Fig 1: miR‐378a‐3p is highly expressed and PDIA4 is poorly expressed in the serum of ovarian cancer patients and ovarian cancer cells. A, RT‐qPCR was performed to determine miR‐378a‐3p expression in normal human ovarian epithelial cells (HOSE) and ovarian cancer cells (OVCAR3, CaOV3, SKOV3, and OV90). B, RT‐qPCR was conducted to determine miR‐378a‐3p expression in serum of ovarian cancer patients and healthy volunteers. C., Survival analysis of miR‐378a‐3p by Kaplan–Meier Plotter online software. D and E, RT‐qPCR and Western blot assay were utilized to detect PDIA4 mRNA and protein expression in normal HOSE and ovarian cancer cells (CaOV3, SKOV3, OVCAR3, and OV90). F, RT‐qPCR was conducted to determine PDIA4 mRNA expression in serum of ovarian cancer patients and healthy volunteers. G, Pearson correlation analysis was performed for the correlation between miR‐378a‐3p and PDIA4. H, TCGA database was implemented to analyze the survival of PDIA4 in ovarian cancer. All data were expressed as mean ± SD (n = 3). *p < .05, **p < .01, ***p < .001 versus HOSE group, ## p < .01 versus HC group. mRNA, messenger RNA; RT‐qPCR, reverse transcription‐quantitative polymerase chain reaction; TCGA, The Cancer Genome Atlas
Fig 2: miR-378a-3p negatively regulates PDIA4. Ovarian cancer cells OVCAR3 and SKOV3 were transfected with miR-378a-3p mimic, miR-378a-3p inhibitor or negative control, respectively. A, PDIA4 mRNA expression in OVCAR3 and SKOV3 cells was determined by RT-qPCR. B, PDIA4 protein expression was detected by Western blot assay. C, The online software Starbase was searched to predict the targeting sites of miR-378a-3p and PDIA4 and the designed PDIA4 mutation sites. The mimic NC or miR-378a-3p mimic, or inhibitor NC or miR-378a-3p inhibitor with WT-PDIA4 or MT-PDIA4 were transfected or cotransfected into HEK-293T cells. D, The luciferase activity was detected by dual-luciferase reporter gene assay. E and F, RIP assay further examined the targeting relationship between miR-378a-3p and PDIA4 in OVCAR3 and SKOV3 cells. All data were expressed as mean ± SD (n = 3). *p < .05, **p < .01, ***p < .001. mRNA, messenger RNA; RT-qPCR, reverse transcription-quantitative polymerase chain reaction
Fig 3: ERp57 down-regulation inhibits HBV entry.(A) Intracellular (upper panels) and cell-surface (lower panels) staining of ERp46, ERp57, and ERp72 protein disulfide isomerase (PDI) members. Huh7-NTCP cells were subjected to flow cytometry analysis, in order to evaluate the expression of the indicated PDIs. Cells stained with secondary antibody only (no primary) were used to provide the background of flow cytometry analyses. (B) Hepatitis delta virus (HDV) or (C) hepatitis B virus (HBV) particles were used to infect Huh7-NTCP cells in which the indicated PDIs were down-regulated by lentiviral vectors carrying shRNA (see Figure 7—figure supplement 1 and Figure 7—figure supplement 2). Naive Huh7-NTCP cells were used as controls. Infected cells were grown for 7 days before total intracellular RNA or DNA was purified. The results of HDV RNA and HBV DNA quantification by quantitative reverse transcription PCR (RTqPCR) and quantitative PCR (qPCR), respectively, are expressed after normalization with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) RNAs as means ± SD (N = 3) per ml of cell lysates containing 106 cells. (D) Huh7 ‘donor’ cells co-expressing HBV glycoproteins (GPs) and a luciferase marker gene driven by the HIV-1 promoter were co-cultured with Huh7-NTCP-tat ‘indicator’ cells that express HIV Tat protein in which the indicated PDIs were down-regulated by lentiviral vectors carrying shRNA. After 24 hr, the cells were treated at pH 4 or pH 7 for 3 min. The luciferase activity induced by the fusion between donor and indicator cells was measured 24 hr later. Fusion mediated by HBV GPs at pH 7 with naive Huh7-NTCP-tat cells (Ctrl) was taken as 100%. A control plasmid that does not allow GP expression (Empty) was used to determine the background of luciferase expression. The bars represent the means (N = 3). Error bars correspond to standard deviations.Figure 7—source data 1.ERp57 down-regulation inhibits HBV entry.The values correspond to the data expressed in the graphs displayed in Figure 7B–D.
Fig 4: miR‐378a‐3p contributes to ovarian cancer cell growth by suppressing PDIA4 expression. Ovarian cancer cells OVCAR3 and SKOV3 were transfected withpcDNA3.1, pcDNA3.1‐PDIA4, mimic NC, or miR‐378a‐3p mimic, respectively. A and B, Viability of OVCAR3 and SKOV3 cells was measured by MTT assay. C and D, Invasion ability of OVCAR3 and SKOV3 cells was detected by Transwell assay. E–G, Migration ability of OVCAR3 and SKOV3 cells was detected by scratch test. All data were expressed as mean ± SD (n = 3). *p < .05, **p < .01. MTT, 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5 diphenyltetrazolium bromide
Fig 5: Uterine gene and protein expression. (a) Uterine tissue gene expression presented as a fold change compared to young mice (n = 10 per group). (b) Protein expression of SOD1 and PDIA4 in uterine tissue by immunohistochemistry. The black arrows point to glandular epithelium; the stars indicate the lumen surrounded by the luminal epithelium (n = 5 per group). Scale bars: 1 mm for the HE images and 500 μm for the IHC images. The gene expression data were analyzed by ordinary one-way ANOVA with Tukey’s multiple comparisons when the overall effect p < 0.05. * p < 0.05 compared to young mice.
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