Effect Of ART1on The Proliferation Of Mouse Colon Carcinoma CT26Cells And Its Possible Mechanism

Objective: To explore the effect and possible mechanism ofArginine-specific ADP-ribosyltransferase1(ART1) on the proliferationability of mouse Colon cancer CT26cells. Methods：Immunofluorescenceassay to confirm that expression of ART1do exists in the CT26cells.Lentivirus of ART1-shRNA,NC-shRNA,ART1-over-expression wereinfected into mouse colon carcinoma CT26cells separately.The expressionof ART1mRNA was detected by RT-PCR,and the expression ofART1,RhoA,c-myc,c-fos,and COX-2proteins of CT26cells were testedby Western-blotting. The difference of ART1expression in four groups ofcells were observed by immunofluorescence.The cell proliferation in eachgroup was measured by cell count kit-8(CCK8) assay.The cell proliferationand cell cycle of CT26cells were measured by cell count kit-8(CCK8)assay and flow cytometry analysis. Tumor xenografts models of mousecolon carcinoma were constructed, growth status (size, weight) of tumorxenografts and survival time of four groups were Observed.The expressionlevel of ART1,RhoA,c-myc,c-fos, and COX-2proteins of tumor xenografts tissues were tested by Western-blotting.Results：1.It Can be determined that ART1exists in the CT26cells.2.Lentivirus of ART1-shRNA,NC-shRNA and ART1-over-expressionwere infected into CT26cells successfully,and the CT26cell line withstable low-expression and over-expression of ART1were successfullyestablished.3.Compared with CT26cells of control groups, the expression ofART1mRNA and proteins were reduced in CT26cells of ART1-shRNAgroup(P＜0.05) and raised in ART1-over-expression group significantly(P＜0.05), but there was no significant difference between NC-shRNA anduninfected group(P＞0.05).4. Immunofluorescence results showed: mean fluorescence intensity ofART1in un-infected group cells was43.51±3.82, much higer than that ofART1-shRNA group which was16.83±2.01(P＜0.05), less than that ofART1overexpression group which was61.41±3.51(P＜0.05),andno obvious difference was observed between un-infected group andNC-shRNA group(P＞0.05).5.In the CCK8assay,when compered with proliferation inhibition rateof control groups cells,that of ART1-shRNA group markedly increased(P<0.05) and ART1overexpression group significantly reduced (P <0.05).6. In ART1-shRNA group, the percentage of cells in G2phase and Sphase and the proliferative indices (PI) decreased when compered with control groups (P<0.05). Oppositely, in the ART1overexpression groupCT26cells, the percentage of cells in G2phase and S phase and theproliferative indices increased (P<0.05).7.The volume and weight of tumor xenografts respectively reduced inART1-shRNA group(P <0.05)and increased in ART1-over-expressiongroup(P <0.05) compered with control groups. Compared with the controlgroup,the average survival time of mice in ART1-shRNA groupsignificantly increased (P <0.05), and the ART1overexpression groupsignificantly reduced (P <0.05).8.The expression of ART1proteins of tumor xenografts significantlydecreased in ART1-shRNA group(P <0.05),but increased inART1-over-expression group(P<0.05).The expression of RhoA, c-myc,c-fos, and COX-2proteins of cells and tumor xenografts in control groupswere weaker than that in the ART1-over-expression group(P <0.05), buthigher than that in the ART1-shRNA group(P <0.05).Conclusion: The results suggest that ART1gene silencing andoverexpression can respectively inhibit and promote the proliferationability of mouse colon carcinoma CT26cells.This effect probablyassociates with the influence of the expression of RhoA and the downstream effectors c-myc and c-fos after changing the expression ofART1gene.