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@#[Abstract] Objective: To investigate the effects of over-expressing VASH1 on the malignant biological behaviors of human colorectal cancer cells. Methods: Lentivirus was packaged and transfected into human colorectal cancer cells SW680 and SW620 to construct an over-expressed VASH1 cell line, the untransfected cells were used as control. qPCR experiment and WB experiment were used to detect the over-expression effect of VASH1. The effects of VASH1 over-expressed on microangiogenesis, proliferation, colony formation and migration of colorectal cancer cells were detected respectively by tubule formation, CCK-8 assay, soft agar assay, Transwell assay and Wound healing assay in vitro. In addition, tumor growth and lung metastasis were detected in NOD-SCID mice subcutaneously injected with VASH1-overexpressing SW620 cells. Results: Successfully constructed SW480 and SW620 cells over-expressing VASH1. Compared with the control group, the abilities of microangiogenesis, proliferation, colony forming and migration were significantly reduced in colorectal cancer cells over-expressing VASH1 (P<0.05) in vitro. The abilities of subcutaneous tumor growth and lung metastasis of colorectal cancer cells over-expressing VASH1 were also significantly reduced (P<0.05) in vivo. Conclusion: Over-expression of VASH1 can suppress the malignant biological behaviors of human colorectal cancer cells.
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@#[Abstract] Objective: To study the effect of micheliolide (MCL) on the sensitivity of colorectal cancer cells to oxaliplatin (OxP) and its possible mechanism. Methods: HCT116 and LoVo cells were treated with 2 μmol/L MCL and 100 μmol/L OxP alone or in combination. The cell viability and colony forming ability in vitro were detected by CCK-8 and plate cloning formation assay, respectively. After being transfected with GFP-LC3 lentivirus, HCT116 cells were respectively treated with 2 μmol/L and 5 μmol/L MCL for 24 h. The aggregation of autophagy bodies in HCT116 cells induced by MCL was observed under fluorescence microscope. The effects of MCL on the expressions of LC3B-Ⅰ, LC3B-Ⅱ, p62 and STAT3 were detected by WB assay; the molecular docking model of MCL and STAT3 was constructed by Autodock version. Results: After the treatment of 2 μmol/L MCL combined with 100 μmol/L OxP, the activity of HCT116 and LoVo cells as well as the colony forming ability of HCT116 cells significantly decreased (all P < 0.01). After HCT116 cells were treated with 2 and 5 μmol/L MCL, the autophagy rate of cells in the treatment groups was significantly higher than that of the control group (all P < 0.01), the LC3B Ⅱ/Ⅰ ratio was 3.25 and 5.78 times that of the control group, the expression level of p62 was 25.5% and 9.8% of the control group, and the phosphorylation level of STAT3 was 2.18 and 3.87 times that of the control group. Molecular docking results showed that MCL might directly bind to STAT3 protein in vivo. Conclusion: MCL may enhance the sensitivity of colorectal cancer cells to OxP by promoting autophagy through STAT3 pathway.
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@#[Abstract] Objective: To observe the pyrolysis of colorectal cancer Lovo cells overexpressing Gasdermin E (GSDME) after the treatment with oxaliplatin. Methods: The expression level of GSDME gene in colorectal cancer Lovo cells and normal colorectal epithelial HCOEPIC cells was detected by qPCR. The GSDME-WT (wild-type GSDME) and GSDME-D270A (mutant GSDME) recombinant plasmids were constructed. The plasmids were packaged as lentivirus and then transfected into Lovo cells to construct Lovo cell line with stable and high expression of GSDME. Western blotting was used to detect the expression level of GSDME in cells of WT, D270A and empty vector groups. Different concentrations of oxaliplatin (0, 4, 8, 16, 32, 64 µg/ml) were applied to treat Lovo cells and HCOEPIC cells in WT and D270A groups, and the morphological changes of the cells were observed under a microscope. Results: The expression of GSDME in HCOEPIC cells was significantly higher than that in Lovo cells (P<0.01). GSDME-WT and GSDME-D270A plasmids with high GSDME expression and the corresponding Lovo cell lines were successfully constructed. Compared with the empty vector group, the expression level of GSDME in Lovo cells of WT and D270A groups were significantly increased (all P<0.05). Observation under the microscope showed that after being treated with 64 µg/ml oxaliplatin for 9 and 12 hours, the volume of Lovo cells and HCOEPIC cells in WT group gradually increased and “blistered” to one side and showed obvious pyrolysis phenomenon. The pyrolysis rate of cells in WT group was significantly higher than that of the control group without oxaliplatin treatment (Lovo cells: [7.405±1.010]% vs [3.441±0.401]%, P<0.05; HCOEPIC cells: [7.203±1.020]% vs [4.201±0.302]%, P<0.05). Conclusion: Oxaliplatin promotes the pyrolysis of colorectal cancer Lovo cells overexpressing GSDME gene.
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@# Objective: To investigate the effects of Wilms’tumor 1-associating protein (WTAP) on proliferation, migration and invasion of human lung adenocarcinoma A549 cells. Methods: Human lung adenocarcinoma cell line A549 and HEK293T cells were chosen for this study. Two sets of shWTAP interference sequences were designed to construct lentiviral vector plasmid. Human lung adenocarcinomaA549 cells were infected after packaging lentivirus in HEK293T cells, and the control group was transfected with 277 empty vector plasmid. The mRNAand protein expression levels of WTAPinA549 cells were detected by qPCR and WB. Changes in proliferation, migration and invasion of A549 cells were detected by BrdU assay, cell scratch healing assay and Transwell assay, respectively. Results: Two plasmids, shWTAP-1 and shWTAP-2, were successfully constructed. Compared with the control group, the mRNA and protein expression levels of WTAP were significantly down-regulated inA549 cells with WTAP knockdown (both P<0.05), and the proliferation, migration and invasion ability of cells were significantly decreased (all P<0.05). Conclusion: Knockdown of WTAP significantly inhibited the proliferation, migration and invasion of human lung adenocarcinoma A549 cells. The expression of WTAP gene is associated with the occurrence and development of lung adenocarcinoma. WTAP may be a potential target for the diagnosis and treatment of lung adenocarcinoma.
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@#Objective: To investigate the inhibitory effect of Triptolide on vasculogenesis of human cervical microvascular endothelial cells, and to explore the mechanism. Methods: Human cervical microvascular endothelial cells (HCerMECs) were used as research subject, and treated with different concentrations of Triptolide (0, 5, 10, 20 and 40 ng/ml) i n v i t r o . The effect of Triptolide on cell proliferation was determined by CCK-8, the cell migration ability was detected by Transwell assay while the expression of vascular endothelial growth factor (VEGF) was examined by western blotting. Results: Triptolide inhibited the proliferation and migration of HCerMECs in a dose-dependent manner ( P <0.05). In addition, Triptolide could inhibit the expression of VEGF in HCerMECs in a concentration-dependent manner. Conclusions: Triptolide could inhibit the proliferation and migration activity of HCerMECs which is related with the suppression of VEGF expression.