Orientin Reduces the Effects of Repeated Procedural Neonatal Pain in Adulthood: Network Pharmacology Analysis, Molecular Docking Analysis, and Experimental Validation.

Background: Premature infants often undergo painful procedures and consequently experience repeated procedural neonatal pain. This can elicit hyperalgesia and cognitive impairment in adulthood. Treatments for neonatal pain are limited. Orientin is a flavonoid C-glycoside that has repeatedly been shown to have pharmacological effects in the past decades. The aim of this study was to systematically explore the effect of orientin on repeated procedural neonatal pain using network pharmacology, molecular docking analysis, and experimental validation. Methods: Several compound-protein databases and disease-protein databases were employed to identify proteins that were both predicted targets of orientin and involved in neonatal pain. A protein-protein interaction (PPI) network was constructed, and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed to explore the potential mechanism of action. Molecular docking analysis was employed to calculate the binding energy and visualize the interactions between orientin and potential target proteins. Finally, a mouse model of repeated procedural neonatal pain was established and orientin was administered for 6 days. The mechanical and thermal pain thresholds were assessed in neonates and adult mice. A Morris water maze was employed to investigate cognitive impairment in adult mice. Results: A total of 286 proteins that were both predicted targets of orientin and involved in neonatal pain were identified. The hub proteins were SRC, HSP90AA1, MAPK1, RHOA, EGFR, AKT1, PTPN11, ESR1, RXRA, and HRAS. GO analysis indicated that the primary biological process (BP), molecular function (MF), and cellular component (CC) were protein phosphorylation, protein kinase activity, and vesicle lumen, respectively. KEGG analysis revealed that the mitogen-activated protein kinase (MAPK) signaling pathway may be the key to the mechanism of action. Molecular docking analysis showed the high binding affinities of orientin for MAPK1, MAPK8, and MAPK14. In mice, orientin inhibited the hyperalgesia in the pain threshold tests in neonates and adult mice and cognitive impairment in adult mice. Immunofluorescence showed that phosphorylated MAPK1 (p-ERK) protein levels in the hippocampus and spinal dorsal horn were downregulated by orientin. Conclusion: The findings suggested that orientin alleviates neonatal pain, and the MAPK signaling pathway is involved.

Hypermethylation-mediated silencing of NDRG4 promotes pancreatic ductal adenocarcinoma by regulating mitochondrial function.

The N-myc downstream regulated gene (NDRG) family members are dysregulated in several tumors. Functionally, NDRGs play an important role in the malignant progression of cancer cells. However, little is known about the potential implications of NDRG4 in pancreatic ductal adenocarcinoma (PDAC). The aim of the current study was to elucidate the expression pattern of NDRG4 in PDAC and evaluate its potential cellular biological effects. Here, we firstly report that epigenetic-mediated silencing of NDRG4 promotes PDAC by regulating mitochondrial function. Data mining demonstrated that NDRG4 was significantly down-regulated in PDAC tissues and cells. PDAC patients with low NDRG4 expression showed poor prognosis. Epigenetic regulation by DNA methylation was closely associated with NDRG4 down-regulation. NDRG4 overexpression dramatically suppressed PDAC cell growth and metastasis. Further functional analysis demonstrated that up-regulated NDRG4 in SW1990 and Canpan1 cells resulted in attenuated mitochondrial function, including reduced ATP production, decreased mitochondrial membrane potential, and increased fragmented mitochondria. However, opposite results were obtained for HPNE cells with NDRG4 knockdown. These results indicate that hypermethylation-driven silencing of NDRG4 can promote PDAC by regulating mitochondrial function and that NDRG4 could be as a potential biomarker for PDAC patients. [BMB Reports 2020; 53(12): 658-663].

Upregulated Long Noncoding RNA UCA1 Enhances Warburg Effect via miR-203/HK2 Axis in Esophagal Cancer.

Reprogrammed glucose metabolism of enhanced aerobic glycolysis, also known as Warburg effect, which exerts a significant contributor to cancer progression, is regarded as a hallmark of cancer. The roles of long noncoding RNAs (lncRNA) in regulating cancer via metabolic reprogramming are mostly unknown, including esophagal cancer (EC). Here, we showed that how the lncRNA urothelial carcinoma associated 1 (UCA1) exerts pro-oncogene in regulating EC glucose metabolism. Firstly, we found that upregulated UCA1 expression enhances the malignant phenotypes of EC, including poor outcome, larger tumor size, positive lymphatic invasion, and advanced pathological stages. UCA1 silencing could suppress EC cell proliferation and metastasis. Following, bioinformatics analyses revealed that UCA1 regulated the HK2 expression through functioning as a competing endogenous RNA (ceRNA). Mechanistically, UCA1 overexpression could elevate the activation of HK2 oncogenes via inhibition of miR-203 activity, as evidenced by the positive correlation of UCA1 with HK2 and inverse correlation with miR-203 expression. Luciferase activity assay further verified the targeting relationship between UCA1, miR-203, and HK2. Upregulated UCA1 in EC cells significantly suppressed the degradation of HK2 by miR-203. Further research showed that upregulated UCA1 effectively increased the rate of glucose uptake, lactate output, and ECAR value, all of which can be attenuate by HK2 interference and 2-DG, whereas knockdown of UCA1 had the opposite effect. In sum, our findings suggest that the UCA1/miR-203/HK2 axis contributes to EC development by reprogramming tumor glucose metabolism, providing new insight into the management of EC patients.

Hypoxia-induced elevated NDRG1 mediates apoptosis through reprograming mitochondrial fission in HCC.

Hypoxia, as a form of stress, plays a critical role in oncogenesis, including metabolic reprogramming. Mitochondrial, the centers of energy production, re-balance mitochondria dynamic to maintain cell survival during high levels of environmental stresses. NDRG1 is a hypoxia-inducible protein that is involved in various human cancers, including HCC. However, little is known about whether NDRG1 participants in the quality control of mitochondrial in times of stress. Here, we firstly showed that how NDRG1 exerted its role through mediating mitochondrial dynamic in HCC cells under hypoxia. Initially, we identified that NDRG1 expression varies with oxygen content. NDRG1 silencing notably induced cell apoptosis under hypoxia, while no obviously change of wildtype cells in hypoxia compared with that in normoxia. Further analysis revealed that NDRG1 silencing in HCC cells led to increase of pro apoptotic protein BAX and decrease in anti-apoptotic proteins Bcl-2 and Bclx, which meant mitochondrial damage were induced. In the analysis of mitochondria, we found that more released cytochrome c located in cytosolic with NDRG1 knockdown in hypoxia, which may be due to mitochondria division. And the following experiment proved that more fragmented mitochondria were presented in NDRG1 silencing cells, as well as destroyed mitochondrial membrane potential with evidence by JC-1 was verified. Moreover, these trends could be reversed by Mdivi1. Further research showed that NDRG1 silencing disrupt hypoxia-enhanced aerobic glycolysis through effectively decreased glucose uptake, lactate output and ECAR value. In sum, we provide the first direct evidence that NDRG1-driven change in mitochondrial dynamics and aerobic glycolysis maintain cells survival in HCC during hypoxia.

SMDT1-driven change in mitochondrial dynamics mediate cell apoptosis in PDAC.

Mitochondrial Ca2+ uptake, an important governing for Ca2+ homeostasis, is catalyzed by the mitochondrial calcium uniporter (MCU) complex. SMDT1, as a subunit of MCU complex, was essential for bridging the calcium-sensing role of MICU1 and MICU2 with the calcium-conducting role of MCU. However, the molecular mechanism and regulatory purpose of SMDT1 remain largely unexplored, especially no study was reported in cancer. Here, we firstly reported that how SMDT1 exerted its role through mediating mitochondrial dynamic in PDAC malignancy. In this study, by screening online of subunit of MCU complex, we confirmed that SMDT1 expression was significantly positive correlated with PDAC prognosis. The GEO datasets showed decreased SMDT1 expression in PDAC tumor compared with non-tumor tissues. SMDT1 overexpression could notably inhibit cell proliferation and induce cell apoptosis. Further analysis demonstrated that up-regulated SMDT1 in ASPC1 and Canpan1 cells led to increased accumulation of pro apoptotic protein BAX and decrease in anti-apoptotic proteins Bcl-2 and Bclx. And more releasing of cytochrome c located in cytosolic. Mechanistically, in the morphological analysis of mitochondria, more fragmented mitochondria were presented in SMDT1 overexpression cells by promting the phosphorylation of Drp1, increasing Fis and decreasing MFN1. Meanwhile, more Drp1 was translocated on the mitochondrial from the cytoplasm in up-regulated SMDT1 cells. On the basis of the evidence above we deduce that SMDT1-driven change in mitochondrial dynamics mediated cells apoptosis in PDAC. And, SMDT1 could serve as an important therapeutic target to normalize mitochondrial dynamic responsible for poor prognosis in PDAC.