Research On The Expression, Function And Mechanism Of Ndrg2 In Colorectal Cancer

Colorectal cancer (CRC) is one of the most common malignant tumors all over the world. The main therapy methods on CRC are radical surgery, neoadjuvant chemotherapy, adjuvant chemotherapy and radiotherapy. Despite earlier diagnosis, progressions in radical surgery, radiotherapy and neoadjuvant chemotherapy, the incidence rate and mortality of CRC remained increased in the last two decades. And the five-year survival rate of patients with TNM stage III and TNM stage IV tumors remained poor. Among all CRC related deaths, the progression, invasion and relapse were the main causes of deaths. Thus, research on the molecular mechanism of CRC will shed light on the prognosis diagnosis, tailored chemotherapy and targeted therapy. N-myc downstream-regulated gene (NDRG) family consists of four members: NDRG1, NDRG2, NDRG3 and NDRG4, which share 57% - 65% amino acid identity. Among the four members, NDRG2 was first cloned and reported by Department of Biochemistry and Melecular Biology in our university. Previous research showed that NDRG2, as one of the Myc downstream-regulated genes, has a decreased expression in a variety of human malignancies such as breast cancer, lung cancer, hepatocellular cancer, gastric cancer, glioma, thyroid cancer and myeloid leukemia. These results suggested that NDRG2 might serve as an tumor suppressor in the modulation of the aggressive behavior of malignant tumor progression. However, the precise molecular and cellular function of NDRG2 has not been fully elucidated.In order to interpret issues raised above, the present study:1. Investigated NDRG2 mRNA expression by RT-PCR in 30 cases of randomized selected CRC samples and matched normal tissues preliminarily.2. Investigated NDRG2 mRNA expression by Real time PCR in 226 cases of CRC samples and matched normal tissues and analyzed its association with clinicopathological data and prognosis of patients with CRC by proper statistical analysis.3. Investigated NDRG2 protein expression by western blot in 86 cases of randomized selected CRC samples and matched normal tissues preliminarily.4. Manufactured tissue microarray by 260 cases of CRC samples recruited in Xijing Hospital and 681 cases of CRC samples recruited in Tianjin Union Medicine Center. Investigated NDRG2 protein expression by immunohistochemistry assay in these cases of CRC and analyzed its association with clinicopathological data and prognosis of patients with CRC by proper statistical analysis.5. Transfected pcDNA3.1-NDRG2 and siRNA-NDRG2 into SW620 cell line by liposome. Investigated NDRG2 mRNA and protein expression in cell by Real time PCR and western blot after transfection. Divided cell into four group: transfected pcDNA3.1-NDRG2, transfected siRNA-NDRG2, blank (untransfected cell) and transfected pcDNA3.1. Investigated cell growth by MTT assay and soft agar colony formation assay; investigated cell cycle and apoptosis by flow cytometry; investigated cell invasion by transwell assay; investigated in vivo tumor formation ability by nude mice.6. Investigated p65 expression, one of the main molecules in NF-κB pathway, in CRC tissue microarray by immunohistochemistry assay and analyzed its association with NDRG2 protein expression by statistical analysis. Investigated p65 mRNA expression in SW620 cell transfected pcDNA3.1-NDRG2, siRNA-NDRG2 respectively.The results of the investigations above showed that:1. Among the 30 cases of randomized selected CRC samples and matched normal tissues, RT-PCR showed that more than 50% decreased expression of NDRG2 mRNA was found in 12 cases of CRC, compared with matched normal tissues, which suggested a decreased expression trend of NDRG2 in CRC.2. Among the 226 cases of CRC samples and matched normal tissues investigated by Real time PCR, statistical analysis showed that NDRG2 mRNA expression was decreased in CRC compared with matched normal tissues (P<0.001). And NDRG2 expression correlated with differentiation status (P<0.001) and TNM stage (P<0.001).3. Among the 86 cases of randomized selected CRC samples and matched normal tissues, western blot showed that decreased expression of NDRG2 protein was found in 52 cases of CRC compared with matched normal tissues, which were in well consistent with results of mRNA level.4. The results of immunohistochemistry assay on tissue microarray showed that NDRG2 protein expression was decreased in CRC compared with matched normal tissues (P<0.001). And the expression of NDRG2 protein was associated with differentiation status (P<0.001) and TNM stage (P<0.001). Univariate and multivariate survival analysis proved that NDRG2 was an independent prognostic factor of patients with CRC.5. Real time PCR and western blot proved that NDRG2 mRNA and protein expression were increased and decreased when transfected pcDNA3.1-NDRG2 and siRNA-NDRG2 respectively. MTT assay and soft agar colony formation assay showed that transfection of siRNA-NDRG2 facilitated cell growth while transfection of pcDNA3.1-NDRG2 inhibited cell growth; flow cytometry showed that transfection of pcDNA3.1-NDRG2 inhibited cell cycle at G1 stage; flow cytometry also showed that transfection of siRNA-NDRG2 inhibited cell apoptosis while transfection of pcDNA3.1-NDRG2 facilitated cell apoptosis; transwell assay showed that transfection of siRNA-NDRG2 facilitated cell invasion while transfection of pcDNA3.1-NDRG2 inhibited cell invasion; transfected cell injection to nude mice showed that transfection of siRNA-NDRG2 facilitated tumor formation while transfection of pcDNA3.1-NDRG2 inhibited tumor formation.6. Immunohistochemistry assay on tissue microarray showed a significant negative correlation between NDRG2 and p65 in CRC. Real time PCR and western blot showed that p65 expression was decreased when transfected pcDNA3.1-NDRG2 while it increased when transfected siRNA-NDRG2.These results above proved that expression of NDRG2 was decreased in CRC and related to tumor differentiation status and progression. NDRG2 could also be an independent prognostic factor for patients with CRC. In CRC, NDRG2 could inhibit cell growth, inhibit cell cycle at G1 stage, facilitate cell apoptosis, inhibit tumor cell invasion and in vivo tumor formation ability. And these functions might rely on the inhibition of NDRG2 on p65, which could inhibit NF-κB pathway.