A Transcription Regulatory Sequence in the 5' Untranslated Region of SARS-CoV-2 Is Vital for Virus Replication with an Altered Evolutionary Pattern against Human Inhibitory MicroRNAs.

Our knowledge of the evolution and the role of untranslated region (UTR) in SARS-CoV-2 pathogenicity is very limited. Leader sequence, originated from UTR, is found at the 5' ends of all encoded SARS-CoV-2 transcripts, highlighting its importance. Here, evolution of leader sequence was compared between human pathogenic and non-pathogenic coronaviruses. Then, profiling of microRNAs that can inactivate the key UTR regions of coronaviruses was carried out. A distinguished pattern of evolution in leader sequence of SARS-CoV-2 was found. Mining all available microRNA families against leader sequences of coronaviruses resulted in discovery of 39 microRNAs with a stable thermodynamic binding energy. Notably, SARS-CoV-2 had a lower binding stability against microRNAs. hsa-MIR-5004-3p was the only human microRNA able to target the leader sequence of SARS and to a lesser extent, also SARS-CoV-2. However, its binding stability decreased remarkably in SARS-COV-2. We found some plant microRNAs with low and stable binding energy against SARS-COV-2. Meta-analysis documented a significant (p < 0.01) decline in the expression of MIR-5004-3p after SARS-COV-2 infection in trachea, lung biopsy, and bronchial organoids as well as lung-derived Calu-3 and A549 cells. The paucity of the innate human inhibitory microRNAs to bind to leader sequence of SARS-CoV-2 can contribute to its high replication in infected human cells.

Salvia miltiorrhiza-derived miRNAs suppress vascular remodeling through regulating OTUD7B/KLF4/NMHC IIA axis.

Objective: Abnormal proliferation and migration of vascular smooth muscle cells (VSMCs) are essential for vascular remodeling. Natural compounds with diterpene chinone or phenolic acid structure from Salvia miltiorrhiza, an eminent medicinal herb widely used to treat cardiovascular diseases in China, can effectively attenuate vascular remodeling induced by vascular injury. However, it remains unknown whether Salvia miltiorrhiza-derived miRNAs can protect VSMCs from injury by environmental stimuli. Here, we explored the role and underlying mechanisms of Salvia miltiorrhiza-derived Sal-miR-1 and 3 in the regulation of VSMC migration and monocyte adhesion to VSMCs induced by thrombin. Methods: A mouse model for intimal hyperplasia was established by the ligation of carotid artery and the injured carotid arteries were in situ-transfected with Sal-miR-1 and 3 using F-127 pluronic gel. The vascular protective effects of Sal-miR-1 and 3 were assessed via analysis of intimal hyperplasia with pathological morphology. VSMC migration and adhesion were analyzed by the wound healing, transwell membrane assays, and time-lapse imaging experiment. Using loss- and gain-of-function approaches, Sal-miR-1 and 3 regulation of OTUD7B/KLF4/NMHC IIA axis was investigated by using luciferase assay, co-immunoprecipitation, chromatin immunoprecipitation, western blotting, etc. Results:Salvia miltiorrhiza-derived Sal-miR-1 and 3 can enter the mouse body after intragastric administration, and significantly suppress intimal hyperplasia induced by carotid artery ligation. In cultured VSMCs, these two miRNAs inhibit thrombin-induced the migration of VSMCs and monocyte adhesion to VSMCs. Mechanistically, Sal-miR-1 and 3 abrogate OTUD7B upregulation by thrombin via binding to the different sites of the OTUD7B 3'UTR. Most importantly, OTUD7B downregulation by Sal-miR-1 and 3 attenuates KLF4 protein levels via decreasing its deubiquitylation, whereas decreased KLF4 relieves its repression of transcription of NMHC IIA gene and thus increases NMHC IIA expression levels. Further, increased NMHC IIA represses VSMC migration and monocyte adhesion to VSMCs via maintaining the contractile phenotype of VSMCs. Conclusions: Our studies not only found the novel bioactive components from Salvia miltiorrhiza but also clarified the molecular mechanism underlying Sal-miR-1 and 3 inhibition of VSMC migration and monocyte adhesion to VSMCs. These results add important knowledge to the pharmacological actions and bioactive components of Salvia miltiorrhiza. Sal-miR-1 and 3-regulated OTUD7B/KLF4/NMHC IIA axis may represent a therapeutic target for vascular remodeling.

Induction of Apoptosis in HeLa by Corn Small RNAs.

Insights in RNA biology have opened up a plethora of opportunities to explore the small regulatory RNAs from various natural and artificial sources. These small RNAs have been suggested to play a role too in tumor progression by either as oncogenic or tumor suppressor small RNAs. In this study, authors have attempted to evaluate the therapeutic potential of small RNAs fractionated from corn (Zea mays) upon growth and survival of HeLa. Here, authors have employed standard cellular-based approaches including microscopy, spectroscopy, and flow cytometry-based staining assays. Our data indicate that corn small RNAs fraction can appreciably decrease HeLa cell proliferation and survival, which is supported by a number of complementary assays such as Trypan blue dye exclusion, MTT, propidium iodide, and Annexin V/PI apoptotic cell death. Taken together, present finding suggests that corn small RNAs fraction may display up to 70% reduction in HeLa cell viability. Furthermore, these data indicate that around 40-50% of HeLa cells become apoptotic due to exogenous use of corn small. Overall, this finding proposes that possibility of cross-kingdom anticancer use of small RNAs from corn and present data need to be explored in depth.

The potential atheroprotective role of plant MIR156a as a repressor of monocyte recruitment on inflamed human endothelial cells.

MicroRNAs have become the spotlight of the biological community for more than a decade, but we are only now beginning to understand their functions. The detection of stably expressed endogenous microRNAs in human blood suggests that these circulating miRNAs can mediate intercellular communication. Our previous study reported the surprising finding that exogenous rice MIR168a could regulate liver low-density lipoprotein receptor adapter protein 1 (LDLRAP1) gene expression in mice. Here, we show that plant MIR156a, which is abundantly expressed in dietary green veggies, also stably presents in healthy human serum. Compared with age-matched individuals, decreased levels of MIR156a are observed both in serum and blood vessel of cardiovascular disease (CVD) patients. In vitro studies demonstrate that MIR156a can directly target the junction adhesion molecule-A (JAM-A), which is up-regulated in atherosclerotic lesions from CVD patients. Functional studies show that ectopic expression of MIR156a in human aortic endothelial cells reduces inflammatory cytokine-induced monocytes adhesion by suppressing JAM-A. These findings offer a novel vasoprotective molecular mechanism of green veggies through plant microRNAs.

Study on the inhibition of Mfn1 by plant-derived miR5338 mediating the treatment of BPH with rape bee pollen.

BACKGROUND: Recent studies have found that plant derived microRNA can cross-kingdom regulate the expression of genes in humans and other mammals, thereby resisting diseases. Can exogenous miRNAs cross the blood-prostate barrier and entry prostate then participate in prostate disease treatment? METHODS: Using HiSeq sequencing and RT-qPCR technology, we detected plant miRNAs that enriched in the prostates of rats among the normal group, BPH model group and rape bee pollen group. To forecast the functions of these miRNAs, the psRobot software and TargetFinder software were used to predict their candidate target genes in rat genome. The qRT-PCR technology was used to validate the expression of candidate target genes. RESULTS: Plant miR5338 was enriched in the posterior lobes of prostate gland of rats fed with rape bee pollen, which was accompanied by the improvement of BPH. Among the predicted target genes of miR5338, Mfn1 was significantly lower in posterior lobes of prostates of rats in the rape bee pollen group than control groups. Further experiments suggested that Mfn1 was highly related to BPH. CONCLUSIONS: These results suggesting that plant-derived miR5338 may involve in treatment of rat BPH through inhibiting Mfn1 in prostate. These results will provide more evidence for plant miRNAs cross-kingdom regulation of animal gene, and will provide preliminary theoretical and experimental basis for development of rape bee pollen into innovative health care product or medicine for the treatment of BPH.

Honeysuckle-encoded microRNA2911 inhibits Enterovirus 71 replication via targeting VP1 gene.

Enterovirus 71 (EV71) is the primary pathogen of hand-foot-and-mouth disease (HFMD) in children and virus infections are associated with severe neurological dysfunctions and even death. MIR2911 is a honeysuckle-encoded atypical microRNA with extreme stability. Here, we report that MIR2911 directly inhibits EV71 replication by targeting the VP1 gene. Bioinformatics prediction and luciferase reporter assay showed that MIR2911 could target the VP1 gene of EV71. Transfection experiments using synthetic MIR2911 and extracted RNA from HS decoction shown that each of these preparations was capable of inhibiting EV71 VP1 protein expression; however, these preparations did not impact EV71 mutants in which the MIR2911-binding sites were mutated. Furthermore, EV71 replication was increased by antagomirs against MIR2911 in the HS decoction, implying that MIR2911 was physiologically functional in controlling EV71 replication in vitro. These results indicated that, by targeting VP1 gene, MIR2911 may effectively inhibit EV71 replication. Our results also provide a potential novel strategy on the therapy and/or prevention of HFMD originating from EV71 virus infection.

MicroRNAs as New Bioactive Components in Medicinal Plants.

Herbal medicine has been used to treat diseases for centuries; however, the biological active components and the mechanistic understanding of actions of plant-derived drugs are permanently discussed. MicroRNAs are a class of small, non-coding RNAs that play crucial roles as regulators of gene expression. In recent years, an increasing number of reports showed that microRNAs not only execute biological functions within their original system, they can also be transmited from one species to another, inducing a posttranscriptional repression of protein synthesis in the recipient. This cross-kingdom regulation of microRNAs provides thrilling clues that small RNAs from medicinal plants might act as new bioactive components, interacting with the mammalian system.In this article, we provide an overview of the cross-kingdom communication of plant-derived microRNAs. We summarize the microRNAs identified in medicinal plants, their potential targets in mammals, and discuss several recent studies concerning the therapeutic applications of plant-based microRNAs. Health regulations of herbal microRNAs in mammals are a new concept. Continuing efforts in this area will broaden our understanding of biological actions of herbal remedies, and will open the way for the development of new approaches to prevent or treat human diseases.

MicroRNA from Moringa oleifera: Identification by High Throughput Sequencing and Their Potential Contribution to Plant Medicinal Value.

Moringa oleifera is a widespread plant with substantial nutritional and medicinal value. We postulated that microRNAs (miRNAs), which are endogenous, noncoding small RNAs regulating gene expression at the post-transcriptional level, might contribute to the medicinal properties of plants of this species after ingestion into human body, regulating human gene expression. However, the knowledge is scarce about miRNA in Moringa. Furthermore, in order to test the hypothesis on the pharmacological potential properties of miRNA, we conducted a high-throughput sequencing analysis using the Illumina platform. A total of 31,290,964 raw reads were produced from a library of small RNA isolated from M. oleifera seeds. We identified 94 conserved and two novel miRNAs that were validated by qRT-PCR assays. Results from qRT-PCR trials conducted on the expression of 20 Moringa miRNA showed that are conserved across multiple plant species as determined by their detection in tissue of other common crop plants. In silico analyses predicted target genes for the conserved miRNA that in turn allowed to relate the miRNAs to the regulation of physiological processes. Some of the predicted plant miRNAs have functional homology to their mammalian counterparts and regulated human genes when they were transfected into cell lines. To our knowledge, this is the first report of discovering M. oleifera miRNAs based on high-throughput sequencing and bioinformatics analysis and we provided new insight into a potential cross-species control of human gene expression. The widespread cultivation and consumption of M. oleifera, for nutritional and medicinal purposes, brings humans into close contact with products and extracts of this plant species. The potential for miRNA transfer should be evaluated as one possible mechanism of action to account for beneficial properties of this valuable species.

Cross-kingdom inhibition of breast cancer growth by plant miR159.

MicroRNAs (miRNAs) are critical regulators of gene expression, and exert extensive impacts on development, physiology, and disease of eukaryotes. A high degree of parallelism is found in the molecular basis of miRNA biogenesis and action in plants and animals. Recent studies interestingly suggest a potential cross-kingdom action of plant-derived miRNAs, through dietary intake, in regulating mammalian gene expression. Although the source and scope of plant miRNAs detected in mammalian specimens remain controversial, these initial studies inspired us to determine whether plant miRNAs can be detected in Western human sera and whether these plant miRNAs are able to influence gene expression and cellular processes related to human diseases such as cancer. Here we found that Western donor sera contained the plant miRNA miR159, whose abundance in the serum was inversely correlated with breast cancer incidence and progression in patients. In human sera, miR159 was predominantly detected in the extracellular vesicles, and was resistant to sodium periodate oxidation suggesting the plant-originated 2'-O-methylation on the 3' terminal ribose. In breast cancer cells but not non-cancerous mammary epithelial cells, a synthetic mimic of miR159 was capable of inhibiting proliferation by targeting TCF7 that encodes a Wnt signaling transcription factor, leading to a decrease in MYC protein levels. Oral administration of miR159 mimic significantly suppressed the growth of xenograft breast tumors in mice. These results demonstrate for the first time that a plant miRNA can inhibit cancer growth in mammals.

Beyond nutrients: food-derived microRNAs provide cross-kingdom regulation.

Food turns out to be not only the nutrient supplier for our body but also a carrier of regulatory information. Interestingly, a recent study made the discovery that some plant/food-derived microRNAs (miRNAs) accumulate in the serum of humans or plant-feeding animals, and regulate mammalian gene expression in a sequence-specific manner. The authors provided striking evidence that miRNAs could function as active signaling molecules to transport information across distinct species or even kingdoms. Although the mechanism of how miRNAs are shuttled between different organisms is still not well characterized, initial results point to the involvement of microvesicles and specific RNA-transporter-like proteins. These findings raise both speculation about the potential impact that plants may have on animal physiology at the molecular level, and an appealing possibility that food-derived miRNAs may offer us another means to deliver necessary nutrients or therapeutics to our bodies.