In recent years, various progresses have been made in identifying microRNAs as significant regulators of gene expression and their relationship of or control of majority of diseases such as cancer, neurodegenerative disorders and infectious diseases. So far, many miRNAs and their targets have been demonstrated and their direct or indirect relation with the infectious diseases has been investigated in a large amount of experimental systems as well as in human tissues. Here we discuss current research of miRNAs and their involvement in infectious diseases. We also debate possible diagnostic and therapeutic values of miRNAs in infectious diseases. The discovery of infectious diseases related miRNAs has been a major breakthrough in research and will provide a reasonable therapeutic strategy for the treatment of bacterial and viral infectious diseases.
Key words miRNAs; bacterial infectious diseases; viral infectious diseases; parasitic infectious diseases
Infectious diseases are caused by pathogens which spread among human, animals or transmit between animals and human. Most of the pathogen are microbes, small part are parasites. After pathogens are discharged from source of infection, they disseminate through single or multiple transmission route, which can be divided into horizontal transmission and vertical transmission. The diagnosis of infectious diseases mainly depend on microculture, however, part of the pathogens can not be artificial cultivation, especially virus and leptospira bacterium. So, many new detection technologies have been developed, for instance, PCR, it is not only rapid but accuracy also increased greatly. Biomarkers are used for differential diagnosis as well, but susceptibility and specificity needs to be improved.
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MicroRNAs (miRNA) are a class of endogenous, 22nt RNAs that play important regulatory roles in animals and plants for degradation or translational repression . At present, approximately 1000 human miRNAs were listed in the miRNA registry which may target about 60% of all mammalian genes, indicating that these small RNAs play fundamental and global functions in human biology, including development , differentiation , apoptosis , cancer  and viral infection . In addition, miRNAs could modulate the innate and adaptive immune responses to pathogens by regulating immune cell differentiation and the development of diseases [8, 9]. MiRNAs were used as diagnostic biomarkers in clinical has already been investigated in all types of cancers [10, 11]. However, compared to their clear role in cancer, their role in susceptibility and resistance to infectious diseases is still lacking.
In spite of the efforts involved in identifying biomarkers of infectious diseases, no such biomarkers have aroused as much interest as the miRNAs. MiRNA was first discovered in Caenorhabditis elegans (C. elegans) in 1993, since then, a large amount of research has been published, demonstrating that the biological function of miRNAs is significant to most infectious diseases. We discuss advances in understanding of the function of miRNAs in infectious diseases; this emerging field is anticipated to profoundly affect clinical research, diagnosis, and therapy of infectious diseases.
1 MiRNAs and bacterial infectious diseases
To date, miRNAs have been confirmed to play key roles in regulating and influencing the important characteristics of bacteria, such as capability of survival, virulence and drug resistance. A variety of bacteria can be regulated by miRNAs, such as mycobacterium tuberculosis, helicobacter pylorus, salmonella, listeria monocytogenes, and so on.
1.1 Perturbation of miRNA expression by bacterium
As a major health issue, there were 8.8 million incident cases of TB, approximately 1.45 million deaths from TB in 2010 . Mycobacterium tuberculosis (MTB), the causative agent for tuberculosis which was identified by Robert Koch in 1882, remains one of the most mysterious bacteria. M.tb-environmental interactions have been widely studied. However, knowledge about potential M.tb-host interaction at the RNA level still needs to be fully studied. So far, several miRNAs have been indicated to be closely related to M.tb survival by inhibiting the formation of cytokines, such as human microRNA mir-125b and mir-144*. Mir-125b could destabilize the transcript of TNF mRNA to interfere TNF biosynthesis in macrophages, thereby allowing MTB to subvert host immunity and potentially increase its virulence . Mir-144* was detected over-expressed in active TB patients, transfection with mir-144* demonstrated that mir-144* could involved in regulation of anti-TB immunity through inhibit TNF-α and IFN-γ production and T cell proliferation .
Helicobacter pylori (H. pylori) are major human pathogenic bacteria in gastric mucosa which is associated with gastric diseases like chronic active gastritis, peptic ulcer, and gastric carcinoma. Several groups have recently examined the miRNAs signatures of H. pylori infection and their results suggested that dysregulation of miRNAs expression is a plausible mechanistic link between H. pylori infection and the development of gastric disease . For example, Matsushima et al demonstrated differential expression of microRNAs between H. pylori positive and negative patients, with restoration of 14 of 30 miRNAs after H. pylori eradication . Meanwhile, altered expression of certain miRNAs such as let-7 family members was dependent upon the presence of the cag pathogenicity island. Furthermore, H. pylori were able to increase the miR-155 expression through NF-κB and AP-1 pathway in gastric epithelial cell lines with subsequent inhibition of IL-8 .Oertli further pointed out that up-regulated miR-155 is required for the Th17/Th1 differentiation which underlies immunity to H. pylori infection, thus induced chronic gastritis and relevant precancerous lesions . Further studies identified Myeloid differentiation protein 88 (MyD88) as a target gene of miR-155, and miR-155 decreased MyD88 expression is a post-transcriptional event . Through the same NF-κB signaling pathway, Liu et al found the expression of miR-146a was up-regulated not only in gastric epithelial cells but also in gastric mucosal tissues after H. pylori infection. Thus, miR-146a may down-regulate the target genes expression, including interleukin-1 receptor-associated kinase 1and TNF receptor-associated factor 6. What's more, miR-146a negatively regulated H. pylori-triggered IL-8, growth-related oncogene-α, and macrophage inflammatory protein-3α through diminishing NF-κB activity .
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Salmonella is an intensely investigated intracellular bacterial pathogen that diverse diseases ranging from mild, self-limiting gastroenteritis to life-threatening systemic infection . A population of pigs was inoculated with Salmonella enterica serovar Typhimurium (ST) and peripheral blood and fecal Salmonella counts were collected. Two groups of pigs with either low shedding (LS) or persistent shedding (PS) phenotypes were identified. ST inoculation triggered substantial gene expression changes and there was differential expression of many genes between two groups. Analysis of the differential profiles of gene expression within and between PS and LS phenotypic classes identified distinct regulatory pathways mediated by IFN-γ, TNF, NF-κB, or one of several miRNAs. They confirmed the activation of SPI1 and CEBPB, and demonstrated that expression of miR-155 was decreased specifically in the PS animals . In addition to pigs, deficient in miR-155 also altered immune response of mice by regulating IL-6 and IL-10 to the facultative intracellular pathogen Salmonella .
Listeria monocytogenes (L. monocytogenes) is an intracellular facultative pathogen that infects a large variety of host cells, including macrophages. L. monocytogenes could avoid phagocytosis by generating a pore-forming toxin (listeriolysin O, LLO) that help the bacterium penetrate into cytoplasm . Izar et al identified and validated five miRNAs (miR-145, miR-146b, miR-155, miR-16 and let-7a1) that were significantly deregulated after listerial infection, and the expression patterns of these microRNAs hinge on pathogen localization and the presence of purified LLO . In addition, L. monocytogenes changed the microRNA expression profile significantly in macrophages, including miR-125a-3p/5p, miR-146a, miR-149 and miR-155, and showed a vacuolar-dependent miRNA signature, LLO independent and MyD88-dependent. These microRNAs are predicted to relevant with immune genes and are likely involved in manipulation of the macrophage innate immune response against infection . Izar said the change of miR-155 depend on LLO while Schnitge argued that it is LLO independent, further insights into the different conclusion of these observations is awaited.
1.2 MiRNAs participate in the transition from latent to active stage
In addition to cause disease, microRNAs participate in the transition from latent to active stage. Take TB for example, the exact underlying mechanisms and its transition to active TB remain to be fully elucidated. Latent TB infection depends on an equilibrium in which the host is capable of controlling the infection but can not eradicate the bacteria completely . Latency may lie on the virulence of M.tb strain  and the host immune response. In order to protect themselves from attack, some bacteria may blunt phagosome and lysosome fusion, antigen presentation, nitric oxide production, or other bactericidal measures of the host, and survive in a phenotype called dormancy . Many studies try to explain the fundamental biology of TB and offer guidelines for diagnosis and treatment in the future, but they do not illuminate the pathogenesis of pathogen transit from latent to active TB. Wang et al used a miRNA microarray to compared the microRNA expression in PBMCs of patients suffered active TB, latent TB, and healthy controls. They found 5 candidate microRNAs regulate the transition from latent TB to active TB infection, and among them, hsa-miR-424 and hsa-miR-365 also exhibited a high level in samples from active TB compare to healthy controls. Using the predicted target genes and genome-wide transcriptional profiles, they described a regulatory network of microRNAs that were differentially expressed in active TB and latent TB infection. The regulatory network uncovered that several microRNAs, with previously established functions in hematopoietic cell differentiation and their target genes may be involved in the transition from latent to active TB. The results increased the understanding of the molecular basis of latent TB infection and confirmed that some microRNA may regulate pathways of gene expression and play key role in pathogenesis of TB .
1.3 MiRNAs as biomarkers for diagnosis and treatment of bacterial infectious diseases
Development of sensitive and specific biomarkers for infectious diseases will improve current management of them. MiRNAs have the following advantages of serving as biomarkers: (a) miRNAs are involved in pathogenesis; (b) miRNAs are tissue, bacteria species or disease stage specific; and (c) some miRNAs are related to treatment response or patients' survival.
Accumulating studies have investigated the diagnostic values of miRNAs in infectious diseases, especially in TB and extrapulmonary tuberculosis, for they are very difficult to diagnose early because of their nonspecific symptoms and slow growth. MiRNAs in circulation is an emerging and interesting research area. It would be of great interest for future studies to correlate miRNAs with bacterial species, stage, chemotherapy resistance and survival of infectious diseases. For instance, miR-155 exhibited a fold change of 1.4 in the healthy control group and 3.7 in the active TB group upon PPD stimulation; besides, miR-155* exhibited a fold change of 1.9 in the healthy control and 4.6 in the active TB group, according to their alteration under the challenge of specific MTB antigens, they can be seen as potential diagnostic markers . Besides, up-regulated miR-29a could distinguish TB patients from healthy controls, and circulating miR-29a has great potential to apply as a marker for the active pulmonary tuberculosis infection detection .
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Good progresses have been made in miRNAs assisted diagnosis of H. pylori infection as well. S. Lario et al confirmed the up-regulation of miR-9, miR-146a, miR-155 and miR-650 and the down-regulation of miR-96 and miR-204 in H. pylori positive patients. These miRNAs can be used as biomarker to assist diagnosis, especially the sensitivities and specificities were improved when the ratio between down-regulated (miR-204 and miR-96) and up-regulated (miR-9, miR-146a, miR-155 and miR-650) miRNAs was used. Among these, the ratio miR-96/miR-155 was the best with a sensitivity of 84.2% and a specificity of 95.5% .
From the correlation of the above four types of bacteria and miRNAs, we could find that miR-155 and miR-146a participate in almost every bacterial infection, especially miRNA-155. Accordingly, we boldly predict that miRNA-155 and miRNA-146a are closely related to bacterial infection, they have a therapeutic potential to bacteria infection and could be an optional treatment for bacterial infectious diseases in the clinical settings.
Since miRNAs gave an extraordinary performance in the diagnosis of TB, both accurate and can distinguish the different stages of disease, what's more, it has a wide application prospect. So some researchers searched the M.tb genome for potential target sites of known human miRNAs using miRNA target prediction method miRanda, and compared three evolutionarily close genomes, M. tuberculosis H37Rv, M. tuberculosis CDC1551 and Mycobacterium leprae (M. leprae) TN, and then, they selected for further analysis only miRNA-target pairs that are conserved across three genomes. They finally gave a list including genes involved in drug-resistance and bacterial survival such as rpsL, hspR, grpE, dnaJ1 and sodC. Many candidate target genes have multiple predicted miRNA binding sites. For example, hspR level can be regulated by binding with has-mir-210, has-mir- 375, has-mir-423 and has-mir-636, bring about the change of M.tb survival . These miRNAs provide us a promising future to diagnosis and treatment of TB efficiently.
2 MiRNAs and viral infectious diseases
Virus is the main pathogenic agent leading to infectious diseases. In order to survive in host cell, viruses have developed a series of strategies to avoid infected cell apoptosis and escape from the host cell attack. Although many macromolecular viruses successful resist the host cell immune attack by their coding protein, but the limited coding ability of genome makes miRNAs become an effective tool. With the identification and functional characterization of virally encoded small regulatory RNAs in Epstein-Barr virus (EBV), it soon became evident that viruses can utilize the RNA silencing machinery to regulate their own transcripts as well as host-derived targets . In this work, five miRNA precursors clustering in two genomic locations of EBV were identified by small-scale cloning of small RNAs from virus infected cells; they are miR-BHRF1-1 to miR-BHRF1-3 and miR-BART1 and miR-BART2 which locate within the mRNA of the Bam HI fragment H rightward open reading frame 1(BHRF1) and the intronic regions of the Bam HI-A region rightward transcript gene (BART) respectively. Subsequent studies demonstrated virally encoded miRNAs in other DNA virus: Hepatitis B virus (HBV). Two of the three reports on viral miRNAs came from an RNA virus, human immunodeficiency virus-1 (HIV-1).
2.1 Perturbation of miRNAs expression by virus
EBV, a B lymphotropic gamma-herpesvirus with potent growth-transforming properties, is etiologically linked to a number of malignancies of lymphoid and epithelial cell origin . In recent years it has been shown that EBV encodes at least 17 miRNAs in latently infected cells. They are arranged in two clusters: 14 miRNAs are located in the introns of the viral BART gene and the other 3 are located next to BHRF1. The 14 miRNAs expression in latently infected epithelial cells is more than that in B cells, while the 3 miRNAs expression is detected at a high level in B cells going through stage III latency but is almost undetectable in B cells or epithelial cells going through stage I or II latency. Activation of EBV replication was found to increase the expression of most of these viral miRNAs . Cellular miRNAs also get changed during infection. MiR-155 is strongly upregulated during latent EBV infection of B cells [39, 40] and is the most abundant miRNA expressed in lymphoblastoid cell lines (LCLs) . These data strongly suggested that this miRNA plays a key role in EBV-associated tumorigenesis. Consequently, several groups have focused on the identification of miR-155 targets toward elucidating its function in the setting of EBV infection. Yin et al analyzed the mRNA expression profile of EBV latency I-expressing Akata cells, which normally lack miR-155 expression, upon reintroduction of this miRNA at levels normally found in LCLs . They found 84 increased mRNAs upon miR-155 expression and 78 repressed mRNAs. Of the suppressed mRNAs, 17 contained miR-155 seed sequences in their 3′UTRs and 8 of these mRNAs were functionally validated as direct miR-155 targets by 3′UTR-luciferase assays. Interestingly, all of them (BACH1, ZIC3, ZNF652, ARID2, SMAD5, HIVEP2, CEBPB, and DET) are transcription factors, indicating that EBV-induced expression of miR-155 likely supports EBV signaling by regulating transcriptional regulatory mechanisms.
Chronic liver diseases caused by hepatitis B (HBV) or C virus (HCV) are common worldwide. HBV is a non-cytopathic, hepatotropic virus which generates extremely large quantities of hepatitis B surface antigen (HBsAg), the outermost structure of both virions and defective particles . Circulating HBsAg was extracted from HBsAg carriers, and 39 human microRNAs were found to be significantly associated with HBsAg. These microRNAs were liver-specific (most frequent = miR-122, miR-126, miR-145, miR-27a and miR-30b) . Same as HBV, certain miRNAs of HCV patient get changed; serum levels of miR-122, miR-34a and miR-16 were significantly higher than in control individuals, what's more, miR-122 and miR-34a levels positively correlated with disease severity . Emerging evidence suggested that HBV and HCV alter miRNAs expression profiles; however, the mechanisms underlying this process needs to be fully elucidated. Some studies showed that subviral HBsAg particles can carry inside microRNAs and associated Argonaute2 (AGO2) protein in the same way as HDV-RNA and HDAg complexes ï¼Œhowever, the auto-antibodies to AGO2 was found in 5% of HCV or HBV+HCV co-infected patients other than in HBV . Moreover, in cells HBV infection, expression of Drosha mRNA and protein were down-regulated by inhibiting the activity of Drosha gene promoter. Interfere with HBx expression significantly restored the Drosha expression, possible factors involved in this process are transcription factors SP1 and AP-2a . Although some microRNA is expressed in different cell types, the expression of most microRNA is restricted to specific organs and tissues [48, 49]. MiR-122, for example, is only expressed in quantity in liver and was not found in other tissues [48, 50, 51].
An understanding of how HIV-1 infection affects the host miRNA pathway could generate new insights into the basic mechanisms. After in vitro HIV-1-infected CD4+ T cells were analyzed by microarray, Sun et al found that miR-223 levels were markedly enhanced in HIV-1-infected CD4+CD8- PBMCs, while miR-29a/b, miR-155 and miR-21 levels were significantly reduced at the peak of HIV-1 infection, suggesting a possible regulatory mechanism at the peak of HIV-1 reproduction which involves the apoptosis of host CD4 cells, up-regulation of microRNA-223, down-regulation of miR-29 and expression of Nef which is a 27-kDa HIV-1 protein that is produced early during infection and translated from multiply spliced viral mRNAs . Under normal conditions, miRNAs function as negative regulators of gene expression by binding to the 3′UTR of the target message. Because the HIV-1 Nef sequence serves as the 3′UTR for most viral transcripts, miRNA binding sites in this region might play a crucial role in HIV-1 infection . It has been shown that Nef plays a positive role in viral replication and pathogenesis. HIV-1 strains with nef gene deletions result in slower progression to AIDS . So, miRNAs targeting the nef gene have the potential to influence HIV-1 pathogenesis. In addition, miR-29a target the HIV-1 nef gene, and ectopic expression of this host miRNA resulted in repression of Nef protein levels and a reduction in viral levels . Other researchers also found that miR-29a can target HIV-1 and repress replication and infectivity. They showed that miR-29a can directly target HIV-1 transcripts to P bodies, and this could be a mechanism for maintaining HIV-1 in a latent state . Swaminathan et al demonstrated that compared to healthy controls and long-term non-progressors, let-7 miRNAs were observably decreased in chronic HIV-1 infected, which may lead to an increase in IL-10 from CD4+ T cells and facilitate the virus with a crucial survival advantage by regulating the host immune response .
2.2 MiRNAs as biomarkers for diagnosis and treatment of viral infectious diseases
Waidmann et al found that the miR-122 serum concentration correlated with the level of alanine aminotransferase, HBV DNA and HBsAg. The miR-122 serum levels discriminated the different patient groups infected with HBV from healthy subjects, and inactive carrier patients with high (>3500 IU/mL) or low (<3500 IU/mL) levels of HBsAg could be differentiated by the miR-122 serum concentration . MiR-122 is liver-specificity and it is well conserved among many species.
In addition, miR-122 function can be specifically inhibited by the allosteric ribozyme in HCV-replicating cells, and HCV replicon replication was efficiently inhibited by the allosteric ribozyme. This ribozyme could be useful for the specific, safe, and efficacious anti-HCV modulation . Beyond that, lentiviral miRNA-based RNAi against the HBsAg gene could be used to inhibit HBV replication . That is, miRNA has a therapeutic potential to HBV and HCV infection and could be a treatment for certain viral infectious diseases in the clinical settings.
All in all, since the first miRNA coded by EBV was identified, scientists have had some knowledge of correlation of viral infection and miRNAs encoded by viral genome and host cell. They demonstrated that miRNAs act as the adjustment factor (activated factor or inhibitory factor), and play an important role in some virus' life cycle which cause fatal infectious diseases, including HCV and HIV-1. To date, most efforts into finding an effective vaccine to combat HIV-1 have not been successful. Understanding the role that miRNAs play in HIV-1 pathogenesis may allow a different approach to targeting key miRNAs or the identification of new important protein targets regulated by miRNAs which may result in a better vaccine construct.
3 MiRNAs and parasitic infectious diseases
A great part of infectious diseases are caused by parasites, such as Schistosoma japonicum, Cryptosporidium parvum, Eimeria papillata, and so on. Researchers have demonstrated that miRNAs related with the parasites above was changed either in parasites themselves or in host cells. Here we summarized as follows.
3.1 MiRNAs regulation system of parasites
Complex living environment, different life forms and cycles and sex differentiation indicate that parasites have extremely complex gene expression regulation system, which is related to essential processing proteins (Argonaute (AGO) and Dicer) in the process of miRNA mature. De et al found the protein-coding genes of AGO and Dicer in Entamoeba histolytica using a bioinformatic approach . P J et al characterized the structure and expression of the Dicer gene from Schistosoma mansoni . The SmAgo2/3/4 and SmDicer1 are differentially expressed during schistosomula growth, suggesting that the miRNA pathway may have effect on gene expression at life cycle of S. mansoni . The Trichomonas genome encodes at least two AGO proteins (Tv_AGO1 and Tv_AGO2), and the expression of Tv_AGO1 gene was significantly higher than Tv_AGO2 gene in the trophozoite stage, these suggested that functional miRNA machinery exists in T.vaginalis. It will be of great interest to examine whether the different types of miRNAs are processed by the same or different AGO proteins at different development stages or experimental conditions .
3.2 Change of miRNA expression by parasites
MiRNAs need to regulate gene expression sensitively if parasites want to quickly adapt to the change of environment and development. Parasites can improve the probability of infection and their proliferation by miRNA regulate genes of their own or hosts, which help escape from immune monitoring [65, 66]. Schistosoma japonicum has a complex life cycle and a unique repertoire of genes expressed at different life cycle stages. Expression of sja-let-7, sja-miR-71 and sja-bantam were analyzed in six stages of the life cycle, i.e. egg, miracidium, sporocyst, cercaria, schistosomulum, and adult worm. The expression patterns of these miRNAs were highly stage-specific. In particular, sja-miR-71 and sja-bantam expression reached their peaks in the cercaria stage and then dropped quickly to the nadirs in the schistosomulum stage, suggesting that they may mediate important roles in Schistosome growth and development .
Parasitic infection could also lead to the change of the host microRNAs, which affects their survival environment. Dkhil et al investigated intestinal infections of male Balb/c mice with the coccidian parasite Eimeria papillata. On day 4 after oral infection, they only present a low inflammatory response of the jejunum. Using microRNA microarray technology, there were significantly up-regulated four miRNA species miR-1959, MCMV-miR-M23-1-5P, miR-203, and miR-21 out of 634 miRNAs, which was also confirmed by quantitative RT-PCR. The data provided evidence that E. papillata parasites are able to induce specific miRNA species in their host target organ, and this might help us to control or cure E. papillata infection . Recent researches reported differential alterations in microRNA expression in cholangiocytes following Cryptosporidium parvum infection [69, 70]. Chen et al reported that cholangiocytes express let-7 family members. Infection of cultured cholangiocytes with Cryptosporidium parvum led to decreased expression of primary let-7i and mature let-7 in a MyD88/NF-κB-dependent manner. The decreased let-7 expression correlate with C. parvum-induced up-regulation of TLR4 in infected cells which is important pathogen recognition molecules and key to innate immune responses to microbial infection. In addition, experimentally induced repression or enhanced expression of let-7i led to reciprocal alterations in C. parvum-induced TLR4 protein expression. The results demonstrate that let-7i regulates TLR4 expression in cholangiocytes and contributes to innate immune responses against C. parvum infection .The apicomplexan parasite Toxoplasma gondii can infect and replicate in various nucleated cell of warm-blooded animals which infects approximately two billion humans . In Toxoplasma-infected primary human cells, levels of miR-17~92 and miR-106b~25 are altered, which are known as crucial roles in mammalian cell regulation and have been multiple reported in plentiful hyperproliferative diseases, however, these altered microRNAs remains unchanged in host cells infected with Neospora caninum. Thus, the Toxoplasma-induced increase of microRNAs is selective and not a general host response to infection .
Conclusions and future perspectives
From the knowledge above, we found that the let-7 family and MyD88 involved time and again in the occurrence and development of the various diseases. Expression of let-7 family members altered after infected by H. pylori, Listeria and HIV-1; infection of cultured cholangiocytes with Cryptosporidium parvum led to decreased expression of primary let-7i and mature let-7 in a MyD88/NF-κB-dependent manner. And likewise, in addition to Cryptosporidium parvum, MyD88 involved in negative regulation of H pylori-induced inflammation, and L. monocytogenes promoted significant changes in the miRNA expression profile is also MyD88-dependent.
Over the past decade, there is a rapid accumulation of evidence on the crucial role of deregulated miRNAs in the pathogenesis and progression of infectious diseases, which holds great potential for new development in current diagnostic and therapeutic strategies in the management of them. Gene profiling studies have demonstrated a significant deregulated miRNAs and identified signatures of both diagnostic and therapeutic value in infectious diseases. However, despite the encouraging results, we are still facing many difficulties in the research field of infection-related miRNAs. The expression of miRNAs is tissue, temporal, and spatial specific, and can be influenced by a variety of factors. In addition, there are several major obstacles to overcome before the application of miRNA-based treatment. Firstly, the multitargeting nature of miRNAs gives the risk of unintended off-target effects that need to be carefully evaluated. Secondly, the expression of target gene may be controlled by several different miRNAs, which may compromise the effect of miRNA-based treatment. Finally, there is still lack of miRNA delivery system with enough specificity and efficacy. Anyway, the small molecules have greatly changed our point of view in pathogenesis and will definitely improve the current management of infectious diseases in the future.