ABSTRACT
Objective To explore the relationship of UHRF1 with the clinicopathological characteristics of colorectal cancer (CRC) patients, as well as the effects of lentivirus transfection overexpression and knockdown of UHRF1 on the proliferation, invasion, and migration of CRC cells and the possible signaling pathways. Methods The expression of UHRF1 mRNA and protein in CRC tissues and adjacent tissues was detected by immunohistochemical staining and RT-PCR. The effects of the constructed UHRF1 overexpression- and knockdown-group cells on the expression of UHRF1, related molecules in the WNT signaling pathway, and MMPR9 were examined by Western blot and RT-PCR. EDU and Transwell assays were used to detect changes in the proliferation, migration, and invasion of CRC cells. Results (1) In the TCGA database and clinical data, the mRNA and protein expression levels of UHRF1 in CRC cancer tissues were significantly higher than those in adjacent normal tissues. UHRF1 expression was closely correlated with TNM stage, N stage, and M stage. Patients with low UHRF1 expression in TCGA had better 5-year OS and disease-specific survival. The area under the ROC curve of UHRF1 for predicting 1-, 3-, and 5-year OS were 0.634, 0.652, and 0.771, respectively. The 3-year OS in the clinical data also showed the same survival benefit. UHRF1 overexpression was a poor prognostic factor for CRC patients. (2) After UHRF1 overexpression, the expression of WNT3a, GSK3β, and MMP9 in SW480 cells significantly increased, whereas the expression of p-β-catenin decreased (P < 0.05). After UHRF1 knockdown, the expression of WNT3a, GSK3β, and MMP9 in HCT116 cells decreased, whereas the expression of p-β-catenin increased (P < 0.05). The "rescue" experiment with IWP-2 and HLY78 can produce consistent results. (3) Compared with the control group, the cell proliferation, migration, and invasion abilities of the UHRF1 overexpression group were enhanced. After IWP-2 treatment, the cell proliferation, migration, and invasion abilities were inhibited. Knockdown experiment exhibited the reverse results to overexpression experiment. Conclusion UHRF1 may play an important role in the occurrence and development of CRC. UHRF1 overexpression may be a poor prognostic factor for CRC patients. UHRF1 may affect the proliferation, migration, and invasion of CRC cells through the WNT/MMP9 signaling pathway.
ABSTRACT
Objective:To explore the effects of KISS1 gene transfected by lentivirus on the proliferation,invasion and migration abilities of the colorectal cancer HCT116 cells,and to clarify their mechanisms.Methods:The human colorectal cancer cells with the lowest expression level of KISS1 gene were selected.The lentiviral vectors were builted and transfected the KISS1 gene,and the cells were divided into control group (treated with PBS),empty vector group (treated with empty vector) and over-expression group(treated with KISS1 gene vector).The multiplicity of infection (MOI) of the cells was detected by fluorescence microscope.Real-time PCR and Western blotting methods were used to detect the expression levels of KISS1 mRNA and protein(metastin);CCK-8 method was used to detect the proliferation ability of the cells;Transwell chambers method was used to detect the invasion and migration abilities of the cells.Results:Among LoVo,SW620,SW480,HCT-116,and HT29 cells,the expression levels of KISS1 mRNA and protein were lowest in HCT116 cells,so they were chosen as the research carrier.After transfected with lentiviral vectors,the HCT116 cells could stably express the enhanced green fluorescent protein(EGFP) gene,and the MOI was over 80%.Compared with control group and empty vector group,the expression levels of KISS1 mRNA and protein in the cells in over-expression group were significantly increased (P0.05).Conclusion:The KISS1 gene transfected by lentivirus vector can over-express KISS1 protein and inhibit the proliferation,invasion and migration abilities of the colorectal cancer cells,and the mechanism may be related to the expression of KISS1 protein.
ABSTRACT
<p><b>OBJECTIVE</b>To investigate the effect of Kiss-1 gene suppression on the metastatic capacity of HCT116 human colorectal carcinoma cells in vitro and the involvement of nuclear factor-κB (NF-κB) signaling pathway.</p><p><b>METHODS</b>A recombinant lentiviral vector of Kiss-1 gene pGC-LV-Kiss-1-EGFP or the empty vector was transfected in HCT116 cells. Cell Counting Kit-8 (CCK8) and Transwell chamber assay were used to detect the changes in cell proliferation, invasion and migration ability after the transfection. Western blotting was used to detect the expression of I-κB, the inhibitive protein of NF-κB signal pathway, and the expression of the downstream effector MMP-9 before and after transfection.</p><p><b>RESULTS</b>In cells over-expressing Kiss-1, I-κB expression increased and MMP-9 expression decreased significantly compared to those in the blank control and vector-transfected cells (P<0.05). Kiss-1 gene over-expression resulted in significant inhibition of HCT116 cell proliferation, invasion, and migration as compared to the control cells (P<0.05).</p><p><b>CONCLUSION</b>Lentivirus-mediated Kiss-1 gene over-expression can inhibit the proliferation, invasion, and migration of HCT116 cells via the NF-B signaling pathway.</p>
Subject(s)
Humans , Cell Movement , Cell Proliferation , Colorectal Neoplasms , Pathology , Genetic Vectors , HCT116 Cells , I-kappa B Kinase , Metabolism , Kisspeptins , Genetics , Lentivirus , Matrix Metalloproteinase 9 , Metabolism , NF-kappa B , Metabolism , Neoplasm Invasiveness , Signal Transduction , TransfectionABSTRACT
Objective To research the effect of the Kiss-1 gene promoter methylation on the Kiss-1 gene expression in colorectal carcinoma tissue,and to analyze the relationship between the Kiss-1 gene methylation and the clinical pathological features of colorectal carcinoma and its clinical significance.Methods The Kiss-1 gene promotor region methylation,Kiss-1 gene mRNA and protein expressions were detected respectively by methylation-specific PCR, Real-time PCR and Western blotting method in 126 cases of colorectal carcinoma tissue and para-cacinoma normal colorectal tissue.Results The positive rate of Kiss-1 gene methylation in colorectal carcinoma tissue (83.33%)was significantly higher than that in normal tissue (30.16%)(P0.05 ). Conclusion The Kiss-1 gene promoter methylation in colorectal carcinoma tissue is associated with the Kiss-1 gene expression level and the malignant characteristics of colorectal carcinoma;Kiss-1 gene promoter methylation may be used as a reference indicator for predicting the risk of metastasis of colorectal carcinoma.