Fig 1: Down-regulated expression of miR-205–3p and up-regulated expression of CXCL11 in GC cellsA: miRNA quantitative RT-PCR analysis of miR-205–3p expression level in eight GC cell lines and one normal gastric cell line. B: Venn diagram of the numbers of overlapping miRNAs from different databases (Targetscan, miRDB, RNA22 and DIANA). C: The miR-205–3p seed sequence is complementary to the 3′-UTR of CXCL11 and is conserved in six different species. D: Quantitative RT-PCR analysis of CXCL11 expression level in eight GC cell lines and one normal gastric cell line. E: CXCL11 expression was analyzed by Western blot in eight GC cell lines and one normal gastric cell line. * p <0.05 and ** p <0.01.
Fig 2: miR-205–3p inducing senescence of gastric cancer cells and secreting SASP factors by regulating CXCL11 and inhibiting Akt activationA, B: AGS cells are transfected with miR-205 alone for 48 h or with addition of CXCL11 (100 ng/mL) after 24 h of transfection for another 24 h, or transfected with CXCL11 siRNA for 48 h. and cell lysates were collected for Western blot analysis. C, D: AGS cells are treated with LY294002 (an Akt inhibitor, 25 µM) for 2 h or transfected with miR-205 for 48 h, The protein levels of indicated genes were measured by Western blot. GAPDH was used as an internal control for the total protein measurement. E: mRNA levels of the indicated genes in AGS cells transfected with negative control or miR-205. Actin was used for normalizing the expression of mRNA. *P<0.05 and **P < 0.01.
Fig 3: Effects of miR-205–3p on cell cycle progression and cell senescence.A, B: Cell cycle analysis shows increase in the G1 phase of AGS cells overexpressing miR-205 as compared with negative control (miR-NC), while additional CXCL11(100 ng/ml) reverses the increase. C, D: SA-ß-gal staining was performed on miR-205, miR-205+CXCL11 and NC groups and light microscope cell images were acquired. The percentage of SA-ß-gal-positive cells is reported (D). ** p <0.01.
Fig 4: miR-205–3p inhibits proliferation and invasion and promotes apoptosis by regulating CXCL11 in gastric cancer cells.A: Putative miR-205–3p binding sequence on CXCL11 wild-type(WT) 3′-UTR and mutant 3′-UTR sequence that abolished binding. B: Luciferase assay shows decreases in reporter activity after co-transfection of CXCL11 wild-type(WT) 3′-UTR with miR-205–3p mimics in AGS cells. C: In CCK8 experiment, the growth rate of AGS cells after miR-205–3p transfection is reduced compared with the NC group(miR-NC), and significantly restores after adding CXCL11(100 ng/ml). D: miR-205–3p overexpression significantly inhibits the clone-forming ability of gastric cancer cells, and the inhibition is counteracted by adding CXCL11(100 ng/ml). E: Apoptotic analysis is performed on miR-205, miR-205+CXCL11 and NC groups. The percentage of apoptotic cells is reported. F: In Transwell invasion assay, miR-205–3p overexpression significantly reduces the number of invasive cells, and the reduction is counteracted by adding CXCL11(100 ng/ml). * p <0.05 and ** p <0.01.
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