Fig 2: miR-520c-3p antagomir inhibition of HBX-induced tumor progression in vivo(A) qPCR and western blot assays showed a significantly higher expression of HBx in a stable HBx-overexpression HepG2 cell line. (B) Mice were imaged with live image equipment. The relative fluorescence intensity of the mice was calculated by using ImageJ, and the scatter diagram was drawn by using GraphPad Prism software. (C) Expression of PTEN and miR-520c-3p in tumors with NC/miR-520c-3p-antagomir-treatment were analyzed by qPCR. (D) Proteins in the AKT/NF-?B pathway were analyzed in tumor tissues by western blot. (E) Representative live images of mouse livers and lungs. (F) A model for HBV-induced cell migration and invasion. HBV, via HBx binding to CREB1, upregulates miR-520c-3p, which in turn downregulates PTEN, thereby leading to the activation of AKT/NF-?B pathway to enhance HCC EMT and thus cell migration and invasion. This study reveals that miR-520c-3p, upregulated by HBx of HBV, targets PTEN, an anti-oncogene, to promote hepatoma cell migration and metastasis through the activation of AKT/NF-?B pathway. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig 3: Schematic representation of the proposed mechanism. In liver tumor tissue, miR-23a upregulation inhibited STAT5A expression, leading to increased AKT phosphorylation to promote cell metabolism and tumorigenesis.

Fig 4: LINC00473 attenuates the Dex-induced inhibition of the viability of hBMSCs by activating the Akt signaling pathway. (A) The viability of hBMSCs in each group was evaluated by CCK-8 assay from 1 to 7 days. (B-a) The apoptotic cells were investigated by a fluorescence microscope through Hoechst 33342 staining (scale bar, 200 µm). White arrows indicate the cells with karyopyknosis. (B-b) The percentage of apoptotic cells was calculated by cell count. A total of 6 randomly selected fields were quantified, and a total of 300 cells in each group were counted. (C-a) Flow cytometric analysis revealed the apoptotic cells with Annexin V-PE and 7-AAD staining: Cells in Q1 represent necrotic cells, cells in Q2 represent late apoptotic cells, cells in Q3 represent early apoptotic cells, cells in Q4 represent normal cells. (C-b) Percentage of Annexin+ cells in Q2 and Q3 in each group. All data are presented as the mean value ± standard deviation of 3 independent experiments. *P<0.05 compared with the normal group, #P<0.05 compared with the Dex group and the Dex + Vector group, &P<0.05 compared with the Dex + LINC00473-up group. LINC00473, lncCRNA-00473; hBMSCs, human bone marrow-derived mesenchymal stem cells; Dex, dexamethasone; Akt, protein kinase B; CCK-8, Cell Counting Kit-8; Q, quadrant.

Fig 5: Molecular mechanisms of intestinal immune defects in AKR1B8 –/– mice. (A) AKR1B8 expression in colonic epithelial cells (ECs) and immune cells from mesenteric lymph nodes (MLN). RNA and proteins were extracted for qRT-PCR (upper panel) and Western blot (lower panel) analyses as described in Materials and Methods. (B) Expression of IL18, CCL20, IL15, IL6, CCL8, and IL1ß cytokines in ECs. Data indicate qRT-PCR results as described in Materials and Methods. (C) AKT and ERK signaling activity. The p-AKT (Ser473), p-AKT (Thr308), t-AKT, p-ERK (1/2), t-ERK, p-p90RSK and p-MSK1 proteins were analyzed by Western blot as described in Materials and Methods. ß-actin was used as an internal control. Left panel, representative images; right panel, quantitative data of band intensity by Image J software. Data were normalized by respective total proteins. (D) NF-?B signal activity. IKKa, IKKß, p-IKKa/ß, p-IKBa, t-IKBa, p-P65 (S536), p-P65 (S276), and t-P65 protein levels were analyzed by Western blot. Left panel, representative images; right panel, quantitative data of band intensity by Image J software. Data of phosphorylated proteins were normalized by respective total proteins. Data of IKKa and IKKß were normalized by ß-actin. WT, wild-type; KO, AKR1B8 –/–. Data represent mean ± SEM of three to four mice per group. *P < 0.05; **P < 0.01.