Fig 1: CD73 is a direct target for miR-30a-5p. a The 3'-UTR of CD73 contains a complementary matching region of miR-30a-5p through predictive analysis by bioinformatics websites. b Transfection of miR-30a-5p mimics or inhibitors was performed, and the efficiency of transfection was examined by qPCR. c, d mRNA and protein expression levels of CD73 in cell lines transfected with miR-30a-5p mimics or inhibitors. e Luciferase activity of the construct containing the CD73 3'-UTR reporter gene in 293 T cells co-transfected with the miR-30a-5p mimics or inhibitors. f The expression level of miR-30a-5p in pancreatic cancer tissue was detected by FISH. Ki-67 was used as cancer cell marker. g miR-30a-5p expression in tumor tissue was lower than that in normal tissue (n = 10, p < 0.05). h The expression levels of miR-30a-5p in pancreatic cell lines with the normal pancreatic duct epithelial cell line HPDE6-C7 as control. i The expression levels of TNFR2, p-AKT, and p-ERK were analyzed by western blotting in cell lines treated with miR-30a-5p mimics or negative control. Data are expressed as mean ± SEM (n = 3). *p < 0.05
Fig 2: Comparative study of purified fruit bromelain (PFB) with bromelain complex. Western blotting analysis of the effects of purified PFB (A), stem bromelain complex (B), and fruit bromelain complex (C) on the protein expression of TNFR1 and TNFR2. Data are expressed as the mean ± SD. Values in normal control (NC) group are set to 100% and other values are given relative to those in the NC group: **P < 0.01 compared with the NC group; #P < 0.05 and ##P < 0.01 compared with LPS control group; &&P < 0.01 compared with as indicated (n = 6 experiments).
Fig 3: Proposed model for the interaction between miR-30a-5p, CD73, and TNFR2 and inhibition of the AKT/ERK pathway to induce G0/G1 phase arrest. Knockdown of CD73 inhibits TNFR2 expression, thus leading to inhibition of AKT/ERK signaling pathway. miR-30a-5p directly targets the CD73 3'-UTR and negatively regulates CD73 and downstream pathways
Fig 4: Differential gene expression analysis by RNA sequencing and validation by real-time PCR and western blot analysis. Heatmap of hierarchical clustering of top 30 most significant differentially expressed genes in OA-MSC vs. OAC (A), and NCSC (B). RNAseq data of OA-MSC2 and NCSC3 were utilized (p < 0.05). Green arrows: chondrogenic genes; red arrows: cytokine genes; brown arrows: fibrosis genes. (C) Heatmap of a group of highly expressed genes in OA-MSC in comparison to OAC and NCSC. The normalized transcript copy number of each gene was shown in triplicated RNA samples. RNAseq data of OA-MSC2 and NCSC3 were utilized (p < 0.05). Validation of RNAseq data by real-time RT-PCR analysis of the group of four highly expressed genes in OA-MSC (D,E). (D) RNA samples were isolated from primary human OA articular cartilage cell cultures (OAC and OA-MSC) at different cell passages as indicated. The information of five OA patients is as follows. Patient 1: Female, Age 56 years; Patient 2: Female, Age 75 years; Patient 3: Female, Age 85 years; Patient 4: Female, Age 73 years; Patient 5: Male, Age 66 years. The value of each RNA sample group = mean ± SD. N = 3 for all groups. **p-value < 0.01. (E) RNA samples were isolated from cell culture of NCSC and OA-MSC cell lines. Real-time RT-PCR data of NCSC1 and five OA-MSC cell lines were utilized as indicated. The value of each RNA sample group = mean ± SD. N = 3 for all groups. ***p-value < 0.001. (F) Western Blot analysis of TNFR2 protein expression in OAC, OA-MSC and various NCSC and OA-MSC cell lines at different cell culture passages as indicated. The data shown is representative of three separate analyses. (G) Quantification of western blot analysis of TNFR2 protein levels in OAC, NCSC, and OA-MSC in low and high passage cell culture. The pixels of TNFR2/b-ACTIN were measured by software Image-Pro Plus. The relative TNFR2 protein expression level of OAC Lower Passage was set as 1. T-test was used for statistical analysis between OA-MSC Low Passage group and OA-MSC High Passage group. **p-value < 0.01.
Fig 5: Serial cell culture passage of OAC induces dedifferentiation into NCSC-like cells and subsequent senescence into pro-inflammatory OA-MSC. (A) FACS analysis of CD166 positive cells during OA chondrocyte passaging in OA-MSC culture medium or OAC medium. The percentage of CD166 positive cells in each passage (x-axis) was indicated in red. The increase of the number of large sized cells (y-axis), a characteristic of senescent cells, was indicated by brackets in Passage 5. The data is representative of three experiments. (B) Cell morphology change of OA chondrocytes during cell culture passages in OAC medium and OA-MSC culture medium. OAC changed its morphology from cuboidal shape typical of chondrocytes (aquamarine arrows) in early passages (2 and 3) to spindle shape typical of fibroblasts (gray arrows) in mid passage (4) to large cells with processes typical of senescent cells (yellow arrows) in late passage (5). Bar = 200 µm. (C) Expression of aging related genes during OAC culture passages in OAC medium and OA-MSC medium. mRNA levels of TNFR2, CXCL1, CXCL8, P16 were quantified by real-time RT-PCR. The value of each sample group = mean ± SD. N = 3 for all groups. *p-value < 0.05; ***p-value < 0.001. (D) The components of OAC medium and OA-MSC medium.
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