TROP2 Affects Gastric Cancer Through Endoplasmic Reticulum Ca²⁺ And Fe²⁺ Exchange Molecular Mechanism Of Invasion

Objective Gastric cancer（GC）is one of the most common malignant tumors in China.According to statistics,in 2020,the number of new cases of gastric cancer and the number of deaths from gastric cancer ranked third among malignant tumors in China.Most patients are already in advanced stage when they are diagnosed,with poor prognosis and a low five-year survival rate.Under the action of phospholipase C（PLC）,the serine site（303 site）of the cytoplasmic tail region of human trophoblast cell surface glycoprotein（TACSTD2/TROP2）binds to Phosphatidy linositol（4,5）bisphosphate（PIP2）,thereby promoting the progression of malignant biological behaviors such as tumor proliferation,invasion,and metastasis.In this study,we investigated the effect of TROP2 on the malignant biological behavior of gastric cancer and the feasibility of TROP2 as a new target for gastric cancer from a cytological perspective.We also elucidated the molecular mechanism of TROP2 affecting the invasion of gastric cancer through endoplasmic reticulum calcium and iron ion exchange.Methods Western blot and RT-q PCR were used to detect the expression of TROP2 protein and m RNA in gastric cancer cells.Stable transfectant cell lines and controls were constructed in cells with relatively low expression of TROP2 and cells with relatively high expression of TROP2,named OE-TROP2,OE-Control,sh-TROP2,sh-Control,and the control was named NC.Western-Blot and RT-q PCR were performed to detect the protein and m RNA expression of TROP2 in the above cells.Transwell assays were performed to detect the invasion and migration ability of the above gastric cancer cells.The migration ability of the above gastric cancer cells was detected by wound healing assays.CCK8 assays were performed to detect the proliferation ability of the above gastric cancer cells.The concentration of IP3（inositol1,4,5-trisphosphate）in the above gastric cancer cell lines was detected by ELISA.Laser Confocal Scanning Microscopy（LCSM）was used to detect the fluorescence localization and fluorescence intensity of Fe²⁺and Ca²⁺in the endoplasmic reticulum of the above gastric cancer cells.Results TROP2 was highly expressed in gastric cancer cells.The expression level of TROP2 protein and m RNA in the OE-TROP2 group were significantly higher than those in the NC group and OE-Control group（P<0.01）,and there was no significant difference between the NC group and OE-Control group（P>0.05）.The expression level of TROP2 protein and m RNA in the sh-TROP2 group were significantly lower than those in the NC group and the sh-Control group（P<0.01）,and there was no significant difference between the NC group and the OE-Control group（P>0.05）.The invasion ability of the OE-TROP2group was higher than that of the NC group and the OE-Control group,and the difference was statistically significant（P<0.001）.There was no significant difference between the NC group and the OE-Control group（P>0.05）.The invasion ability of the sh-TROP2 group was lower than that of the NC group and the sh-Control group,and the difference was statistically significant（P<0.001）.There was no significant difference between the NC group and the OE-Control group（P>0.05）.After 24 hours,the migration ability of the OE-TROP2 group was significantly higher than that of the NC group and the OE-Control group（P<0.001）,and there was no significant difference between the OE-Control group and the NC group（P>0.05）.After 48 hours,the migration ability of the sh-TROP2 group was significantly lower than that of the NC group and the sh-Control group（P<0.001）,and there was no significant difference between the NC group and the sh-Control group（P>0.05）.After 96 hours,the cell growth inhibition rate of the OE-TROP2 group was lower than that of the NC group and the OE-Control group,and the difference was statistically significant（P<0.05）.There was no significant difference between the NC group and the OE-Control group（P>0.05）.After 96hours,the cell growth inhibition rate of the sh-TROP2 group was higher than that of the NC group and the sh-Control group,and the difference was statistically significant（P<0.05）.There was no significant difference between the NC group and the OE-Control group（P>0.05）.IP3 concentration levels were measured at 0,2,4,6,8,10,and 12h.The concentration of IP3 in the OE-TROP2 group was significantly higher than that in the NC group and the OE-Control group（P<0.01）,and there was no significant difference between the NC group and the sh-Control group（P>0.05）.The concentration of IP3 in the sh-TROP2group was significantly lower than that in the NC group and the sh-Control group（P<0.01）.There was no significant difference between the NC group and the sh-Control group（P>0.05）.Laser confocal microscopy further observed that the endoplasmic reticulum Ca²⁺fluorescence intensity of the OE-TROP2 group was higher than that of the OE-Control group and the NC group,and the difference was statistically significant（P<0.001）There was no significant difference between the OE-Control group and the NC group（P>0.05）.The fluorescence intensity of Fe²⁺in the endoplasmic reticulum of the OE-TROP2 group was lower than that of the OE-Control group and the NC group,and the difference was statistically significant（P<0.01）.There was no significant difference between the OE-Control group and the NC group（P>0.05）.The fluorescence intensity of endoplasmic reticulum Ca²⁺in the sh-TROP2 group was significantly lower than that in the OE-Control group and the NC group（P<0.01）,and there was no significant difference between the sh-Control group and the NC group（P>0.05）.The fluorescence intensity of Fe²⁺in endoplasmic reticulum in the sh-TROP2 group was significantly higher than that in the sh-Control group and the NC group（P<0.05,P<0.01）,and there was no significant difference between the sh-Control group and the NC group（P>0.05）.Conclusion TROP2 affects endoplasmic reticulum"Ca²⁺-Fe²⁺exchange"through regulating IP3 to promote gastric cancer invasion and metastasis.TROP2 overexpression can increase endoplasmic reticulum Ca²⁺content and decrease endoplasmic reticulum Fe²⁺content,which can promote migration and proliferation of gastric cancer cells and ultimately improve the invasive ability of gastric cancer cells.TROP2 knockdown reduces endoplasmic reticulum Ca²⁺content and increases endoplasmic reticulum Fe²⁺content,which can inhibit migration and proliferation of gastric cancer cells and ultimately reduce the invasive ability of gastric cancer cells.The development of TROP2-targeted therapy and rational combination with several anti-cancer drugs may be a new direction for future non-surgical precise guidance of individualized treatment of advanced gastric cancer.