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@# Objective: To investigate whether inhibitor of differentiation 1 gene (Id1) and Id3 gene can synergistically promote epithelial-mesenchymal transition (EMT), invasion and migration of colon cancer SW620 cells and to explore its underlying mechanisms. Methods: The SW620 cell strain with Idl or Id3 gene knockdown and the SW620 cell strain with Id1/Id3 gene double-knockdown were constructed by lentiviral vectors transfection. The SW620 cells were divided into four groups, which included SW620-Sh-Id1 group (transfected with shRNA-Id1), SW620-Sh-Id3 group (transfected with shRNA-Id3), SW620-Sh-Id1-Id3 group (transfected with shRNAId1 plus shRNA-Id3) and SW620-NC group (transfected with negative lentivirus). The efficiency of knockdown was detected by Realtime qPCR and Western blotting. The influence of stable knockdown of Idl or Id3 on cell morphological change was observed under a microscope. The changes of migration and invasion abilities of the SW620 cells were determined by wound healing assay and Transwell assay. EMT, invasion and migration related proteins were measured by Western blotting. Results: The SW620 cell strains with Idl and/or Id3 gene knockdown were successfully constructed. Idl and Id3 knockdown induced the epithelial-like to the mesenchymanl-like transformation of SW620 cells. (1) Compared with the control group, the invasion and migration abilities of the SW620 cells were significantly decreased in the SW620-Sh-Id1 group and SW620-Sh-Id3 group (all P<0.05). (2) Meanwhile, the invasion and migration abilities in the SW620-Sh-Id1-Id3 group were obviously weaker than the SW620-Sh-Id1 group and SW620-Sh-Id3 group (all P<0.05). (3) Compared with the control group, the SW620-Sh-Id1 group and SW620-Sh-Id3 group had a reduction in the protein expressions of βcatenin, snail1 and MMP2, and an increase in the protein expressions of E-cadherin and TIMP2 (all P<0.05). (4) Compared with the SW620-Sh-Id1 group and SW620-Sh-Id3 group , the protein expressions of β-catenin, snail1 and MMP2 were reduced, and the protein expressions of E-cadherin and TIMP2 were increased in the SW620-Sh-Id1-Id3 group (all P<0.05). Conclusion: Id1 and Id3 could synergistically influence invasion and migration of SW620 cells, possibly through inducing EMT.
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Objective The inhibitor of differentiation 3 (Id3) is an important transcriptional regulation factor, which participates in tumorigenesis, cell proliferation, and cell apoptosis.β-catenin, as a central molecule of the Wnt signaling pathway, is critical for tumor development.This study aimed to evaluate the expressions of these two molecules and the regulatory effect of Id3 on β-catenin in different tumor cells.Methods Total RNA was extracted using the Trizol Reagent.The relative mRNA expression levels of Id3 and β-catenin in tumor cells were detected by quantitative real-timePCR(qRT-PCR).The recombinant eukaryotic expression vector pEGFP/Id3 with the human Id3 gene was transfected into A549, A549/ DDP and SW-480 cells using the non-liposome-mediated method.The protein expressions of Id3 and β-catenin were determined by Western blot.Results The expression of Id3 was significantly lower in the colorectal cancer cell lines SW-480 and HT-29 than in A549 and other tumor cells (P0.05).Western blot showed the same results.Conclusion The expression levels of Id3 and β-catenin vary in different tumor cell lines.Anabnormally high level of β-catenin is an important risk factor for colorectal cancer, and the down-regulatedexpression of β-catenin after eogenous transfection of Id3 may provide some new ideas for target gene therapies of colorectal cancer.
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Objective:To investigate the inhibitory effect of inhibitor of differentiation 3(Id3)on growth of human lung adenocarcinoma cell line A549.Methods:Recombinant eukaryotic expression vector pEGFP/Id3 was constructed and transfected into A549 cells by liposome-mediated method.Expression of pEGFP/Id3 in A549 cells was analyzed by flow cytometry(FCM),fluorescence microscopy,semi-quantitative RT-PCR and immunocytochemistry.The growth inhibitory rate of A549 cells was examined by MTT assay;cell cycle change was evaluated by PI(propidium iodide)staining method.Cell apoptotic rate and nuclear morphology were detected by Annexin V/7-AAD and Hoechst33258 staining. Results:The recombinant eukaryotic expression vector pEGFP/Id3 was successfully constructed.The expression of EGFP reached the peak 48-72 h after transfection;the expresion of pEGFP-transfected group was higher than that of the pEGFP/ Id3 group.RT-PCR and immunocytochemistry staining showed that Id3 mRNA and protein were effectively expressed in pEGFP/Id3-transfected A549 cells.The growth of cells in pEGFP/Id3 transfeeted cells was significantly inhibited 48-72 h after transfection(P
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Objective: To express and purify the inhibitor of differentiation(Id3) in E.coli.Methods: The DNA fragment in the coding region of the human Id3 gene was amplified by PCR and cloned into the pGEM-T Easy vector for sequencing.The Id3 cDNA fragment was then subcloned into the prokaryotic expression vector(pET-32a)(+) and transformed into E.coli BL 21(DE3).The expression of the histidine-tagged(His-Tag) fusion protein was induced with isopropy-?-D-thiogalactoside(IPTG),confirmed by Western blot and purified by the immobilized Ni2+ absorption chromatographic column.Results: The prokaryotic expression vector of Id3 was successfully constructed.Western blot confirmed the expression of the His-Tag fusion protein in E.coli BL 21(DE3) and that of the Id3 fusion protein with the relative molecular mass size of 33 000 after purified by the affinity chromatographic column. Conclusion: Recombinant Id3 can be expressed in E.coli BL 21(DE3) and the obtained fusion protein can be purified by the Ni-affinity chromatography column.