miR-34a

The most advanced miRNA therapies for cancer are miR-34 mimics encapsulated in lipid nanoparticles, which are now being investigated in the first phase of the clinical study (NCT01829971) in many hematological cancers. Many preclinical investigations have shown that miR-34 mimics have the potential to be an anticancer treatment. In mice models of the liver [28], prostate [29], and lung cancer [30], for example, the miR-34 mimic which was encapsulated in lipid nanoparticle exhibited assuring action. Systemic distribution of miR-34a in carriers of liposomes resulted in lower growth of the tumor, enhanced tumor cell death, and reduced CD44⁺ cell numbers in a model of orthotopic pancreatic cancer employing cells of MiaPaca2, depicting a reduction in metastatic cells [31]. In a prostate cancer mouse model, neutral-lipid emulsion (NLE)-based administration of miR-34a resulted in only a minor decrease in tumor development. However, due to a decrease in metastatic dissemination to the lung and other organs, a considerable improvement in survival periods was observed [29]. In an aggressive NSCLC KRAS; p53 tumor suppressor gene (Trp53) mouse model, therapy with a miR-34a mimic MRX34 which was coated in nanoparticles of lipid that are currently licensed for human clinical trials resulted in considerable tumor reduction [28]. Furthermore, in this model, a combined strategy employing the same lipid nanoparticle carrier to deliver let-7 and miR-34 resulted in a substantial decrease in nodules of tumor and a prolonged viability benefit. Also, in vitro, a combination of the epidermal growth factor receptor (EGFR) inhibitor erlotinib with the miR-34a and let-7 inhibitors exhibited coordinated effects in suppressing the development of cell lines of NSCLC [32].

miR-200 family

miR-200c targets the interleukins, and delivery of the mimics of miR-200 family members utilizing the 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC) lipid nanoparticles resulted in fewer tumor nodules and distant metastasis in an orthotopic mice models of ovarian (miR-200a/b), basal-like breast cancer (miR-141), and lung (miR-200a/b) cancers [33].

miR-26a

miR-26a levels were considerably shown lower in a wide range of RNA samples taken from individuals with HCC of n = 455 as compared to normal tissues [34]. Low levels of miR-26a were also linked to poor patient survival [35]. The specific targeting of mRNAs encoding for the cell cycle regulators cyclin D2 (CCND2) and cyclin E2 (CCNE2) by expression of adeno-associated virus mediated by miR-26a resulted in considerable tumor reduction in a mouse model of HCC [34].

miR-506 and miR-520d-3p signaling

In two researches, DOPC liposomes involving miR-506 mimics which is a regulator of DNA damage response, and epithelial-mesenchymal transition (EMT) phenotype [36] or miR-520d-3p mimics which target the Ephrin type-A receptor 2 (EPHA2) and EPHB2 oncogenes [37] were delivered to orthotopic mouse models of ovarian cancer, suppression of ephrin signaling by miR-520d-3p resulted in remarkable tumor suppression and reduced expression in the respective mRNA targets in vivo.

miR-15a/16-1

In MEG01 subcutaneous model of leukemia, the ectopic expression of clusters of miR-15a/16-1 via viral vectors resulted in a considerable decrease in tumor volume and growth [38]. Furthermore, administration of miR-16-1 via a nano-cell delivery system which is an EGFR-targeted Delivery Vehicle (EDV) of EnGeneIC in NSCLC xenograft mice models and malignant pleural mesothelioma, led to the targeted delivery in tumor and considerable tumor repression [39].

miR-10b

Anticancer techniques on the basis of OncomiRs suppression by utilizing antimiRs based on antagomirs, LNAs, and ASOs have been examined in several preclinical investigations [12]. ASOs were utilized to successfully inhibit miR-10b in an orthotopic breast cancer model in an early evaluation of the therapeutic value of antimiRs [40]. Metastasis was reduced because of the expression of the homeobox D10 (HOXD10) anti-metastatic gene being rescued by this antimiR. However, the authors found no reduction in original tumor development, indicating that the first combinations of chemotherapy and tumor reduction surgery are required. It would be fascinating to observe if miR-10b inhibitors have an effect on long-term survival. Delivery of an antagomir against miR-10b utilizing a polyethyleneimine (PEI) delivery technology which was a in vivo-jet PEI, resulted in a substantial decrease in the tumor development in an orthotopic glioblastoma mouse model [41]. Given that HOXD10 is not considered to play a role in the original tumor’s development, this finding requires more investigation. It also shows that miR-10b has significance other than downregulating HOXD10.

miR-221

In HCC, miR-221 is one of the most dramatically elevated miRNAs, downregulating important tumor suppressors such as p27KIP1, phosphatase and tensin homolog (PTEN), and tissue inhibitor of metalloproteinase 3 (TIMP3) [42, 43]. A cholesterol-modified version of antimiR-221, given intravenously to HCC xenografts, was found to have considerable effectiveness in downregulating miR-221 and raising the levels of its mRNA targets [44]. Tumors were reduced in antimiR-221-treated animals, and they lived substantially more than the control mice. The utilization of this cholesterol-modified antimiR for future research is currently limited due to a lack of comprehensive toxicity data.

miR-155

It was demonstrated via the model of a mouse miR-155-induced with lymphoma in which the expression of miR-155 was controlled by doxycycline (miR-155^LSLtTA), that removing doxycycline caused miR-155 expression to shut down and tumor reduction beyond detectable limits [45]. The introduction of antimiR-155 nanoparticles packed in nanoparticles of polylactic-coglycolic acid resulted in a lower burden of tumor in the same mouse model, indicating that miR-155 inhibition may have therapeutic potential. pH low insertion peptide (pHLIP)-antimiR-155, a pH-sensitive antimiR-155 conjugate, was recently investigated in this model [46]. A short peptide called pHLIP creates a helix of transmembrane within acidic conditions [47]. Because the tumor milieu is acidic, a pHLIP and antimiR-155 combination boosted antimiR distribution to cancer cells. Mice that were given pHLIP–antimiR-155 had a considerable decrease in tumor burden, which resulted in a longer survival time. There was no notable toxicity, indicating that clinical translation of this method is possible [46].

miR-630

miR-630 is an OncomiR that is elevated in response to hypoxia in a tumor-specific environment. In an ovarian cancer orthotopic model, it was observed that via using the antimiR that was against the miR-630 through the DOPC delivery system there was a substantial decrease in the metastasis and the growth of the tumor [48].

Repositories for miRNA discovery

For the identification of miRNAs in the laboratory carrying out the experiments like cloning, microarray, and in-situ hybridization methods are all very much time-consuming and also expensive methods. However, in recent years, next generation sequencing (NGS) has made miRNA detection simple and less costly. As a result, hundreds of miRNA sequences for animals, plants, and viruses have been identified. Many techniques have been designed which utilize the high throughput sequencing data to find out new miRNAs. These methods are based upon a sequence-structures factor that is unique to miRNAs, such as the composition of the sequence, the conservation across the species, hairpin loop location and number, the number of base pairs and bulges, and all the structural parameters such as the Gibbs free energy and secondary structures [49]. As a result, a miRNA Registry was created to meet the storage needs of these miRNA sequences. Over 24,000 miRNA genes are represented in miRBase [50, 51], with over 30,000 mature miRNAs and extensive annotations from over 200 species [52]. There are more than 1,800 pre-miRNAs in humans and more than 2,500 mature miRNAs that are included in the current edition. Plant microRNA database (PMRD) [53], a repository containing miRNAs from plants, is an example of a species-specific database that provides more extensive coverage of experimentally confirmed miRNA sequences. Similarly, viral miRNA (VIRmiRNA) is the first dedicated resource to archive experimentally proven virus-encoded miRNAs, their targets, and antiviral miRNAs in three sub-databases. Epimir is a broad resource of common regulations between miRNAs and epigenetic alterations [54], and AVIRmiR, is a sub-database of VIRmiRNA that encompasses host’s encoding miRNAs that are reported to have anti-viral effects [55], these are the two examples of repository which holds miRNAs according to particular conditions or experimental principles used to extract them. Some databases provide miRNA annotations as well as visualization tools, such as miRviewer, which is a Web service that visualizes miRNAs via miRBase and also their homologs which are identified by utilizing miRNAminer [56]. Moreover, the miRBase tracker, from the name itself, explains that it keeps the researchers up to date on changes in miRNA annotation and makes it easier to annotate [57]. MirGeneDB, on the other hand, employs a historical hierarchy to provide comprehensive annotations of miRNA:miRNA^* duplex [58]. Others, such as miROrtho, which is an online program that gives predictions of pre-miRNA from genomes of animals in conjunction with their orthology [59], and also focuses on both the evolutionary and storage aspects of miRNA. CoGemiR [60] is an online database that proposes conservation across the evolution. Furthermore, mESAdb is an online database that contains expression data of miRNAs from a variety of species [61]. As a result of advancements in NGS-based approaches, several other depositories for archiving predicted miRNAs and multiple other aspects of NGS-based information, such as YM500v2—which is an archive for predicting and quantifying miRNA and related isomiRs from short-RNA sequencing data sources, have been established. In its current edition [62], cancer miRNAome research is also included. miRNEST is a database that contains experimentally predicted miRNAs from a variety of animals [63]. MirPub, a comprehensive database with a search capability that provides researchers with papers relevant to specific miRNAs [64], also, in the field of miRNA, is one of the literature searches enhancing depositories.

Differential expression of miRNAs

miRNAs are known to be gene expression regulators, and their altered expression has been linked to a variety of illnesses. Real-time polymerase chain reaction (PCR), southern blotting, and NGS-based methods have recently been utilized to characterize the differential expression of miRNAs using several miRNA profiling methodologies. Because of their precision and speed, NGS-based approaches have grown popular. As a result, methods that reveal differentially expressed miRNAs will be tremendously valuable in discovering biomarkers for a wide range of diseases. Scientists have created a unique category of resources for storing and analyzing DEmiRs [49]. MirEX 2.0 database [65] and human miRNA expression database (HMED) [66] are two computational tools that include miRNA expression profiles. MirEX and HMED are two powerful tools for analyzing miRNA expression data. DEmiRs may also be found in several databases, such as bloodmiRs, which include miRNAs that are cell-specific in the peripheral blood of humans [67]. Likewise, ExcellmiRDB [68] has been the first database specialized in DEmiRs in biofluids with vetted data. miRandola, on the other hand, gives extensive information on numerous extracellular circulating miRNAs [69]. During every disease, environmental variables, as well as miRNAs, play an important influence. A database called miREnvironment was created to hold empirically verified data revealing correlations between environmental factors, miRNAs, and their associated phenotypes [70]. Some sites are dedicated to storing differentially expressed miRNAs from the tissues of the plant; for example, PmiRExAt is a repository in the early stages of development that has a complete view of plant miRNA expression profiles across a number of tissues. They also looked at the expression patterns of miRNAs using data from publicly available short RNA datasets [71]. HPVbase [72] summarizes the expression patterns of differentially controlled miRNAs associated with HPV infection in great detail.

Bioinformatics databases and tools enclosing targets of miRNAs

The finding of a vast amount of miRNAs expressed in different species raised a critical question concerning their function. In order to answer this question, cloning and computational techniques were developed to find their targets. TarBase [73] is the most comprehensive library of experimentally confirmed miRNA targets from a variety of species. miRTarBase [74, 75] is a database that tracks the target interactions of miRNAs. miRGate is a database that is manually maintained and it examines miRNA and its gene targets, and also isoforms, from humans, mice, and rats 3’ (untranslated region) UTR regions [76]. miRNA targets that are conserved in five vertebrates are provided by TargetScanS. Further, the plant non-coding RNA database (PNRD) is a specialized tool for miR-targets and other non-coding RNAs (ncRNAs) in plant species. It now has over 25,000 entries of around 10 different types of ncRNAs from over 150 plant species [77]. Likewise, VIRmirTar is a subdivision repository of the VIRmiRNA coordinated resource that contains a wealth of knowledge on the cellular and viral targets of miRNAs expressed by viruses [55]. Both experimental and computationally anticipated data may be found in certain well-known archives. miRecords is a comprehensive repository of miRNA-target interactions. It is made up of two sub-databases. The former includes carefully selected, empirically confirmed miRNA targets gleaned from thorough literature searches, whereas the other includes targets that are computationally predicted. These forecasts are governed by eleven well-known prediction systems [78]. Moreover, miRWalk is a free, all-encompassing resource that provides empirically validated and projected miRNAtarget interaction pairings [79]. Seven commonly known miRNA target gene prediction methods have been added to miRSystem: PicTar, DIANA, TargetScan, PITA, miRBridge, RNA22, and miRanda [80]. There exists computational prediction of miRNA-binding regions in the sequences of the promoter of ideal sequences; for instance, microPIR2 is a repository of computationally determined miRNA target sites in human and mouse genome promoter sequences. It also includes investigative information on expected targets. [81]. Similar to miRBase [82], ViTa volunteers scientifically identified host miRNA targets.

miRTar is a repository to observe miRNA-target relations [83], while DIANA microT-coding sequence (CDS) which is also an online database for predicting miRNA-target relations [84]. Depositories containing projected miR-target interactions are typically of two different types: those that carry information from numerous species and/or those that are specialized to a single species. MicroCosm contains miRNA targets that have been computationally predicted for a variety of taxa. More than of about 2 million putative gene targets are controlled via more than 6,500 miRNAs, according to miRDB [85, 86]. The EMBL’s miRNA—Target Gene Prediction repository contains information on miRNA-targets gene prediction for the fruit flies. Some of the computational data sources, such as multiMiR, a catalog-cum-miRNA-target R program, allow target prediction by combining accessible techniques with other connected functionalities. This portal provides access to over 50 million human and mouse records from a variety of databases [87]. Similarly, the CSmiRTar database, which stands for the condition-specific miRNA target database, makes predictions that are based on various situations [88]. Single nucleotide polymorphism in the miRNA-binding sites of the genes has been shown to affect the miRNA target repertoire. miRSNP, miRdSNP, and PolymiRTS were created to cope with the existing biological data in these factors. miRSNP is a collection of human miRNAs predicted single nucleotide polymorphisms (SNPs). Likewise, miRdSNP provides a complete database of single nucleotide polymorphism on human genes’ 3’ UTRs [89]. PolymiRTS is a database-as-a-service that may be used to investigate the impact of SNPs in miRNA seed sequences. Users can investigate the relationships between SNPs and gene expression features, pathways, and disease phenotypes [90, 91].

Bioinformatics tools for miRNA disease associations

Additionally, archives containing miRNAs organized by illness or experimental systems used to elicit them are given. HMED, for example, is a database that includes human miRNA expressions [66]. EpimiR is a comprehensive database of epigenetic alterations and miRNA associations. CoGemiR is another online server that tracks the development of miRNAs in diverse animal species. OncomiRD, miRCancer, and dbDEMC are catalogues of differentially expressed miRNAs from different kinds of malignancies [92, 93]. Further, PhenomiR is an online resource that provides data regarding DEmiRs and their roles in numerous diseases and biological processes [94]. PMTED was created to assess miRNA expression patterns and also their targets which are from the published plant micro-array research [95]. Furthermore, miRStress is an online database that contains miRNAs linked to stress [96]. The Dietary MiRNA Database (DMD) contains reported miRNA from human meals [97]. PASmiR was created with the goal of creating software for collecting, standardizing, and querying data on miRNA-stress regulations in the plant. Likewise, EpimiRBase is a handy database that contains data on miRNAs that have been linked to epilepsy [98]. Similarly, antiviral miRNAs during viral infections are covered by AVIRmiR, a VIRmiRNA type of database, some of the resources are described in Table 2 [55].

In the bloodstream, unmodified miRNAs are rapidly eliminated and degraded

Maintaining stability and uniformity of miRNAs in circulations is a difficulty in miRNA therapies. In seconds, nucleases in the blood, such as serum RNase A-type nucleases, degrade naked miRNAs with an unaltered 2’-OH in the ribose moiety [128]. Furthermore, naked miRNAs are promptly excreted by the kidneys, which results in a shorter half-life in the systemic circulation. The chemically designed miRNA alterations on the phosphodiester-backbone and on the 2 of the ribose that prevents the miRNA from degrading and boosting its long-term efficacy are the solution to this problem [128, 129]. Phosphodiester links, ribose-backbone, 2’-O-Methyl, 2’-O-(2-Methoxyethyl), 2-Fluoro, and 2-locked-nucleic acid are some of the chemical changes that have been discovered. These changes enhance the stability of oligonucleotides while simultaneously increasing the binding-affinity towards the target and assisting loading it into the miRNA-induced silencing complexes (miRISCs), of which both boost miRNA function.

In vivo and in vitro penetration of miRNA is limited

Hundreds of miRNAs have been discovered to have a role in the genesis and progression of illness, including cancer. Despite significant advancements in miRNA delivery, the primary difficulty of miRNA therapies is effectively delivering it to the targeted tissue with an efficient payload penetration onto a particular spot [130]. Poor blood perfusion is caused by the leaky nature of tumor blood vessels in cancer, which lowers the transport effectiveness of bare miRNAs.

Escape of endosome is difficult

miRNA trafficking initiates in the primary compartment of the endosome, whether the miRNAs are supplied by nanoparticles, cell-type specific, or cationic lipid delivery vehicles. The contents of the early endosomes are then transferred to the late endosomes. The environment of late endosomal vesicles is acidic, i.e., around pH 5 to 6. The contents of the endosomes are subsequently transported to lysosomes, which are acidified with a pH of 4.5 and contain numerous nucleases which promote the breakdown of oligonucleotides-miRNA mimic. miRNA mimics should escape into the cytoplasm from the endosome, where they may interact with the RNA-interference machinery, to avoid lysosomal destruction. The endosomal uptake mechanism is recognized to represent a rate-limiting barrier in miRNA distribution. Endosomal vesicles capture bioactive compounds, which are then destroyed in the lysosomal compartment, requiring the development of efficient ways to enable endosomal escape and increase cytosolic bioavailability [142]. The endosomal release can be aided by a variety of methods: (1) lipoplexes with a pH sensitivity [124]; (2) polyplexes with a pH sensitivity [143]; (3) molecules that are photosensitive [144]; and (4) also with the use of cationic nanoparticles [145].

There are various obstacles to extrahepatic-delivery

The well-known trivalent GalNAc compound binds with the receptor of asialo-glycoprotein on hepatocytes with great specificity and affinity, which results in targeted delivery of oligonucleotides and the gene-silencing in the hepatocytes. Further, the platform of GalNAc performance illustrates that efficient delivery of tissue of the therapeutic oligonucleotide is the cornerstone of each clinical trial. However, lipids in conjugation with miRNAs have been reported to improve the delivery of miRNA [146]. The liver accumulates the bulk of lipid conjugated miRNAs examined, such as cholesterol conjugated miRNAs (60–80%). Extrahepatic tissues, on the other hand, show accumulation and productive silencing of cholesterol-modified miRNAs. Furthermore, local injections of siRNA which are cholesterol-modified result in functional gene-silencing in the skin, brain, and vaginal canal [146]. Extra-hepatic tissue targeting of miRNAs remains a challenge, restricting the application of miRNA-based therapeutics. Despite the fact that polymer nanoparticles and lipids may be designed for effective endosomal escape and cellular absorption, they nonetheless aggregate in the spleen, liver, and kidney. Extra hepatic scaffolds, such as peptides, lipids, and antibodies that are coupled to particles, or aptamers, are used to overcome this difficulty, resulting in targeting by recognition of particular receptors [134, 147].

Unwanted on-target effects and off-target effects

One of the primary challenges with miRNA treatment is the off-target effects of the miRNAs after they are transported in the cytoplasm and are released from the endosomes. miRNAs are made to particularly target certain pathways via faulty hybridization with the 3’ UTRs, but they might silence the other genes unintentionally. The off-target gene-silencing could result in toxicity and a reduction in the therapeutic efficacy. Besides, the potential of a single miRNA that could target several mRNAs warrants critical consideration since it suggests the risk of unforeseeable side effects. Moreover, even if a single miRNA is targeted adequately, undesired on-target effects may occur [148]. To solve such a barrier, one of the strategies is the utilization of modest dosages of coupled miRNAs that influence the expressions of the same targeted genes synergistically [149]. The co-transfections of the miR-15a/16 and miR-34a, for example, resulted in enhanced cell-cycle arrest in the NSCLC because of the fact that miR-15a and miR-16 selectively downregulate the CCNE1 and the CCND3, respectively. Such kind of gene-regulation has a complimentary impact towards the miR-34-mediated cell-cycle regulations [150].

miRNAs have the ability to trigger the immune system

The host system may see double-stranded RNAs as pathogens, and the innate immune system may identify those and gets activated. Such as, like other forms of nucleic acids, systemic miRNA administration could trigger the innate immune system, resulting in undesirable and major toxic side effects. Through Toll-like receptors (TLRs), systemic injection of miRNA duplexes can activate the release of inflammatory cytokines and type I interferons (IFNs). Single or double-stranded RNAs could activate TLRs3, 7, and 8 to promote adaptive and innate immune responses. TLRs detect double-stranded RNA molecules in the cellular endosomes and lysosomes, triggering the IFNs pathway as well as cytokines synthesis [151]. Scientists have been currently researching immunological responses to miRNAs, but it appears that the chemical alterations also diminish immune system recognition [122]. The reaction of the immune system towards the delivery vehicles which are highly positively charged, which could also be toxic and might activate the immune system is also of great importance. Interactions with the immune systems are unavoidable when nanoparticles enter into the body. Furthermore, the surface modifications, size, hydrophobicity, and shape of the nanoparticles all have an influence on their interactions with the immune system [152]. As a result, both the miRNAs and the delivery system must be investigated. Reduced dose and hence presumably reduced immune responses will be possible if the carrier is targeted to certain organs [153].