chuka_lis (chuka_lis) wrote,
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chuka_lis

МикроРНК как мишень для лечения при ковиде

Интересная статья, о том, что при ковиде, в крови и тканях появляется избыточное количество определенной микроРНК- miR-2392, в то время как 8 других  присутствуют в сниженных количествах, чем у здоровых. Такая под действием вируса возникает дисрегуляция в этой области. МикроРНК могут выполнять разные регуляторные функции, и  влиять на  работу  разных типов РНК,  ДНК и белков. Избыточная miR-2392 а клеточной модели показала картину патологии, схожую с нарушениями, которые наблюдаются в организме при ковиде - стимуляция воспаления, снижение работы митохондрий, окислительный стресс, метаболические нарушения, все вместе приводящее в проблемам в сердце, почках, иммунной системе (кроме легких) итд. И даже предсказала некоторые симптомы-синдромы-последствия, которые только иногда случаются при ковиде, но выразительные по неприятности. Так же, согласно моделям, особенно велико влияние дисрегуляции miR-2392 у тех, кто  находится в группе риска ( гипертензия, диабет, рак, возраст, этс). Они собственно и являются "группой риска" тяжелого ковида. Авторы предложили вещество, которое могло бы (в теории) отрегулировать miR-2392, и оно показало себя неплохо на клеточной модели; но  в экспперименте на хомячках результат оставляет желать лучшего. Тем не менее, в этом направлении стоит продолжать работу- и теоретические расчеты на моделях позволили выявить  десятки веществ- которые имело бы смысл проверить. Часть из них уже применяется (дексаметазон) или проходит испытания  (клинические).
Так же изболие этой miR-2392 может использоваться и как маркер диагностики системного ковида и его последствий.
Попутно, кстати, там обговаривается защитная роль Н-ацетилцистеина. В общем,  полезно прочесть, выглядит убедительно, хотя, не уверена, что  можно будет что-то быстро найти, чтобы "отладить" ответ организма по микроРНК - уж слишком широко эта микроРНК может прикладываться, много мишеней. 
Выдержки:

One potential avenue for an alternative antiviral agent is treatment against specific microRNAs (miRNAs) associated with SARS-CoV-2 infection and subsequent manifestation of COVID-19. MicroRNAs (miRNAs) are non-coding RNAs that are involved with regulation of post-transcriptional gene expression and can impact entire pathways related to viruses and diseases (Jiang et al., 2009; Trobaugh and Klimstra, 2017). Each miRNA can target multiple messenger RNAs (mRNAs) and taken together, miRNAs are predicted to regulate over half of the human transcriptome (Friedman et al., 2009). Recent evidence has shown different diseases, including COVID-19, leads to distinct complements of miRNAs in the blood (Mishra et al., 2020; Nersisyan et al., 2020; Portincasa et al., 2020; Sacar Demirci and Adan, 2020; Sardar et al., 2020; Teodori et al., 2020; Widiasta et al., 2020; Zhang et al., 2021). These circulating miRNAs are highly stable and have the potential to be used for minimally invasive novel detection, potential biomarkers, and therapeutic targets (Tribolet et al., 2020). Research on the interactions between miRNAs and viruses have revealed a multifaceted relationship. Specifically, viruses have been shown to avoid the immune response by leveraging cellular miRNAs to complete their replication cycle (Trobaugh and Klimstra, 2017). The following mechanisms are central to the interaction of viruses and miRNAs: 1) miRNA processing pathways can be blocked or inhibited by viruses interacting with key proteins such as Dicer and associated proteins, 2) viruses can sequester miRNAs resulting in dysregulation of specific target mRNAs, 3) viruses can utilize miRNAs to redirect regulatory pathways of other miRNA targets to provide survival advantages, and 4) viruses can directly encode miRNA precursors that are processed by the canonical miRNA cellular pathway and have well-defined functions to specifically target and regulate the viral replicative cycle (Schult et al., 2018; Trobaugh and Klimstra, 2017).
Here, we report on a miRNA, miR-2392, that may directly regulate and drive a COVID-19 response. This miRNA was initially predicted from COVID-19 patient data that consisted of multiple miRNAs being suppressed/inhibited (miR-10, miR-10a-5p, miR-1-3p, miR-34a-5, miR-30c-5p, miR-29b-3p, miR-155-5p, and miR-124-3p) and one miRNA being upregulated (miR-2392). With further examination, we discovered miR-2392 to be a key miRNA involved with COVID-19 progression. Specifically, miR-2392 drives downstream suppression of mitochondria activity while increasing inflammation, glycolysis, and hypoxia. MiR-2392 upregulation was concomitant with symptoms associated with COVID-19 infection in the host. We found that miR-2392 was circulating in COVID-19 infected patients and increased as a function of viral load. Our results demonstrate that miR-2392 may be utilized as an effective biomarker of COVID-19. Furthermore, we have developed a miR-2392 inhibitor and provide evidence that its use reduces SARS-COV-2 viability in targeted viral screens with A549 cells and reduces the impact of infection in COVID-19 animal models. With further development this miR-2392 inhibitor may represent an effective antiviral therapeutic towards inhibiting the virus and limiting a negative host response from COVID-19.
Eight miRNAs were predicted to drive significant changes in COVID-19 positive patients with the downregulation of seven miRNAs (miR-10, miR-1, miR-34a-5p, miR-30c-5p, miR-29b-3p, miR-124-3p, and miR-155-5p) and upregulation of a single miRNA, miR-2392 (Fig. 1A). Using IPA’s downstream effects analysis to predict biological processes from the combined suppression of the seven miRNAs and the upregulation of miR-2392 resulted in increased inflammation, immune suppression, and suppression of mitochondrial activity in the BALF dataset
hough limited, the existing literature on miR-2392 demonstrates it is related to mitochondrial suppression and increased glycolysis (Fan et al., 2019) and circulating factors related to negative health risks (Chen et al., 2013; Fan et al., 2019; Hou et al., 2019; Li et al., 2017; Park et al., 2014; Yang et al., 2019).
Pathway analysis was performed with targets and pathways for miR-2392 to determine its impact on the host when upregulated. We observed that the upregulation of miR-2392 in the RNA-seq dataset impacted many downstream targets and pathways related to negative health outcomes (Fig. 1C). In addition to mitochondrial suppression, we also predicted activation of factors related to reactive oxygen species (ROS). Alternatively, since it is known that miR-2392 directly interacts with the mitochondrial DNA (mtDNA) to inhibit the levels of many of the mtDNA coded oxidative phosphorylation transcripts, this could be a compensatory response to the inhibition of mitochondrial bioenergetics.Glycolytic pathways are also upregulated in association with increased miR-2392. MiR-2392 drives both hexokinase 2 (HK2) and pyruvate kinase (PKM) which are both positive regulators of glycolysis. HK2 phosphorylates glucose to produce glucose-6-phosphate and is a primary regulator of glycolysis. HK2 further enhances GDP-glucose biosynthesis. GDP-glucose is a nucleotide sugar which an essential substrate for all glycosylation reactions (i.e. glycosylation of viral spike proteins). Pyruvate kinase is essential for the production of ATP in glycolysis as this enzyme catalyzes the transfer of the phosphate group from phosphoenolpyruvate to ADP to make ATP. The mechanism of how miR-2392 is driving these pathways is not clearly understood, but one possibility could be due to the stabilization of glycolytic transcripts. Overall, the miR-2392 observed upregulation of glycolysis and antiviral effects related to miR-2392 suppression are consistent with the recently documented role of glucose metabolism in the progression of viral infection and poor outcome of COVID-19 (Ardestani and Azizi, 2021). It is also consistent with the reported effects of suppression of glycolysis by inhibitors like the glucose analog, 2-deoxy-D-glucose (2-DG), that was shown to suppress SARS-CoV-2 replication in in vitro models (Ardestani and Azizi, 2021; Bojkova et al., 2020; Codo et al., 2020).Targets related to the goals of antioxidant N-acetyl cysteine (NAC) therapy are also observed to be upregulated. These include activated endothelial cell increasing their expression of numerous adhesion molecules, including intercellular adhesion molecule 1 (ICAM1), vascular cell adhesion molecule 1 (VCAM1), and E-selectin, which allow attachment of hematopoietic immune and non-immune cells to the endothelial surface, and thus, contribute to inflammation and activation of the coagulation cascade. Powerful antioxidants such as NAC counteract COVID19 infections by potentially suppressing viral replication via improving intracellular thiol redox ratio as a precursor for major thiol antioxidant glutathione (Ho and Douglas, 1992) and inhibiting the NF-kB pathway (Poppe et al., 2017). Inhibition of the NF-κB pathway has been shown to reduce inflammatory damage by altering the glutathione and glutathione disulfide ratio (Aykin-Burns et al., 2005; Griffin et al., 2003; Jia et al., 2010). Because NAC can also modulate oxidative burst and reduce cytokine storm without weakening the phagocytizing function of neutrophils (Allegra et al., 2002; Kharazmi et al., 1988; Sadowska et al., 2006), its use in COVID-19 patients as a single agent or in combination with other antioxidants are being conducted in clinical trials (Alamdari et al., 2020). A recent study has shown noteworthy benefits of NAC in patients with severe COVID-19 infection (Ibrahim et al., 2020). Major mechanisms proposed for these favorable patient outcomes were NAC’s ability to reduce IL-6 induced mitochondrial oxidative stress via Complex I inhibition as well as to prevent increased inflammation due to uncontrolled activation of mTORC1. These results were in line with the role of miR-2392 in reducing the activities of electron transport chain complexes and enhancing glycolysis, which is known to be induced by mTORC1 activation. The same study also speculated that NAC could inhibit SARS-CoV-2 binding to ACE2 by reducing disulfide bonds in its receptor-binding domain. Inflammatory pathways and others that are observed with COVID-19 infection were also seen to be activated downstream of miR-2392.
Viral miRNAs can play a role in interspecies transmission due to the high conservation of miRNAs among species and the ability of viruses to integrate miRNAs into its own genome (Sacar Demirci and Adan, 2020; Schult et al., 2018). In addition, such integration of miRNAs within the virus has been shown to assist viruses to replicate and evade the immune system (Islam and Islam, 2021). To determine if miR-2392 might be capable of driving the observed COVID-19 health risks and symptoms in the host, we analyzed the conservation of human miR-2392 across species and the integration of miR-2392 into the SARS-CoV-2 genome
A base wise evolutionary conservation comparison demonstrated that miR-2392 is highly conserved among non-human primates. In addition, conservation of miR-2392 is evident in dogs, cats, and ferrets, species known to be infected with SARS-CoV-2 while mice and rats, species not impacted by COVID-19 (Johansen et al., 2020), have poor conservation with miR-2392.
...miR-2392 does not seem to significantly affect normal tissues.
We found that the miR-2392 seeding region is heavily integrated within SARS-CoV-2 and conserved in different viral strains. The three best hits from the miRanda scores are located in the NSP2, NSP3, and E-genes. Notably, these regions were conserved among 6 variants and lineages of concern each represented by 14 recent genomes from the respective lineage available from the Global Initiative on Sharing All Influenza Data (GISAID, (Shu and McCauley, 2017)).
The majority of these upregulated miR-2392 targets are involved in immune and inflammatory pathways. The downregulated miR-2392 targets were involved in mitochondrial function, oxidative stress, cell cycle, developmental biology, and ubiquitin binding which are pathways recently associated with the SARS-CoV-2 infection process (Hemmat et al., 2021). This data demonstrates miR-2392 may target several gene pathways related to perpetuating SARS-CoV-2 infection. For the majority of the tissues (excluding the lymph nodes), higher viral loads are associated with greater miR-2392 gene targets being regulated. Interestingly, the lymph nodes show an inverse relationship with viral loads compared to other tissues.
Because miR-2392 was recently shown to directly target the transcription of mitochondrial DNA genes (Fan et al., 2019), we evaluated the impact on expression of the mitochondrial miR-2392 targets in our datasets.
Hence, SARS-CoV-2 seems to downregulate nuclear mitochondrial gene transcription in the more oxidative heart and kidney, as well as in nasal tissues.Since inflammation is a key component of COVID-19 infection, we also overlaid the standard known inflammatory genes determined from Loza et al. (Loza et al., 2007) to the miR-2392 targets \The analysis reveals that, at the mRNA level, most of the complement pathway genes are upregulated in the tissue samples analyzed. These changes could be compensatory, as proteins encoded by the genes could be downregulated as a function of traditional miRNA effects. The responses reflect the importance of degrees of inflammation for mediation of disease severity in COVID-19 patients and a key modulatory role of miR-2392 in this context.
Proteomic and transcriptomic analysis on miR-2392 targets on blood from COVID patients utilizing COVIDome (Sullivan et al., 2021) revealed interesting patterns between RNA and protein levels for miR-2392 targets
In the blood, the miR-2392 targets CXCL10, STAT1, IFIT3, and C1QC were positively regulated at both the protein and gene levels. This upregulation was also observed in all other tissues
Overexpression of miR-2392 simulates a phenotype similar to COVID-19 infection
we found miR-2392 overexpression impacted genes involved with mitochondria, and inflammation
To determine if there was a direct correlation between miR-2392 overexpression and SARS-CoV-2 infection, comparisons were made using gene expression fold-change values or overlap in statically significant curated gene sets from canonical pathways determined by our fGSEA analysis.
Using previously published data from Blanco-Melo et al. (Blanco-Melo et al., 2020), showed there was a statistically significant and positive correlation of the miR-2392 treatment compared to both the A549 and Calu-3 cell culture models infected with SARS-CoV-2as well as in lung biopsies post-mortem from two COVID-19 positive patients . Using nasal swab samples, a significant and positive correlation was determined between patients with medium- and low-viral loads compared to non-infected patients. Further identification of miR-2392 correlation to SARS-CoV-2 infections was made using RNA-seq from multiple tissues (heart, kidney, liver, lymph node, and lung) obtained during autopsies of COVID-19 patients with high or low viral loads . There was a positive correlation to lung and lymph node tissues with miR-2392 expression.
It was observed that the miR-2392 treatment induced pathway response that was significantly related to SARS-CoV-2 pathways. One obvious relationship shows that the Reactome SARS-CoV-2 pathways were significantly activated for the miR-2392 treated cells compared to the controls
We observed a statistically significant increase of miR-2392 in COVID-19 positive patients from both the serum and urine samples  In addition, Receiver Operating Characteristic (ROC) curve analysis revealed that miR-2392 is significantly associated with SARS-CoV-2 infection in patients in all tissues. Lastly, when dissecting the amounts of miR-2392 with specific conditions associated with infection, we observe that more severely affected patients (i.e. intubated patients or patients in ICU), had higher presence of miR-2392
Since we hypothesize that miR-2392 is a primary initiator for systemic impact of the infection, this might indicate that miR-2392 does not strongly appear until the virus has established its presence in the body.
The link that we found between miR-2392 and COVID-19 infection prompted us to ask whether we could develop effective antivirals for COVID-19 by inhibiting miR-2392. We used the Facile Accelerated Specific Therapeutic (FAST) platform to develop an effective antisense-based therapeutic against human miR-2392 (Aunins et al., 2020; Eller et al., 2021), termed SBCov207, for the treatment of COVID-19
The anti-miR-2392 FASTmer was evaluated for efficacy and toxicity against a SARS-CoV-2 infection of the human lung cell line A549
Treatment of uninfected A549 cells showed no cytotoxicity up to 20 µM. The control nonsense FASTmer (SBCoV208) showed no toxicity even up to 40 µM. Treatment of A549 cells infected with SARS-CoV2 showed drastic improvement in cell viability with an average of 85% viral inhibition at 10 µM (IC50 of 1.15 ± 0.33 µM). In contrast, the control nonsense FASTmer showed significantly lower viral suppression
Human cell line-based infection models reaffirm that the anti-miR-2392 (SBCov207) is effective in inhibiting SARS-CoV2, while not exhibiting toxicity at the concentrations tested.
In a separate in vivo model, the anti-miR-2392 FASTmer was evaluated in a Syrian hamster infection model
The anti-miR-2392 FASTmer treatment was given by intraperitoneal (IP) injection or intranasal (IN) instillation 24 hours before viral inoculation or both 24 hours before and 24 hours after viral inoculation. Each FASTmer dose was at a concentration of 10 µM in a 100 µL volume (approximately 0.13 mg/kg). Правда, особых отличий с контролем вырявить не удалось- ни в кратине болезни, и поражениях тканей, ни в вирусно репкликации.
To predict whether miR-2392 might have a direct relationship to COVID-19 symptoms in the host, we determined the pathway and disease relevance of miR-2392
mong the diseases predicted to be associated with miR-2392 were a surprising number of clinical observations present in individuals with COVID-19 infection (Fig. 7A). These include heart or cardiovascular disease and failure, both known to heavily contribute to morbidity and mortality in patients with COVID-19 (Nishiga et al., 2020), hyperesthesia (Krajewski et al., 2021), as well as less common COVID-19 symptoms, such as lymphadenopathy and pharyngitis related to sore throat (Edmonds et al., 2021; Walsh-Messinger et al., 2020), liver dysfunction (Portincasa et al., 2020), splenomegaly (Malik et al., 2020), CNS (Mahajan and Mason, 2021; Rodriguez et al., 2020) and kidney failure (Hultstrom et al., 2021).
It is interesting to note that miR-2392 was also predicted to affect diseases that appeared not to be associated with COVID-19 infection, but literature searches reveal these pathologies do occur in some COVID-19 patients. For example, azoospermia, which is linked to male infertility, has been shown to occur in some male patients (Younis et al., 2020). The menstrual cycle in females have been reported to be deregulated for months after COVID-19 infection (Li et al., 2021). Association with dental damage has also been observed in COVID-19 patients (Sirin and Ozcelik, 2021), also deafness or hearing loss (Koumpa et al., 2020). We used the tool Kaplan-Meier Plotter (Nagy et al., 2018) to associate miR-2392 expression with pan-cancer patient survival . We observed that the high expression of miR-2392 is generally related to poor prognosis with the majority of cancer types (p-value < 0.05). If miR-2392 is associated with COVID-19, as we are hypothesizing, and is persistent after the virus clears the host, then the implications for the potential long-term impact on the millions of people infected with COVID-19 could be devastating. Intriguingly, one of the miR-2392 predicted consequences in the immune category is decreased antibody levels in the blood; this might account for the reported loss of the antibodies overtime (Gudbjartsson et al., 2020; Self et al., 2020).
Using computational prediction models, we also predicted small molecules, including FDA-approved drugs that could inhibit miR-2392 from two different approaches. The first approach employed a state-of-the-art machine learning method that we recently developed for predicting missing drug targets (Galeano et al., 2021).
A list of the top-20 predicted small molecules for miR-2392 (Fig. 7B) includes Dexamethasone, the first drug known to save lives in critically ill COVID-19 patients (Ledford, 2020), and Atorvastatin, that has shown similar protective role in COVID-19 patients (Rossi et al., 2020). The second approach follows ideas first presented in Sirota et al. (Sirota et al., 2011)and consists on analyzing the genomic signature of miR-2392 (i.e. significant up and down-regulated genes) and predicting small molecules that can reverse it.
The top-20 small molecules predicted by our approach (Sirota et al., 2011) includes the androgen receptor antagonist Enzalutamide and the insulin sensitizer Pioglitazone (Carboni et al., 2020) both of which are in clinical trials for COVID-19; Clinical Trial #NCT04475601 and NCT04604223). We also found literature evidence for the leukotriene inhibitor ubenimex (Asai et al., 2020), and the bacterial DNA inhibitor metronidazole (Gharebaghi et al., 2020).
(табличка)


Tags: биология, коронавирус, лечение, статьи
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