The Regulatory Effects Of SATB2 On Biological Characteristics And Stemness Of Colorectal Caner Cells And Its Mechanisms

In the process of the occurrence and development of tumors, cancer stem cells play a very important role. Just after breast cancer and lung cancer, Colorectal cancer is the third highest incidence of tumors in China. Therefore, it’s very meaningful to illuminate the process and the influencing factors of colorectal cancer stem cells for better clinical treatment and prognosis.SATB2, as the homologous gene of SATB1,is a nuclear matrix binding protein involving in remodeling chromatin and regulating gene expression, which is associated with development of brain and bone and stem cell differentiation. Our previous research has found that SATB2 is associated with stem cell correlated genes and has predicted the genes’promoter regions that SATB2 can combine with.The effect and its mechanism of SATB2 on colorectal cancer stem cells still need further study.Methods1.Effects of knocking down of SATB2 gene on the biological behaviors of human CRC cellsWe infected SW480 cells and DLD1 cells with virus packed with Plko.l-TRC to knock down expression of SATB2. The effects of knocking down of SATB2 on the proliferation, adhesion, migration and tumorigenesis capacity of SW480 cells and DLD1 cells were detected by CCK8, adhesion experiments, Transwell chamber,plate colony formation assays and experiment of tumor formation subcutaneously in nude mice.2. The effects and mechanism of SATB2 on cancer stem cell of CRC cellsThe features of Cancer stem cell include cell self-renew capacity and differentiation ability.We observed the cell self-renew capacity of cancer stem cell of CRC cells through stem cell balls formation experiment.We then detected the expression of CD133 of cells in which SATB2 was knocked down by Western Blot, immunohistochemistry and cell immunofluorescence. Using bioinformatics software,we predicted the binding sites of SATB2 and promoter regions of stem cell corelated genes.Then we validated that SATB2 binds with promoter regions of stem cell corelated genes by chromatin immune co-precipitation technology (CHIP).3. SATB2 regulates expression of Dicer through miR202 in CRC cellsWe found Dicer, the potential target protein of miR202,by bioinformatics software and validated it by Western blot.We transfected CRC cells with pCAG-SATB2 and detected changes of miR202 in cells by QPCR and changes of Dicer by Western blot. We then transfected CRC cells with pCAG-SATB2 and miR202 mimics or miR202 inhibitors and detected changes of Dicer in cells by Western blot.Results1.Effects of knocking down of SATB2 gene on the biological behaviors of human CRC cellsAdhesion experiments showed that the adhesive ability of SW480-2 and SW480-5 cells were enhanced obviously compared with SW480-NC (F=71.123, P= 0.000).And the adhesive ability of DLD1-5 was enhanced obviously compared with DLD1-NC (F=39.280,P=0.000).CCK8 assays showed that the proliferation ability of SW480-2 and SW480-5 cells were enhanced obviously compared with SW480-NC cells (F=6.301, P= 0.000), while the proliferation ability of SW480-5-C2 cell was decreased significantly (F=25.907, P=0.000).And the proliferation ability of DLD1-2, DLD1-5 cells were enhanced obviously compared with DLD1-NC cells (F=3.807, P=0.000), while the proliferation ability of DLD1-5-C11 cell was decreased significantly (F=8.774, P= 0.000).Cell plate clone formation experiment showed that compared with SW480-NC cells, SW480-2 and SW480-5 cells had an enhanced plate clone formation (F=5.832, P=0.001), while a significantly decreased plate clone formation for SW480-5-C2 cell(t=3.216, P=0.015). Compared with DLD1-NC cells, DLD1-2 and DLD1-5 cells had an enhanced plate clone formation (F=24.066, P=0.000), however a significantly decreased plate clone formation for DLD1-5-C11 cell (t=2.650, P=0.045).Transwell chamber Movement experiment showed that, compared with SW480-NC cells,SW480-2 and SW480-5 cells had an enhancement on migration ability (F= 32.281, P=0.000) the same as SW480-5-C2 cells (t=3.833, P=0.004). So were DLD1-2 and DLD1-5 cells when compared with DLD1-NC cell (F=28.901, P= 0.000).When compared with DLD1-NC cell, DLD1-5-C11 cell also had an enhancement on migration ability significantly(t=3.579, P=0.006).The experiment of tumor formation subcutaneously in Nude mice showed that tumor formation ability of SW480-5-C2 and SW480-5-C8 cells were weakened obviously compared with SW480-NC cells (F=3.480, P=0.003).And so was DLD1-5-C11 cell(F=5.876, P=0.023) when compared with DLD1-NC cell,while tumor formation ability of DLD1-5 cell was enhanced significantly compared with DLD1-NC cell (F=14.172, P=0.001).2. The effects and mechanism of SATB2 on cancer stem cell of CRC cellsStem cell balls formation test showed that the self-renew ability of SW480-5-C2 and SW480-5-C8 cells were enhanced obviously compared with SW480-NC cells (F =5.722, P=0.040).And it’s the same with DLD1-5 and DLD1-5-C11 cells when compared with DLD1-NC cell (F=6.600, P=0.031).The expression of CD 133 was enhanced after SATB2 was knocked down in both SW480 and DLD1 cells which was deteced by Western blot, immunohistochemistry and cell immunofluorescence.Chromatin immune co-precipitation experiments(CHIP) have shown that gene SATB2 can regulate expression of stem cell related genes,such as CD133, CD44, Dicer, Meis, Axin2 and PRL-1,by binding with regions on their promoter.3. SATB2 regulates expression of Dicer through miR202 in CRC cellsThe expression of Dicer was lowered in SW480,SW620,HCT116 and DLD1 cells after they were transfected with miR202 mimics,compared with control groups,which was detected by Western blot.While the expression of Dicer did not increase in SW480,SW620,HCT116 and DLD1 cells after they were transfected with miR202 inhibitors when compared with control groups.Compared with control groups,the expression of miR202 was enhanced significantly in SW480 (1=3.301,P=0.021),SW620 (t=3.201, P=0.015),HCT116 (t= 3.371, P=0.012) and DLD1 (t=2.658, P=0.045)cells after their overexpression of SATB2 which was detected by QPCR.While the expression of Dicer in cells with overexpression SATB2 was lower than those of control groups which was detected by Western blot.And we detected an lower expression of Dicer in SW480, HCT116, and DLD1 cells when we transfected pCAG-SATB2 and miR202 mimics at the same time.Conclusions1. Cell invasion ability of colorectal cancer cells was enhanced after knocking down of SATB2. The expression of SATB2 and SATB1 regulate the proliferation, clone formation in vitro and in vivo tumor origination capacity of colorectal cancer cells together.2. Knocking down of SATB2 in colorectal cancer cells can increase the self-renew ability and the expression of CD 133 of cells. SATB2 regulates stem cell related genes,such as CD133, CD44, Dicer, MEIS, Axin2 and PRL-1, by binding with the regions of their promoters.3. SATB2 and miR202 can both cause a decrease of expression of Dicer in colorectal cancer cells.SATB2 can regulate the expression of Dicer by adjusting the level of miR202 in colorectal cancer cells.