| Colon cancer is a common malignant tumor, and the incidence of colon cancer in women and men, respectively, ranked second and third.With the development of early intervention, the incidence of colon cancer has stabilized or declined, but in developing countries the figure is still rising. In China,with the changes in the way of life of the population and the aging of the population,colon cancer has become one of the important diseases that threaten people’s health, which the incidence rate is increasing year by year, and the age of onset is ahead of time.In the past few years, the treatment of colorectal cancer has made considerable progress. However, due to the occult onset and atypical symptoms, many patients with colorectal cancer treatment has reached the advanced stage,resulting in high mortality rate, and the main reason is the tumor invasion and metastasis. The development of colon cancer involves multiple genetic changes, therefore, how to screen for colorectal cancer prognostic biomarkers and targeted intervention has become a pressing matter at the moment. The ATPase family AAA domain-containing protein 2（ATAD2）regulates the transcription of its target genes, participates in cell cycle regulation and signal transduction, and its abnormal expression is usually associated with the occurrence and development of malignant tumors.Studies have demonstrated that compared with the adjacent tissues,ATAD2 showed abnormal high expression in colon cancer tissues, and RNA interference（RNAi）-mediated ATAD2 down-regulation has been shown to inhibit the proliferation and invasion of a variety of cancer cells.However, the role of ATAD2 in the progression of colon cancer is unclear.In order to study the role of ATAD2 in the development and progression of colon cancer, this study intends to silence the expression of ATAD2 in human colon cancer cells Caco-2 and SW480 by RNA interference, then investigate the effect and relevant mechanisms of down-regulation of ATAD2 on the proliferation, invasion and metastasis of colon cancer cells and the ability of tumor formation in vivo, and provide the therapeutic targets and theoretical basis for the study of pathogenesis and gene therapy of colon cancer. Our research mainly includes the following aspects:Effect of ATAD2 gene silencing on proliferation and cell cycle of colon cancer cells1、The construction of ATAD2 sh RNA recombinant expression vector According to the ATAD2 gene（XM XM011516994） conserved sequence from Gen Bank and reference the RNAi design software from Ambin Corporation website, we identified the ATAD2 target sites and designed the ATAD2-sh RNA（sh ATAD2） oligonucleotide. The obtained sh RNA and vector p RNA-H1.1 were digested and ligated, and the recombinant vector was sequenced.2、Screening and identification of ATAD2 gene silencing stable clones p RNA-H1.1-sh ATAD2 and control NC plasmid were transiently transfected into Caco-2 and SW480 cells by liposome method. The stably transformed cell lines were obtained by G418 resistance screening.Fluorescence quantitative PCR and Western blot were utilized to detect the ATAD2 m RNA and protein in expression, and the results showed that ATAD2 gene silencing stable clone screening success.3、Effect of ATAD2 gene silencing on cell proliferation and cell cycle Detected cell proliferation and colony forming ability respectively by CCK8 and flat cloning experiments. The results showed that compared with non transformed cells Caco-2-wt, SW480-wt and transfection control group Caco-2-sh Ctr, SW480-sh Ctr, the cell proliferation ability of ATAD2 gene silencing group Caco-2-sh ATAD2, SW480-sh ATAD2 cells were significantly down regulated and their clone formation ability were inhibited（P<0.05）; The cell cycle was detected by flow cytometry,and the experimental results showed that in ATAD2 gene silence group, the G1 phase of Caco-2-sh ATAD2 and SW480-sh ATAD2 cells increased significantly, and the S-phase percentage decreased, which indicated that the ATAD2 gene silencing colon cancer cell cycle arrested in G1 phase.4、The related mechanism of ATAD2 gene silencing on cell proliferation and cell cycle Effects of ATAD2 gene silencing on sell proliferation and cyclin-related genes and proteins were detected by the fluorescent quantitative PCR and western blot. The experimental results showed that in the gene ATAD2 silencing group Caco-2-sh ATAD2, SW480-sh ATAD2 group, the expression levels of cell proliferation related factor PCNA,C-myc and Ki67 and m RNA and protein were decreased, Akt phosphorylation was reduced, and signal pathway activity was inhibited;the cell cycle related factor of Cyclin D1, Cyclin E1, CDK2 m RNA expression levels were significantly down regulated, and the anti-oncogene p21 m RNA expression level was increased significantly.The results suggest that ATAD2 gene silencing can inhibit the expression of cell proliferation-associated genes and the activation of related signaling pathways.The influence of ATAD2 gene silencing on invasion and migration of colon cancer cells1、ATAD2 gene silencing inhibits the migration and invasion of colon cancer cells The migration and invasion of colon cancer cells after the ATAD2 gene silencing were analyzed by cell wound healing and transwell chamber. The results showed that: compared with untransfected group and transfected control group, the ATAD2 silencing group Caco-2-sh ATAD2, SW480-sh ATAD2 scratch healing ability and invasion ability were significantly decreased. The result suggested that ATAD2 gene silencing inhibited colon cancer cell invasion and migration.2、Effect on colon cancer cells EMT process and mechanism by ATAD2 gene silencing Fluorescence quantitative PCR, Western blot and immuno fluorescence were used to detect the EMT related index changes, to analyze ATAD2 gene silencing effect on colon epithelial mesenchymal transition process. The results showed that compared with the control groups, in the ATAD2 silencing group Caco-2-sh ATAD2, SW480-sh ATAD2, the expression levels of intercellular cytoplasmic marker and cell transcription factor related protein, includes Vimentin, Slug, Snail,and the other indexs were fallen down, and the expression level of epithelial cell marker E-cadherin was increased significantly（P<0.01）.These results suggest that ATAD2 gene silencing may inhibit the biological behavior of epithelial-mesenchymal transition of colon cancer cells and inhibit the malignant transformation of tumor cells.3、The possible mechanism of ATAD2 gene silencing on cell invasion In order to explore the molecular mechanism of ATAD2 gene silencing on invasion of colon cancer cells, the expressions of matrix metalloproteinases MMP2, MMP9 and MMP13 were detected by western blot, and the activities of MMP2 and MMP9 were detected by gelatin zymography.The results showed that ATAD2 gene silencing could decrease the expression of MMP2, MMP9 and MMP13, and inhibit the activities of MMP2 and MMP9.Effect of ATAD2 silencing on colon cancer xenograft tumor formation1、The establishment of nude mice model When the SW480-sh ATAD2 and control SW480-sh Ctr cells were grown to logarithmic growth phase, we subcutaneously injected1×107/0.2ml tumor cells respectively to the armpit of nude mice. From the date of inoculation, observe the state of nude mice, tumor formation time and tumor growth every day.2、General observation of transplantation tumor formation Both the two groups began to show palpable tumor induration in about 9 days after inoculation of the cells. Compared with the control group（sh Ctr）, tumor volume growth of silencing group（sh ATAD2） is relatively slow, after four weeks the mice were killed, tumors were removed for weighing. The result showed that tumor weight of silencing group（sh ATAD2） decreased significantly.3、Tumor growth curve From the observation of tumor formation in nude mice, tumor nodules were measured by vernier calipers every three days to calculate the tumor volume and draw the tumor growth curve. The results showed that tumor tissue in nude mice of the control group（sh Ctr） increased rapidly. Compared with the control group（sh Ctr）, the tumor volume growth in silencing group（sh ATAD2） is slower, suggesting that ATAD2 gene silencing could inhibit the growth of colon cancer cells in the formation of the xenografts in vivo.4、Detection of tumor proliferation related factor The m RNA levels of ATAD2 and proliferating genes PCNA, C-myc and Ki67 were detected by real-time PCR. The experimental results show that, compared with the group sh Ctr, the expression of ATAD2, PCNA,C-myc and Ki67 in sh ATAD2 cells was significantly reduced（P < 0.01）.Immunohistochemistry was used to detect the expression of ATAD2 and Ki67 m RNA in the tumor tissues. The results showed that, compared with group sh Ctr, expression level of ATAD2 and Ki67 in tumor tissue was significantly down-regulated in sh ATAD2 group.Conclusion: In this paper, the cell level and in vivo level experimental study shows that ATAD2 gene silencing can inhibit the ability of tumor cell with proliferation, invasion and metastasis,suggesting that ATAD2 expression plays an important role in the development of tumor growth. The study provides additional candidate target points and the theory basis for the colon cancer gene therapy. |
