Ensembles of endothelial and mural cells promote angiogenesis in prenatal human brain.

Interactions between angiogenesis and neurogenesis regulate embryonic brain development. However, a comprehensive understanding of the stages of vascular cell maturation is lacking, especially in the prenatal human brain. Using fluorescence-activated cell sorting, single-cell transcriptomics, and histological and ultrastructural analyses, we show that an ensemble of endothelial and mural cell subtypes tile the brain vasculature during the second trimester. These vascular cells follow distinct developmental trajectories and utilize diverse signaling mechanisms, including collagen, laminin, and midkine, to facilitate cell-cell communication and maturation. Interestingly, our results reveal that tip cells, a subtype of endothelial cells, are highly enriched near the ventricular zone, the site of active neurogenesis. Consistent with these observations, prenatal vascular cells transplanted into cortical organoids exhibit restricted lineage potential that favors tip cells, promotes neurogenesis, and reduces cellular stress. Together, our results uncover important mechanisms into vascular maturation during this critical period of human brain development.

Effects of spinal cord injury associated exosomes delivered tRF-41 on the progression of spinal cord injury progression.

BACKGROUND: Spinal cord injury (SCI) is a devastating neurological and pathological condition. Exosomal tsRNAs have reported to be promising biomarkers for cancer diagnosis and therapy. This study aimed to investigate the roles of SCI-associated exosomes, and related tsRNA mechanisms in SCI. METHODS: The serum of healthy controls and SCI patients at the acute stage were collected for exosomes isolation, and the two different exosomes were used to treat human astrocytes (HA). The cell viability, apoptosis, and cycle were determined, and the expression of the related proteins were detected by western blot. Then, the two different exosomes were sent for tsRNA sequencing, and four significant known differentially expressed tsRNAs (DE-tsRNAs) were selected for RT-qPCR validation. Finally, tRT-41 was chosen to further explore its roles and related mechanisms in SCI. RESULTS: After sequencing, 21 DE-tsRNAs were identified, which were significantly enriched in pathways of Apelin, AMPK, Hippo, MAPK, Ras, calcium, PI3K-Akt, and Rap1. RT-qPCR showed that tRF-41 had higher levels in the SCI-associated exosomes. Compared with the control HA, healthy exosomes did not significantly affect the growth of HA cells, but SCI-associated exosomes inhibited viability of HA cells, while promoted their apoptosis and increased the HA cells in G2/M phase; but tRF-41 inhibitor reversed the actions of SCI-associated exosomes. Additionally, SCI-associated exosomes, similar with tRF-41 mimics, down-regulated IGF-1, NGF, Wnt3a, and ß-catenin, while up-regulated IL-1ß and IL-6; but tRF-41 inhibitor had the opposite actions, and reversed the effects induced by SCI-associated exosomes. CONCLUSIONS: SCI-associated exosomes delivered tRF-41 may inhibit the growth of HA through regulating Wnt/ ß-catenin pathway and inflammation response, thereby facilitating the progression of SCI.

Elucidation of pericholangitis and periductal fibrosis in cholestatic liver diseases via extracellular vesicles released by polarized biliary epithelial cells.

Cholestatic liver diseases causes inflammation and fibrosis around bile ducts. However, the pathological mechanism has not been elucidated. Extracellular vesicles (EVs) are released from both the basolateral and apical sides of polarized biliary epithelial cells. We aimed to investigate the possibility that EVs released from the basolateral sides of biliary epithelial cells by bile acid stimulation induce inflammatory cells and fibrosis around bile ducts, and they may be involved in the pathogenesis of cholestatic liver disease. Human biliary epithelial cells (H69) were grown on cell culture inserts and stimulated with chenodeoxycholic acid + IFN-γ. Human THP-1-derived M1-macrophages, LX-2 cells, and KMST-6 cells were treated with the extracted basolateral EVs, and inflammatory cytokines and fibrosis markers were detected by RT-PCR. Highly expressed proteins from stimulated EVs were identified, and M1-macrophages, LX-2, KMST-6 were treated with these recombinant proteins. Stimulated EVs increased the expression of TNF, IL-1ß, and IL-6 in M1-macrophages, TGF-ß in LX-2 and KMST-6 compared with the corresponding expression levels in unstimulated EVs. Nucleophosmin, nucleolin, and midkine levels were increased in EVs from stimulated cells compared with protein expression in EVs from unstimulated cells. Leukocyte cell-derived chemotaxin-2 (LECT2) is highly expressed only in EVs from stimulated cells. Stimulation of M1-macrophages with recombinant nucleophosmin, nucleolin, and midkine significantly increased the expression of inflammatory cytokines. Stimulation of LX-2 and KMST-6 with recombinant LECT2 significantly increased the expression of fibrotic markers. These results suggest that basolateral EVs are related to the development of pericholangitis and periductal fibrosis in cholestatic liver diseases.NEW & NOTEWORTHY Our research elucidated that the composition of basolateral EVs from the biliary epithelial cells changed under bile acid exposure and the basolateral EVs contained the novel inflammation-inducing proteins NPM, NCL, and MK and the fibrosis-inducing protein LECT2. We report that these new results are possible to lead to the potential therapeutic target of cholestatic liver diseases in the future.

Intercellular crosstalk in adult dental pulp is mediated by heparin-binding growth factors Pleiotrophin and Midkine.

BACKGROUND: In-depth knowledge of the cellular and molecular composition of dental pulp (DP) and the crosstalk between DP cells that drive tissue homeostasis are not well understood. To address these questions, we performed a comparative analysis of publicly available single-cell transcriptomes of healthy adult human DP to 5 other reference tissues: peripheral blood mononuclear cells, bone marrow, adipose tissue, lung, and skin. RESULTS: Our analysis revealed that DP resident cells have a unique gene expression profile when compared to the reference tissues, and that DP fibroblasts are the main cell type contributing to this expression profile. Genes coding for pleiotrophin (PTN) and midkine (MDK), homologous heparin-binding growth-factors, possessed the highest differential expression levels in DP fibroblasts. In addition, we identified extensive crosstalk between DP fibroblasts and several other DP resident cells, including Schwann cells, mesenchymal stem cells and odontoblasts, mediated by PTN and MDK. CONCLUSIONS: DP fibroblasts emerge as unappreciated players in DP homeostasis, mainly through their crosstalk with glial cells. These findings suggest that fibroblast-derived growth factors possess major regulatory functions and thus have a potential role as dental therapeutic targets.

Identification and requirements of enhancers that direct gene expression during zebrafish fin regeneration.

To identify candidate tissue regeneration enhancer elements (TREEs) important for zebrafish fin regeneration, we performed ATAC-seq from bulk tissue or purified fibroblasts of uninjured and regenerating caudal fins. We identified tens of thousands of DNA regions from each sample type with dynamic accessibility during regeneration, and assigned these regions to proximal genes with corresponding expression changes by RNA-seq. To determine whether these profiles reveal bona fide TREEs, we tested the sufficiency and requirements of several sequences in stable transgenic lines and mutant lines with homozygous deletions. These experiments validated new non-coding regulatory sequences near induced and/or essential genes during fin regeneration, including fgf20a, mdka and cx43, identifying distinct domains of directed expression for each confirmed TREE. Whereas deletion of the previously identified LEN enhancer abolished detectable induction of the nearby leptin b gene during regeneration, deletions of enhancers linked to fgf20a, mdka and cx43 had no effect or partially reduced gene expression. Our study generates a new resource for dissecting the regulatory mechanisms of appendage generation and reveals a range of requirements for individual TREEs in control of regeneration programs.

Evaluation of Midkine levels in pregnant women with preterm premature rupture of membranes: A cohort study from a tertiary hospital.

OBJECTIVE: To assess midkine (MK) levels in pregnant women with preterm premature rupture of membranes (PPROM) and compare them to healthy pregnant women. We also assessed the performance of the maternal serum MK level in predicting neonatal intensive care unit (NICU) requirement in the PPROM group. METHODS: Forty pregnant women who presented to our clinic at 24-37 gestational weeks and were diagnosed with PPROM were included in the study group. During the same period, 40 healthy pregnant women at similar gestational weeks were randomly selected as the control group. Clinical characteristics, inflammatory markers, and serum MK levels were compared between the groups. The same parameters were then compared between the PPROM cases with and without NICU requirement. Finally, the receiver operating characteristic (ROC) analysis was performed to assess the predictive value of MK for NICU requirement. RESULTS: The PPROM and control groups were similar in terms of demographics. The MK level of the pregnant woman with PPROM was significantly higher than that of the controls. No statistically significant difference was found between the MK levels of the cases with and without NICU requirement in the PPROM group. In the ROC analysis, the optimal cut-off value of was found to be 0.287, at which it had 63 % sensitivity and 65 % specificity (area under the curve(AUC): 0.78, 95 % confidence interval(CI): 0.683-0.881, p < 0.001) for the prediction of NICU requirement in cases with PPROM. In the same analysis performed for the prediction of PPROM, when the optimal cut-off value was taken as 0.298, MK had 56 % sensitivity and 60 % specificity (AUC: 0.65, 95 % CI: 0.522-0.770, p = 0.037). CONCLUSION: Serum MK seems to be associated with complicated inflammatory processes leading to PPROM, and this novel marker has the potential to predict NICU requirement in PPROM cases.

Overexpression and potential roles of midkine via regulation of vascular endothelial growth factor A in psoriasis.

Midkine plays a critical role in angiogenesis by regulating the vascular endothelial growth factor (VEGF) signalling pathway, which is known to be associated with psoriasis pathogenesis. However, research on midkine-psoriasis relationship remains limited. The objective of this study was to detect midkine expression in psoriasis and investigate its potential role in the disease. Midkine expression was measured using immunohistochemistry and ELISA. Effects of midkine on HaCaT cell proliferation, VEGF-A production and signalling pathways were assessed using CCK8, RT-PCR and WB. Scratch and in vitro tube formation tests were used to evaluate the effects of HaCaT-cell-activated midkine on the migration and tube formation of human dermal microvascular endothelial cells. Murine psoriasiform models were injected with midkine recombinant protein and midkine monoclonal antibody to investigate skin lesions, tissue sections and dermal microvessel density. Levels of midkine significantly increased in both lesions and serum of patients with psoriasis. Serum expression of midkine decreased after treatment and a positive correlation was found between midkine and disease severity. Midkine promoted HaCaT cell proliferation and VEGF-A production. The Notch2/HES1/JAK2-STAT5A pathway expression increased after midkine treatment of HaCaT cells. The supernatant of HaCaT cells treated with midkine promoted HMEC-1 migration and angiogenesis in vitro. Recombinant midkine protein exacerbated psoriasiform lesions with increased expressions of VEGF-A and microvessel density, while midkine monoclonal antibody alleviated psoriasis lesions. Midkine may have a significant impact on psoriasis angiogenesis by regulating VEGF-A expression through the Notch2/HES1/JAK2-STAT5A pathway, highlighting a potential therapeutic target for psoriasis treatment.

Secreted proteins MDK, WFDC2, and CXCL14 as candidate biomarkers for early diagnosis of lung adenocarcinoma.

BACKGROUND: Early diagnosis of lung adenocarcinoma (LUAD), one of the most common types of lung cancer, is very important to improve the prognosis of patients. The current methods can't meet the requirements of early diagnosis. There is a pressing need to identify novel diagnostic biomarkers. Secretory proteins are the richest source for biomarker research. This study aimed to identify candidate secretory protein biomarkers for early diagnosis of LUAD by integrated bioinformatics analysis and clinical validation. METHODS: Differentially expressed genes (DEGs) of GSE31210, gene expression data of early stage of LUAD, were analyzed by GEO2R. Upregulated DEGs predicted to encode secreted proteins were obtained by taking the intersection of the DEGs list with the list of genes encoding secreted proteins predicted by the majority decision-based method (MDSEC). The expressions of the identified secreted proteins in the lung tissues of early-stage LUAD patients were further compared with the healthy control group in mRNA and protein levels by using the UALCAN database (TCGA and CPTAC). The selected proteins expressed in plasma were further validated by using Luminex technology. The diagnostic value of the screened proteins was evaluated by receiver operating characteristic (ROC) analysis. Cell counting kit-8 assay was carried out to investigate the proliferative effects of these screened proteins. RESULTS: A total of 2183 DEGs, including 1240 downregulated genes and 943 upregulated genes, were identified in the GSE31210. Of the upregulated genes, 199 genes were predicted to encode secreted proteins. After analysis using the UALCAN database, 16 molecules were selected for further clinical validation. Plasma concentrations of three proteins, Midkine (MDK), WAP four-disulfide core domain 2 (WFDC2), and C-X-C motif chemokine ligand 14 (CXCL14), were significantly higher in LUAD patients than in healthy donors. The area under the curve values was 0.944, 0.881, and 0.809 for MDK, WFDC2, and CXCL14, 0.962 when combined them. Overexpression of the three proteins enhanced the proliferation activity of A549 cells. CONCLUSIONS: MDK, WFDC2, and CXCL14 were identified as candidate diagnostic biomarkers for early-stage LUAD and might also play vital roles in tumorigenesis.

Expression of the pleiotrophin-midkine axis in a sheep tooth socket model of bone healing.

OBJECTIVE AND BACKGROUND: Resorption of alveolar bone after tooth extraction is a common problem often requiring bone grafting. The success of the grafting procedures is dependent on multiple factors including the presence of growth factors. This is the first in vivo study to investigate the role of the pleiotrophin family of cytokines in alveolar bone regeneration. This research investigated the role of the pleiotrophin-midkine (PTN-MDK) axis during osteogenesis, with and without a grafting material, after tooth extraction in a sheep model. METHODS: Thirty Romney-cross ewes were anesthetized, and all premolar teeth on the right side were extracted. The sockets were randomized to controls sites with no treatment and test sites with Bio-Oss® graft material and Bio-Gide® membrane. Samples were harvested after sacrificing animals 4, 8, and 16 weeks post-grafting (n = 10 per time-point). Tissue for qRT2 -PCR gene analysis was recovered from the socket next to the first molar using a trephine (Ø = 2 mm). Each socket was fixed, decalcified, paraffin-embedded, and sectioned. Immunohistochemistry was conducted to localize both PTN and MDK along with their receptors, protein tyrosine phosphatase receptor type Z1 (PTPRZ1), ALK receptor tyrosine kinase (ALK), and notch receptor 2 (NOTCH2). RESULTS: Within the healing sockets, high expression of genes for PTN, MDK, NOTCH2, and ALK was found at all time-points and in both grafted and non-grafted sites, while PTPRZ1 was only expressed at low levels. The relative gene expression of the PTN family of cytokines was not statistically different at the three time-points between test and control groups (p > .05). Immunohistochemistry found PTN and MDK in association with new bone, NOTCH2 in the connective tissue, and PTPRZ1 and ALK in association with cuboidal osteoblasts involved in bone formation. CONCLUSIONS: The PTN-MDK axis was highly expressed in both non-grafted and grafted sockets during osteogenesis in a sheep model of alveolar bone regeneration with no evidence that grafting significantly affected expression. The activation of NOTCH2 and PTPRZ1 receptors may be important during bone regeneration in vivo. The discovery of the PTN-MDK axis as important during alveolar bone regeneration is novel and opens up new avenues of research into these stably expressed highly active cytokines. Growth factor supplementation with PTN and/or MDK during healing may be an approach for enhanced regeneration or to initiate healing where delayed.

Whole-body imaging of lymphovascular niches identifies pre-metastatic roles of midkine.

Cutaneous melanoma is a type of cancer with an inherent potential for lymph node colonization, which is generally preceded by neolymphangiogenesis. However, sentinel lymph node removal does not necessarily extend the overall survival of patients with melanoma. Moreover, lymphatic vessels collapse and become dysfunctional as melanomas progress. Therefore, it is unclear whether (and how) lymphangiogenesis contributes to visceral metastasis. Soluble and vesicle-associated proteins secreted by tumours and/or their stroma have been proposed to condition pre-metastatic sites in patients with melanoma. Still, the identities and prognostic value of lymphangiogenic mediators remain unclear. Moreover, our understanding of lymphangiogenesis (in melanomas and other tumour types) is limited by the paucity of mouse models for live imaging of distal pre-metastatic niches. Injectable lymphatic tracers have been developed, but their limited diffusion precludes whole-body imaging at visceral sites. Vascular endothelial growth factor receptor 3 (VEGFR3) is an attractive 'lymphoreporter' because its expression is strongly downregulated in normal adult lymphatic endothelial cells, but is activated in pathological situations such as inflammation and cancer. Here, we exploit this inducibility of VEGFR3 to engineer mouse melanoma models for whole-body imaging of metastasis generated by human cells, clinical biopsies or endogenously deregulated oncogenic pathways. This strategy revealed early induction of distal pre-metastatic niches uncoupled from lymphangiogenesis at primary lesions. Analyses of the melanoma secretome and validation in clinical specimens showed that the heparin-binding factor midkine is a systemic inducer of neo-lymphangiogenesis that defines patient prognosis. This role of midkine was linked to a paracrine activation of the mTOR pathway in lymphatic endothelial cells. These data support the use of VEGFR3 reporter mice as a 'MetAlert' discovery platform for drivers and inhibitors of metastasis.

Maternal serum midkine level in fetal growth restriction: a case-control study.

OBJECTIVES: To compare maternal serum midkine (MK) level in pregnant women with idiopathic fetal growth restriction (FGR) and healthy. In addition, we assessed the value of maternal serum MK level in predicting neonatal intensive care unit (NICU) admission. METHODS: A total of 144 pregnant women were included, 72 with idiopathic FGR and 72 healthy in this study. The control group was matched for the mother's age, parity, gestational age, and pre-pregnancy body mass index (BMI) with the idiopathic FGR group at the time of recruitment into the study and sample collection. RESULTS: Serum MK level is higher in the idiopathic FGR than the control group (0.24 ng/mL (0.19-0.32) vs. 0.18 ng/mL (0.14-0.23), p<0.001). In addition, we compared the maternal serum MK level of those with and without NICU admission in the FGR group (0.25 ng/mL (0.19-0.37) vs. 0.21 ng/mL (0.18-0.28), p=0.014). We performed ROC curve analysis to serum MK level predicting NICU admission in the FGR group (AUC: 0.668, %95 CI [0.550, 0.785], p=0.014). A sensitivity of 63% and a specificity of 62% for the serum MK level were achieved with a cut-off value of 0.22 for NICU admission. CONCLUSIONS: To the best of our knowledge, this study is the first to compare maternal serum MK level in pregnant women with idiopathic FGR and healthy. We showed that maternal serum MK level was significantly elevated in pregnant women with FGR than healthy.

Total synthesis of chondroitin sulfate E oligosaccharides and biological study.

A total synthesis approach of CS-E oligosaccharides was established and a series of derivatives were synthesized. These oligosaccharides were evaluated for a glycosaminoglycan (GAG)-binding protein interaction against cytokines, midkine, and pleiotrophin, by surface-plasmon resonance (SPR) assay. The binding epitopes of oligosaccharides to midkine were mapped using a saturation transfer difference (STD) NMR technique. The groups on the reducing end contributed to binding affinity, and should not be ignored in biological assays. These findings contribute to the structure and activity relationship research and a foundation of understanding that will underpin potential future optimization of this class of oligosaccharides as pharmaceutical agents.

Elevated Midkine Serum Levels Are Associated with Long-Term Survival in Critically Ill Patients.

Midkine (Mdk) is a multifunctional protein involved in inflammatory processes. Hence, circulating Mdk is increased in sepsis and has been previously suggested as a potential biomarker in these patients. The aim of this study was to elucidate the role of Mdk serum concentrations in critical illness and sepsis and to verify its value as a prognostic biomarker. Thus, we analyzed the Mdk serum concentrations of 192 critically ill patients on admission to the medical intensive care unit (ICU). While the serum levels of Mdk at admission were similar in septic and nonseptic critical illness (362 vs. 337 ng/L, p = 0.727), we found several interesting correlations of Mdk to laboratory and clinical markers associated with ischemia or hypoxia, e.g., to renal failure and hepatic injury. Mdk serum concentrations at admission did not differ between various causes of sepsis or other critical illness. Most noticeable, we observed upregulated Mdk serum concentrations at admission in patients surviving in the long-term, which was only seen in nonseptic critical illness but not in sepsis. Our study suggests a relevant role of Mdk in critically ill patients in general and highlights the possible protective features of Mdk in critical illness.

Spermidine-Eugenol Supplement Preserved Inflammation-Challenged Intestinal Cells by Stimulating Autophagy.

Increases in non-communicable and auto-immune diseases, with a shared etiology of defective autophagy and chronic inflammation, have motivated research both on natural products in drug discovery fields and on the interrelationship between autophagy and inflammation. Within this framework, the tolerability and protective effects of a wheat-germ spermidine (SPD) and clove eugenol (EUG) combination supplement (SUPPL) were investigated on inflammation status (after the administration of lipopolysaccharide (LPS)) and on autophagy using human Caco-2 and NCM460 cell lines. In comparison to the LPS treatment alone, the SUPPL + LPS significantly attenuated ROS levels and midkine expression in monocultures, as well as occludin expression and mucus production in reconstituted intestinal equivalents. Over a timeline of 2-4 h, the SUPPL and SUPPL + LPS treatments stimulated autophagy LC3-11 steady state expression and turnover, as well as P62 turnover. After completely blocking autophagy with dorsomorphin, inflammatory midkine was significantly reduced in the SUPPL + LPS treatment in a non-autophagy-dependent manner. After a 24 h timeline, preliminary results showed that mitophagy receptor BNIP3L expression was significantly downregulated in the SUPPL + LPS treatment compared to the LPS alone, whereas conventional autophagy protein expression was significantly higher. The SUPPL shows promise in reducing inflammation and increasing autophagy to improve intestinal health.

Role of Midkine in Cancer Drug Resistance: Regulators of Its Expression and Its Molecular Targeting.

Molecules involved in drug resistance can be targeted for better therapeutic efficacies. Research on midkine (MDK) has escalated in the last few decades, which affirms a positive correlation between disease progression and MDK expression in most cancers and indicates its association with multi-drug resistance in cancer. MDK, a secretory cytokine found in blood, can be exploited as a potent biomarker for the non-invasive detection of drug resistance expressed in various cancers and, thereby, can be targeted. We summarize the current information on the involvement of MDK in drug resistance, and transcriptional regulators of its expression and highlight its potential as a cancer therapeutic target.

Identification and validation of a poor clinical outcome subtype of primary prostate cancer with Midkine abundance.

Recent studies identified Midkine (MDK) as playing a key role in immune regulation. In this study, we aimed to discover the clinical significance and translational relevance in prostate cancer (PCa). We retrospectively analyzed 759 PCa patients who underwent radical prostatectomy from Huashan Hospital, Fudan University (training cohort, n = 369) and Chinese Prostate Cancer Consortium (validation cohort, n = 390). A total of 325 PCa patients from The Cancer Genome Atlas (TCGA) database (external cohort) were analyzed for exploration. Immune landscape and antitumor immunity were assessed through immunohistochemistry and flow cytometry. Patient-derived explant culture system was applied for evaluating the targeting potential of MDK. We found that intratumoral MDK expression correlated with PCa progression, which indicated an unfavorable biochemical recurrence (BCR)-free survival for postoperative PCa patients. Addition of MDK expression to the postoperative risk assessment tool CAPRA-S could improve its prognostic value. Tumors with MDK abundance characterized the tumor-infiltrating CD8+ T cells with less cytotoxicity production and increased immune checkpoint expression, which were accompanied by enriched immunosuppressive contexture. Moreover, MDK inhibition could reactivate CD8+ T cell antitumor immunity. MDK mRNA expression negatively correlated with androgen receptor activity signature and positively associated with radiotherapy-related signature. In conclusion, intratumoral MDK expression could serve as an independent prognosticator for BCR in postoperative PCa patients. MDK expression impaired the antitumor function of CD8+ T cells through orchestrating an immunoevasive microenvironment, which could be reversed by MDK inhibition. Moreover, tumors with MDK enrichment possessed potential sensitivity to postoperative radiotherapy while resistance to adjuvant hormonal therapy of PCa. MDK could be considered as a potential therapeutic target for PCa.

Serum midkine level might be a diagnostic tool for COVID19 disease in pregnancy: From the disease severity, hospitalization and disease progression respects.

BACKGROUND: New biomarkers for diagnosis and monitoring the COVID-19 disease are the most important topics to be studied recently. We aimed to investigate the association between midkine levels and disease severity in pregnant women with COVID-19. METHODS: Totally 186 pregnant women were participated in this study. 96 of them were healthy pregnant women, 90 of them were pregnant women with COVID19. Pregnant women were evaluated according to their trimesters. Serum midkine level, biochemical profile clinical and disease severity outcomes of pregnant women were obtained. RESULTS: Our results showed that pregnant women with COVID19 have significantly increased serum midkine level compared to healthy pregnant women (1.801 ± 0.977 vs 0.815 ± 0.294 ng/dL). According to the data among each trimester, it was shown that there were significant increase in serum midkine level during all pregnancy trimesters (1st trimester Control Group: 0.714 ± 0.148, COVID-19 group 1.623 ± 0.824, p < 0.0001; 2nd trimester Control Group: 0.731 ± 0.261, COVID-19 group 2.059 ± 1.146, p < 0.0001; 3rd trimester Control Group: 1.0 ± 0.35, COVID-19 group 1.723 ± 0.907, p = 0.001). Serum midkine levels were significantly different between disease severity subgroups of pregnant women with COVID19; moderate and severe/critic groups had significantly higher serum midkine level than mild group. There was also significant correlation between serum midkine level and severity status (p:0.0001, r: 0.468). The most striking results of serum midkine levels were corelation between length of hospitalization (p: 0.01, r: 0.430) and O2 saturation (p < 0.0001, r: -0.521). ROC curve analysis showed that serum midkine level might be a tool for predicting COVID-19 in pregnant women with COVID-19 (AUC: 0.912, 95% CI: [0.871, 0.952], p < 0.0001) CONCLUSION: Our data showed that there is an obvious relation between COVID19 progression and serum midkine level for the first time which might be used for monitoring the disease process.

CAF-derived midkine promotes EMT and cisplatin resistance by upregulating lncRNA ST7-AS1 in gastric cancer.

This study aimed to investigate the role of cancer-associated fibroblast (CAF)-derived midkine (MK) in cisplatin (DDP) resistance. The primary cultures of CAFs and non-cancer fibroblasts (NFs) were isolated and purified. The DDP-resistant gastric cancer (GC) cells were cultured with CAF-conditioned medium. QRT-PCR and Elisa assays were employed to determine MK expression. The expression of ST7-AS1 was measured by qRT-PCR. The impact of CAFs, MK, and ST7-AS1 silencing on DDP resistance was determined by MTT and Annexin V/PI staining assay. Expression of EMT markers and PI3K/AKT was determined by Western blot and qRT-PCR. The role of MK in DDP resistance was confirmed in a xenograft model. Incubation with CAF-conditioned medium increased the IC50 to DDP. Also, incubation with CAF-conditioned medium increased cell viability, reduced cell apoptosis, and promoted EMT in DDP-resistant GC cells, which were all blocked with MK neutralization antibody treatment. MK increased the DDP resistance and upregulated the expression of ST7-AS1 in DDP-resistant GC cells. Additionally, ST7-AS1 knockdown increased the sensitivity to DDP by inhibiting EMT. Moreover, ST7-AS1 knockdown significantly decreased the phosphorylation of PI3K and AKT, and suppressed EMT, which were restored by MK addition. Finally, MK promoted tumor growth and DDP resistance in a mice model bearing the SGC-7901/DDP xenografts. CAF-derived MK promotes EMT-mediated DDP resistance via upregulation of ST7-AS1 and activation of PI3K/AKT pathway.

Midkine promotes breast cancer cell proliferation and migration by upregulating NR3C1 expression and activating the NF-κB pathway.

BACKGROUND: Breast cancer (BC) is the most common malignancy in females and is the second leading cause of cancer-related death among women worldwide. Midkine (MDK) is a heparin-binding growth factor that is abnormally expressed at high levels in various human malignancies. We aimed to uncover the biological function and molecular mechanism of MDK in BC cells. METHODS AND RESULTS: MDA-MB-231-shMDK and T47D-shMDK BC cells were established. The in vitro biological functions of MDK were demonstrated by CCK-8 assays, Transwell assays and Western blotting, whereas qPCR pathway arrays were implemented to explore the mechanism of MDK in BC cells. Functionally, we verified that silencing MDK significantly suppressed BC cell proliferation and migration by inhibiting the activation of the nuclear factor kappa B (NF-κB) pathway and the nuclear distribution of NF-κB. Meanwhile, Ingenuity Pathway Analysis (IPA) and a qPCR pathway array revealed that silencing MDK decreased the expression of NR3C1, a potential downstream target of the NF-κB pathway. We also confirmed that treatment with an NF-κB inhibitor suppressed NR3C1 expression in BC cells. Finally, we demonstrated that silencing NR3C1 repressed BC cell proliferation and migration. CONCLUSIONS: Our findings highlight a novel mechanism by which MDK influences BC progression via regulation of the NF-κB-NR3C1 pathway.

Insulin-Like Growth Factor 1 Receptor Drives Hepatocellular Carcinoma Growth and Invasion by Activating Stat3-Midkine-Stat3 Loop.

BACKGROUND: Activation of the insulin-like growth factor 1 receptor (IGF-1R)-mediated Janus kinase (JAK)1/2-Stat3 pathway contributes to hepatocarcinogenesis. Specifically, a previous study showed that IGF-1R inhibition downregulated Midkine expression in hepatocellular carcinoma (HCC). AIMS: The present study investigated the role of IGF-1R-JAK1/2-Stat3 and Midkine signaling in HCC, in addition to the molecular link between the IGF-1R-Stat3 pathway and Midkine. METHODS: The expression levels of IGF-1R, Stat3, and Midkine were measured using reverse transcription-quantitative PCR, following which the association of IGF-1R with Stat3 and Midkine expression was evaluated in HCC. The molecular link between the IGF-1R-Stat3 pathway and Midkine was then investigated in vitro before the effect of IGF-1R-Stat3 and Midkine signaling on HCC growth and invasion was studied in vitro and in vivo. RESULTS: IGF-1R, Stat3, and Midkine mRNA overexpressions were all found in HCC, where the levels of Stat3 and Midkine mRNA correlated positively with those of IGF-1R. In addition, Midkine mRNA level also correlated positively with Stat3 mRNA expression in HCC tissues. IGF-1R promoted Stat3 activation, which in turn led to the upregulation of Midkine expression in Huh7 cells. Similarly, Midkine also promoted Stat3 activation through potentiating JAK1/2 phosphorylation. Persistent activation of this Stat3-Midkine-Stat3 positive feedback signal loop promoted HCC growth and invasion, the inhibition of which resulted in significant antitumor activities both in vitro and in vivo. CONCLUSIONS: Constitutive activation of the IGF-1R-mediated Stat3-Midkine-Stat3 positive feedback loop is present in HCC, the inhibition of which can serve as a potential therapeutic intervention strategy for HCC.