Effects And Mechanisms Of Trop2 On Chemotherapy Resistance Of Gastric Cancer Cells

BACKGROUNDGastric cancer?GC?is a common malignant tumor in the digestive system,representing the fourth most common malignancy in the world.GC is also the second most common tumor and causes the second-most cancer deathes in our country.The great mass of Chinese GC patients have entered advanced stage at initial diagnosis due to atypical symptoms at early stage and undistributed GC prevention systems.Chemotherapy plays an important role in perioperative and palliative treatment to GC,but primary drug resistance or multidrug resistance is one of major factors affecting overall survival and life quality of GC patients.Human trophoblast cell surface antigen 2?Trop2/TACSTD2/M1S1/GA733-1?is a cell-surface glycoprotein originally identified in human placental trophoblastic tissue.Trop2 can regulate intracellular calciumion concentration and influence signal pathways like MAPK and IGF-1R.Our laboratory has found that GC tissues expresse high level of Trop2 compared with adjacent tissues of cancer,and GC patients with high Trop2 expression are related to poor prognosis.Trop2 is correlated with CREB,P27 and etc in function,which may influence the therapeutic effects of tamoxifen and gemcitabine.However,little is known about the role of Trop2 in GC chemotherapy resistance and the molecular mechanism it works.This study detected the effects of Trop2 on GC chemotherapy resistance by constructing Trop2 shRNA stable cell line,and explored the underlying molecule mechanisms.The study provides a theoretical basis for reversing GC drug resistance.METHODS1.The construction of stable cell line transfected with Trop2 shRNAWestern bolt was performed to detect the expressions of Trop2 in human gastric epithelial cell GES-1 and gastric cancer cells MGC803,BGC823,MKN45,MKN28,HGC27 and SGC7901.Trop2 short hairpin RNA?shRNA?was constructed.BGC823 cell line which expresses relative high expression of Trop2 was transfected with the lentivirus encoding shRNA against Trop2?marked with BGC823-shTrop2?and the scrambled shRNA lentivirus?marked with BGC823-shNC?;qRT-PCR,Western blot and immunofluorescence were performed to confirm the efficient of transfection;Transwell assay was performed to detect migration and invasion of GC cells.2.The effects of Trop2 on GC chemotherapy resistanceCCK-8 proliferation test was utilized to detect the effects of Trop2 downregulation on cell proliferation after DDP or daunorubicin treatment.Flow cytometry and Hoechst 33342 staining were performed to detect the effects of Trop2downregulation on cell apoptosis after DDP or daunorubicin treatment.Subcutaneous xenograft models were generated.Tumour growth curves and histological observation were performed to analysis the function of Trop2 in growth inhibition of tumours after chemotherapy in vivo;Immunohistochemical staining was performed to detect Trop2 expression of tumour tissues.3.The mechanisms of Trop2 in GC chemotherapy resistanceqRT-PCR?Western blot were performed to detect mRNA and protein expressions of resistance-related genes MRP1 and Topo??;Western blot was performed to detect variations in Notch1 signaling pathway.RESULTS1.Compared with BGC823-shNC and BGC823,BGC823-shTrop2 expresses low mRNA and protein levels of Trop2?P<0.05?;Migration and invasion of BGC823-shTrop2 were weaker than that of BGC823-shNC and BGC823?P<0.05?;2.BGC823-shTrop2 showed higher proliferation inhibition rates after DDP or daunorubicin treatment than these in BGC823-shNC,and the IC₅₀0 value of the BGC823-shTrop2 was lower than that in BGC823-shNC?P<0.05?;The chemotherapy-induced cell apoptosis rates in BGC823-shTrop2 were higher than that in BGC823-shNC?P<0.05?;Compared with BGC823-shNC groups,tumours in BGC823-shTrop2 group exhibited more obvious growth inhibition effects after DDP treatment in vivo.3.Trop2-knockdown induced decreasing mRNA and protein expressions of MRP1?P<0.05?,and the mRNA and protein expressions of Topo??in BGC823-shTrop2increased than that in BGC823-shNC?P<0.05?.The protein expression of Notch1 in BGC823-shTrop2 decreased than that in BGC823-shNC?P<0.05?.CONCLUSIONS1.The stable cell line BGC823-shTrop2 transfected with Trop2 shRNA was constructed successfully.2.Trop2 could enhance the tolerance of GC cells to chemotherapeutic drugs in vitro and reduce the inhibition effects of DDP to tumors in vivo.3.Trop2 could promote the expression of MRP1 by regulating Notch1 signaling pathway and inhibit the expression of Topo??,resulting in intensive GC chemotherapy resistance.