The role of Gastrokine 2 in regulating the JAK2/STAT3 pathway in the migration and invasion of gastric cancer
Objective To investigate the role of Gastrokine 2(GKN2)in gastric cancer(GC)and its associated molecular mechanisms.Methods Immunohistochemistry was used to detect the expression of GKN2 and TFF1 in 90 gastric cancer tissues(GC),48 adjacent gastric tissues(PT),and 22 distal gastric mucosa tissues(DGM).A GKN2 overexpression vector was constructed and transfected into human gastric cancer cells MKN28 and SGC7901.Transfection efficiency was validated by qRT-PCR and Western blot.The experiment included three groups:GKN2 group,NC group,and blank group.CCK-8,transwell migration and invasion assays were used to determine the cell proliferation,migration,and invasion abilities,and Western blot was employed to observe changes in JAK2,STAT3,p-JAK2,and p-STAT3 protein expression after GKN2 transfection.Results The expression of GKN2(6.67%)and TFF1(21.11%)was down-regulated in gastric cancer tissues(P<0.05)compared with paracancerous gastric tissues(GKN2,43.75%;TFF1,62.50%)and distal gastric mucosal tissues(GKN2,86.36%;TFF1,81.82%);however,there was no correlation between GKN2 and TFF1 expression in gastric cancer tissues(r≈0.23,P=0.074).Compared with the NC group(0.25±0.03)and the blank group(0.24±0.03),the OD570 of GKN2-transfected MKN28 cells decreased to(0.15±0.02)after 24 h,and the OD570 of GKN2-transfected MKN28 cells(0.22±0.06)after 48 h was also lower than the corresponding OD570 of the NC group(0.49±0.06)and the blank group(0.44±0.02)at 72 h(P<0.05),and OD570(0.25±0.05)after 72 h was reduced(P<0.05)compared with the corresponding NC group(0.65±0.21)and blank group(0.63±0.03).OD570(0.721±0.014)after 24 h of GKN2 transfection in SGC7901 cells was lower than that in the NC group(1.352±0.168)and blank group(1.381±0.168),and OD570(0.674±0.028)after 48 h was also lower than that of the corresponding NC group(1.459±0.211)and blank group(1.565±0.351),and the OD570 of GKN2 after 72 h was lower than that of the NC group(1.745±0.194)and blank group(1.792±0.385)decreased(P<0.05).Transwell migration assay revealed that the number of cancer cells crossing the membrane in MKN28 cells in the GKN2 transfected group(26±5.01)was significantly lower than that in the NC group(109±7.10)and the blank group(110±4.04)(P<0.05);compared with that in the NC group(94±6.00)and the blank group(75+7.05),high GKN2 expression significantly reduced the number of cells crossing the membrane(37±3.11)(P<0.05);Transwell invasion assay confirmed that GKN2 high expression significantly decreased the number of MKN28 cells crossing the stromal gel(28±4.10)compared with the NC group(67±5.02)and the blank group(66±5.16)(P<0.05);and the number of cancer cells crossing the stromal gel in the GKN2 transfected group in the SGC7901 cells was much lower(10±2.06)than that in the NC group(58±5.13)and blank group(55±3.17)(P<0.05).Western blot results showed that high expression of GKN2 significantly down-regulated the expression levels of total JAK2 and STAT3 proteins,as well as the phosphorylated forms of p-JAK2 and p-STAT3 in MKN28 and SGC7901 cells(P<0.05).Conclusion High expression of GKN2 inhibits gastric cancer cell migration and invasion by blocking the JAK2/STAT3 pathway.
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