Abstract:

Objective To explore the mechanism of the long non-coding RNA （lncRNA） FGD5-AS1/miR-154-5p/WNT5A signaling pathway in regulating the proliferation and migration of paclitaxel （PTX）-resistant gastric cancer cells. Methods The PTX-resistant gastric cancer cell line HGC-27/PTX was divided into sh-NC group （transfected with FGD5-AS1 shRNA negative control），sh-FGD5-AS1 group （transfected with FGD5-AS1 shRNA），sh-NC+PTX group （transfected with FGD5-AS1 shRNA negative control and then incubated with 8 nmol/L PTX for 24 hours），sh-FGD5-AS1+PTX group （transfected with FGD5-AS1 shRNA and then incubated with 8 nmol/L PTX for 24 hours），miR-NC group （transfected with miR-154-5p mimic negative control），miR-154-5p mimic group （transfected with miR-154-5p mimic），si-NC group （transfected with WNT5A siRNA negative control），si-WNT5A group （transfected with WNT5A siRNA），and si-WNT5A+PTX group （transfected with WNT5A siRNA and then incubated with 8 nmol/L PTX for 24 hours）. The relative expression level of FGD5-AS1 was detected by quantitative real-time PCR，the median inhibition concentration （IC₅₀） of PTX on HGC-27/PTX cells was determined by the CCK-8 assay，the apoptosis ability of cells was detected by flow cytometry，and the migration and invasion abilities of cells were assessed using the Transwell chamber assay. The expression of LC3-Ⅱ/LC3-Ⅰ and P62 in cells was detected by Western blotting. The targeting relationships between FGD5-AS1 and miR-154-5p，as well as between miR-154-5p and WNT5A，were verified by dual-luciferase reporter gene assay. Results The IC₅₀ values of HGC-27/PTX cells in the sh-NC group and sh-FGD5-AS1 group were （81.10±1.46），（36.61±2.05） nmol/L，with a statistically significant difference （t=17.45，P<0.001）. The apoptosis rates of HGC-27/PTX cells in the sh-NC group，sh-FGD5-AS1 group，sh-NC+PTX group，and sh-FGD5-AS1+PTX group were （0.24±0.04）%，（6.41±0.90）%，（0.20±0.06）%，and （7.86±1.07）%，respectively，with a statistically significant difference （F=110.40，P<0.001）； there were statistically significant differences between the sh-NC group and sh-FGD5-AS1 group，as well as between the sh-NC+PTX group and sh-FGD5-AS1+PTX group （both P<0.001）. The numbers of cell migration in the four groups of HGC-27/PTX cells were 140.30±15.95，68.00±3.61，124.30±7.02，and 15.00±2.00，respectively，and the numbers of cell invasion were 115.70±6.11，51.33±4.62，114.70±11.24，and 12.33±3.22，respectively，with statistically significant differences （F=122.00，P<0.001； F=161.10，P<0.001）； there were statistically significant differences between the sh-NC group and sh-FGD5-AS1 group，as well as between the sh-NC+PTX group and sh-FGD5-AS1+PTX group （both P<0.001）. The LC3-Ⅱ/LC3-Ⅰ values of the four groups of HGC-27/PTX cells were 1.66±0.11，1.04±0.07，1.64±0.15，and 0.99±0.05，respectively，and the relative expression levels of P62 were 0.24±0.06，0.78±0.08，0.20±0.09，and 1.01±0.08，respectively，with statistically significant differences （F=36.31，P<0.001； F=79.68，P<0.001）； there were statistically significant differences between the sh-NC group and sh-FGD5-AS1 group，as well as between the sh-NC+PTX group and sh-FGD5-AS1+PTX group （both P<0.05）. The dual-luciferase reporter gene assay results showed that，miR-154-5p was a target gene of FGD5-AS1，and WNT5A was a target gene of miR-154-5p. The LC3-Ⅱ/LC3-Ⅰ values of HGC-27/PTX cells in the NC group，si-WNT5A group，si-WNT5A+PTX group were 2.21±0.09，1.96±0.06，and 0.96±0.06，respectively，the relative expression levels of P62 were 0.06±0.02，0.18±0.02，and 0.68±0.04，respectively，with statistically significant differences （F=245.90，P<0.001； F=378.40，P<0.001）； there were statistically significant differences between the si-NC group and the si-WNT5A group，as well as between the si-WNT5A group and the si-WNT5A+PTX group （both P<0.05）. Conclusions The lncRNA FGD5-AS1/miR-154-5p/WNT5A signaling pathway promotes the proliferation and migration of PTX-resistant gastric cancer cells by regulating autophagy.

Key words: Stomach neoplasms, Taxol, Drug resistance，multiple, Wnt-5a protein, Cell proliferation, Neoplasm metastasis