| Objective: 1. To observe the expressions of tumor-associated calcium signal transducer-2(TROP-2)and epithelial-mesenchymal transition(EMT)associated proteins(E-cadherin, vimentin, etc.)as well as matrix metalloproteinase-9(MMP-9)in colorectal cancer tissue samples, and to investigate their relationships as well as their clinical significance. 2. To observe the effect of TROP-2 gene overexpression on cell proliferation, migration, invasion and EMT of colon tumor cell line SW480. 3. To explore the potential mechanisms of TROP-2 mediated EMT.Methods: 1. Eighty colorectal cancer patients with a follow-up period longer than 5 years were consecutively included and the resected colorectal cancer tissue samples were collected. All cases were not treated with chemotherapy, radiotherapy and targeted therapy before operation. The expression levels of TROP-2, EMT associated proteins and MMP-9 were detected by immunohistochemistry. The correlations between the expressions of these molecules and clinicopathological parameters were analyzed. The relationship between the expression of TROP-2 and EMT associated proteins as well as MMP-9 was further explored. 2. SW480 cells infected with TROP-2 lentivirus vector served as TROP-2 lentivirus group. SW480 cells without any pretreatment served as control group. SW480 cells infected with empty vector served as empty vector control group. Each group of the cells was collected and the expressions of TROP-2,E-cadherin,vimentin and MMP-9 m RNA and protein level were measured by real-time PCR and Western Blot, respectively. The effect of the overexpression of TROP-2 on SW480 cell morphology was observed under microscope. The capability of cell clone formation, cell migration and cell invasion was measured by plate clony formation assay, wound healing assay as well as Transwell experiment, respectively. 3. To further explore the potential mechanisms of TROP-2, SW480 cells was either treated with NF-κB inhibitor pyrrolidine dithiocarbamate(PDTC)or not after TROP-2 overexpression, which was respectively served as PDTC group or TROP-2 lentivirus control group. The expressions of E-cadherin,vimentin and MMP-9 m RNA level and protein level were detected by real-time PCR and Western Blot, respectively.Results: 1. In colorectal cancer tissues, the rates of positive expression of TROP-2, E-cadherin, vimentin and MMP-9 were 81.25%, 16.25%, 48.75% and 72.5%, respectively. The expression of TROP-2 was significantly correlated with TNM staging,the depth of tumor invasion,lymph node metastasis and 5-year survival rate(P < 0.05). E-cadherin expression was correlated with tumor location, the depth of tumor invasion and 5-year survival rate(P < 0.05). Vimentin expression was correlated with TNM staging, the depth of tumor invasion, lymph node metastasis and 5-year survival rate(P < 0.05). MMP-9 expression was correlated with the depth of tumor invasion, lymph node metastasis, and 5-year survival rate(P < 0.05). In colorectal cancer tissues, the expression of TROP-2 was positively correlated with the expression of vimentin and MMP-9(r = 0.276, P < 0. 05;r = 0.421, P < 0. 05), respectively, while the expression of TROP-2 was negatively correlated with the expression of E-cadherin(r =-0.628, P < 0. 05). 2. Compared with the control group and the empty vector control group, TROP-2 lentivirus group showed significantly elevated level of TROP-2 m RNA and protein. Morphology study demonstrated that TROP-2 lentivirus group had a long and irregular cell type with fusiform, while the control group and the empty vector control group had a cobblestone or polygon cell type. The morphological change suggested the possibility of EMT. Real-time PCR and Western Blot analysis showed that, the expressions of vimentin and MMP-9 m RNA level and protein level were increased significantly in TROP-2 lentivirus group, while the expressions of E-cadherin m RNA level and protein level were significantly decreased in TROP-2 lentivirus group, compared with control group and empty vector control group, respectively. In plate clony formation assay, the colony formation was 75±6, 76±5 and 119±3 in the control group, empty vector control group and TROP-2 lentivirus group, respectively(P < 0.05). In wound healing assay, the scratch gap was 389 ± 5 μm, 395 ± 3μm and 158±5 μm in the control group, empty vector control group and TROP-2 lentivirus group, respectively(P < 0.05). In the transwell assay, the cell numbers migrated through permeable membrane were 89±4, 92±5 and 139±6 in the control group, empty vector control group and TROP-2 lentivirus group, repectively(P < 0.05). 3. Real-time PCR and Western Blot analysis showed that, expressions of vimentin and MMP-9 m RNA level and protein level were decreased significantly in PDTC group,while expressions of E-cadherin m RNA level and protein level were significantly increased in PDTC group, compared with TROP-2 lentivirus control group.Conclusions: 1. Expression of TROP-2 and EMT associated proteins in colorectal cancer tissues are closely related with tumor invasion and metastasis. 2. TROP-2 may potentiate the migration and invasion capability of human colon cancer cells via the induction of EMT. 3. NF-κB/MMP-9 signal is one of the vital pathways in colon cancer cells through which TROP-2 regulates EMT. |
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