| Objective:To study the effect of activation and blockade of transforming growth factor-beta(TGF-β)pathway on the proliferation and invasion of human gastric cancer cell line HGC-27 in vitro and in vivo,and to explore the relationship of this pathway to the Slug and E-cad gene expression.Methods:(1)Part of in vitro experiment: after culturing the human gastric cancer HGC-27 cell line,it was divided into: blank control group,agonist group(25ng/m L TGF-β1),blocker group(2μg/m LSB431542),agonist+ low concentration blockers group(25ng/m LTGF-β1+2μg/m LSB431542)and agonist + high concentration blockers group(25 ng/m LTGF-β1+4μg/m L SB431542).Transwell was used to detect the invasion ability of cells in each group;CCK8 was used to detect the proliferation activity of cells in each group,and Elisa was used to detect the protein expression of epithelial-mesenchymal transition-related genes Slug and E-cad in the supernatant after cell lysis.(2)Part of in vivo experiment: after successful establishment of a subcutaneous tumor model of human gastric cancer nude mice,the mice were divided into 5 groups:(1)blank control group;(2)agonist group: subcutaneous injection of TGF-β1(1μg/mice);(3)blockers group:intraperitoneal injection of SB431542(10mg/kg);(4)agonist+low concentration blockers group:subcutaneous injection of TGF-β1(1μg/mice)+intraperitoneal injection of SB431542(10mg/kg);(5)agonist+ high concentration blockers group:subcutaneous injection of TGF-β1(1μg/mice)+intraperitoneal injection of SB431542(15mg/kg);6 nude mice in each group,after feeding for 24 days,3 nude mice in each group were anesthetized and killed,and tumor tissues were taken out and placed in nitrogen for later use.The remaining 3nude mice continued to be fed until cachexia appeared due to rapid weight loss.The time required for cachexia and the tumor mass and volume were recorded.RT-PCR,Elisa and Western blot were used to detect the m RNA and protein expression of Slug and E-cad genes in all tumor tissues;tumor tissue was digested with trypsin to extract primary cells,CCK8 was used to detect the proliferation activity of primary cells,and Elisa was used to detect the protein expression of Slug and E-cad genes in primary cells.Results:In vitro experiments:(1)After using the agonist TGF-β1 to activate the TGF-βpathway,the proliferation and invasion abilities of human gastric cancer HGC-27 cells were stronger than those of the other groups(P<0.05).The expression level of Slug protein was higher than that of other groups(P<0.05),while the expression level of E-cad protein was lower than that of other groups(P<0.05).(2)After blocking the TGF-β pathway with the blockers SB431542,the proliferation and invasion abilities of human gastric cancer HGC-27 cells were weaker than those of the other groups(P<0.05),and the expression level of Slug protein in the cells detected by Elisa was lower than that of the other groups(P<0.05),while the expression level of E-cad protein was higher than that of the other groups(P<0.05).(3)In the agonist +high-concentration blockers group and the agonist + low-concentration blockers group,the proliferation,invasion ability,Slug and E-cad protein expression levels of the two groups of cells were compared with each other.Compared with the blank control group,the difference was not statistically significant(P>0.05).In vivo experiment:(1)After injecting agonist TGF-β1 to activate the TGF-βpathway in nude mice,the tumor volume and mass were larger than those of the other groups(P<0.05),and the time of cachexia in nude mice was longer than that in the other groups(P<0.05),the m RNA and protein expression levels of Slug gene in tumor tissues detected by RT-PCR,Elisa and WB were higher than those in the other groups(P<0.05),while the m RNA and protein expression levels of E-cad gene lower than the other groups(P<0.05).Tumor tissue blocks were digested with trypsin,primary cells were cultured,and Slug and E-cad protein expressions in tumor cells were detected by Elisa.The expression level of cad protein was lower than other groups(P<0.05).The proliferation ability of primary cells extracted from tumor tissues in each group was detected by CCK8,and its proliferation activity was higher than that of other groups within 24 hours(P<0.05).(2)After blocking the TGF-β pathway with the blockers SB431542,the tumor volume and mass were smaller than those of the other groups(P<0.05),and the time of cachexia in nude mice was later than that in the other groups(P<0.05).The m RNA and protein expression levels of Slug gene in tumor tissue detected by RT-PCR,Elisa and Western blot were lower than those in the other groups(P<0.05),and the protein and m RNA expression levels of E-cad were higher than those in the other groups(P<0.05).Primary cells were cultured after trypsinization of the tumor mass,and Elisa was used to detect the protein expression of Slug and E-cad in tumor cells.The protein expression level of Slug gene was lower than that of the other groups(P<0.05),and the expression level of E-cad protein was higher than that of other groups(P<0.05).The proliferation ability of primary cells extracted from tumor tissues in each group was detected by CCK8,and its proliferation activity was lower than that of the other groups within 24 hours(P<0.05),and after 48 hours,there was no significant difference compared with the blank control group(P>0.05).(3)In the agonist+high-concentration blockers group and the agonist + low-concentration blockers group,tumor mass and volume,time required for cachexia in nude mice,tumor tissue and primary cells,The m RNA and protein expression levels of Slug and E-cad in the tissue were not significantly different from those in the blank control group(P>0.05).The proliferation ability of primary cells extracted from tumor tissues in each group was detected by CCK8.Within 48 hours,the proliferation activity of primary cells was not significantly different from that of the blank control group(P>0.05).Conclusions:1.Activation of TGF-β pathway can lead to the increase of Slug m RNA and protein expression in human gastric cancer HGC-27 cells and transplanted tumor tissue,and the inhibition of E-cad gene m RNA and protein expression.2.After activating the TGF-β pathway,the high expression of Slug gene can promote the occurrence of EMT by inhibiting the expression of E-cad gene,and enhance the proliferation and invasion ability of human gastric cancer HGC-27 cells in vitro and in vivo.3.After activating the TGF-β pathway,the time required for cachexia in nude mice with gastric cancer xenografts with high Slug gene expression was shortened.suggesting that Slug can be used to judge the development of gastric cancer to a certain extent. |
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