In vivo and in vitro Experiment of E74-Like Factor 5 Overexpression Inhibiting the Biological Behavior of Colon Cancer Cells

  Objective  To investigate the effect of E74-like factor 5 (ELF5) overexpression on the growth and invasion ability of colorectal cancer cells and its effect on tumor formation in nude mice.

  Methods   Human colorectal cancer SW480 and HT-29 cells were divided into 5 groups: the lentivirus (LV)-GFP group transfected with empty vector LV-GFP, the LV-ELF5 group transfected with recombinant LV-ELF5, the shRNA-NC group transfected with empty vector shRNA-NC, the shRNA-ELF5 group transfected with recombinant shRNA-ELF5, and the control group, not transfected with any vector. Seventy-two h after transfection, the cell supernatant containing lentivirus was collected. The mRNA expression level of ELF5 in each group was examined by real-time fluorescent quantitative PCR (RT-qPCR). The protein expression levels of ELF5, apoptosis-related cleaved Caspase-3/Caspase-3 and cleaved Caspase-9/Caspase-9, and invasion-related E-cadherin and N-cadherin were checked with Western blot. CCK-8 was used to check cell viability. Colony formation experiment was done to evaluate colony formation rate. Flow cytometry was used to assess cell apoptosis. Transwell migration assay was used to examine cell invasion. TUNEL assay was used to examine the apoptosis of tissues cells. Immunohistochemistry test was done to determine the expression of E-cadherin and N-cadherin in tissues. 20 BALB/c nude mice were put into 4 groups (5 in each group): LV-GFP group, shRNA-NC group, LV-ELF5 group, and shRNA-ELF5 group. Recombinant lentiviral SW480 cell supernatants were subcutaneously injected into nude mice to construct nude mice tumorigenesis models and the volume changes of transplanted tumors were monitored. On the 30th day, transplanted tumor tissues from the nude mice were extracted and the tumor mass was measured. Western blot was done to measure the expression of ELF4 protein in the transplanted tumors. TUNEL staining was used to check cell apoptosis in the tissues, and the positive expression of N-cadherin in the transplanted tumor was measured by immunohistochemical tests.

  Results  Compared with the control group, there was no statistically significant difference in the indicators of the two cell lines in the LV-GFP group and shRNA-NC group. The results of Western blot and RT-qPCR showed that the ELF5 protein and mRNA of the LV-ELF5 group of the two cell lines were up-regulated (P<0.05, compared with those of the LV-GFP group), and the ELF5 protein and mRNA of the shRNA-ELF5 group were down-regulated (P<0.05). The ELF5 overexpression system and interference system were successfully constructed. Compared with the LV-GFP group, data from the LV-ELF5 group showed that cell viability and colony formation rate (P<0.05) were reduced, SW480 and HT-29 cell apoptosis was promoted, cleaved Caspase-3/Caspase-3 and cleaved Caspase-9/Caspase-9 protein expression was up-regulated (P<0.05), cell invasion was inhibited, and the expression of E-cadherin protein was up-regulated while the expression of N-cadherin protein was down-regulated (P<0.05). After ELF5 interference, the above-mentioned expression of cells demonstrated an opposite trend (P<0.05, comparing shRNA-ELF5 group with shRNA-NC group). In vivo experimental results indicated that ELF5 overexpression reduced tumor volume and tumor mass (P<0.05), promoted cell apoptosis in tissues (P<0.05), and inhibited N-cadherin protein expression (P<0.05). When ELF5 expression was inhibited, the above mentioned experimental results showed the opposite trend.

  Conclusion  In vivo and in vitro experiments showed that ELF5 overexpression could promote the apoptosis of colorectal cancer cells and inhibit the growth and invasion of colorectal cancer cells.