Collapsin Response Mediator Protein 4 Isoforms Have Divergent Effects On Cell Proliferation, Migration, And Invasion In Gastric Cancer

Objective To study the expression of CRMP 4a and CRMP 4b in the gastric cancer tissues and cell lines, and analyze CRMP 4a and CRMP 4b functions in regulating gastric cancer cell occurrence and development.Methods Part One Thirty gastric cancer patients who underwent curative resection at Zhongnan Hospital of Wuhan University were enrolled in the study. The expression of CRMP 4a and CRMP 4b mRNA were detected by qPCR. The expression of CRMP 4a and CRMP 4b protein were detected by western blot. The expression of CRMP 4a and CRMP 4b protein in the gastric cancer cell lines BGC823, GC981, HGC27, MGC803, NCI N87 were detected western blot. Part two:CRMP 4a-overpression plasmid and CRMP 4b-shRNA was generated. For transfection,0.5 mL of lentiviral suspension and tumor cells were then incubated at 37?. And MGC803 and BGC823 cells were passaged twice per week with culture medium containing puromycin to select for cells overexpressing CRMP 4a. Part three:Cultured the stable cells. Cell proliferation was measured using MTT assay kit. Annexin V-FITC apoptosis detection and cell cycle detection kits were used to analyze the apoptosis rate and cell cycle distribution. Cell migration and invasion were assessed using a transwell assay. Clone formation were detected by plate colony. Cells adhesion were detected using FN/LN protein. Apoptosis-related protein were detected by western blot. Part four:Six-week old male athymic nude mice were subcutaneously injected in the right armpit region with 4×106 cells in 0.2 mL of PBS.Four groups of mice injected stable cells were tested. Tumor size was measured using calipers. Tumor volume was calculated using the formula (L×W2)/2, where L is the length and W is the width of the tumor.Results Part one A significant reduction in CRMP 4a mRNA and protein expression was identified in tumor tissue samples as compared to paired non-tumor tissue samples (P<0.05).A significant increase in CRMP 4b mRNA and protein expression was identified in tumor tissue samples compared to paired non-tumor tissue samples (P<0.05). CRMP 4a mRNA and protein expression is reduced in BGC823? GC981?HGC27?MGC803 and NCI N87 gastric cancer cells as compared to normal gastric epithelial cells (P<0.05);CRMP 4a mRNA and protein expression was lowest in MGC803 cells. CRMP 4b mRNA and protein expression is increased in the above gastric cancer cell lines as compared to normal gastric epithelial cells (P<0.05);CRMP 4b mRNA and protein expression was highest in BGC823 cells. Part two:CRMP 4a-overpression cell lines and CRMP 4b-shRNA transfected cells were successfully established. A significant increase in CRMP 4a mRNA and protein expression was identified in MGC803-CRMP 4a compared to MGC803-NC (P<0.05), whereas the expression of CRMP 4b had no significant change (P>0.05).A significant reduction in CRMP 4b mRNA and protein expression was identified in BGC823 sh-CRMP 4b compared to BGC823 sh-NC (P<0.05), but the expression of CRMP 4a had no significant change (P>0.05). Part three:CRMP 4b silencing significantly suppressed BGC823 cell proliferation, migration, invasion, and adhesion (P<0.05). CRMP 4b silencing did not significantly affect the percentage of apoptotic BGC823 cells and the protein expression of Bcl 2, Caspase 3, or Caspase 8 in BGC823 cells (P>0.05). CRMP 4a overexpression significantly suppressed proliferation, migration, invasion, and adhesion of MGC803 cells (P<0.05). CRMP 4a overexpression did not significantly affect the percentage of apoptotic BGC823 cells (P>0.05). Moreover, CRMP 4a overexpression did not affect the protein expression of Bcl2, Caspase 3, or Caspase 8 (P>0.05). Part four:The results showed that sh-CRMP 4b and CRMP 4a-overexpression can significantly inhibit the growth speed, volume and weight of gastric cancer (P<0.05).Conclusion The short CRMP 4 isoform, CRMP 4a, had a low expression level and acted as a tumor suppressor in gastric cancer. Conversely, the long CRMP 4 isoform, CRMP 4b, had a high expression level and acted as an oncoprotein in gastric cancer. These two isoforms of CRMP 4 exhibited opposing functions in regulating gastric cancer cell proliferation, migration, and invasion.