The Role Of NDRG2in Proliferation And Differentiation Of Colon Cancer Cells

Colorectal cancer (CRC), which is one of the most common carcinoma, isthe third most commonly diagnosed cancer worldwide every year. In2008, therewere1.2million new cancer cases and608,700people died of this disease. Bynow, the treatment for CRC is mainly dependent on surgeryor the combinationwith chemotherapy and radiotherapy. Although the early diagnosis, new surgeryprocedures, radiotherapy and neoadjuvant therapy are more popular, theprognosis of CRC is poor, especially the advanced CRC. Recently, moleculartargeted therapy has been becoming new model and the trend, providing moreeffective methods for CRC treatment. To improve the early diagnosis and moreeffective strategy, it`s important to investigate the molecular mechanism of CRCdevelopment and find the new molecular target.Human NDRG2was firstly discovered and reported as a new tumorsuppressor gene by the Department of Biochemistry and Molecular Biology inour university in1999. Previous study showedthe decreased expression ofNDRG2in many cancers, such as CRC, gastric cancer, esophagus cancer andprostate cancer, and was associated with the poor prognosis. NDRG2can notonly inhibit the cell proliferation, but also promote the cell differentiation. Innervous system, NDRG2expression increased with the differentiation of PC12cells，and overexpression of NDRG2promoted NGF induced neurite outgrowth of PC12cells. In CRC, the expression of NDRG2was closely correlated to thedifferentiation of CRC. We used sodium butyrate to induce the differentiation ofcolon cancer cells, and both of the mRNA and protein level of NDRG2increasedwith the process of differentiation, suggesting NDRG2might involved in thedifferentiation process of colon cancer cells.To study the function of NDRG2in the development of CRCand elucidatethe signaling pathway, we constructed the lentivirus vectors of NDRG2overexpression or NDRG2shRNA to produce the lentiviral particles, and theninfected the colon cancer cells. With long term selection of blasticidin orpuromycin respectively, we established the stable cell lines with NDRG2overexpression or NDRG2knockdown.To study the effect of NDRG2in the proliferation of colon cancer cells, weused the MTT, EDU, plate colony formation assay and nude mice to test thegrowth of colon cancer cells with NDRG2overexpression. The results showedthat overexpressed NDRG2resulted in proliferation inhibition of colon cancercells and tumor formation inhibition in nude mice. Flow cytometric analysis ofthe cell cycle showed that NDRG2overexpression caused cell cycle arrest in G1phase, suggesting that NDRG2inhibited the growth of colon cancer cells throughcell cycle arrest.To test the effect of NDRG2on the differentiation of colon cancer cells, we usedsodium butyrate to induce the differentiation of colon cancer cells, and detect thechanges of morphological structure and differentiation marker with electronmicroscopy (EM), AKP test and RT-PCR. The results showed that ectopicexpression of NDRG2could promote the differentiation of colon cancer cells,and NDRG2silence impaired the differentiation of colon cancer cells, suggestingthat NDRG2played an important role in differentiation. To clarify the mechanism of NDRG2induced cell differentiation, we tested thecell cycle related molecules with western-blot. The results showed that the levelof p21and p27were elevated, indicating that NDRG2promoted thedifferentiation of colon cancer cells by up-regulation of p21and p27. We furtherknockdown the expression of p21and p27in NDRG2overexpressed cells withPLKO system, and found that the differentiation marker was significantlyreduced.In this study, we showed a NDRG2’s important role in colon cancer celldifferentiation, and NDRG2promoted the differentiation depending on the p21and p27. We supply a new target for molecule diagnosis and therapy in CRC andupdate the signaling pathway of NDRG2.