Triazolo[4,5-d]pyrimidin-5-amines based ERK3 inhibitors fail to demonstrate selective effects on adipocyte function.

Extracellular signal-regulated kinase 3 (ERK3 also designated MAPK6 - mitogen-activated protein kinase 6) is a ubiquitously expressed kinase participating in the regulation of a broad spectrum of physiological and pathological processes. Targeted inhibition of the kinase may allow the development of novel treatment strategies for a variety of types of cancer and somatic pathologies, as well as preserving metabolic health, combat obesity and diabetes. We chose and synthesized three triazolo [4,5-d]pyrimidin-5-amines proposed previously as putative ERK3 inhibitors to assess their selectivity and biological effects in terms of metabolic state impact in living cells. As it was previously shown that ERK3 is a major regulator of lipolysis in adipocytes, we focused on this process. Our new results indicate that in addition to the previously identified lipolytic enzyme ATGL, ERK3 also regulates hormone-sensitive lipase (HSL) and monoglyceride lipase (MGL). Moreover, this kinase also promotes the abundance of fatty acid synthase (FASN) as well as protein kinase cAMP-activated catalytic subunit alpha (PKACα). To investigate various effects of putative ERK3 inhibitors on lipolysis, we utilized different adipocyte models. We demonstrated that molecules exhibit lipolysis-modulating effects; however, the effects of triazolo [4,5-d]pyrimidin-5-amines based inhibitors on lipolysis are not dependent on ERK3. Subsequently, we revealed a wide range of the compounds' possible targets using a machine learning-based prediction. Therefore, the tested compounds inhibit ERK3 in vitro, but the biological effect of this inhibition is significantly overlapped and modified by some other molecular events related to the non-selective binding to other targets.

miR-144 affects the immune response and activation of inflammatory responses in Cynoglossus semilaevis by regulating the expression of CsMAPK6.

MicroRNAs are increasingly recognized for their pivotal role in the immune system, yet the specific regulatory functions of fish-derived microRNAs remain largely unexplored. In this research, we discovered a novel miRNA, Cse-miR-144, in the Chinese tongue sole (Cynoglossus semilaevis), characterized by a 73-base pair precursor and a 21-nucleotide mature sequence. Our findings revealed that the expression of Cse-miR-144 was notably inhibited by various Vibrio species. Utilizing bioinformatics and dual-luciferase assay techniques, we established that the pro-inflammatory cytokine gene CsMAPK6 is a direct target of Cse-miR-144. Subsequent in vitro and in vivo western blotting analyses confirmed that Cse-miR-144 can effectively reduce the protein levels of CsMAPK6 post-transcriptionally. Moreover, CsMAPK6 is known to be involved in the activation of the Nuclear Factor kappa-light-chain-enhancer of activated B cells (NF-kB). Additional investigations using qPCR and ELISA demonstrated that suppression of Cse-miR-144 leads to an upsurge in the liver mRNA levels of various immune genes (including MYD88, TRAF6, NF-κB, TRAF2, TRAF3, and TNF), alongside a marked increase in the production and secretion of pro-inflammatory cytokines (IL-1ß, IL-6, and IL-8) in the bloodstream of C. semilaevis. These findings collectively underscore the potential of Cse-miR-144 as a key inhibitor of CsMAPK and its crucial role in modulating the immune and inflammatory responses in teleost fish. Compared to the siRNA, miRNA is a better tool in controlling the expression of target gene with a lower cost.

Long non-coding RNA DANCR increases spinal cord neuron apoptosis and inflammation of spinal cord injury by mediating the microRNA-146a-5p/MAPK6 axis.

OBJECTIVE: This research was to unravel the impact of the lncRNA differentiation antagonizing non-protein coding RNA (DANCR)/microRNA (miR)-146a-5p/mitogen-activated protein kinase 6 (MAPK6) axis on spinal cord injury (SCI). METHODS: SCI mouse models were established and injected with si-DANCR or miR-146a-5p agomir. The recovery of motor function was assessed by Basso Mouse Scale. SCI was pathologically evaluated, and serum inflammatory factors were measured in SCI mice. Mouse spinal cord neurons were injured by H2O2 and transfected, followed by assessment of proliferation and apoptosis. DANCR, miR-146a-5p, and MAPK6 in tissues and cells were detected, as well as their relationship. RESULTS: DANCR increased and miR-146a-5p decreased in SCI. Silencing DANCR or enhancing miR-146a-5p stimulated the proliferation of mouse spinal cord neurons and reduced apoptosis. DANCR was bound to miR-146a-5p to target MAPK6. DANCR affected the proliferation and apoptosis of spinal cord neurons by mediating the miR-146a-5p/MAPK6 axis. Downregulating DANCR or upregulating miR-146a-5p improved inflammation, the destruction of spinal cord tissue structure, and apoptosis in SCI mice. CONCLUSION: DANCR affects spinal cord neuron apoptosis and inflammation of SCI by mediating the miR-146a-5p/MAPK6 axis.

CircDNAJC11 interacts with TAF15 to promote breast cancer progression via enhancing MAPK6 expression and activating the MAPK signaling pathway.

BACKGROUND: Breast cancer (BC) is a common malignant tumor in women worldwide. Circular RNA (circRNA) has been proven to play a critical role in BC progression. However, the exact biological functions and underlying mechanisms of circRNAs in BC remain largely unknown. METHODS: Here, we first screened for differentially expressed circRNAs in 4 pairs of BC tissues and adjacent non-tumor tissues using a circRNA microarray. Functionally, gain- and loss-of-function experiments in vitro and in vivo showed that circDNAJC11 promoted BC cell proliferation, migration, invasion, and tumor growth. Mechanistically, RNA pull-down, mass spectrum, RNA immunoprecipitation, fluorescence in situ hybridization assays, and rescue experiments were executed. RESULTS: We found that circDNAJC11 was significantly upregulated in triple-negative breast cancer tissues and cells. Clinical data revealed that the high expression of circDNAJC11 was closely correlated with a poor prognosis of BC patients and could be an independent risk factor for BC prognosis. Functionally, gain- and loss-of-function experiments in vitro and in vivo showed that circDNAJC11 promoted BC cell proliferation, migration, invasion, and tumor growth. Mechanistically, RNA pull-down, mass spectrum, RNA immunoprecipitation, fluorescence in situ hybridization assays, and rescue experiments were executed. We demonstrated that circDNAJC11 combined with TAF15 to promote BC progression via stabilizing MAPK6 mRNA and activating the MAPK signaling pathway. CONCLUSIONS: The circDNAJC11/TAF15/MAPK6 axis played a crucial role in the progression and development of BC, suggesting that circDNAJC11 might be a novel biomarker and therapeutical target for BC.

Targeting ERK3/MK5 complex for treatment of obesity and diabetes.

Kinases represent one of the largest druggable families of proteins. Importantly, many kinases are aberrantly activated/de-activated in multiple organs during obesity, which contributes to the development of diabetes and associated diseases. Previous results indicate that the complex between Extracellular-regulated kinase 3 (ERK3) and Mitogen-Activated Protein Kinase (MAPK)-activated protein kinase 5 (MK5) suppresses energy dissipation and promotes fatty acids (FAs) output in adipose tissue and, therefore promotes obesity and diabetes. However, the therapeutic potential of targeting this complex at the systemic level has not been fully explored. Here we applied a translational approach to target the ERK3/MK5 complex in mice. Importantly, deletion of ERK3 in the whole body or administration of MK5-specific inhibitor protects against obesity and promotes insulin sensitivity. Finally, we show that the expression of ERK3 and MK5 correlates with the degree of obesity and that ERK3/MK5 complex regulates energy dissipation in human adipocytes. Altogether, we demonstrate that ERK3/MK5 complex can be targeted in vivo to preserve metabolic health and combat obesity and diabetes.

Long noncoding RNA NEAT1 sponges miR-495-3p to enhance myocardial ischemia-reperfusion injury via MAPK6 activation.

The biological function of long noncoding RNA NEAT1 has been revealed in a lot of diseases. Nevertheless, it is still not yet clear whether NEAT1 can modulate the process of myocardial ischemia-reperfusion injury (M-I/R). Here, we reported that NEAT1 was able to sponge miR-495-3p to contribute to M-I/R injury through activating mitogen-activated protein kinase 6 (MAPK6). First, elevated expression of NEAT1 was revealed in M-I/R injury mice, meanwhile, lactate dehydrogenase (LDH) and creatine kinase-muscle/brain (CK-MB) were also upregulated in the serum. Meanwhile, as previously reported, miR-495 serves as a tumor suppressor or an oncogenic miRNA in different types of cancer. Currently, we found miR-495-3p was remarkably reduced in M-I/R mice. Additionally, NEAT1 was significantly induced whereas miR-495-3p was greatly reduced by H2 O2 treatment in H9C2 cells. Moreover, loss of NEAT1 in H9C2 cells could repress the viability and proliferation of cells. For another, overexpression of NEAT1 exhibited an opposite phenomenon. Furthermore, LDH release and caspase-3 activity were obviously triggered by upregulation of NEAT1 while suppressed by NEAT1 knockdown. miR-495-3p was indicated and validated as a target of NEAT1 using the analysis of bioinformatics. Interestingly, we observed that miR-495-3p mimics repressed tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), and IL-18 protein expression while their levels were enhanced by the inhibition of miR-495-3p in H9c2 cells. Subsequently, it was manifested that MAPK6 was a target of miR-495-3p, which could exert a lot in the NEAT1/miR-495-3p-mediated M-I/R injury. Overall, our results implied that NEAT1 contributed to M-I/R injury via the modulation of miR-495-3p and MAPK6.

Crystal Structure and Inhibitor Identifications Reveal Targeting Opportunity for the Atypical MAPK Kinase ERK3.

Extracellular signal-regulated kinase 3 (ERK3), known also as mitogen-activated protein kinase 6 (MAPK6), is an atypical member of MAPK kinase family, which has been poorly studied. Little is known regarding its function in biological processes, yet this atypical kinase has been suggested to play important roles in the migration and invasiveness of certain cancers. The lack of tools, such as a selective inhibitor, hampers the study of ERK3 biology. Here, we report the crystal structure of the kinase domain of this atypical MAPK kinase, providing molecular insights into its distinct ATP binding pocket compared to the classical MAPK ERK2, explaining differences in their inhibitor binding properties. Medium-scale small molecule screening identified a number of inhibitors, several of which unexpectedly exhibited remarkably high inhibitory potencies. The crystal structure of CLK1 in complex with CAF052, one of the most potent inhibitors identified for ERK3, revealed typical type-I binding mode of the inhibitor, which by structural comparison could likely be maintained in ERK3. Together with the presented structural insights, these diverse chemical scaffolds displaying both reversible and irreversible modes of action, will serve as a starting point for the development of selective inhibitors for ERK3, which will be beneficial for elucidating the important functions of this understudied kinase.

The C-Terminus Tail Regulates ERK3 Kinase Activity and Its Ability in Promoting Cancer Cell Migration and Invasion.

Extracellular signal-regulated kinase 3 (ERK3) is an atypical member of the mitogen-activated protein kinase (MAPK) family. It harbors a kinase domain in the N-terminus and a long C-terminus extension. The C-terminus extension comprises a conserved in ERK3 and ERK4 (C34) region and a unique C-terminus tail, which was shown to be required for the interaction of ERK3 with the cytoskeletal protein septin 7. Recent studies have elucidated the role of ERK3 signaling in promoting the motility and invasiveness of cancer cells. However, little is known about the intramolecular regulation of the enzymatic activity and cellular functions of ERK3. In this study, we investigated the role of the elongated C-terminus extension in regulating ERK3 kinase activity and its ability to promote cancer cell migration and invasion. Our study revealed that the deletion of the C-terminus tail greatly diminishes the ability of ERK3 to promote the migration and invasion of lung cancer cells. We identified two molecular mechanisms underlying this effect. Firstly, the deletion of the C-terminus tail decreases the kinase activity of ERK3 towards substrates, including the oncogenic protein steroid receptor co-activator 3 (SRC-3), an important downstream target for ERK3 signaling in cancer. Secondly, in line with the previous finding that the C-terminus tail mediates the interaction of ERK3 with septin 7, we found that the depletion of septin 7 abolished the ability of ERK3 to promote migration, indicating that septin 7 acts as a downstream effector for ERK3-induced cancer cell migration. Taken together, the findings of this study advance our understanding of the molecular regulation of ERK3 signaling by unraveling the role of the C-terminus tail in regulating ERK3 kinase activity and functions in cancer cells. These findings provide useful insights for the development of therapeutic agents targeting ERK3 signaling in cancer.

Mitogen-activated protein kinase 6 integrates phosphate and iron responses for indeterminate root growth in Arabidopsis thaliana.

MAIN CONCLUSION: A MAPK module, of which MPK6 kinase is an important component, is involved in the coordination of the responses to Pi and Fe in the primary root meristem of Arabidopsis thaliana. Phosphate (Pi) deficiency induces determinate primary root growth in Arabidopsis through cessation of cell division in the meristem, which is linked to an increased iron (Fe) accumulation. Here, we show that Mitogen-Activated Protein Kinase6 (MPK6) has a role in Arabidopsis primary root growth under low Pi stress. MPK6 activity is induced in roots in response to low Pi, and such induction is enhanced by Fe supplementation, suggesting an MPK6 role in coordinating Pi/Fe balance in mediating root growth. The differentiation of the root meristem induced by low Pi levels correlates with altered expression of auxin-inducible genes and auxin transporter levels via MPK6. Our results indicate a critical role of the MPK6 kinase in coordinating meristem cell activity to Pi and Fe availability for proper primary root growth.

The circ_0003928/miR-31-5p/MAPK6 cascade affects high glucose-induced inflammatory response, fibrosis and oxidative stress in HK-2 cells.

BACKGROUND: Diabetic nephropathy (DN) is a severe diabetic complication disorder. Circular RNAs (circRNAs) actively participate in DN pathogenesis. In this report, we sought to define a new mechanism of circ_0003928 in regulating high glucose (HG)-induced HK-2 cells. METHODS: To construct a DN cell model, we treated HK-2 cells with HG. Cell viability and apoptosis were detected by CCK-8 and flow cytometry, respectively. The inflammatory cytokines were quantified by ELISA. Protein analysis was performed by immunoblotting, and mRNA expression was detected by quantitative PCR. The circ_0003928/miR-31-5p and miR-31-5p/MAPK6 relationships were validated by RNA pull-down and luciferase assays. RESULTS: HG promoted HK-2 cell apoptosis, fibrosis and oxidative stress. Circ_0003928 and MAPK6 levels were enhanced and miR-31-5p level was decreased in HK-2 cells after HG treatment. Circ_0003928 disruption promoted cell growth and inhibited apoptosis, inflammatory response, fibrosis and oxidative stress in HG-induced HK-2 cells. Circ_0003928 targeted miR-31-5p, and MAPK6 was a target of miR-31-5p. Circ_0003928 regulated MAPK6 expression through miR-31-5p. The functions of circ_0003928 disruption in HG-induced HK-2 cells were reversed by miR-31-5p downregulation or MAPK6 upregulation. CONCLUSION: Circ_0003928 exerts regulatory impacts on HG-induced apoptosis, inflammation, fibrosis and oxidative stress in human HK-2 cells by the miR-31-5p/MAPK6 axis.

Increased methylation of ZNF671 suppresses tumor progression by promoting MAPK6 transcription in laryngeal carcinoma.

Background: Laryngeal squamous cell carcinoma (LSCC) is a malignant tumor of the head and neck, the exact mechanism of which has not been explored. Methods: By analyzing the GEO data, we found the highly methylated and low expression gene ZNF671. The expression level of ZNF671 in clinical samples was verified by RT-PCR, western blotting and methylation-specific PCR. The function of ZNF671 in LSCC was detected by cell culture and transfection, MTT, Edu, TUNEL assays and flow cytometry analysis. The binding sites of ZNF671 to MAPK6 promoter region were detected and verified by luciferase reporter gene and chromatin immunoprecipitation. Finally, the effect of ZNF671 on LSCC tumors was tested in vivo. Results: In this study, by analyzing GEO data GSE178218 and GSE59102, we found that zinc finger protein (ZNF671) expression was decreased, and DNA methylation level was increased in laryngeal cancer. Moreover, the abnormal expression of ZNF671 was associated with poor survival prognosis of patients. In addition, we found that overexpression of ZNF671 could inhibit the viability, proliferation, migration and invasion of LSCC cells, while promoting cell apoptosis. In contrast, the opposite effects were observed after knockdown of ZNF671. Through the prediction website and chromatin immunoprecipitation and luciferase reporter experiments, it was found that ZNF671 could bind to the promoter region of MAPK6, thereby inhibiting the expression of MPAK6. In vivo experiments confirmed that overexpression of ZNF671 could inhibit tumor growth. Conclusion: Our study found that ZNF671 expression was down-regulated in LSCC. ZNF671 up-regulates the expression of MAPK6 by binding to its promoter region, thus participating in cell proliferation, migration and invasion in LSCC. Our study may provide new ideas for early prediction and treatment of LSCC.

CircGAB1 Facilitates Podocyte Injury Through Sponging miR-346 and Activating MAPK6 in Diabetic Nephropathy.

BACKGROUND: Podocyte injury is very important process in diabetic nephropathy (DN) progression. Circular RNA (circRNA) takes part in regulating the advancement of DN. Herein, we explored the role and mechanism of circGAB1 in DN progression. METHODS: The abundances of circGAB1, microRNA-346 (miR-346) and mitogen-activated protein kinase 6 (MAPK6) were detected by qRT-PCR in DN serum samples and podocyte HGPC. Moreover, cell viability and apoptosis were determined using CCK8 assay and flow cytometry. Also, the protein levels of MAPK6, proliferation-related markers and apoptosis-related markers were analyzed by western blot. ELISA assay was used to measure the levels of inflammatory factors, and corresponding kits were used to detect the levels of oxidative stress-related markers. The relationship between miR-346 and circGAB1 or MAPK6 was distinguished by dual-luciferase reporter assay. RESULTS: CircGAB1 expression was increased in DN serum samples and HG-treated HGPC cells. CircGAB1 knockdown inhibited HG-induced apoptosis, inflammatory response and oxidative stress in HGPC cells. In terms of mechanism, circGAB1 sponged miR-346, and miR-346 targeted MAPK6. The inhibition effect of circGAB1 knockdown on HG-induced podocyte injury could be reversed by miR-346 inhibitor. Moreover, miR-346 overexpression repressed HG-induced podocyte injury by targeting MAPK6. CircGAB1 served as miR-346 sponge to positively regulate MAPK6. CONCLUSION: CircGAB1 contributed to podocyte injury through mediating miR-346/MAPK6 axis, suggesting that circGAB1 might promote DN progression.

HNF4G increases cisplatin resistance in lung adenocarcinoma via the MAPK6/Akt pathway.

Background: Lung adenocarcinoma is one of the most common tumors, and cisplatin is frequently used in treating lung adenocarcinoma patients. This study aimed to look into the roles and mechanisms of HNF4G in cisplatin resistance of lung adenocarcinoma. Materials & Methods: Cisplatin resistance and gene expression data of 542 cell lines from the CTRP and CCLE databases were analyzed. HNF4G expression was detected in the lung adenocarcinoma cell lines after treatment with various concentrations of cisplatin. Cisplatin sensitivity curves were detected in cells that overexpressed or knocked down HNF4G. The ChIP-Seq data were then analyzed to identify the targets of HNF4G involved in cisplatin resistance. Expression and phosphorylation of the MAPK6/Akt pathway were detected after HNF4G was overexpressed or knocked down. Finally, ChIP-qPCR and dual-luciferase assays were used to investigate the regulation of HNF4G on MAPK6. Results: In cell lines, high expression of HNF4G was significantly positively correlated with cisplatin resistance, and lung adenocarcinoma patients who had high HNF4G expression had a poor prognosis. Cisplatin treatment increased HNF4G expression, and overexpression of HNF4G significantly increased the resistance to cisplatin in A549 and HCC827 cells, whereas knockdown of HNF4G had the opposite effect. HNF4G overexpression increased MAPK6 expression and activated the MAPK6/Akt pathway, while an Akt inhibitor reduced the effects of HNF4G on cisplatin resistance. HNF4G bound to the MAPK6 promoter region, promoting MAPK6 expression, according to ChIP-qPCR and luciferase assays. Conclusion: By binding to the MAPK6 promoter region, HNF4G promotes MAPK6 expression and subsequent Akt phosphorylation, resulting in resistance to cisplatin in lung adenocarcinoma.

Rab31 promotes the invasion and metastasis of cervical cancer cells by inhibiting MAPK6 degradation.

Persistent infection with high-risk human papillomavirus (HPV) is the main risk factor for cervical cancer. Our mass spectrometry data showed that the Ras-associated binding protein Rab31 was upregulated by HPV; however, little is known regarding the role of Rab31 in the metastasis of cervical cancer cells. In this study, we showed that Rab31 was highly expressed in cervical cancer tissues and cells, and both HPV E6 and E7 promoted the expression of Rab31. Rab31 knockdown inhibited while Rab31 overexpression promoted the migration and invasion capabilities of cervical cancer cells. Additionally, Rab31 knockdown inhibited the epithelial-mesenchymal transition (EMT) and cytoskeletal rearrangement in cervical cancer cells. Furthermore, Rab31 interacted with mitogen-activated protein kinase 6 (MAPK6), and Rab31 knockdown inhibited the expression of MAPK6, which was mainly localized in the cytoplasm. More importantly, Rab31 knockdown promoted and Rab31 overexpression inhibited MAPK6 degradation. Accordingly, MAPK6 overexpression restored the decreased migration potential caused by Rab31 knockdown. Finally, a xenograft mouse model showed that Rab31 knockdown in cervical cancer cells led to reduced tumor growth and impaired lung and liver metastasis in vivo. In conclusion, Rab31 plays a crucial role in cervical cancer metastasis by inhibiting MAPK6 degradation. Thus, Rab31 may serve as a novel therapeutic target to manage cervical cancer.

Docking simulation and ADMET prediction based investigation on the phytochemical constituents of Noni (Morinda citrifolia) fruit as a potential anticancer drug.

Morinda citrifolia is a traditional plant used in Asian and African countries for its wide nutraceutical and therapeutic effects for the treatment of various ailments. The fruit of M. citrifolia has various biological properties such as anti-bacterial, anti-oxidant, anti-cancer. Using the molecular docking based investigation; we explored around twenty three bioactive phytochemicals in M. citrifolia fruit against human cancer. MAPK6 (mitogen-activated protein kinase 6) was selected as target protein and these twenty three phytochemicals along with a known MAPK6 inhibitor were docked against the target protein. The docking scores of the bioactive phytochemicals against MAPK6 protein range between - 4.5 kcal/mol to - 7.9 kcal/mol and the docking score of the standard drug (CID: 447077) was - 7.3 kcal/mol. Based on the binding affinity five phytochemicals asperuloside (- 6.7 kcal/mol), asperulosidic acid (- 7.2 kcal/mol), deacetylasperulosidic acid (- 7.0 kcal/mol), eugenol (- 6.8 kcal/mol) and rutin (- 7.9 kcal/mol) were chosen for further evaluation. These five compounds were further investigated through RC plot analysis, density function theory and ADMET properties. Stable linkage of protein-ligand interaction was observed through RC plot, density function theory showed the structural stability and reactivity of bioactive compounds through the energy gap between HOMO and LUMO and the ADMET (adsorption, distribution, metabolism, excretion and toxicity) studies showed the safety profile of the bioactive compounds. These in silico results support the utilization of M. citrifolia fruit in the traditional medication and the initiation for the development of new drug against human cancer through in vivo and in vitro evaluation. Supplementary Information: The online version contains supplementary material available at 10.1007/s40203-022-00130-4.

Exosomes from miR-374a-5p-modified mesenchymal stem cells inhibit the progression of renal fibrosis by regulating MAPK6/MK5/YAP axis.

Chronic kidney disease (CKD) in clinical is defined as a gradual loss of kidney function for more than 3 months. The pathologic course of CKD is characterized by extensive renal fibrosis; thus, preventing renal fibrosis is vital for the treatment of CKD. It has been reported that microRNA (miR)-374a-5p was under-expressed in renal venous blood samples from patients with CKD. In addition, it exhibited anti-apoptotic effects in renal tissues suggesting that miR-374a-5p may play an important role in CKD. However, it is not clear whether miR-374a-5p could be delivered to renal cells by exosomes and exerts anti-renal fibrosis effects. To mimic renal fibrosis in vitro, human renal tubular epithelial cell lines (HK-2 cells) were treated by transforming growth factor-ß (TGF-ß) 1. Reverse transcription-quantitative polymerase-chain reaction (RT-qPCR) or Western blot was carried out to evaluate the mechanism by which miR-374a-5p regulated the development of renal fibrosis. Next, exosomes were isolated using with ultracentrifugation method, and the relationship between miR-374a-5p and MAPK6 was evaluated using dual-Luciferase a reporter assay system. The results indicated TGF-ß1 significantly down-regulated the expression of miR-374a-5p in HK-2 cells and miR-374a-5p agomir remarkably inhibited the progression of fibrosis in vitro. In addition, exosomal miR-374a-5p could be internalized by HK-2 cells and obviously enhanced the level of miR-374a-5p in HK-2 cells. Furthermore, exosomal miR-374a-5p prevented the progression of renal fibrosis in vivo by regulating MAPK6/MK5/YAP axis. In conclusion, exosomal miR-374a-5p inhibited the progression of renal fibrosis by regulating MAPK6/MK5/YAP axis.

LncRNA GAS6-AS2 promotes non-small-cell lung cancer cell proliferation via regulating miR-144-3p/ MAPK6 axis.

The function of a new long non-coding RNA GAS6-AS2 in non-small cell lung cancer (NSCLC) is not fully understood. In this study, GAS6-AS2 was identified, and its roles as well as mechanisms in regulating proliferation of NSCLCs cells were investigated. qRT-PCR was used to analyze GAS6-AS2, miR-144-3p, and MAPK6 expression. Protein expression was detected by Western blotting. Cell Counting Kit-8 (CCK8) assay was used to examine the cell proliferation ability. The interaction between GAS6-AS2 and miR-144-3p was confirmed by dual-luciferase reporter assay and RNA pull down assay. A xenograft model was constructed to monitor the mice NSCLC tumor growth in vivo. GAS6-AS2 was up-regulated, while miR-144-3p was suppressed in NSCLC cells compared with normal lung cells. GAS6-AS2 suppression could inhibit the progression of NSCLC cells, and miR-144-3p could attenuate the effect. GAS6-AS2 could function as a competitive endogenous RNA (ceRNA) via direct sponging miR-144-3p-3p, which further regulating the expression of MAPK6. The knockdown of GAS6-AS2 could greatly suppress the tumor growth of NSCLC in vivo. GAS6-AS2 up-regulated MAPK6 by sponging miR-144-3p in NSCLC tissues and cells. Thus, GAS6-AS2 is an effective therapeutic target in NSCLC.

let7f­5p attenuates inflammatory injury in in vitro pneumonia models by targeting MAPK6.

Pneumonia accounts for ~1.3 million mortalities in children per year worldwide. MicroRNAs are implicated in several diseases, including cancer and pneumonia; however, the role of let7f­5p in pneumonia is not completely understood. In the present study, lipopolysaccharide (LPS) was used to establish an in vitro pneumonia model in A549 and WI­38 cells. The reverse transcription­quantitative PCR (RT­qPCR) and western blotting results demonstrated that let7f­5p expression levels were significantly decreased, whereas MAPK6 expression levels were significantly increased in the peripheral venous blood of patients with pneumonia and in LPS­induced A549 and WI­38 cells compared with healthy volunteers and control cells, respectively. Furthermore, the dual­luciferase reporter assay demonstrated that let7f­5p targeted the 3'­untranslated region of MAPK6. The ELISA and RT­qPCR results demonstrated that let7f­5p mimic ameliorated LPS­induced inflammatory injury in A549 and WI­38 cells, as demonstrated by decreased expression levels of proinflammatory cytokines, including TNF­α and IL­6. In addition, the Cell Counting Kit­8 assay results indicated that let7f­5p mimic ameliorated LPS­induced reductions in cell viability, and the western blotting results demonstrated that let7f­5p mimic reversed LPS­induced activation of the STAT3 signaling pathway. Notably, the aforementioned let7f­5p­mediated effects were reversed by MAPK6 overexpression. Collectively, the results of the present study suggested that let7f­5p inhibited inflammation by targeting MAPK6 in the in vitro pneumonia model, thus let7f­5p may serve as a potential novel therapeutic target for pneumonia.

Computationally predicted SARS-COV-2 encoded microRNAs target NFKB, JAK/STAT and TGFB signaling pathways.

Recently an outbreak that emerged in Wuhan, China in December 2019, spread to the whole world in a short time and killed >1,410,000 people. It was determined that a new type of beta coronavirus called severe acute respiratory disease coronavirus type 2 (SARS-CoV-2) was causative agent of this outbreak and the disease caused by the virus was named as coronavirus disease 19 (COVID19). Despite the information obtained from the viral genome structure, many aspects of the virus-host interactions during infection is still unknown. In this study we aimed to identify SARS-CoV-2 encoded microRNAs and their cellular targets. We applied a computational method to predict miRNAs encoded by SARS-CoV-2 along with their putative targets in humans. Targets of predicted miRNAs were clustered into groups based on their biological processes, molecular function, and cellular compartments using GO and PANTHER. By using KEGG pathway enrichment analysis top pathways were identified. Finally, we have constructed an integrative pathway network analysis with target genes. We identified 40 SARS-CoV-2 miRNAs and their regulated targets. Our analysis showed that targeted genes including NFKB1, NFKBIE, JAK1-2, STAT3-4, STAT5B, STAT6, SOCS1-6, IL2, IL8, IL10, IL17, TGFBR1-2, SMAD2-4, HDAC1-6 and JARID1A-C, JARID2 play important roles in NFKB, JAK/STAT and TGFB signaling pathways as well as cells' epigenetic regulation pathways. Our results may help to understand virus-host interaction and the role of viral miRNAs during SARS-CoV-2 infection. As there is no current drug and effective treatment available for COVID19, it may also help to develop new treatment strategies.

Circ_0000285 promotes podocyte injury through sponging miR-654-3p and activating MAPK6 in diabetic nephropathy.

Recently, increasing evidence has reported that circRNAs are non-coding RNAs and they bind with the corresponding miRNAs to modulate the target genes. However, the detailed role of circRNAs in the pathogenesis of DN still remains poorly known. Currently, we aimed to study how circ_0000285 functions in DN development. We found that circ_0000285 was significantly increased in DN mice models and mouse podocytes incubated with HG. Then, circ_0000285 was overexpressed in mouse podocytes and we observed that overexpression of circ_0000285 promoted podocytes injury. Moreover, miR-654-3p was precited as a target of circ_0000285. It was shown that circ_0000285 was strongly pulled down by circ_0000285 specific probe and circ_0000285 specific probe was used to successfully enrich miR-654-3p. In addition, we reported that miR-654-3p was obviously down-regulated in DN. Inhibitors of miR-654-3p greatly reversed the effects of circ_0000285 siRNA on podocytes injury. Moreover, the inflammation release was restrained by loss of circ_0000285, while induced by miR-654-3p inhibitors. IL-6, L-1ß and TNF-α level was remarkably depressed by the knockdown of circ_0000285 and miR-654-3p inhibitors induced that. Furthermore, MAPK6 was confirmed as a direct downstream target of miR-654-3p. As shown, MAPK6 was markedly suppressed by circ_0000285 siRNA, which was rescued by the decrease of miR-654-3p. These findings revealed that circ_0000285 promoted podocyte injury via sponging miR-654-3p and activating MAPK6 in DN.