Fig 2: Downregulation of miR-362-5p inhibits the SW780 xenograft tumor growth. (A) A hairpin sequence containing the 100% complementary nucleotide sequence of miR-362-5p was constructed into pRNA-H1.1/Adeno vector. The SW780 cells were transfected with vector containing anti-miR-362-5p (2 µg) or miR-NC. Then transfected SW780 cells were selected using 300 µg/ml G418. The Balb/c nude mice were injected subcutaneously into the left flank with 1×107 SW780 cells stable transfected cells (100 µl PBS) to form xenograft tumors. The tumor volumes were measured on day 25. (B) The relative expression of miR-362-5p in xenograft tumor tissues from anti-miR-362-5p or miR-NC injected mice were analyzed by qRT-PCR. The expression was displayed as fold of NC. (C) The tumor growth curves were determined by assessing the tumor volumes every 3 days from day 7 to day 25 by measuring two perpendicular diameters and the tumor volumes were calculated according to the following formula: [(width)2 × length]/2. (D) The xenograft tumor weight was measured on day 25. (E) The expression levels of Ki-67 in the xenograft tumor tissues were determined using immunohistochemical assay. (F) The protein levels of QKI, Cyclin D, MacroH2A1.1, MacroH2A1.2, p27, c-Fos, PARP-1, and E2F1 in xenograft tumor tissues were detected by western blot. GAPDH was used as an internal control in western blot. *p < 0.05 vs. NC controls.

Fig 3: Regulation of CA2 and osteoblast differentiation-related proteins by HSYA. (A) The relative expression levels of CA2, OPG, RANK, RANKL, CTSK, MMP-9, c-fos, NFATc1, and TRAF6 proteins were detected by western blotting. (B–J) The gray values of CA2, OPG, RANK, RANKL, CTSK, MMP-9, C-FOS, NFATc1, and TRAF6 proteins were quantified by ImageJ software. “**” indicates p < 0.01, “*” indicates p < 0.05, and the difference was statistically significant when compared with the control group. Statistical significance. “#” indicates p < 0.05, and the difference was statistically significant in the HSYA treatment group compared with the model group.

Fig 4: The effects of transcription factor FOS on acute and persistent FMDV infection. The RNA and protein expression levels of FOS in BHK-21 after transfected with shRNA were verified by RT-qPCR (A) and Western blot (B). The infectious FMDV titers in BHK-21 cells (C) and BHK-VEC cells (D) were verified by TCID50 assay. The RNA and protein expression levels of FOS and FMDV 3D in BHK-21 after downregulation of FOS in BHK-21 cells were verified by RT-qPCR (E) and Western blot (F). The RNA and protein expression levels of FOS and FMDV 3D in BHK-21 after upregulation of FOS in BHK-21 cells were verified by RT-qPCR (G) and Western blot (H). The RNA and protein expression levels of FOS and FMDV 3D in BHK-VEC after downregulation of FOS in BHK-VEC cells were verified by RT-qPCR (I) and Western blot (J). The RNA and protein expression levels of FOS and FMDV 3D in BHK-VEC after upregulation of FOS in BHK-VEC cells were verified by RT-qPCR (K) and Western blot (L). Sh, shRNA; OE, over expression; NC, negative control. GAPDH was served as the internal reference to normalize data. Three independent replicates were conducted for each sample. Data are expressed as the means ± SE (n = 3). *P < 0.01.

Fig 5: Characterization of the functional enrichment response during FMDV-persistent infection. Bar (A) and dot (B) diagram of the GO enrichment of BHK-Op. The dot from small to big corresponds to a small to large amounts of gene expression, and the color from blue to red corresponds to a low to high abundance of gene expression. Chord diagram (C) showed the relationship between genes and individual GO items. Bar (D) and dot (E) diagram of the KEGG enrichment of BHK-Op. The dot from small to big corresponds to a small to large amounts of gene expression, and the color from blue to red corresponds to a low to high abundance of gene expression. Scatterplot of gene dispersion (F) labeled the top fifteen genes with the greatest amount of variation. In two dimensions, the expression levels of FOS (G), JUN (H), and four other randomly selected genes (I) were displayed. The color light to dark corresponds to a low to high abundance of gene expression.