| Background and purposeColorectal cancer is the fourth of the most common malignant tumor in our country, the incidence increased year by year and more young people got this disease. Metastasis is the main cause of death in patients with colorectal cancer. Metastasis is a multi-step, multi-stage complex process, each stage is regulated by many genes or proteins, which change the tumor cell motility and athletic ability. Peritoneal carcinomatosis (PC) is a common mode of metastasis from colorectal cancer (CRC). PC is found in approximately7%of patients at the time of their primary surgery and in approximately4%to19%of patients during follow-up after curative surgery. Due the poor prognosis for CRC patients, who have a mean survival between5and9months, it is vital to identify biologic predictors for early diagnosis and prognosis. Therefore, finding the effective and specific metastasis-related genes and investigating its function not only help elucidate the molecular mechanism of colorectal cancer metastasis, but also find new targets for clinical diagnosis.With the gradual implementation of human genome plan and rapid development of molecular biology disciplines, more and more gene sequence of animal, plant and microorganism have been to be definite, and the gene sequence data are growing at an unexpected rate. Gene chip (also known as DNA chip) technology is the product of the scientific development requirements. Because of its high-speed, high-throughput, intensive and low-cost characteristics, Gene chip has been widely used in screening of differentially expressed genes of different cancer tissue, such as pancreatic cancer, ovarian cancer, breast cancer, gastric cancer and colorectal cancer. Similarly, there are a large number of differentially expressed genes between colorectal primary tumor and the corresponding liver, lymph node metastasis foci, but the differentially expressed genes between the peritoneal metastases and primary tumor tissue has rarely been reported around the world. Thus, colorectal cancer peritoneal metastasis related genes can be found by comparing the different mRNA expression level between the primary tumor tissues of colorectal cancer and peritoneal metastasis.To this end, we screened the four paired primary tumor tissues and the corresponding peritoneal tissues with gene chip to compared the differences genome-wide mRNA expression level. Then we identify the differentially expressed genes by multiples method and analysis these genes with functional annotation clustering analysis. Through analysis of gene functional classes of tumor progression, we explore the molecular mechanism of peritoneal metastasis of colorectal cancer. The gene chip technology has a certain false-positive rate, and therefore we validate part of the filtered differentially expressed genes by RT-PCR. According to the these results, we finalized choose collagen triple helix repeat protein (Collagen Triple Helix Repeat Containing CTHRC1) as the experiment target in the future.Human CTHRC1was first found in a screen for differentially expressed sequences in balloon-injured versus normal rat arteries, and increased expression of CTHRC1in rat fibroblasts promoted cell migration and inhibited collagen I synthesis in these cells. Aberrant expression of CTHRC1is widely present in solid human tumors and seems to be associated with cancer tissue invasion and metastasis. Recently, some studies that focused on digestive tract tumors found that CTHRC1is associated with Barrett’s esophagus and esophageal adenocarcinoma, and up-regulated CTHRC1in gastric carcinogenesis contributed to tumor cell invasion and metastasis.CTHRC1is a cofactor of wnt protein. CTHRC1can activate wnt/pep pathway selectively by stabilizing the membrane receptor complex. Activation of the Wnt/pcp Pathway enhanced its downstream small GTPases activity, especially Racl and RhoA. The Ras GTPases Superfamily contains Rho GTPases subsets, Racl (Ras-related C3botulinum. Toxin Substrate) and Cdc42(the Cell Division CYCLE42) are the most widely studied of all RhoGTPases. Activation of Racl promotes cancer cell migration, invasion, activation of RhoA promotes cancer cell migration and metastasis. by reducing cancer cell adhesion ability.MiRNA is21to25bp non-coding small RNA. It is mainly through two kinds of mechanism to regulate gene expression:When the miRNA and the purpose mRNA completely or almost completely complement, the purpose mRNA degradated; if not completely complement, miRNA regulate the translation process negatively, hinder the protein translation, but not affect the purpose mRNA. Current study has found there were700kinds of human miRNAs, which involved in nearly1/3of the human gene expression process. Abnormal expression of miRNA exist in numbers of human cancers, miRNA plays a role like tumor suppressor or oncogenes. Recent research has further proved that miRNA is related to tumor invasion and metastasis, which provides a new way for diagnosis and treatment of tumor metastasis. There are few studies about MiR-520family, and its main research focused on breast cancer.Some literatures indicate that miR-520d is related with HER2/nue receptor in breast cancer.The other literatures indicate that miR-520c play a role of a tumor suppressor gene in breast cancer because of regulation between TGF-P and NF-κB pathway with miR-520d.The purpose of this study is to study the characteristics of CTHRC1expression in colorectal and explore the mechanisms of CTHRC1promoting colorectal cancer metastasis, investigate the relationship between miR-520d and CTHRC1in colorectal.Methods and results(A) Gene chip screening the differentially expressed genesFour paired primary colorectal cancer and the corresponding peritoneal metastasis tissues of the four patients who underwent surgical treatment of colorectal adenocarcinoma in Nanfang Hospital of Southern Medical University from April2010to June2009were collected. Samples were soaked in the RNAlater then stored at-80℃until further use. The total RNA were extracted by Trizol reagent and subsequently inversely transcribed into cDNA, and in vitro RNA syntheses was applied to synthetize aRNA, then Cy3dye was applied for labeling reaction of aRNA, after which, the Cy3labeled aRNA samples were hybridized with the Whole Human Genome Oligo Microarray. Hybridized slides were scanned using the LuxScan3.0Scanner (Beijing CapitalBio Biotech, Ltd, China), and images were acquired by GenePix Pro v6.0software and transferred into digital signal then analyzed. The Empirical Bayes method was used to screen differentially expressed genes(p≤0.05), and the GO Term Finder analysis software was applied to functional annotations. GENEMANIA software was applied to constuct co-expression network, and evently the selected genes were verified by RT-PCR.According to experiments results and some related articles, collagen triple helix overlapping protein1(CTHRC1) was chosen as the research target for further study. (B) A preliminary study for biological function of CTHRC11. The influence of CTHRC1on colorectal cancer cell biological functions1.1Western Blot was used to detect protein expression level of CTHRC1in SW480, SW620, HCT116, LS174, HT29and LOVO,6human colorectal cancer cell lines. GAPDH is used as an internal reference; result of the Western Blot is analysis with Gelpro32software. The optical density (IOD) value of strips presents the expression level of the corresponding strip.The results showed that the expression of CTHRCl was significantly lower in low metastatic potential cell lines SW480(1.455±0.21) than in high metastatic potential cell lines SW620(5.224±0.053)(P<0.001)1.2CTHRC1transfection efficiency detection by ImmunoblotGV287empty vector (control group, NC) and GV287-CTHRC1recombinant vectors were transfected in SW480and SW620cells. Western Blot was used to detect protein expression level of CTHRC1of these cell lines after transfection and the results showed that the expression level of CTHRC1in GV287-CTHRC1group was higher than that in the NC group (GV287empty vector).1.3Influence of CTHRC1on biological characteristics of colorectal cancer cells.1.3.1Cells showed a decreased proliferation after transfected with CTHRCl by in vitro CCK-8assay. The results showed that SW480cells over-expression group and the control group was statistically significant differences appeared in the fourth day (P=0.001), proliferation and interaction time with statistics differences (F=16.528, P<0.001), the different proliferation ability within the group of over-expression group and the control group was statistically significant (P <0.001); SW620cells over-expression group and the control group was statistically significant differences appeared in the3rd day (P=0.004), proliferation and interaction time with statistics differences (F=2529.379, P<0.001), the different proliferation ability within the group of over-expression group and the control group was statistically significant (P<0.001)1.3.2colony-forming experiments results showed that:colony formation rate of CTHRC1overexpression group was lower than the NC group. The difference of SW480cells was statistically significant (P<0.001), and the difference of SW620cells was statistically significant too (P=0.001).1.3.3The results of in vitro invasion assay showed that cells had significant enhanced invasiveness after transfected with the CTHRC1, SW480cells were significantly different in groups (P<0.001); SW620cells were significantly different in groups (P=0.001).2. CTHRC1expression in colorectal cancer tissues and its clinical significance2.1For immunohistochemistry, all141tissues samples from111CRC patients who underwent surgical resection at the Nanfang Hospital of Southern Medical University (Guangzhou, China) were enrolled. Fifty-seven paraffin-embedded tissue samples of primary CRC and the corresponding30peritoneal metastases were obtained from57of these CRC patients. ALL these CRC patients who underwent surgical resection between the April2003and June2009suffered from PC and have been followed up after the surgery once6months a time. The remaining54primary CRC tissue samples were obtained from the patients who underwent surgical resection between April2003and June2006, and all of these patients were free from PC after five years’ follow-up.2.2CTHRC1was predominantly positive in the cancer cell cytoplasm. The mean AOD of the primary tumors with PC group, the primary tumors without PC group and the PC group were0.25±0.04,0.19±0.03and0.26±0.04, respectively, and the expression of CTHRC1was different between the three groups (P<0.001). Furthermore, the expression of CTHRC1was higher in primary CRC with peritoneal metastases and peritoneal metastases than in primary CRC without peritoneal metastases (both P<0.001).2.3All of the enrolled patients were divided into two groups according to the mean AOD (0.103to0.396, mean value=0.224). As expected, Kaplan-Meier curves revealed that patients with higher expression of CTHRC1had a significantly poor survival than those with low CTHRC1expression (31.36±5.63months vs.57.84±3.69months, P<0.001)2.4The Cox regression analysis showed that CTHRC1expression status was an independent negative prognostic factor along with sex, tumor invasion depth, tumor size and lymph node metastasis, with a hazard ratio (HR) of2.754between groups with high and low CTHRC1expression (P<0.001,95%CI1.731to4.383).(C) Preliminary study of the relationship between gene CTHRC1and hsa-mir-520d1. Predict and characterize the target miRNA of CTHRC11.1Predict target miRNA with the index CTHRC1in the microRNA.org, TargetScan, Pictar and miRanda prediction database, the four results will be intersected, which predicted a higher reliability and accuracy of target miRNA.1.2Real-time PCR was used to detect the expression of hsa-mir-520d in four colorectal cancer cell lines SW620, SW480, HT29, and LOVO. The results showed that the expression of hsa-mir-520d was significantly lower in low metastatic potential cell lines SW480cells than in high metastatic potential cell lines SW62(178.493±7.867, SW620为7.09±2.73, P<0.001).2. Analysis of correlation between CTHRC1and mir-520d-5p2.1Real-time PCR was used to detect the expression of hsa-mir-520d in23colorectal paired tissues (tumor and normal tissues). The results showed that mir-520D-5P expression in normal tissues was significantly higher than the paired cancer tissues (P=0.001) 2.2Western blot was used to detect CTHRC1protein expression levels in23paired colorectal cancer tissues. The results showed that CTHRC1expression in cancer tissues was significantly higher than that in paired normal tissues (1.3018±1.016vs0.6567±0.552, P=0.003).2.3Impact of miR-520d-5p on changes of CTHRC1protein expression levels in cells of colorectal cancer2.3.1Real-time PCR was used to detect the expression of hsa-mir-520d after colorectal cancer cells was transiently transfected with mimics and inhibitor of miR-520D-5P. The results showed that hsa-mir-520d expression was significantly higher after transfected with mimics than the cells transfected with NC (161.167±16.06vs19.456±3.53, P0.001). And hsa-mir-520d expression was significantly lower after transfected with inhibitor than the cells transfected with NC (P=0.002).2.3.2Western blot was used to detect CTHRC1protein expression levels after colorectal cancer cells transfected with mimics and inhibitor of hsa-mir-520d. The results showed that CTHRC1protein expression level was lower in the hsa-mir-520d mimics group than the NC group. And meanwhile, CTHRC1protein expression level was higherer in the hsa-mir-520d inhibitor group than the NC group.Conclusion1. CTHRC1is the colorectal cancer peritoneal metastasis-related gene.2. CTHRC1is related with peritoneal metastasis in colorectal cancer and indicates a bad prognosis.3. CTHRC1can depress proliferation but promote immigration in colorectal cancer cells.4. CTHRC1protein expression level may regulated by miR-520d-5p in colorectal cancer. |
