles, which enrich the tumor microenvironment. There is a strong view that dissecting out the mechanistic and regulatory aspects of miRNA export and role may uncover many prospects for overcoming the signaling defects and thereby controlling aberrant cell division. The detection of circulating miRNAs associated with breast carcinoma can also be used as biomarkers for early diagnosis. This review article is an attempt to provide updated knowledge on implications of short RNAs and their transport in the breast cancer pathophysiology.

The success or failure of these pathways is a key to regulate cell growth, proliferation, differentiation, apoptosis, and resistance against genotoxic insults (17). Small non-coding RNAs are a group of highly conserved molecules responsible for controlling gene expression and dictating cellular signaling. microRNAs (miRNAs) as a type of short non-coding RNAs with a sequence of 21-25 nt have widely been appreciated for their role in transcriptional repression, mRNA degradation, and extra-environmental signaling (13,18,19). Recently, there are sufficient data pointing to upregulation and downregulation of miRNAs in setting up the platform for augmented pathophysiology of breast carcinoma (20). The concerted efforts of protein family cause the miRNA to be stabilized in the exterior region of the cells, making it accessible to get transported to local vicinity (13,14,19). Currently, signaling pathways mediated by miRNAs have garnered wide attention in breast cancer pathophysiology. The abnormal exchange of signals involving non-coding short RNAs, including miRNAs, are found to create tumor-friendly microenvironment (21). One of the approaches to establish bridges between breast carcinoma and their microenvironment cellular community is exocytosis of tightly packed miRNAs (22)(23)(24)(25). The current understanding and consensus have pointed out that distribution and expression of miRNAs are of utmost importance for disease prognosis and potential target in cancer therapy (26,27).
In this review, we summarize biogenesis of miRNAs and their export mediated by exosome to target the neighboring cells in the microenvironment. The focus of this paper is to mark the miRNAs as a potent extracellular signaling molecule responsible for breast carcinoma pathophysiology.

CeLL SiGNALiNG OveRview iN BReAST TUMOR
Signaling is the key element for any cell to survive, grow, and proliferate. The mechanism of signaling is categorized into four types, autocrine, paracrine, juxtacrine, and endocrine signaling, which is described in the Section "Introduction. " Signaling pathways are based on the ligand-receptor binding complex, which leads to a much stronger downstream signal, thereby regulating the genetic expression. Signaling upon binding of a ligand in the cells mostly involves phosphorylation of the receptor at specific amino residues, which recruits various intracellular proteins amplifying the intensity toward the nucleus. There are a number of signaling pathways that are responsible for the cells growth, differentiation, proliferation, and also for resisting apoptosis and further metastasizing (28).
One of the signaling pathways is Notch pathway that is responsible for survival, growth, and proper development of cells. Notch is a transmembrane receptor, which has a strong affinity for the delta ligands and with its proteolytic cleavage travels to the nucleus and acts as transcriptional regulator activating gene expression. Some miRNAs either act as the notch receptor inhibitors or as the nuclear transcriptional inhibitors. As such, notch receptor inhibitor is miRNA-141, whereas Snail and zinc finger E-box binding homeobox 1 (ZEB1) transcriptional factors are inhibited by miRNA-34a (11). miRNA-374a is found to be overexpressed in Wnt/β-catenin pathway. For example, miRNA-451 acts as a potent inhibitor for the downstreaming signaling of the Wnt/β-catenin, decreasing the levels of β-catenin (29). Hedgehog is a developmental signaling pathway, which focuses on the embryogenesis, carcinogenesis, and tissue repair caused due to chronic inflammation. The oncogenic miRNA cluster family (miRNA17-92) regulates the Hedgehog signaling (9). Signaling pathway NF-κB plays a crucial role in the cell proliferation, tumor development, inflammation, and also regulation of the innate and adaptive immune response (8). The pathway is also known to be guided by the expression of different miRNAs. There are several mechanisms by which NF-κB pathway is regulated. miRNA-21 targets signal transducer and activator of transcription 3 (STAT3), which is downregulated. Furthermore, miRNA-21 targets phosphatase and tensin homolog (PTEN), thereby inhibiting Akt phosphorylation and promoting NF-κB activation (30).
Cancer-associated fibroblasts are the stromal cells, nonmalignant in nature, which arise from the differentiation of the progenitor cells to promote tumor health by satisfying their nutrient needs (31,32). Highly activated growth and proliferation of the cancer cells is due to unregulated signaling pathways. The tumor microenvironment helps the cancer cells for this aberrant signaling, as these cells are highly dependent on the microenvironment for their increased need of energy for their proliferation. This tumor microenvironment is solely responsible for causing apoptosis and drug resistance by cell-cell communication via some matrix soluble factors enhancing the cellular growth and its development (33). Overexpression of soluble growth factor such as cytokines, mediated by cancer-associated fibroblast cells, acting as nutrient benefactor for the cells can decrease the integrin expression, further leading to metastatic progression (33).

TYPeS OF NON-CODiNG RNAs
Short non-coding RNAs consist of 1% of the genome (34), with size ranging from 20 to 25 nt. Small non-coding RNAs consist of miRNA, small interfering RNA (siRNA), piwi-interacting RNA (piRNA), small nucleolar RNA (snoRNA), and extracellular RNA (exRNA) (35). These classes of short RNAs differ in the biogenesis and their mechanism of action (36). There is another category of non-coding RNA that is the long non-coding RNA (lncRNA). Short non-coding RNAs are directed by protein family, Argonaute (Ago), which guide the RNAs to their specific targets, thereby reducing the expression of the target gene (36). Among the class of non-coding RNA, miRNAs are known to play a pivotal role in the extracellular signaling of the cells. The flow diagram of different small non-coding RNAs is depicted in Figure 1.
Small interfering RNAs (siRNAs) are small (20-25 nt) doublestranded RNAs that act through the RNA interference (RNAi) pathway to interfere with the expression of a specific gene containing a complementary sequence. RNAi can downregulate expression by inducing degradation of the targeted RNA, interfering with transcription or inducing epigenetic changes to the gene (37).
Piwi-interacting RNAs also known as piRNA is a category of small non-coding RNAs with length slightly greater than miRNAs of about 26-31 nt. These categories of RNAs act as gene silencers. piRNA carries an anti-sense sequence to the transposons, thereby targeting the silencing of the transposons (38).
Small cajal body RNA (scaRNA) is an important player in the splicing mechanism, which regulates the spliceosome machinery by biochemically modifying specific nucleotide (39).
The extracellular miRNAs (exRNA) are one of the forms by which non-coding RNAs are exported. These exRNAs are loaded onto high-density lipids (HDLs) or bound by AGO2 protein outside the donor cell, thereby protecting miRNA from RNA degrading enzymes. These are known to regulate cell-cell communication and are used as biomarkers in various diseases as these RNAs circulate stably in the blood stream (40,41).
Small nucleolar RNA generally range from 60 to 300 nt in length and guide the site-specific modification of nucleotides in target RNAs via short regions of base pairing. There are two major classes, the box C/D snoRNAs, which guide 20-O-ribosemethylation, and the box H/ACA snoRNAs, which guide pseudouridylation of target RNAs. These RNAs take part in the development of cancer, where they promote transformation, tumorigenesis, and metastasis (42).
Small nuclear RNA (snRNA) is one of the many small RNA species confined to the nucleus; several of the snRNAs are involved in splicing or other RNA processing reactions. The snRNAs are extremely conserved in both primary and secondary structure. Moreover, some residues seem to be completely invariant between organisms. They promote exon ligation and intron excision by acting as a catalyzing agent in the spliceosome machinery and thereby aiding the protein translation (43).
Long non-coding RNAs have received wide attention in gene regulation and are considered as non-protein coding transcripts longer than 200 nt. This nucleotide length limit differentiates long ncRNAs from small regulatory RNAs, such as miRNAs, short interfering RNAs (siRNAs), piRNAs, snoRNAs, and other short RNAs (44).

BiOGeNeSiS OF microRNA
First miRNA discovered was lin-4 of 22-nt length and was identified in a gene responsible for postembryonic development in C. elegans carrying a complementary sequence in the 3′ UTR of its regulatory target gene (34,45,46). There are about 6930 miRNAs discovered in animals and their viruses (47). The miRNA gene is transcribed by RNA polymerase II to produce a primary microRNA (pri-miRNA) precursor molecule whose length varies greatly may be up to 3-4 kb (13,19,34,45,46). Furthermore, pri-miRNA undergoes nuclear cleavage with sequential actions by nuclear RNase III Drosha and a partner called DGCR8 with double-stranded RNA-binding domain forming a pre-miRNA, 60-70 nt long, as a hairpin structure with two overhangs at their 3′ and 5′ phosphate groups (34,45,46). Exportin-5 recognizes the 2-nt overhang generated by RNase III enzyme Drosha at the 3′ end of pre-miRNA hairpin. The protein Exportin-5 (XPO5) plays a crucial role in escorting the pre-miRNA bound to Ran-GTP from the nucleus to the cytoplasm through the nuclear pore (13,19,45). Cytoplasmic cleavage of the pre-miRNA occurs with cytoplasmic RNase III Dicer into ~22-nt miRNA duplexes. The Dicer, an endoribonuclease, interacts with the ends and cuts away the loop joining it, generating an imperfect duplex of miRNA:miRNA. In cytoplasm, the Dicer and TAR RNA-binding protein (TRBP) separates the two strands which are incorporated into the RNA-induced silencing complex (RISC), also known as miRISC (45). The strand with higher stability and the stronger base pairing is associated with the silencing complex and other proteins like Argonaute. The weaker strand in aspect of the two criteria can be either degraded or can act as a functional unit, further recruiting the RISC and protein complex (48).
The outline of miRNA biogenesis is illustrated in Figure 2. miRNA plays an important role in the posttranscriptional regulation by recognizing the specific mRNA and incompletely pairing with the 3′ untranslated region causing translational suppression and inhibition of protein synthesis or by degrading the mRNA. They play a major role in making diverse biological decisions such as proliferation, differentiation, and apoptosis. miRNA can be secreted as circulating miRNA (49) that can either exist outside the cells with the help of various binding proteins or can be exported outside by extracellular vesicles such as exosomes, microvesicles, or apoptotic bodies (50). RNA-binding protein (RBP), Ago-1, and high-density lipopolysaccharides have been reported to assist miRNA out of the cells and prevent its degradation. miRNAs present in the blood plasma are most stable in the microenvironment carrying the property of resisting high and low temperature and varied pH (40). Among all the non-coding RNAs, only miRNAs are seen to play a pivotal role in tumor signaling and also in cardiovascular, neurological, and other developmental diseases (51,52). The pre-miRNAs exit from the nucleus to the cytoplasm by exportin 5 (XPO5). In the cytoplasm, it is further processed by DICER1, a ribonuclease III (RIII) enzyme, that gives rise to the mature miRNAs. One strand of the mature miRNA (the guide strand) is loaded into the miRNA-induced silencing complex (miRISC) comprising DICER1 and Argonaute (AGO) proteins. The binding of miRNA to target mRNAs is facilitated by sequence complementary binding, leading to translational repression. This schematic diagram is modified from Lin and Gregory (42). PACKAGiNG AND SHiPPiNG OF miRNAs microRNAs are protected from the potent degrading factors such as RNase during circulation and signaling through the microenvironment. The diagram showing mode of miRNA export is provided in Figure 3. These miRNAs are shielded by two ways, one being encapsulation in the extracellular vesicles, namely, exosomes, microvesicles, or apoptotic bodies (53) and the other being complexing with number of RBPs, such as Ago protein family, or HDL, that occurs in the blood plasma (54). All the three categories of the extracellular vesicles share the same membrane composition, thereby making it tedious to separate out, but they carry different pathways of biogenesis and secretary mechanism (55). Exosomes are lipid vesicles ranging between 40 and 100 nm in diameters, which arise from the inward budding of plasma membrane of the cell forming an intraluminal vesicle (56). They are associated with the plasma membrane through ligand-receptor interactions (57,58) or lipids. Exosomes carry a property of taking up various miRNAs, mRNAs, and proteins without eliciting any immune response (59). These vesicles are relatively stable in the cell's external environment and can easily cross the blood-brain barrier. Exosomes are composed of numerous protein families such as tetraspanin proteins, CD63, CD9, and CD81. Primarily, the cell membrane is internalized to form an endosome, followed by the formation of small vesicles inside the endosomes. These endosomes are then called multivesicular bodies, and they transfer the vesicles into the extracellular space in the form of exosomes (60). The exosome membrane contains lipid rafts containing cholesterol, sphingolipids, glycerophospholipids, and ceramide. The protein content is also significant for the vesicle to travel and target the cell membrane. Various proteins, such as integrin family, cytoskeletal proteins, heat-shock protein family, and vesicle trafficking proteins, play a role in making the vesicular environment habitual for shipping the RNAs and proteins (61).
The levels of signaling via exosomes or vesicular transport have seen to be elevated in tumor cells as compared with the normal cells. There are lots of compounds which are studied to act on a target molecule to inhibit the formation of the extracellular vesicles carriers, thereby creating a signaling gap within the tumor microenvironment. Molecules, such as proton pump inhibitors, methyl-β-cyclodextrin, and annexin, are known to inhibit the vesicle formation by targeting the Na + reabsorption, cholesterol, and phosphatidylserine, respectively, and thereby blocking the membrane fusion, lipid raft-mediated endocytosis, and phagocytosis (62).
Microvesicles also referred as shedding microvesicles or ectosomes are small membranous sacs, which are the product of the budding and blebbing of plasma membrane (56,63). Microvesicles are large vesicles, which are 50-1000 nm in diameter. Microvesicles participate in the cells growth, proliferation, and its death. There are tumor-originated microvesicles, which are secreted into the tumor microenvironment, which helps the neighboring cells to secrete cytokines, which can help the tumor replenish and can mask the immune response (64).
Apoptotic bodies also called apo-bodies arise from the cells undergoing cell death by apoptosis. These are the largest of the extracellular vesicles of 400-4000 nm in diameter. These bodies consist of the condensed chromatin and cytoskeletal proteins (65). As these arise from the shrinkage of the cell, there are many RNA and DNA fragments present whose uptake can result into the transcriptional repression (66).
Extracellular miRNAs are associated with lipid-based carriers and lipid-free proteins (62). Circulating miRNAs are stable when they are associated with plasma, which serves as the disease biomarkers (67). The miRNA-223 is found to be transported using HDL molecule, and the Ago protein family is associated with the extracellular miRNA transport accompanying both in and out of the cell (67). Exosome circulate for a short time in the body fluids before getting internalized into the recipient cell, a process called free floating. Furthermore, adhesion requires a conformational change in the status of the low affinity to high affinity binding to integrin-bound exosome (68). Uptake of exosome is dependent upon the signaling status of the recipient cell and the protein composition of the vesicle (69). Exosome internalization involves various mechanisms such as membrane fusion, soluble signaling, juxtacrine signaling, lipid raft-mediated endocytosis, and macropinocytosis (68). These mechanisms are either receptor-based or involve actin filament protrusions, which engulf the vesicle dispatching the cargo in the cytoplasm (70).
Release of exosomes or microvesicles occurs in the similar fashion like the viral budding (63,71). The property of the plasma membrane forming a protrusion is driven by a number of proteins and lipids associated with the membrane. The budding of the exosomes via endocytosis and membrane trafficking is mediated via various coat proteins like clathrins and others like annexing, a Ca 2+ binding protein, which are present in the cell and which has the ability to recruit phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2], sphingolipids, and cholesterol (72). A protein motif called amphipathic helix that acts on the integral site and membrane proteins that bind on the periphery senses the membrane curvatures and are inserted into the membrane, facilitating the budding, and thereby the vesicle formation (69). Lipid rafts play a major role in the promotion of signaling mediated through proteins anchored by glycophosphatidylinositol and help in sorting and membrane trafficking in the secretory pathways (62,73).

COMMUNiCATiON BRiDGe BeTweeN miRNAs TO NeiGHBORiNG FiBROBLAST
Specific targeted therapies can be achieved by encapsulating the drug into the exosomes with a clear prospect of the therapeutic cargo, the peptide or DNA target of the drug, mode of uptake, and mechanism of their action (74). Some miRNA-targeted therapies are also being designed to deliver antitumor miRNA to the breast tumor cells (75). Exosomes have natural ability to transfer genetic material both locally and systemically, and, therefore, many research groups investigated these vesicles as therapeutic agents. As an instance, microvesicles isolated from endothelial progenitor cells (EPCs) contained proangiogenic miRNA-126 and miRNA-296 (76), and transfer of these miRNAs triggered the activation of the PI3K/Akt signaling pathway and phosphorylation of endothelial nitric oxide synthases and directed endothelial cells to undergo angiogenic and antiapoptotic program (77).
The pivotal role played by the microenvironment-associated resident cells in breast tumor progression has been widely appreciated. In a recent attempt to emphasize the give and take type of exosome-based connection between breast carcinoma and preadipocyte, a type of cellular community in the tumor microenvironment has been reported with clear role of miR-140/SOX2/SOX9 axis that may regulate tumor microenvironment signaling and communication (78). Intentionally diverted glucose metabolisms are a trademark of breast carcinoma. In recent findings, several research groups have attempted to provide evidence that breast carcinoma cells use the secreted vesicle-contained miRNAs as a tool to deprive of the neighboring cells for their food availability, in particular, they target the glucose uptake (79). They have also convincingly mentioned that breast carcinoma may use such strategy to reprogram and progress with destructive metastasis goal (80).
As an evidence of influencing the progression of breast phyllodes tumor xenografts, miRNA-21 enhanced tumor growth and facilitated metastasis by inducing myofibroblast differentiation (81). The recurrence and relapse of breast carcinoma after removal of primary tumor may be driven by well-communicated cell-cell signaling, leading to cancer cell dormancy. In view of the role of secreted miRNAs in maintaining the dormancy of breast cancer cells, evidence has explained that exosomal transfer of miRNA-23b from the bone marrow may promote breast cancer cell dormancy in metastatic settings (82). Such observation will lead to insights for therapeutic approaches by blocking the miRNA-23b-mediated dormancy control in breast cancer cells so that further cell cycling stages of cancer cells will succumb to the drug regimen therapy (82).
The differential expressions of miRNAs in the context of contribution of cancer-associated fibroblast in breast cancer progression are being unraveled. In the same line, the experimental evidence points out that miRNA-26b is highly aberrant miRNAs found in cancer-associated fibroblast and may be implicated in breast carcinoma progression, growth, and recurrence (83).
There is growing view that the cancer-associated fibroblasts and normal fibroblasts being in the neighborhood of breast tumor act differently with the first responsible for inducing breast tumor progression and second engaged in tumor blockade. In this context, about 11 dysregulated miRNAs in cancer-associated fibroblasts have been pinpointed that include three upregulated (miRNA-221-5p, miRNA-31-3p, and miRNA-221-3p) and eight downregulated (miRNA-205, miRNA-200b, miRNA-200c, miRNA-141, miRNA-101, miRNA-342-3p, let-7g, and miRNA-26b) (84). Furthermore, such observation accentuates the notion that miRNAs derived from breast tumor neighboring cells may influence the status of breast tumor through cell-cell communication (85).
The collaboration between stromal cell community and breast tumor cell is being overwhelmingly recognized as a factor behind the tumor progression and development. In recent evidence, there are highlighted reports that suggest that the miRNA-320 is a vital component of the phosphatase and tensin homolog deleted on chromosome 10 (Pten) tumor suppressor axis that works in stromal fibroblasts to manipulate the tumor microenvironment and restrain tumor progression (86). The inducement of senescence has been considered as a blockade for different cancer cell progression, including breast carcinoma. There has been report on the contribution in tumor suppression by senescence-associated microRNAs (SA-miRNAs), miRNA-22, which is overexpressed in human senescent fibroblasts and epithelial cells. Since, such reports validate the idea of communication bridge between stromal cell and tumor cell progression, further studies will provide better understanding and platform for therapy approaches by manipulating the miRNA level in the tumor neighborhood cell community (87).

The Communication Bridge between Carcinoma and Surrounding Macrophage
In an effort to delineate the collaboration between breast carcinoma and their cellular community in microenvironment, authors have reported that miRNA-21 and miRNA-29a expression are upregulated in tumor-infiltrated myeloid cells and macrophages. Furthermore, authors have suggested that these upregulated miRNAs act as helping hand for the breast tumor for their progression and metastasis (88). In recent time, there are emerging evidence of close association between breast carcinoma Circulating miRNAs Circulating miRNAs: novel breast cancer biomarkers and their use for guiding and monitoring response to chemotherapy (116) miRNA-10b Evaluating the expression levels of microRNA-10b in patients with gliomas (117) and carcinoma-trained or -taught macrophages, in tumor microenvironment. Furthermore, the miRNA profiling data corroborated the observation that deregulated miRNAs expression level is linked with breast tumor progression and prognosis (89). Recent convincing view is that breast tumor-associated macrophages are being extensively causative in the trademark settings of tumor microenvironment. To understand such phenomena, one study suggested that a crucial extra player, miRNA-19a-3p, highly expressed in tumor-associated macrophages may be able to retard breast cancer progression and metastasis through the downregulation of Fra-1 proto-oncogene (90). The friendly promotion of breast cancer progression and metastasis by tumor-associated macrophages has extensively been highlighted in recent times. In the same direction, there are reports on macrophage-secreted exosome-encapsulating miRNAs such as miRNA-223 that are packed to deliver breast carcinoma and upon entry into the cancer cells resulted into better potentiation of carcinoma cells for their progression and metastasis (91). Recently, appreciation has been received to find the clue behind friendly neighborhood cell community in supporting the survival strategy of breast carcinoma cells.

Crosstalk between Carcinoma and Stromal Cells
In the line of reported macrophages, fibroblast, and epithelial cells, there are findings on the contribution of human mesenchymal stem/stromal cells (hMSCs) to upkeep breast cancer survival advantages mediated by the secretome. Furthermore, Vallabhaneni et al. (92) have also demonstrated and characterized the role of miRNA-21 and 34a as tumor-friendly miRNAs. Being heterogeneous within the breast tumor milieu, the breast cancer cells are always receptive toward any signals or messages coming from the outside stromal cells populations. To investigate such cell-cell crosstalk, the facts have established that communication between stromal stem cells with the breast carcinoma cells mediated by Twist1 protein player leads to aberrant expression of miRNAs, specifically led by miRNA-199a, which ultimately landed in repression of Forkhead box protein P2 (FOXP2) transcription factor, that may be responsible to encourage breast cancer survival and progression (93).
There has been interest in the clues or signals contributing to molecular alterations in breast tumor stromal environment that actually contribute in the betterment of breast carcinoma. To follow such unrevealed, it was pointed out that upregulation of miRNA-21 in stromal cells may be helping breast tumor for progression and linked it as a prognostic factor (94). The release of extracellular vesicular cargo containing vital messenger in the form of proteins, miRNAs, or tRNAs has been reported from both breast carcinoma and their neighborhood cells, and such cargo gifs are released in the microenvironment to guide or mentor each other type of cells (95). In future, better understanding of molecular interplay involved in the breast tumor microenvironment supporting the breast tumor progression and metastasis may open up the therapeutic intervention strategy or early detection (95,96).
The contributions of stromal cells in the breast tumor progression are being established through multiple molecular mechanisms, such as augmented invasiveness, angiogenesis, and immunosurveillance masking. The recent experimental data report that miRNA-126/miRNA-126(pair) overwhelms the sequential recruitment of mesenchymal stem cells and inflammatory monocytes into the tumor stroma, leading to the breast tumor metastasis. In other words, downregulation of miRNA-126/miRNA-126(pair) in the breast carcinoma cells may be a strategy to avoid immunosurveillance in tumor (97)

The Summary of Therapeutic Relevant miRNAs
Oncogenic microRNAs (OncomiRNAs) dysregulation causes the onset of cancer. OncomiRs act as negative regulators of the tumor suppressor genes. The most screened miRNAs as prognostic markers of breast cancers are miRNA-10b, miRNA-21, miRNA-373, and miRNA-27a (20,98). The summarized reviews on oncogenic miRNAs are presented in Table 1.

Tumor Suppressor miRNAs
Tumor progression can lead to increase and decrease of some exosome-mediated miRNAs. The decreased miRNAs are known to play a role as suppressor of the tumor. These miRNAs inhibit the growth of tumor by negatively regulating the OncomiRNAs (15,26,27). In wide prospects as oncogenic, tumor suppressor, prognosis, and diagnosis tools in cancer, there are appreciable efforts that have been reported in the form of clinical trails, and few examples have been listed in Tables 2 and 3.

CONCLUSiON AND FUTURe ASPeCTS
Short RNAs participate in the intracellular as well as intercellular metabolic and developmental signaling pathways in several types ReFeReNCeS of cancer, including breast carcinoma. Decades of research has provided in-depth information regarding the non-coding RNA pathways. miRNAs are known for their ability to communicate with the extracellular microenvironment comprising of cells in its proximity via vesicle-derived cargo or conjugated proteins. Levels of various miRNAs differ according to the progression and pathophysiology of breast cancer. Consequently, therapeutic agents such as tumor suppressor miRNA can be designed based on encapsulation in exosome-like vesicle to the neighboring cells of breast tumor for reduced communication. In future, drugs/ inhibitors to disrupt friendly communication between breast carcinoma and neighboring cells should be appreciated. Oncogenic miRNAs that are seen to be upregulated in breast tumor and released in their surroundings may used as a biomarkers and diagnostic tools. This review provides the summary in the field of breast cancer pathophysiology focusing on the role of small non-coding RNAs, including miRNA.

AUTHOR CONTRiBUTiONS
NS contributed by proposing the concept of the title of review paper, designing the abstract, and drafting the manuscript. DJ and SP contributed by reviewing the literature and converting it into the manuscript form, and drawing of flow diagrams and figures. All authors listed, have made substantial, direct and intellectual contribution to the work, and approved it for publication.