Role of Exosome in Delivery of MicroRNA Into Cells As a Major Tool of Disease Resistance


 Exosomes are small-sized extracellular vesicles. Their size ranges from 30-150 nm and contains proteins, lipids, nucleic acids along other bioactive molecules. It is now also considered as communicating mediators between cells where their content varies with the microenvironment of the cells which may ultimately change numerous physiological and pathological functions of the cells. Because of their small size, safety, biocompatibility, biorecognition, high stability, target specificity, and ability to cross the blood-brain barrier, the exosomes have recently gained attention as a potential nano delivery system. However, the focus of this review article is to cover the latest updates regarding the role of exosomal delivery of different micro RNAs which have an important role in disease control. Micro RNAs, which are of utmost importance as an exosomal cargo, has been reported to show both positive and negative impact on the cell it is targeted or delivered to.


Introduction
Several studies reported that miRNA carries via exosome from a parental cell to a target cell can play an immensely positive role in disease control and different miRNA families are responsible for repressing different diseases when delivered via exosomes (Barile and Vassalli, 2017). The functions of exosomal miRNAs can be generally classi ed into two types. One is the conventional function, i.e., miRNAs perform negative regulation and confer characteristic changes in the expression levels of target genes and may be responsible for disease development. The other is the building of defence mechanisms against different diseases. Till now there are several miRNAs reported as a controversial role in both disease development as well as in disease prevention mechanisms. Based on these two functions we have selected ten miRNAs for this review article.
Role of different miRNAs in disease development and prevention mechanisms against diseases: The miRNAs are the key elements of the regulation of many diseases. This is due to their exclusive property of binding to the 3´ UTR region of the target messenger RNA, which eventually results in posttranscriptional down-regulation of the expression of that gene (Haug et al, 2015). For this, sometimes the miRNAs cause the development of the disease by silencing a gene that may act as a suppressor of the disease. Such as, miR-128, which targets PPARγ (peroxisome proliferator-activated receptor-gamma) leading to the development of Alzheimer's disease in mice . On the other hand, they may sometimes act as a suppressor of a gene that may be associated with the development of the disease. For example, miR-27a-5p and miR-34b-3p were found to act as tumor-suppressor micro RNAs in small cell lung cancer, targeting topoisomerase 2 alpha (TOP2A), maternal embryonic leucine zipper kinase (MELK), centromere protein F (CENPF), and SRY-box 1 (SOX1), which are identi ed as oncogenes (Mizuno et al, 2017). Thus, exosomal delivery of these various micro RNAs plays a very crucial role in disease regulation as exosomes are recognized as one of the media of communications between two or more neighbouring cells (Fig.2).
Micro RNA-21: The micro RNA 21 (miR-21) has been shown to play a pivotal role in the development as well as disease defence. Several studies have revealed that exosomal miR-21 has a role in the development of chemoresistance in different carcinomas like neuroblastoma, ovarian cancer, gastric cancer, pancreatic cancer, hepatocellular carcinoma, etc (Challagundla et. al, 2015). Exosomes isolated from ovarian cancer-associated broblasts (CAFs) and cancer-associated adipocytes (CAAs), carry miRNA-21 which could not only increase chemoresistance to paclitaxel through downregulating their direct target Apoptotic protease activating factor 1 (APAF1), but also could suppress the apoptosis of ovarian cancer cells (Yeung et al., 2016). It has been seen that transfecting cisplatin-resistant oral squamous cell carcinoma (OSCC) derived exosomes can transform non-resistant parental OSCCs into resistance to cisplatin targeting phosphatase and tensin homolog (PTEN) and programmed cell death 4 (PDCD4) . Transfection of miRNA-21 loaded exosomes into human gastric cancer cell line acted as a promoter of the tumor by targeting the PDCD4 gene, causing inhibition of PDCD4 expression and hence apoptosis in gastric cancer cells .
Environmental factors are also found to be effective in disease development via the exosomal transfer of miRNAs. Exosomes derived from arsenite-transformed human bronchial epithelial (HBE) cells can deliver miRNA-21 to normal HBE cells resulting in their transformation into malignant cells .
The miRNA-21 has been found to play a progressive role in breast cancer development (Hannafon et. al, 2016). Such as it was found to be present in extracellular vesicles derived from serum-deprived human mesenchymal stem/stromal cells (SD-MSCs), playing a tumor-promoting role in breast cancer (MCF-7) or osteosarcoma (KHOS) cells (Vallabhaneni et. al, 2015). Exosomes derived from Cancer-associated broblasts (CAFs) carry mir-21 which is mainly responsible for a signi cant increase in capacity of the breast cancer cells to form mammospheres, increasing stem cell and epithelial-mesenchymal transition (EMT) markers, and anchorage-independent growth of the cells (Donnarumma et. al, 2017).
The gastric cancer cell-derived exosomes were found to be carrying miR-21-5p, which is considered as the guide strand of miR-21, that prompted mesothelial-to-mesenchymal transition of peritoneal mesothelial cells rendering peritoneal metastasis . A signi cantly enhanced expression of miR-21 is considered as a marker for the detection of aggressiveness of pancreatic cancer (Goto et. al, 2018). It was observed that level of exosomal miR-21 was higher in hepatocellular carcinoma cell-derived exosomes and when hepatic stellate cells (HSC) were treated with these exosomes, they were activated through the PTEN/PDK1/Akt pathway, converting HSCs into cancer-associated broblast cells, that promotes angiogenesis, and this was regulated by miR-21 . Bioinformatics analysis revealed that miR-21 owned binding sites on PTEN, PTENp1, and TET (ten-eleven translocation) family proteins (TET1, TET2, and TET3), decreased PTENp1 and PTEN expression, thereby signi cantly promoting the proliferation of hepatocellular carcinoma cells (HCC), inhibiting their apoptosis, as well as promoting cell invasion ). In the same study, it was seen that the direct cecal coimplantation of colorectal cancer cells (SW620) with MRC5 broblasts, stably overexpressed miR-21 and as a result, there was a greater amount and size of metastatic tumor accumulation in the liver in an orthotopic colorectal cancer model, and it was supported by histological analysis exhibiting the presence of colorectal adenocarcinoma in the liver metastases (Bhome et. al, 2017). In patients with TNM (Tumor Nodes and Metastasis) stage of colorectal cancer miR-21, which was upregulated in the exosomes, showed a signi cant association with liver metastasis and TNM stage, also worsening the overall survival rates and disease-free survival rates of the patients (Tsukamoto et. al, 2017). When packed into M2 macrophage-derived exosomes (MDE), miR-21a-5p shows a high expression level and promotes migration and invasion of colorectal cancer cells by downregulating BRG1 expression when shuttled from M2 macrophages to colorectal cancer cells via exosomes (Lan et. al, 2019).
The miR-21 exhibited a 1.318-fold increase in the urinary exosomes from renal brosis patients compared to the controls also showing a signi cantly positive relationship with the tubulointerstitial damage index in the disease cases, suggesting them to be an important diagnostic tool in the diagnosis of the disease (Lv et. al, 2018). A miRNA pro ling of urinary exosomes in active lupus nephritis patients showed upregulated expression of miR-21, which played an important role in increasing the synthesis of pro brotic molecules which in turn affects renal brogenesis (Solé et . al, 2019). On the other hand, in another study, a down-regulation of miR-21 was observed in urinary exosomes of active lupus nephritis patients concerning inactive ones. It had a negative correlation with both protein leakages and glomerular ltration rate (Tangtanatakul et. al, 2019). A remarkably interesting nding was shown that Endometrium mesenchymal stem cell (EnMSCs) derived exosomal miR-21 when delivered to infarcted rat myocardium, it acts as a principal effector of cardioprotection by contributing to the enhanced paracrine activity of EnMSCs, involving reduced expression of PTEN and increased phosphorylation of Akt in the exosome-treated target cells . Primary human TAM (Tumor-associated Macrophages)-derived exosomes were enriched with miRNA-21-5p, which synergistically induced Treg/Th17 cell imbalance by targeting STAT3 directly in CD4p T cells, which in turn generates an immune-suppressive microenvironment facilitating the progression of epithelial ovarian cancer .
This micro-RNA also exhibited some disease-developing characteristics in bone metastases cases. A miRNA pro ling of human osteosarcoma cells-derived exosomes revealed that the metastatic osteosarcoma cell (SAOS2) derived exosomes contained a 3 fold higher level of miR-21-5p than the nonmetastatic osteosarcoma cell (MG63) derived exosomes. Also, that may be due to this high content of miR-21-5p in the SAOS2 exosomes, they act as paracrine agents and could induce metastatic behaviour The role of this miR-21 is remarkably interesting in the case of cardiac diseases. The role of this miRNA is mostly protective towards cardiac diseases. Being able to better survive in ischemic myocardium, induced pluripotent stem cell-derived exosomes were used in a study to treat myocardial disease. It was seen that these exosomes contain a high amount of miR-21 and when they are transported into cardiomyocytes, they result in the inhibition of cardiomyocyte apoptosis by suppressing the expression of caspase3 protein . MiR-21 was signi cantly upregulated in exosomes derived from oxidative stress-induced cardiac progenitor cells. The expression of this micro RNA seemed to be downregulated in cardiomyocytes under oxidative stress but regained their expression when these cardiomyocytes were treated with the Cardiac Progenitor Cells (CPC)-derived exosomes. Not only that but this miRNA results in a decrease of caspase3 and PDCD4, leading to the inhibition of apoptosis of the cardiomyocytes (Xiao et. al, 2016). Delivery of Mesenchymal stem cell-derived exosomes carrying highly expressed miR-21-5p, to cardiomyocytes (H9c2), exhibit protective effects by preventing cardiac cell death from oxygen-glucose deprivation, basically by targeting and downregulating pro-apoptotic gene products, such as PDCD4, PTEN, Peli1 and FasL (Luther et. al, 2018). Despite its protective activities in cardiac cases, it was seen that miR-21-3p, which is the passenger strand of miR-21, was present in an enriched amount in the exosomes derived from nicotine-treated macrophages, stimulated the development of atherosclerosis and induced migration of vascular smooth muscle cells by targeting PTEN (Zhu et. al, 2019).
This miRNA was also found to have a speci c role in viral diseases. Human papilloma viruses (HPVs) are one of the causative agents of cervical cancer. According to an analysis, the upregulation of miR-21-5p in HPV-infected cervical cancer cell-derived exosomes was dependent upon the silencing of the E6/E7 oncogene (Honegger et. al, 2015). A lower expression of miR-21 in plasma-derived exosomes seemed to be associated with the declination of CD4 T cells in HIV-1-infected elite controllers (Ruiz-de-Leo´n et. al, 2019).
All the above, studies were e cient in suggesting the differential role of the exosomal miR-21 family, in the development or/and the treatment of different diseases.
Micro RNA-19: The role of the miR-19 family is seen most signi cantly in colorectal carcinoma cases and identi ed as a potential marker for this disease (Eylem et. al, 2020). Not only in cases of colorectal cancer but also the differential expression of exosomal miR-19 was observed in patients with gastric cancer , pancreatic cancer (Zou et. al, 2019), bladder cancer (Amuran et. al, 2020) and in patients with chronic lymphocytic leukemia (CLL) (Jurj et. al, 2018). Therefore, exosomal miR-19 can act as a potential diagnostic marker for these cancers too. Additionally, miR19a exhibit a disease-resistant role in cardiac diseases. miR-19a reduced the expression of apoptosis regulatory genes PTEN and BIM (Bcl2l11), directly participating in the regeneration of the myocardium, thus providing cardio protection in post-ischemic conditions .
Exosomal miR-19b has an interesting role in the recovery of diabetic neuropathy (Fan et. al, 2020), Parkinson's disease (Cao et. al, 2017) and Alzheimer's disease (Gui et. al, 2015) and be a potential biomarker of these diseases.
Micro RNA-let-7 group: The infection of the Japanese Encephalitis virus, resulted in an increase of let-7a/b in the microglia-derived exosomes. This elevated amount of let-7a/b, by collaborating with TLR7 and NOTCH signaling pathway increased the secretion of TNFα from microglia. Not only that, but these miRNAs also promoted neuronal damage by activating caspase, a mediator of in ammation and apoptosis (Mukherjee et. al, 2019). Although, this group of miRNAs has been also found to show the bene ciary effect on neuron-related diseases. As, in a certain study, it was found that mouse mesenchymal stem cell-derived exosomes contained highly expressed miRNAs including let-7a, let7e, and let-7d, which downregulated TLR4/NF-κB signaling pathway, the main element involved in diabetic peripheral neuropathy and hence contribute to improvement of neurovascular function and recovery from the disease (Fan et. al, 2020).
Cigarette smoke is the main causative agent of chronic obstructive pulmonary disease (COPD). The primary human lung microvascular endothelial cells release exosomes (termed as circulating endothelial microparticles i.e., EMPs) enriched with several different micro RNAs including let-7d, which signi cantly increased upon exposure to cigarette smoke. It was assumed in this study, that the uptake of these exosomes by distant endothelial cells may in ect their phenotype, thus contributing to the development of COPD (Serban et. al, 2016). On the other hand, the downregulation of let-7a-5p, in the exosomes isolated from venous blood of lung adenocarcinoma patients, alongside, an upregulation of BCL2L1, were responsible for the poor survival of the patients . Exosomes derived from menstrual blood-derived stem cells (MenSCs) contained miR let-7, which by being transported into alveolar epithelial cells suppress the activation of Reactive Oxygen Species (ROS) and Mitochondrial DNA (mtDNA) damage by regulating NLRP3 signaling, using LOX1 as a target and thus improved the damaged condition of the injured lung epithelial cells, exhibiting a protective effect on lung brosis .
In cardiac disease-related cases, this miRNA shows positive contribution, playing a role as disease therapeutic. The transfer of pericardial uid derived exosomal let-7b-5p, to the endothelial cells, results in vascular repair. Being a pro-angiogenic miRNA, let-7b-5p downregulated the anti-angiogenic gene TGFBR1, thus promoting post-ischemic angiogenesis (Beltrami et. al, 2017). The level of let-7i was found to be signi cantly increased in plasma exosomes of multiple sclerosis patients. This elevated amount of let-7i repressed Foxp3+ regulatory T cells, which is the suppressor of the proliferation of in ammatory T cells and thus helps in the development of this T-cell mediated autoimmune disease (Kimura et. al, 2018). The expressions of let-7d-5p and let7c-5p were found to be signi cantly reduced in exosomes of multiple myeloma patients than in those of unaffected individuals and it was assumed that this downregulation may alter the expression of IL-6, contributing to the progression of the disease .
In urinary exosomes of lupus nephritis patients, the level of let-7a was signi cantly downregulated during the disease outbreak. From the references of some previous studies and based on the outcomes of this study, it was predicted that this downregulation may have a contribution to the failure of epigenetic regulatory systems of the kidney (Tangtanatakul et. al, 2019). Cervical cancer caused by human papilloma viruses (HPVs) depends upon the expression of E6/E7 oncogene for the cancer progression. In an experiment, it was seen that the undisrupted expression of E6/E7 oncogene results in the increased expression of let-7d-5p in cervical cancer cell-derived exosomes, and the silencing of this oncogene leads to downregulation of exosomal let-7d-5p. This made clear that the expression of exosomal let-7d-5p was dependent upon the E6/E7 expression and this relationship between the two can be used for prediction and early diagnosis of cervical cancer (Honegger et. al, 2015). Interestingly, the miR-let-7f, which was highly expressed in umbilical mesenchymal stem cell-derived exosomes, could reduce the replication of hepatitis C virus RNAs (Qian et. al, 2016). In a study, tumor-derived exosomes (isolated from breast cancer cell line, 4T1) were modi ed with some micro RNAs, including let-7i, which could downregulate TGF-β, SOCS1, and KLRK1 mRNA expression, at the same time overexpressing IL-6, IL-17, IFNγ, IL-1b, and TLR4 mRNA and boosted maturation of dendritic cells, changing the immune-suppressive nature of tumorderived exosomes into immune responsive one (Taghikhani et. al, 2018).
Micro RNA-29: The micro RNA 29 family also deserves to be mentioned as it is one of those miRNAs who exhibit their disease developing as well as disease resistance potential for a variety of diseases. It has immense importance as a marker of advanced hepatocellular carcinoma (HCC). Exosomes isolated from HCC patient-derived cells (PDCs) showed an own regulation of mir-29b-3p, in the case of fast-growing PDCs, compared to slow-growing cells which improve overall survival. Hence this group of miRNAs acts as a potential biomarker for HCC cell metastasis . Signi cantly low expression of miR-29c in HCC derived exosomes were also observed in a comparative study between hepatocellular carcinoma, Hepatitis B virus (HBV), and hepatocirrhosis patients (Lin and Zhang, 2019). However, this miRNA has been found to act against hepatitis C virus (HCV) infection in hepatocytes. TLR3-activated macrophages secrete exosomes that carried signi cantly increased level of miR-29a, miR-29b, and mir-29c and on treating HCV infected hepatocytes with exosomes containing miR-29 members, replication capacity of HCV is inhibited, which is reversed with miR-29 inhibitor. This indicates that the miR-29 family; in this case, acts as an anti-HCV agent (Zhou et. al, 2016). A high expression of miR-29a-3p was observed in exosomes isolated from oral squamous cell carcinoma cells (OSCC). This also resulted in the M2 subtype polarization of macrophages, reinforcing the proliferation and incursion of the OSCC cells (Cai et al, 2019).
There are several contributions of the miR-29 family in renal diseases also. A downregulation of miR-29c was observed in urinary exosomes of renal brosis patients and this was negatively associated with estimated glomerular ltration rate and tubulinterstitial damage index (Lv et. al, 2018). The primary human tumor-associated macrophage (TAM) derived exosomes showed an upregulation of hsa-miR-29a-3p. This was found to be responsible for Tregs/Th17 cells imbalance in CD4 + T cells obtained from mice model with metastasizing ovarian cancer. This imbalance of Tregs/Th17 cells is a vital factor associated with the overall survival of epithelial ovarian cancer patients. The transfer of exosomal hsa-miR-29a-3p caused suppression of STAT3 expression thus causing immunosuppression, which triggered the Tregs/Th17 cells imbalance and this, in turn, leads to the progression of Epithelial Ovarian Cancer (EOC) (Zhou et.al, 2018).
To observe the effect of the exosomal miR-29 family on age-related insulin resistance, an experiment was performed, where bone marrow mesenchymal stem cells (BM-MSCs) derived exosomes collected from aged mice showed a signi cantly increased level of miR-29b-3p, which was higher in aged mice compared to young mice. This correlation of increased expression of miR-29b-3p with increasing age was also observed in exosomes derived from human BM-MSCs, suggesting the association of miR-29b-3p activity with aging. Administration of these exosomes into insulin-sensitive mice model, resulted in the alleviation of insulin-mediated glucose uptake by the adipocytes and myocytes, by targeting SIRT1 mRNA in the insulin-sensitive mice model and thus created insulin resistance in them (Su et. al, 2019).
The inactivation of Matrix Metalloprotease 9 (MMP9) and thus improvement of cardiovascular functions during the onset of diabetes could be attained by the highest expression of miR-29b in exosomes isolated from cardiac tissues and serum of diabetic mice model (Chaturvedi et. al, 2015). In another investigation also, among some other miRNAs, the elevated expression of miR-29 in Mesenchymal stem cell-derived exosomes was found to play a cardioprotective role by arousing cardiomyocyte proliferation and inhibiting cardiac cell apoptosis (Shao et. al, 2017). Hence, it can be assumed that in the case of cardiac dysfunctions the exosomes carrying miR-29 play a disease-resisting role.
Glioma-derived exosomes exhibit expression of miR-29a due to hypoxic conditions. Transfer of this exosomal miRNA into myeloid-derived suppressor cells (MDSCs) induces their proliferation and activity by downregulation of Hbp1 and Prkar1a gene expressions, which leads to the development of an immunosuppressive microenvironment (Guo et . al, 2019).
Micro RNA-146 group: The exosomal miR-146 family also exhibits some cardioprotective functions as reported in some previous studies. Patients with heart failure were subjected to an experiment, where it was observed that the exosomes isolated from cardiomyocytes of the patients exhibited overexpression of miR-146a in response to in ammation resulted from the heart failure. Not only that, but this elevated exosomal miR-146a could debilitate this in ammation, functioning as a cardioprotective cargo of exosomes of patients with heart failure (Beg et. al, 2017). Exosomes isolated from miR-146a overexpressed adipose-derived stem cells could put down EGR1 thus recovering myocardial injury, as well as myocardial brosis and apoptosis after acute myocardial infarction in both rat models and hypoxiainduced cardio myoblast cells (H9c2) (Pan et . al, 2019). On the contrary, there are some studies where this exosomal miR-146 family has been found to play role in cardiac disease development. For example, exosome isolated from endothelial colony-forming cells (ECFCs) with coronary artery disease contained overexpressed miR-146a-5p and miR-146b-5p, which when suppressed, resulted in the build-up of migration and microtubule formation activity of those cells. Whereas, after being carried via exosomes, an elevation of these micro RNAs in healthy ECFCs brought about an inhibition of the normal migration and tube formation capacity of these healthy cells, by targeting Rho-related GTP-binding protein RHOJ (Chang et al, 2017). Exosomes isolated from Treponema pallidum showed high expression of miR-146a-5p, which by being transported into the endothelial cells, downregulated JAM-C expression and decreased trans endothelial movement of monocytes and endothelial permeability, and hence inhibited the recovery from the injury caused by Treponema pallidum .
To investigate the role of exosomal miRNAs in systemic lupus erythematosus (SLE), urinary exosomes were isolated from patients, in which a signi cantly increased level of miR-146a was observed in the SLE patients with active lupus nephritis, compared to control and this signi cant increase was about 8 fold in case of SLE patients with inactive lupus nephritis. These observations indicated that miR-146a in urinary exosomes of SLE patients could be used as a marker to differentiate the SLE patients with active lupus nephritis from those without lupus nephritis (Perez-Hernandez J et. al, 2015). While, in another study, serum exosomes collected from SLE patients showed a signi cantly low expression of miR-146a. The delivery of these exosomes into the bone marrow mesenchymal stem cells (BM-MSC) lowered the level of miR-146a in them, leading to the increase of senescence of the BM-MSC by activating TRAF6/NF-κβ signaling pathway and this is a cause of SLE progression. Thus, lower expression of miR-146a in serum exosomes of SLE patients increased the senescence of BM-MSc, which was con rmed by observing the inhibition of BM-MSC senescence treated with SLE serum exosomal miR-146a mimic with its high expression (Dong et. al, 2019). Exosomes were isolated from the urine of hypertensive patients with and without albuminuria, and the level of miR-146a was found to be signi cantly low in the urinary exosomes of hypertensive patients with albuminuria compared to the hypertensive patients of the non-albuminuria group. Also, it was seen that the severity of albuminuria was associated with a lower expression of miR-146a in the urine exosomes of the patients, suggesting that this micro RNA can serve as a promising marker for detecting renal injury by observing the intensity of albuminuria in the hypertensive patients As a result of the signi cantly exalted expression of these cytokines and chemokines in the healthy MSCs, increased migration and proliferation of the multiple myeloma cells were observed (Veirman et. al, 2016). Exosomal expressions of miR-146a-5p and miR-146a-3p of trophoblast cells were stimulated by the antiphospholipid antibody. There was no effect seen in the secretion of IL-8 due to miR-146a-5p, while miR-146a-3p seemed to increase the IL8 secretion in the trophoblast cells. Incubating TLR8 dominantnegative trophoblast cells with normal trophoblast cells seemed to attenuate the IL-8 secretion implying that this exosomal miR-146a-3p mediated IL8 secretion was due to activation of TLR8, thus leading to the generation of in ammatory response in the trophoblast cells (Gysler et. al, 2016). Some studies are implying the direct contribution of exosomal miR-146a in disease defence. Dementia due to type 2 diabetes is a result of the aggregation of cellular prion proteins. In general, miR-146a was seen to be downregulated in brain endothelial cell-derived exosomes of type 2 diabetic mice with excessive expression of prion proteins. The binding of externally elevated exosomal miR-146a to prion proteins in the brain of Type 2 diabetic mice leads to its suppression and thus the revival of memory (Kalani et. al, 2017).
Exosomes isolated from chronic lymphocytic leukemia patients were found to contain a high amount of miR-146a, which they transferred into endothelial and mesenchymal stem cells stimulating the secretion of in ammatory cytokines in them and in ecting the surrounding stromal cells to exhibit characteristics of cancer-associated broblasts (Paggetti et. al, 2015). Conversely, upregulation of miR-146a in exosomes isolated from TNF-α treated human umbilical cord-derived mesenchymal stem cells lead to the amelioration of broblast activation and in ammatory response correlated with urethral brosis when taken up by the broblasts of TGFβ1 injected urethral stricture tissues (Liang et . al, 2019). A study conducted to observe the role of virus-infected cell-derived exosomes showed that incorporation of EV71 viral RNA along with miR-146a leads to the restraining of type I interferon response by the exosomal miR-146a, resulting in increased replication of the viral RNA, as observed in several cell lines (Fu et. al, 2017).
The upregulation of miR-146a in the exosomes isolated from interleukin-1β treated human umbilical cordderived mesenchymal stem cells, when injected into CLP-induced sepsis mouse model, the exosomal miR-146a downregulated IRAK1, TRAF6, and IRF5, contributing to M2 polarization of macrophages and recovering of sepsis. This is an interesting disease protective aspect of this micro-RNA (Song et. al, 2017).
Signi cant overexpression of miR-146a in exosomes derived from vitreous humor as well as serum of uveal melanoma patients could serve as a potential biomarker for the disease (Ragusa et. al, 2015).
Micro RNA-181 group: The role of exosomal miR-181has been observed both in disease development as well as in disease resistance. In a recent study, mi-RNA pro ling of serum exosomes from hepatocellular carcinoma patients exhibited that, miR-181a can be considered as a potential non-invasive biomarker of the disease due to its stable expression . There is evidence showing the role of exosomal miR-181 in disease resistance. For example, adipose-derived mesenchymal stem cells release exosomal miR-181-5p, which after being transferred to hepatic stellate cells induces autophagy by downregulating STAT3 and Bcl-2, and alleviated liver brosis (Qu et. al, 2017). Human liver stem-like cell-derived exosomes carry miR-181b, which suppressed the angiogenesis of tumor-derived endothelial cells, by downregulating the expressions of ITGB3, FGF1, EPHB4, and PLAU angiogenic genes (Lopatina et. al, 2019). Signi cant downregulation of miR-181b-5p and miR-181d was observed in exosomes derived from gastric cancer-associated-ascites than in those derived from liver cirrhosis-associated ascites. Moreover, the combination of miR-181b-5p and carcinoembryonic antigen showing signi cantly high speci city and sensitivity towards gastric cancer which was proved to be acting as the greatest diagnostic marker (Yun et al, 2019). Hypoxic colorectal cancer cell-derived exosomes contained miR-181a-5p, which was recovered in high-risk locally advanced rectal cancer acting as a marker of the disease (Bjørnetrø et. al, 2019). Mir-181a-5p was found to be upregulated in plasma exosomes of several other cancers also, hence used as a biomarker for detection of this disease (Samsonov et. al, 2016). An overexpression of exosomal hsa-miR-181a/b derived from human melanoma cells was found to suppress the TNFα secretion and T-cell receptor signaling and thus support the tumor progression (Vignard et. al, 2020). This re ects the disease resistance capability of this mi-RNA.
The high accumulation of miR-181b-5p in tumor-derived exosomes of adenocarcinoma patients could be assumed to serve as a potential biomarker for early detection of non-small cell lung cancer, adenocarcinoma . Exosomes isolated from Adipose-derived stem cells (ADSCs) contained miR-181b-5p, which was further outstandingly increased by exposing the ADSCs to brain extracts of rat model with middle cerebral artery occlusion. These micro RNAs were transferred to oxygenglucose deprived brain microvascular endothelial cells (BMECs) resulting in endorsement of tube formation and migration of the BMECs through the downregulation of TRPM7, thus stimulating the angiogenesis of the BMECs .
In the case of renal diseases, miR-181a was signi cantly decreased in the urinary exosomes of early as well as late chronic kidney disease patients by about 200-fold, thus serving as a strong potential biomarker of the disease (Khurana et. al, 2017). Whereas, in another study, it was seen that the increased expression of miR-181 in serum exosomes due to acupuncture with low-frequency electrical stimulation in the hindlimb of mouse model seemed to increase renal blood ow, by downregulating the expression of angiotensinogen (Su et. al, 2018).
Micro RNA-1246: There are several reports available that studied the role of exosomal miR-1246 in breast cancer and assumed to be a factor that makes it a potential diagnostic marker for breast cancer (Hannafon et. al, 2016). A high expression of miR-1246 was observed in metastatic breast cancer cellderived exosomes, which after being transferred to non-malignant human mammary epithelial cells (HMLE) induces cell proliferation, migration and generates a chemotherapy-resistant capacity in them by downregulating CCNG2 . A signi cantly high expression of exosomal miR-1246 was observed in other cancer patients also such as ovarian cancer (Kanlikilicer et. al, 2018), prostate cancer (Bhagirath et. al, 2018), colorectal cancer (Eylem et. al, 2020), and gastric cancer (Shi et. al, 2020), etc.
The level of miR-1246 was found to be signi cantly higher in serum exosomes of both early and advanced hepatocellular carcinoma patients, than in those of liver cirrhosis patients and normal control individuals, which help to detect hepatocellular carcinoma in both early and advanced stages, thus improving the possible treatments of the disease . The upregulated miR-1246 in highly metastatic human oral squamous cell carcinoma (OSCC) cells, when get transferred into poorly metastatic OSCC cells, they increased the motility and invasion of the recipient cells by downregulating the expression of domain-containing 2D genes (Sakha et. al, 2016). This study indicates the association of exosomal miR-1246 with OSCC progression.
Mutant p53 acquire some gain-of-function due to which they act differently from wild-type p53, which acts as a tumor-suppressor. In a certain study, it has been found that colon cancer cells accommodating GOF mutp53, secreted exosomes with highly expressed miR-1246, which by being carried into neighbouring macrophages, resulted in their transformation into tumor-associated macrophages, by the increased expression of IL-10, VEGF, CCL2 and TGF-β and the decreased expression of IL-8 and TNF-α. It has been also observed that these phenomena are associated with the poor survival of colon cancer patients (Cooks et. al, 2018). Interestingly, in a study, it was found that pulmonary artery smooth muscle cells (PASMC) secreted exosomes in which miR-1246 was downregulated due to platelet-derived growth factor (PDGF) stimulation applied on the PASMCs. These exosomes when get transferred into pulmonary artery endothelial cells (PAEC) due to the downregulated expression of miR-1246, an elevated migrating capacity of the PAECs was observed. This study explored the role of exosomal miR-1246 in maintaining vascular homeostasis and pathology (Heo et. al, 2020).
Mir-1246 was elevated in exosomes secreted by Rhabdomyosarcoma cells, and they combined with another elevated micro RNA targeted pathways associated with tumorigenesis, particularly, Wnt and GnRH signaling pathways but also included angiogenesis signaling pathway and apoptotic signaling pathway. These exosomes were seemed to increase proliferation and angiogenesis in recipient broblast cells (Ghayad et. al, 2016). An interesting observation implied that miR-1246 released from lung cancer cells with ionizing radiation, both in exosomal and non-exosomal forms, but their expression was much less in exosomes than in extracellular space, that is their expression was higher in non-exosomal form and they resulted in proliferation and radio resistance of the recipient cells being in their non-exosomal form .
Increased expression of exosomal miR-1246 isolated from culture media of BEP2D cells with 2 Gyirradiation induces DNA damage in non-irradiated cells by the increased expression of 53BP1 foci, micronuclei, and comet tail (Mo et. al, 2018). Exosomal miR-1246 also serves as a potential biomarker for detecting acute myeloid leukemia (Hornick et. al, 2015).
Micro RNA-122: The most signi cant role of exosomal miR-122 was observed in the case of hepatic disease. The level of miR-122 was signi cantly reduced in exosomes isolated from hepatocellular carcinoma patients, compared to those isolated from chronic hepatitis B patients and liver cirrhosis patients, due to which it could serve as a potential serological marker for hepatocellular carcinoma . In another study, the role of exosomal miR-122 was analyzed in hepatocellular carcinoma patients and it was found that the level of exosomal miR-122 was signi cantly decreased after transarterial chemoembolization (TACE) treatment, where before TACE the level of exosomal miR-122 was found to be signi cantly associated with aminotransferase and alanine aminotransferase levels.
Therefore, these ndings suggested that exosomal miR-122 could serve as a biomarker for hepatocellular carcinoma with liver cirrhosis after TACE treatment (Suehiro et. al, 2018). However, in another study, it was seen that transfecting adipose tissue-derived mesenchymal stem cells (AMSCs) with miR-122, results in secretion of exosomes carrying miR-122 which after transfer to hepatocellular carcinoma cells increases their sensitivity towards chemotherapy (Lou et. al, 2015). This study highlights the diseaseresistant capacity of exosomal miR-122 in the case of hepatocellular carcinoma. It was also shown that the level of exosomal miR-122 was signi cantly lower in hepatitis C infected patients compared to healthy individuals except for 1b genotype of hepatitis C patients (Fan et. al. 2017). Acetaminophen (APAP) induced liver injury, elevates miR-122 and glutamate dehydrogenase level in 5-fold compared to the hepatic injury biomarker, alanine aminotransferase, and a part of this elevated miR-122 resides in the plasma exosomes released from the injured liver cells, suggesting that exosomal miR-122 can also act as a potential biomarker for drug-induced liver injury (Thulin et. al, 2016). In the case of thioacetamideinduced injury, the level of exosomal miR-122 decreased than the control after 12 h of administration in the injured rat model and then increased at 24 h (Motawi et. al, 2018). All implied that exosomal miR-122 is a promising biomarker for acute liver injury. The level of exosomal miR-122 derived from hepatocytes of the mouse model was found to be signi cantly increased after alcohol consumption, the same was observed in the case of human hepatocytes. The exosomal miR-122 being transferred into monocytes, decrease the level of HO-1, resulting in the increased expression of IL-β and increase of in ammation favourable activities of the monocytes (Momen-Heravi et. al, 2015). The level of hsa-122-5p in serum exosomes collected from patients with chronic atrophic gastritis was signi cantly higher compared to chronic non-atrophic gastritis, which indicated that exosomal hsa-miR-122-5p was also a potential biomarker for chronic atrophic gastritis analysis . It was also identi ed to be a promising biomarker for colorectal cancer, as the level of exosomal miR-122 signi cantly increased in the case of colorectal cancer patients than in healthy patients (Sun et. al, 2020). There are some signi cant roles of exosomal miR-122 in breast cancer disease. Breast cancer cells were found to secrete exosomes carrying elevated level of miR-122, which is transferred into lung broblasts, brain astrocytes, and neurons put down the level of GLUT1 and PKM2, also putting down the glucose consumption by the cells and accelerated their metastasis (Fong et. al, 2015). It was found that miR-122-5p was one of those mi-RNAs which was signi cantly elevated in plasma exosomes of breast cancer patients than in normal individuals, offering it to be a potential biomarker for breast cancer . Even liver cells carried exosomal miR-122-5p in a huge amount that could suppress syndecan-1, a heparan sulfate proteoglycan, associated with several malignant carcinomas, that further lead to the increased motility of breast cancer cells (Uen et. al, 2018).
Interestingly, exosomal miR-122-5p can also be used for the treatment of osteonecrosis of the femoral head (ONFH) by generating osteoblast cells, as observed in rabbit models (Liao et. al, 2019). This is strong evidence indicating the defensive role of exosomal miR-122 in bone-related hemorrhages. The hsa-miR-122 was one of those mi-RNAs which were present in a huge amount in exosomes isolated from melanoma tumor cells and associated with the immune evasion of tumor growth (Vignard et. al, 2020). Additionally, the incorporation of miR-122-5p into melanoma cells is in uenced by lysophosphatidic acid receptor-3 (LPAR3) and the partial involvement of PGC-1a (Byrnes et. al, 2019).
Plasma exosomal rno-miR-122-5p was downregulated in rats with the transient ischemic attack, than in control rats, thus acting as a potential biomarker for the analysis of this disease .  (Kia et. al, 2018). M2 macrophage-derived exosomes exhibited signi cantly higher expression of miR-155-5p, which transported them into colorectal cancer cells, where this exosomal miR down regulatesBRG1, the regulatory factor of colorectal cancer cell metastasis (Lan et. al, 2019). Additionally, exosomal miR-155-5p derived from paclitaxel-resistant gastric cancer cells when transferred to the paclitaxel sensitive gastric cancer induces epithelial-mesenchymal transition and drug-resistant capacity in those recipient cells through downregulation of GATA3 and TP53INP1 expression . The level of miR-155 in acute myeloid leukemia (AML) exosomes were found to be increased with disease development and can be considered as a minimally invasive biomarker for early detection of AML (Hornick et. al, 2015). A similar nding was observed in the case of chronic lymphocytic leukemia (CLL) (Yeh et. al, 2015). Therefore, these studies implied that exosomal miR-155 is a strong candidate for tracing the disease pro le of different types of cancers and leukemia. It is also reported that miR-155 has a role in the development of type 1 diabetes. T-lymphocytes of both human and non-obese diabetic mice secrete exosomes that carried miR-155. On being transferred to β cells these exosomal miRNAs result in the induction of β cell apoptosis, also upregulating the expression of CCL2, CCL7, and CXCL10 (Guay et. al, 2019). In another study, it was found that cytokine-treated islet cell-derived exosomes, collected from nondiabetic cadaveric donors carried highly upregulated hsa-miR-155-5p. The treatment of islets by the cytokines such as IL-1β and IFN-γ was useful in creating a mimic of the pro-in ammatory ambiance of type 1 diabetes and thus the differential expression of exosomal miR-155-5p was assumed to serve as a marker for type 1 diabetes (Krishnan et. al, 2019).
Interestingly, the disease resisting role of this exosomal miR-155 was also observed in the inhibition of Enterovirus A71 (EV-A71) infection by suppression of VP1 protein and virus genomic RNA . It was observed that the serum exosomes isolated from the rheumatic heart disease (RHD) rat model carried signi cantly upregulated miR-155-5p which downregulated the expression of S1PR1 and SOCS1 and activated STAT3 phosphorylation along with IL-6 expression leading to the valvular damage.
But, inhibiting the expression of exosomal miR-155-5p, could mitigate the damages in RHD cases . The level of exosomal miR-155 was found to be signi cantly upregulated in rheumatoid arthritis (RA) patients infected with hepatitis C virus (HCV) than in RA patients without HCV infections, but this expression of miR-155 suppressed HCV replication which could relapse upon rituximab therapy (Liao et. al, 2018). This study indicated the disease recovery role of exosomal miR-155.
Coming back to the disease developing aspects, it was seen that melanoma derived exosomal miR-155 downregulates cytokine signaling 1 (SOCS1) expression, leading to the activation of JAK2/STAT3 signaling pathway and the induction of vascular endothelial growth factor A, broblast growth factor 2, and matrix metalloproteinase 9 expressions, thus generating angiogenesis in the normal broblast cells and transforming them into cancer-associated broblasts . Co-culturing melanoma cells with neuroblastoma cells lead to the increased expression of miR-155 in human monocytes and it is believed to be caused by neuroblastoma-derived exosomes carrying upregulated miR-155. It was also observed that, in a co-culture of monocytes and neuroblastoma, human monocytes secreted exosomal miR-155, causes downregulation of TERF1 expression and increases telomerase activity in the recipient neuroblastoma cells, and makes them drug-resistant. This study was an example of how exosomal miR-Page 16/31 al, 2015). The transfer of exosomal miR-155 from Epstein-Barr virus-infected Burkitt's lymphoma cells to the retinal pigment epithelial (RPE) cells resulted in an elevated level of miR-155 in the RPE cells also leading to the induction of VEGF-A expression, thus bringing about angiogenic properties in them (Yoon et. al, 2016). The exosomal level of miR-155 was found to be elevated in the case of pregnant women with preeclampsia, than in women with normal pregnancy. Moreover, it was observed that the transfer of this miR-155 from placenta associated serum of PE patients to the primary human umbilical vein endothelial cells via exosomes, resulted in reduced nitric oxide (NO) production and eNOS expression in the recipient cells, thus inducing a PE favorable environment in the endothelial cells (Shen et. al, 2018).
Micro RNA-150: There are several experimental pieces of evidence suggesting the differential role of miR-150 in leukemia. The plasma exosomes of chronic lymphocytic leukemia cells were found to deliver miR-150 to recipient bone marrow mesenchymal stromal cells, which would develop in ammatory characteristics and cancer-associated broblast-like phenotype in these cells (Paggetti et. al, 2015). The differential expression of miRNA-150 in serum exosomes of mice with acute myeloid leukemia (AML)can act as a potential biomarker for detecting the disease (Hornick et. al, 2015). Moreover, there is also a signi cant correlation between B cell receptor signaling pathway activation and increased secretion of exosomal miR-150 in Chronic Lymphocytic Leukaemia (Yeh et. al, 2015).
It is already reported that exosomal miR-150 could serve as a biomarker for different renal diseases, bladder cancer, triple-negative breast cancer patients, non-small cell lung cancer patients than in those of healthy individuals which were useful in the detection and diagnosis of these cancers ( of mTOR, p53, and AMPK pathways, exosomal miR-150 is considered as a potential biomarker of type 2 diabetic nephropathy . In another article, it was revealed that mesenchymal stem cellderived exosomal miR-150-5p served as a therapeutic for decreasing the severe effect of arthritis in rheumatoid arthritis patients .
From the overall discussion, we get to know that the differential expressions of various exosomal micro RNAs may serve as either a disease mediator or a biomarker or a therapeutic. The diseases mostly included different types of carcinoma, though there are also some unusual as well as well-known diseases that seemed to be affected by the different types of miRNA cargo of the exosomes. The search of such roles of these exosomal micro RNAs in disease development or disease defence is still going on which will provide new openings for the treatment procedures of the diseases.

Conclusion
Exosome has been established as a potential carrier of different types of cargos, speci cally miRNAs.
The emerging eld of exosome-related research works is offering more consequences to use them as a carrier of therapeutic cargos. The review enlightens the aspects of using exosome as a communicating vehicle to treat a broad number of diseases, as well as the negative impact of the exosome-mediated 134. Zhou Y, Wang X, Sun L, Zhou L, Ma TC, Song L, Wu JG, Li JL, Ho WZ (2016) Toll-like receptor 3activated macrophages confer anti-HCV activity to hepatocytes through exosomes.  Figure 1