Effect Of Inhibitor Of Differentiation-1(Id-1) On Differentiation Induction Of Malignant Solid Tumors

Neoplasms could be an error in the control of cell differentiaion, showing changes in cell morphology, increased nuclear/plasma ratio, abnormal cell cycle, excess proliferation and loss of normal functions. But with the effect of some interior or exterior factors, the malignant phenotypes of these tumor cells can be reversed. Up to now there are only few drugs used for the induction-differentiation therapy for leukemia. Few studies are reported on the phenotypic reversion of solid tumors. Inhibitor of Differentiation (Id) family is an important class of protein, which plays key roles in the regulation of lineage commitment, cell fate decisions and in the timing of differentiation during embryo development. Its abnormal expression is related to tumorigenesis, tumor cell proliferation, angiogenesis and tumor invasion. Since these proteins are related with differentiation, their effect on differentiation of malignant tumors is unclear. Among Id proteins, Id-1 is expressed in all tumor tissues ever detected. So in this study we take Id-1 as the molecular target, decrease its expression in tumor cells using RNAi and try to find its effect on the differentiation induction of solid tumors.First we investigated the effect of Id-1 on the differentiation of malignant tumor cells in vitro. By tissue chip technique and immunochemical methods, our laboratory has proved that Id-1 protein is highly expressed in lung cancer, colon cancer pancreas cancer and stomach cancer. Furthermore, the level of Id-1 expression is negatively correlated with the differentiation of tumor cells. In order to identify its effect on differentiation of malignant tumor cells, we took the mouse Dendritic Cell Sarcoma (DCS) cell line as the cell model, which can form solid tumor in vivo, have definite differentiation markers, and express high level Id-1 protein. Then we designed and chemically synthesized three pieces of small interfering RNA (siRNA) to aim directly at mouse Id-1 mRNA. Packaging with cationic lipid, we transfected the DCS cells, and screen for the most effective siRNA, the optimal concentration and the optimal time by Western Bloting, then verified the level of Id-1 mRNA by real-time PCR.When the expression of Id-1 in DCS cells was inhibited by RNAi technique, we investigated the changes of DCS cells in biological characters, differentiation properties and malignant behaviors. Meanwhile we took no treatment group and no-target siRNA group as negative control, and induction-differentiation drug sodium butyrate (SB) as positive control. When the expression of Id-1 was down regulated, the DCS cells showed mature morphs of dendritic cells: cell stretch, more and longer branches; the cells became bigger, and the nuclear/plasma ratio diminished. In addition, there were great changes in cell cycle: if processed by RNAi technique, 24h and 48h later, more cells in phase G₀/G₁ and less in phase S, p＜0.01, suggested significant difference. Furthermore, DCS cells showed some mature markers, such as increased DC differentiation markers Id2 and CD86, the latter doesn't express in normal DCS, but was positive after RNAi. We also found that the abilities of cell proliferation, colony formation and invasion in vitro were decreased respectively by MTT, colony formation assay and transwell assay. Result of statistical analysis showed p＜0.01. The above datas suggested that when decreased the expression of Id-1 protein, malignant solid tumor cells can be induced to differentiate, and the malignant phenotype can be reversed.In order to explore which signal pathway Id-1 participated in inducing differentiation of tumor cells, we used Western Bloting technique to investigated the changes in expression of E2A, which is the direct downstream molecular of Id-1, and take part in MAPK and Notch signal pathway to regulate differentiation of many cells, such as B cells and myogenic cells. Result showed that inhibiting Id-1 protein, the expression of E2A increased, suggesting that Id-1 may function through regulating E2A pathway.For the purpose of detecting the therapeutic effect in vivo by decreasing the expression of Id-1, we proceeded to the second part. We employed mouse H22 ascites tumor model. H22 is mouse liver cancer cells, and highly expressed Id-1 protein. We made methyl modification of siRNA which was verified effective in the first part, packaged with cationic lipid, and administrated by intraperitoneal injection to evaluate the therapeutic effect by diminishing abdomen circumference and body weight. Meanwhile, we wanted to check the feasibility of RNAi used in tumor therapy in vivo. Results showed that the growth of body weight and abdomen circumference of mice in high-dose-interference group and low-dose-interference group was lower than that of mice in no-target group, especially for the high-dose-interference group. Ascites tumor in this group was greatly inhibited, the relative inhibition rate was more than 50%, and the difference was significant compared with that in no-target group (P＜0.05). These results suggest that siRNA decreasing the expression of Id-1 protein can inhibit the proliferation of tumor cells in vivo, and slow the growth of ascites. More important, the therapeutic effect is dose dependent.In conclusion, decreasing the expression of Id-1 protein by RNAi can induce the solid tumor cells to differentiate in vitro, and the abilities of cell proliferation, tumorigenesis and invasion are decreased. In vivo, it can inhibit the growth of ascites tumor cells, slow down the increase of ascites. So solid tumor can be induced to differentiate, and RNAi technique and Id-1 protein could be the new method and the new target in neoplasm therapy in clinic.