Study On E3Ubiquitin Ligase Nrdp 1 Inhibiting Colorectal Cancer Cell Invasion Throughmatrix Metallopoteinase 7(MMP7)Signaling Pathway

Background and significanceColorectal carcinoma (CRC) is a common disease influenced by inter and external factors and severely threaten the health of human beings. Study on molecular mechanism for CHC will help to explain its development mechanism and provide a tool for early detecting. Besides, it will provide some prospective markers and some hypermutation and common signal path may be the target for therapy.Ubiquitination is an important post-translational modification way for protein in human body, which is correlated with stability and function of the modified protein. Ubiquitination could effectively regulate the apoptosis and immune inflammatory reaction process. E3 ligase is one of the ubiquitin-proteasomes with most kinds. At present, the ubiquitin system not only acts as the protein degradation system, but only participate in the down-regulating the activity of protein, and many kinds of important occurrence and development process. The abnormal ubiquitin system is correlated with inflammatory disease and tumor. Neuregulin receptor degradation protein-1 (NrdP1) was an E3 ligase, which not only participates in apoptosis and inflammatory reaction, but also could inhibit growth and reproduction of tumor cells. At present, Nrdp1 has been proved to inhibit the growth of tumor cells by down-regulating the expression of p27. However, whether Nrdp1 could regulate the invasion ability of colorectal carcinoma cells is still unknown.In this study, we aimed at investigating the invasion ability and mechanism of Nrdp1 in colorectal carcinoma. In vitro experiments, the relationship between Nrdpland MMP7 in colorectal carcinoma Caco-2 cells lines using RT-PCRand Western blotting method, and the invasion ability and mechanism of Nrdpl was investigated by siRNA knocking-down Nrdp1, MMP7. Western blotting method was used to evaluate the expression level of Nrdpl in colorectal carcinoma tissue. Eventually, we firstly illustrated the pathway for Nrdpl inhibiting the expression of MMP7 in colorectal carcinoma and invasion abilities through ERK/MAPK. This study will provide new theoretical basis for better understanding action of Nrdpl in tumor.ObjectiveThe expression level of Nrdpland MMP7 at colorectal carcinoma tissue (CR) and normal tissue (NT) was determined and the correlation was analyzed. The influence of Nrdpl on MMP7 invasion abilities was explored.Method20 cases of tissue samples (colorectal carcinoma tissue and normal tissue) were collected from patients diagnosed as colorectal carcinoma form 2008 to 2014 in our hospital. The expression level of Nrdp1 and MMP7 at colorectal carcinoma tissue (CR) and normal tissue (NT) was determined and the correlation was analyzed, and the correlation between Nrdpland MMP7 was analyzed. On the basis of colorectal carcinoma Caco-2 cells, the over-expressed Nrdpl cells (Caco-2-nrdpl) was built usingliposome mediated gene transfer, blank plasmid transfected (Caco-2null) as control. Moreover, Nrdp1 siRNA transfected Caco-2 cells was used as low-expressed Nrdp1 cells (Caco-2-shndp1). The expression level of MMP7 colorectal carcinoma cells with different MMP7 expression level, which was further proved by Western Blotting. In order to whether Nrdpl could regulate the invasion ability of colorectal carcinoma cells, we firstly transfected Caco-2 cells using MMP7siRNAand Ncontrol siRNA, and the MMP7 mRNA and protein level after 48 h was investigated using Real Time PCR and Western blotting. The MMP7 expression level between Nrdp1/MMP7 knocked-down cells and MMP7 knocked-down cells was comparatively studied. The invasion abilities of Nrdpl/MMP7 knocked-down cells and MMP7 knocked-down cells was comparatively studied. The invasion abilities of Caco-2 cells with different level of Nrdpl was comparatively studied. The pathway for Nrdpl inhibiting the expression of MMP7 in colorectal carcinoma and invasion abilities was screened. The reproduction abilities of Caco-2 cells with different level of Nrdpl was comparatively studied. The apoptosis ratio of Nrdpl/MMP7 knocked-down cells and MMP7 knocked-down cells was comparatively studied.ResultsThe expression level of Nrdpl at colorectal carcinoma tissue was significantly lower than that of normal tissue (P＜0.05), while expression level of MMP7 at colorectal carcinoma tissue was significantly higher than that of normal tissue (P<0.05). The expression level of Nrdp1 was negatively correlated with MMP7 expression level (r=-0.82, P＜0.001). The expression level of Nrdpl in Caco-2-nrdpl cells significantly increased, while at Caco-2-shnrdpl significantly decreased (P＜0.05). Meanwhile, the expression level of MMP7 in Caco-2-nrdp1 cells significantly decreased, while at Caco-2-shnrdpl significantly increased (P＜0.05), which was confirmed with Western Blotting experiment. The expression level of MMP7 in MMP7siRNA transfected Caco-2 cells significantly decreased. The expression level of MMP7 in Nrdpl knocked-down cells significantly increased and that of Nrdp1/ MMP7 knocked-down cells was significantly higher that of MMP7 knocked-down cells (P＜0.05). Transmembrane cell number of Ncontrol siRNA, MMP7 siRNA and Nrdp1/MMP7 siRNA transfected Caco-2 was 252.67±14.16, 56.00±5.00, 156.00±10.00, respectively, indicating that an increased invasion abilities of MMP7 knocked-down cells and a significantly increased invasion abilities of Nrdp1/ MMP7 siRNA transfected Caco-2 compared with MMP7 knocked-down cells. This results showed thatNrdpl could inhibit the invasion abilities of MMP7. Transmembrane cell number of Caco-2-Nrdp1、Caco-2null、Caco-2-shNrdpltransfected Caco-2 was 89.60±12.10,152.008.00 and 589.00±12.00, respectively. After inhibition MMP7 expression, transmembrane cell number of Caco-2-shNrdpl-shMMP7 transfected Caco-2 was 126.40±4.35. This results demonstrated thatCaco-2-Nrdpl showed the weakest invasion abilities, and decreased invasion ability was seen after inhibition MMP7 expression of MMP7. PD98059 did not influence the expression of Nrdpl, but inhibited the expression of MMP7 (P<0.05), leading to a decreased invasion ability (P<0.05), which indicated that Nrdpl may inhibit the expression of MMP7 through ERK/MAPK signal pathway. The reproduction abilities of MMP7 knocked-down Caco-2 significantly decreased. The apoptosis ratio of Ncsi-Caco-2, MMP7si-Caco-2and Nrdpl/MMP7si-Caco-2 was 0.8%,1.6%and 1.9%, respectively. Due to the low apoptosis ratio of control group, we inferred that Caco-2 cells were at reproduction stage and knocking-down MMP7 maybe nor influence apoptosis.Conclusion1. In colorectal carcinoma tissue, expression level of Nrdpl increased and MMP7 decreased, and a negative correlation between them was found;2. A significantly increased invasion abilities, reproduction ability of Nrdpl/MMP7 siRNA transfected Caco-2 compared with MMP7 knocked-down cells was observed, while no for apatosis; After inhibition MMP7 expression, transmembrane cell number of Caco-2-shNrdpl-shMMP7 transfected Caco-2 was 126.40±4.35. This results demonstrated thatCaco-2-Nrdpl showed the weakest invasion abilities, and decreased invasion ability was seen after inhibition MMP7 expression of MMP7;3. Nrdpl may inhibit the expression of MMP7 through ERK / MAPK signal pathway.To sum up, we proved thatNrdp1/MMP7 new pathway could inhibit the invasion ability, indicating that Nrdpl showed diverse function. Therefore, Nrdpl could ubiquitylate some protein, which provided new research orientation for oncomolecularbiology.