The Role Of NIK And IKKβ Binding Protein (NIBP) In Invasion And Metastasis In The Colorectal Cancer

Background and AimColorectal cancer is a common cancer in gastrointestinal tract; its incidence is increased in recent years. This cancer is the fourth most common cancer cause of death globally, and invasion and metastasis of cancer cells is the main reason of the high mortality. In recent studies, the molecular mechanism of invasion and metastasis has become the main focus in colorectal cancer. NF-κB signal pathway is a ubiquitous transcriptional pathway in the body, and is involved in every aspect of intestinal carcinogenesis, from initiation to metastasis. NIK-and IKKβ-Binding Protein (NIBP), a novel NF-κB regulatory protein, which directly interacts with NIK and IKKβ and is required for cytokine-induced NF-κB signal pathway activation. Recently, NIBP has been found in several cancer tissues, such as human breast, colon cancer, osteosarcoma, and lymphoma, and NIBP may be a new biomarker to predict cancer development and target for therapy. However, the roles and mechanisms of NIBP in the invasion and metastasis of human colorectal cancer were rarely reported. In present study, the roles of NIBP in the invasion and metastasis of colorectal carcinoma and its mechanisms on regulation of NF-κB signal pathway activity and the effects on expression of MMP2 and MMP9 were measured from three levels, the clinical pathology and clinic data, cell experimental studies in vivo and in vitro.Methods1. Cancer tissues:114 human colorectal cancer tissues and 21 colorectal adenoma tissues were collected from department of colorectal surgery and endoscopy department, the First hospital of GuangXi medical university. The expression of NIBP, p-p65, MMP2 and MMP were measured by immunohistochemistry, and the improved immune response score (IRS) was used to analysis the expression of these proteins. The TNM stage, age, gender, the pathologic type of tumor, cancer location, cancer diameter and the metastasis were collected, and the different on the expression of NIBP, p-p65, MMP2, MMP in the TNM stages was compared, as well as other clinic data. The relationship of the expression of NIBP and p-p65, MMP2, MMP9 was analyzed by Pearson test.2. Construction of stably transfected NIBP over-expression of HT29 cell line: The whole gene was synthesized by a chemical process, after digestion with endonucleases, it was then linked to the recombinant pcDNA3.1(+) NIBP over-expression vector. Then, the products were transformed into coli DH5a competent cells, coated plates, after the plasmid was extracted, enzyme digestion and target fragment PCR detection results show that the expression vector was constructed successfully. The pcDNA3.1(+) NIBP over-expression vector transfect HT29 cells mediated by lentivirus, transfected cells were selected by Blasticidin to become stably transfected cells by using the biotechnologies of cloning, and the NIBP expression was measure by qPCR and westernblot.3. Construction of stably transfected NIBP knock-down of HCT116 cell line: Three NIBP interference target sequences were designed and synthesized, which were annealed to connect the linear miRNA vector with the oligo double strand and then transformed to the plasmid containing the bacteria. After digestion with endonucleases, it was then linked to the recombinant pLenti6.3-EGFP-NIBP-miR NIBP knock-down vectors. The products were transformed into coli DH5a competent cells, coated plates, after the plasmid was extracted, enzyme digestion and target fragment PCR detection results show that the expression vector was constructed successfully. Difference pLenti6.3-EGFP-NIBP-miR NIBP knock-down vectors transfect the HCT116 cells mediated by lentivirus, transfected cells were selected by Blasticidin to become stably transfected cells by using the biotechnologies of cloning. The NIBP expression was measure by qPCR and westernblot, and the most efficient vector was selected for the next experiments.4. Detection of migration and invasion of human colorectal cancer cell line in different expression of NIBP in vitro:Cell migration and invasion in not transfected HT29 cells, HT29+NC cells, stable over-expression of NIBP HT29 cells and not transfected HCT116 cells, HCT116 NC cells and stable NIBP knock-down HCT116 cells were measured by the boyden chamber transwell.5. The metastasis of human colorectal cancer cell line in different expression of NIBP in vivo:not transfected HT29 cells, stable over-expression of NIBP HT29 cells, not transfected HCT116 cells and stable NIBP knock-down HCT116 cells was transplanted subcutaneously into nude mice, when the tumor reached the size of 1 cm in diameter, cut it off and into pieces of 1mm in diameter, then it was orthotopically implanted into the colons of other nude mice. Each mouse in the experimental group was observed for up to 4 to 6 weeks, and the primary cancer, metastatic tumor, and serum were snap frozen in liquid nitrogen for subsequent experiments.6. Relationship between activation of NIBP and classical NF-κB signaling pathway, expression of MMP2 and MMP9, as well as the effects on invasion and migration of cancer cells:the human colorectal cancer cells (not transfected HT29 cells and stable over-expression of NIBP HT29 cells) were treated by PDTC (5μg/mL), a inhibitor of the canonical NF-κB signal pathway; and other cancer cells (not transfected HCT116 cells and stable NIBP knock-down HCT116 cells) were treated by TNF-a (20ng/mL), a activator of the canonical NF-κB signal pathway; then p65 and IκBβ and their phosphorylation, the downstream factors of canonical NF-κB signal pathway, and the expression of MMP2 and MMP9 were detected by westernblot. The invasion and migration of cancer cells were measured by the boyden chamber transwell in vitro.7. The relationship between different expression of NIBP and canonical NF-κB signal pathway activity, expression of MMP2 and MMP9 in the liver metastasis of the orthotopic transplantation tumor in nude mice:The expression of p-p65, MMP2 and MMP9 were measured by immunohistochemistry, and the improved immune response score (IRS) was used to analysis the expression of these proteins in the liver metastasis tissues.8. Statistical analysis:All data are presented as the mean±SD. The statistical significance of differences between the means was evaluated using the unpaired Student’s t test or the one-way analysis of variance test (if variance not neat, using the Dunnctt’s T3 test; if variance neat, using the Bonferroni test). The frequencies in different groups were compared using Fisher’s exact test. The correlation of immunohistochemistry score between NIBP and other proteins was detected Pearson correlation analysis. Statistical analysis was performed using SPSS 20.0. A value of P<0.05 was considered significant.Results1. The expression of NIBP, p-p65, MMP2 and MMP9, the relationship between these proteins and clinical pathology:Twenty-one cases were colorectal adenoma, there were 19 cases in TNM I stage,44 cases in TNM Ⅱ stage,30 cases in TNM Ⅲ, 21 cases in TNM Ⅳ; and 93 cases of tubular adenocarcinoma,21 cases of mucinous adenocarcinoma. Liver metastasis was found in 12 cases (57.14%) and other organ metastasis in 9 patients with distant metastasis, including 8 peritoneal metastasis (38.10%) and one lung metastasis (4.76%). There was no significant difference between NIBP and p-p65 expression in colorectal adenoma tissues, TNM Ⅰ and Ⅱ TNM stage (P>0.05). Both in TNM Ⅲ stage and TNM Ⅳ stage colorectal cancer tissues were significantly higher than that in colorectal adenoma, TNM Ⅰ and TNM Ⅱ, the difference was statistically significant (P<0.05). The NIBP IRS was similar in TNM Ⅲ and TNM Ⅳ stage, the difference was not statistically significant (P>0.05); but p-p65 IRS was significantly increased in TNM Ⅳ stage compared with in TNM Ⅲ stage, the difference was statistically significant (P<0.05). The MMP2 IRS in TNM Ⅰ stage and TNM Ⅱ stage in the colorectal cancer tissue is similar, MMP9 IRS in TNM Ⅰ stage and colorectal adenoma is similar, the difference was not statistically significant (P> 0.05); expression of MMP2 and MMP9 was significantly higher in Ⅲ TNM than in colorectal adenoma, TNM Ⅰ and TNM Ⅱ stage colorectal cancer tissues, and the difference was statistically significant (P<0.05). There was a slight decrease of MMP2 and MMP9 IRS in TNM Ⅳ stage, but it was still significantly higher than that in colorectal adenoma tissues and TNM I stage, the difference was statistically significant (P<0.05). NIBP IRS, p-p65, MMP2 and MMP9 were the lower in tubular adenoma than that in cancer tissues, the difference was statistically significant (P<0.05). However, there was no significant difference between tubular adenocarcinoma and mucinous adenocarcinoma (P>0.05). There were no differences in p-p65, MMP2 and MMP9 IRS in different gender, cancer location and cancer diameter, and the difference was not statistically significant (P>0.05). NIBP IRS was similar in colorectal cancer tissues at different gender and location (P<0.05), but was lower in the cancer diameter (<2cm), the difference was statistically significant (P<0.05). NIBP and p-p65 IRS was significantly increased in the cancer tissues which present liver metastasis compare with that lymph node metastasis, the difference is statistically significant (P<0.05), but was not significantly increased compared to that with other organ metastasis (P>0.05); and MMP2 and MMP9 IRS in all tissues had no significant difference (P>0.05).2. Construction of NIBP over expression of HT29 cell line was successful:The whole gene was synthesized by a chemical process, after digestion with endonucleases, it was then linked to the recombinant pcDNA3.1(+) NIBP over-expression vector. Then, the products were transformed into coli DH5a competent cells, coated plates, after the plasmid was extracted, enzyme digestion and target fragment PCR detection results show that the expression vector was constructed successfully. The pcDNA3.1(+) NIBP over-expression vector transfect HT29 cells mediated by lentivirus, transfected cells were selected by Blasticidin to become stably transfected cells by using the biotechnologies of cloning, and the NIBP expression was measure by qPCR and westernblot. The results showed that the construction of NIBP over expression of HT29 cell line was successful, and named as HT29+NIBP cell; the cell transfected with empty plasmid was named as HT29+NC cell.3. Construction of NIBP knock down of HCT116 cell line was successful:Three NIBP interference target sequences were designed and synthesized, which were annealed to connect the linear miRNA vector with the oligo double strand and then transformed to the plasmid containing the bacteria. After digestion with endonucleases, it was then linked to the recombinant pLenti6.3-EGFP-NIBP-miR NIBP knock-down vectors. The products were transformed into coli DH5a competent cells, coated plates, after the plasmid was extracted, enzyme digestion and target fragment PCR detection results show that the expression vector was constructed successfully. Difference pLenti6.3-EGFP-NIBP-miR NIBP knock-down vectors transfect the HCT116 cells mediated by lentivirus, transfected cells were selected by Blasticidin to become stably transfected cells by using the biotechnologies of cloning. The NIBP expression was measure by qPCR and westernblot. The most efficient interference target sequence was GGAAGCTGTCCTGAATTTCAA. The results showed that the construction of NIBP knock down of HCT116 cell was successful, and named as HCT116+NIBP shRNA cell; the cell transfected with empty plasmid was named as HCT1I6+NC cell.4. NIBP regulated the migration and invasion of colorectal cancer cell in vitro: Cell migration and invasion was in a low level in HT29 and HT29+NC cells, and was markedly raised in HT29+NIBP cell by the transwell experiments, the difference is statistically significant (P<0.05). On the other side, the migration and invasion was statistically inhibited in the HCT116+NIBP shRNA cell compare to that in HCT116 and HCT116+NC cell, the difference is statistically significant (P<0.05).5. NIBP regulated the ability of liver metastasis in colorectal cancer cell in vivo: There was only one in five was confirmed to have metastasis to the liver in HT29 cell mice, and four in six was found the liver metastasis in HT29+NIBP cell mice. The number of liver metastasis was higher in HT29+NIBP cell mice, but the liver metastasis rate of the two groups was similar, as well as the weight of primary tumor tissues, the difference was not statistically significant (P> 0.05). All the HCTU6+NIBP shRNA cell mice were found the liver metastasis (4/4), and there was only one in six was confirmed to have metastasis to the liver in HCT116 cell mice, the rate of liver metastasis was higher in the former, also the weight of the primary tumor tissues, the difference is statistically significant (P<0.05).6. Increased expression of NIBP could promote the activation of classical NF-κB signaling pathway, expression of MMP2 and MMP9, and then raised the migration and invasion of colorectal cancer:The total p65 and IκBP, and their phosphorylation were similar in HT29 and HT29+NC cells, and the expression of MMP2 and MMP9 was in a low level. The total p65 and IκBP were not changed, but the phosphorylation of p65 and IκBβ, and expression of MMP2 and MMP9 were inhibited in HT29 cell which treated by the 5ug/mL PDTC for 48 hours. The total p65 and IκBP, and their phosphorylation, and the MMP2 and MMP9 were increased in the HT29+NIBP cell, and the expression of these proteins was not decreased in the HT29+NIBP cell which treat by PDTC. Furthermore, the migration and invasion were highest in the HT29+NIBP cell, and the inhibition of PDTC was not obvious, the difference is statistically significant (P<0.05).7. Inhibition of NIBP could reduce the activation of classical NF-κB signaling pathway, expression of MMP2 and MMP9, and then dicreased the migration and invasion of colorectal cancer:The total p65 and IκBP, and their phosphorylation were similar in HCT1 16 and HCT1 16+NIBP shRNA cells, and the expression of MMP2 and MMP9 was in a high level. The total p65 and IκBP were not changed, but the phosphorylation of p65 and IκBP, and expression of MMP2 and MMP9 were increased in HCT1 16 cell which treated by the 20ng/mL TNF-a for 48 hours. The total p65 and IκBP, and their phosphorylation, and the MMP2 and MMP9 were decreased in the HCT1 16+NIBP shRNA cell, and the expression of these proteins was not raised in the HCT1 16+NIBP shRNA cell which treat by TNF-a. Furthermore, the migration and invasion were highest in the HCT1 16+NIBP shRNA cell, and the promotion of TNF-a was not obvious, the difference is statistically significant (P<0.05).8. NIBP regulated the ability of liver metastasis by effect on the activation of classical NF-κB signaling pathway and expression of MMP2 and MMP9 in vivo: The expression of p-p65, MMP2 and MMP9 in the liver metastatic tissue of HT29+NIBP mice was higher than that in HT29 mice. By contrast, the expression of these proteins was lower in the liver metastatic tissue of HCT116+NIBP shRNA mice than that in HCT116 mice.Conclusion1. NIBP may be a novel molecular marker that reflects the growth and metastasis of human colorectal cancer, which increased in later stages of CRC and that with liver metestasis.2. NIBP directly regulates the activation of classical NF-κB signaling pathway, and the expression of MMP2 and MMP9, then affects the invasion of human colorectal cancer in vitro, and may be play a part in the metastasis of CRC in vivo.