| ObjectiveTo explore the role of gastrokine 2(GKN2)on migration and invasion in human gastric cancer cell line SGC7901,and investigate the regulatory effect of GKN2 on the JAK2/STAT3 pathway.Methods1.The constructed GKN2 eukaryotic vector plasmid was transiently transfected into human gastric cancer SGC7901 cells,and then the transfection efficiency was observed under the fluorescent inverted microscope,and Western Blot was performed to verify the success of GKN2 transfection.2.The effects of GKN2 transfection on migration and invasion of SGC7901 cells were analyzed on using MTT assay,wound-healing assay,transwell migration,and transwell invasion assay.3.Western Blot was performed to observe the expression of JAK2,STAT3,p-JAK2,and p-STAT3 after GKN2 transfection.4.The expression of GKN2 in serum of the patients with gastric cancer and healthy controls was analyzed by ELISA.Results1.The transfection efficiency of the cells was observed under fluorescence microscope.No fluorescence was expressed in the untransfected group.The green fluorescence could be seen clearly in the empty vector group and the GKN2 transfection group.The transfection efficiency of the empty vector group and GKN2 transfection group was approximately 91% and 90%,respectively.Western Blot experimental results showed that the relative expression of GKN2 protein in GKN2 vector transfected group(1.6678 ± 0.0008)was significantly increased compared with untransfected(0.2290 ± 0.0007)and empty vector group(0.2210 ± 0.0008)(P<0.05).It was suggested that GKN2 was transfected successfully.2.MTT showed that compared with the non-transfected group and the empty vector group,the transfected group with GKN2 could inhibits the proliferation of SGC7901 at 24 hrs,48hrs and 72 hrs,and its inhibitory effect was time-dependent(P < 0.05).3.The wound-healing assay show that compared with untransfection group(71.46±15.50)and empty vector group(70.63±9.05),the cell migration relative distance decreased significantly(P < 0.05)in SGC7901 cells with GKN2 vector transfected group(20.03±6.35).4.Transwell migration assay showed that the migration of cell number in GKN2 vector transfected group(37±3)was significantly lower than that in untransfection group(75±7)and empty vector group(94±6)(P < 0.05).Similarly,transwell invasion assay showed that the invasion of cell number in transfected GKN2 vector transfected group(10±2)was significantly lower than that in untransfection group(55±3)and empty vector group(58±5)(P < 0.05).5.Western Blot showed that the expression of JAK2 protein(0.36030 ± 0.0005)in GKN2 vector transfected group was significantly lower than that in the untransfected group(0.6462 ± 0.006)and empty vector group(0.6661 ± 0.0005)(P<0.05);p-JAK2 was expressed in GKN2 vector transfection group(0.1271 ± 0.0058)was significantly down-regulated(P<0.05)compared with the untransfected group(0.2014 ± 0.0051)and the empty vector group(0.2458 ± 0.0053).The expression of STAT3 protein after GKN2 transfection(0.4934 ± 0.0006)was significantly lower than that of the untransfected group(0.9115 ±0.0009)and empty vector group(0.9611 ± 0.0008)(P<0.05);similarly,with the untransfected group(0.6575 ± 0.0008)Compared with the empty vector group(0.6984 ± 0.0006),GKN2 transfected(0.3894± 0.0005)inhibited the expression of p-STAT3(P<0.05).6.The ELISA results showed that there was no difference in serum GKN2 concentration between the gastric cancer group and the normal control group(P=0.241).Conclusion1、GKN2 overexpression inhibited the migration and invasion of SGC7901 cell line,and was related to the blocking of JAK2/STAT3 pathway.2、GKN2 is not a potential biomarker for gastric cancer. |
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