Research Paper Volume 13, Issue 16 pp 20302—20318

Regulation of follistatin-like 3 expression by miR-486-5p modulates gastric cancer cell proliferation, migration and tumor progression

FSTL3 overexpression or knockdown modulates gastric cancer cell viability, proliferation and migration. (A) Western blot analysis of FSTL3 protein levels in GES1, MGC-803, SGC-7901, BGC-823, HGC-27 and AGS gastric cell lines; blotting for the housekeeping protein β-actin was used as a loading control in this experiment. (B) Analysis of relative FSTL3 mRNA levels in GES1, MGC-803, SGC-7901, BGC-823, HGC-27 and AGS gastric cell lines using qRT-PCR (see Materials and Methods). (C) Overexpression of FSTL3 in gastric cancer cell line SGC-7901 monitored by Western blotting and qRT-PCR. (D) FSTL3 knockdown in gastric cancer cell line MGC-803 monitored by Western blotting and qRT-PCR. (E) FSTL3 overexpression (SGC-7901) or knockdown (MGC-803) cell lines were analyzed for cell viability compared to controls. (F) FSTL3 overexpression (SGC-7901) or knockdown (MGC-803) cell lines were assayed for cell proliferation using EdU incorporation. (G) Wounded cell monolayer assays on FSTL3 overexpression (SGC-7901) or knockdown (MGC-803) cell lines (48h). (H) Transwell cell migration assay on FSTL3 overexpression (SGC-7901) or knockdown (MGC-803) cell lines followed by staining of Transwell filters to detect migrated cells. (I) FSTL3 knockdown and effects on MGC-803 cell colony formation. Quantification in panels (B–I) were carried out as described in Materials and Methods and compared to control cell lines. Error bars indicate +SEM; significance indicated by asterisks, *PPP

Figure 3. FSTL3 overexpression or knockdown modulates gastric cancer cell viability, proliferation and migration. (A) Western blot analysis of FSTL3 protein levels in GES1, MGC-803, SGC-7901, BGC-823, HGC-27 and AGS gastric cell lines; blotting for the housekeeping protein β-actin was used as a loading control in this experiment. (B) Analysis of relative FSTL3 mRNA levels in GES1, MGC-803, SGC-7901, BGC-823, HGC-27 and AGS gastric cell lines using qRT-PCR (see Materials and Methods). (C) Overexpression of FSTL3 in gastric cancer cell line SGC-7901 monitored by Western blotting and qRT-PCR. (D) FSTL3 knockdown in gastric cancer cell line MGC-803 monitored by Western blotting and qRT-PCR. (E) FSTL3 overexpression (SGC-7901) or knockdown (MGC-803) cell lines were analyzed for cell viability compared to controls. (F) FSTL3 overexpression (SGC-7901) or knockdown (MGC-803) cell lines were assayed for cell proliferation using EdU incorporation. (G) Wounded cell monolayer assays on FSTL3 overexpression (SGC-7901) or knockdown (MGC-803) cell lines (48h). (H) Transwell cell migration assay on FSTL3 overexpression (SGC-7901) or knockdown (MGC-803) cell lines followed by staining of Transwell filters to detect migrated cells. (I) FSTL3 knockdown and effects on MGC-803 cell colony formation. Quantification in panels (BI) were carried out as described in Materials and Methods and compared to control cell lines. Error bars indicate +SEM; significance indicated by asterisks, *P<0.05, **P<0.01, ***P<0.001; number of experiments, n=3.