Bioinformatic Analysis And Functional Studies Of Colo Cancer Cells Proliferation And Migration Related Genes

Microarray technology offers a high thoughput, fast and repeatable opportunity to gain insight into a series of complex life phenomenon, such as occurrence, development, differentiation, aging, disease and so on. Used the microarray expression profiles, we can discussed the different gene expression and precise regulation in gene network between biological physiology and pathology from mRNA level. Genes often appear oscillation expression under stimulating with growth and development signal, including collaborative oscillation and antagonist oscillation. Cells response to growth and development by cytokine at first, then transmit signal to transcription factor and mediate downstream events, which play the role of signal amplification and make signaling cascades. At present, multiple cytokines and transcription factors were oscillating in expression and regulation, but little is known in aging or disease organs.Here, we defined age as a variable, and screened oscillation genes followed with age changes from intestinal tisse microarray; Then related genes were verified by expression analysis in colon cell lines, and further analyzed the influence of cytokines (IL-8, IL33) and transcription factor (STAT6) on colon cells proliferation and migration; At last, we maked a guess about the mechanism and a possible functional model, these results are as follows: According to oscillation genes expression followed with age change in data set (GSE7821), we found that genes expression were significant difference in two samples at age of24years old. Compared with other age groups,2356gene probes displayed oscillation expression during aging; Some genes significantly up-regulated (153gene probes), while the majority of genes were down-regulated (1396+572gene probes). The results from gene function clustering indicated that transcription factors (STAT6, SOX4, SKI, Rest, TFCP2L1and SMARCA1/4), cytokines (IL-8, IL-33), and nuclear receptor (RARA) were significant up-regulated, while SP1, MCM4and P53were apparently down-regulated. Further, we analyzed expression level of these genes in colon cancer data set (GSE8671). The results show that STAT6, SOX4, SP1, IL-8, IL-33, RARA and MCM4were up-regulated in adenoma, while Rest, TFCP2L1, SMARCA1/4, SKI and NFIX were down-regulated.In vitro experiments, IL-4, IL-8or IL-33has less influence on promoting proliferation of migration of SW480cells; In SW620cells, IL-8and IL-33promoted cells proliferation, but not migrated significantly; In RKO cells, IL4, IL-8and IL-33promoted cells proliferation and migration significantly.Expression analysis of related genes in three colon cancer cell lines (SW480, SW620, and RKO) shows that:Genes of IL-4/STAT6signal pathway can activate in all cell lines; Expression of SKI and SKIL were induced by IL-4, DOX or DOX+IL-4at the same time; In SW620cells, SKIL was up-regulated while SKI was down-regulated; DOX could decrease expression of SKIL, but IL-4or IL-8not; In RKO cells, IL-8increased STAT6and RARA levels, and SKI maintained low expression, but activated by adding IL-4、DOX or DOX+IL-4, while SKIL was down-regulated.We constructed the lentivirus eukaryotic expression vector of human STAT6open reading frame and STAT6RNAi segment, which were transfected into kidney cell line293T cells by lipofectamine2000to produce the lentiviral particles, then collected and concentrated the lentiviral particles to infect three colon cell lines; After that we got the transgene cell lines by tetracycline and fluorescence screening. The mRNA and protein expression of STAT6were identified by RT-PCR, Q-PCR and Western Blot in SW480, SW620and RKO transgene lines. Cell proliferation more faster in SW620and RKO cells, which constitute expression of STAT6compare with control transgene lines, while cell proliferation were inhibit and went to die after RNA silence of STAT6. Interestly, it has no influence on cell proliferation or apoptosis when overexpression or interference STAT6in SW480cell. In vitro culture, SW480and SW620cells did not showed apparently migration through transwell experiment, while RKO cells migrated remarkably. On the contrary, the ability of cell migration was increased by overexpression STAT6in SW620and RKO lines, but not increased obviously in transgene lines of SW480. Meanwhile, RKO cells decreased the migration rate after RNA interference STAT6expression.To sum up, we did data mining and screening the significant differently expression genes from intestinum tenue and colon tissue, and further analyzed the function of IL-8, IL-33and STAT6and so on. These results will be good for understand the mechanism of gene expression caused cancer cell migration, and setting the stage for researching the relationship between inflammation and cancer.