Experimental Study Of Effect Of Wnt Antagonist Genes Methylation On Laterally Spreading Tumor(LST)

1. Objectives and significanceLaterally spreading tumors(LST) is a kind of flat colon tumor, which is charactered with laterally spread along the surface of colon membrana mucosa, and highly carcinogenesis potency. Till now, research about LST is limited in clinic endoscopy diagnosis and treatment.Colorectal adenomas can be divided into two groups in morphology, protruded-type and flat-type. However, the accurate frequencies of genetic and epigenetic alterations in flat-type colorectal advanced adenomas (laterally spreading tumors) have remained largely unknown.Growing evidence suggests that flat colorectal cancers (CRC) account for 10% to 20% of all CRCs and that these are frequently associated with more advanced pathologies. However, controversy exists as to the origin and progression of flat CRCs compared with the more common polypoid-type morphology.Supporters of the adenoma-carcinoma sequence posit that flat carcinomas originate through an adenoma intermediate, either by ulceration of a polyp or through the formation of a flat adenoma. Others have asserted that the identification of flat carcinomas with no observable adenoma component suggests that flat carcinomas arise de novo without progressing through an adenomatous intermediateThe Wnt signaling pathway, which plays important roles in embryogenesis, development and homeostasis, is also closely linked to carcinogenesis. The Wnt ligands bind to Frizzled receptors and low-density lipoprotein receptor-related protein (Lrp) 5/6 co-receptors located at the plasma membrane and activate both canonical and non-canonical pathways. Activation of the canonical Wnt pathway leads to stabilization ofβ-catenin and its accumulation in the cytoplasm. This is followed by translocation to the nucleus, whereβ-catenin associates with T-cell factor (TCF)/lymphocyte enhancer factor family transcription factors to stimulate the expression of target genes. Constitutive activation of the canonical Wnt pathway resulting from mutation of APC, AXIN1 and CTNNB1 (β-catenin) has been observed in various human cancers.The non-canonical Wnt pathways are known to transduce signals independent ofβ-catenin and to include the planar cell polarity, Wnt/Ca2+ and c-jun N-terminal kinase pathways, yet much about their roles in cancer remains unknown.Today more and more attention is payed to Wnt pathways.In our study,we investigate effect of Wnt antagonist genes methylation on Laterally spreading tumors(LSTs).2. Methods and project1. A total of 103 tumors (consisted of 52 LSTs and 51 PAs) were collected from individuals who underwent endoscopic resection under total colonoscopy at Nanfang Hospital from 2006 to 2009. All of 103 tumor tissues were fixed in 10% formalin and embedded in paraffin.There are 59 tumors (consisted of 32 LSTs and 27 PAs) have-collected fesh-specimens.We have collected tumor and adjacent normal tissues in fresh ones. All of the fresh tissues were frozen immediately in liquid nitrogen and stored at -80℃until required.2. We investigated the mRNA levels of key molecules (SFRPs) in the wnt signaling by semi-quantitative RT-PCR in tumor specimens. CRI micro color system was applied to observe and take photos. The professional image analysis software, Image Pro plus 6.0 was used to analyse the difference of integrated optical density (IOD) and stained area (SA) in the two groups, mean density(MD)= IOD/SA, and the mean concentration of the two groups was compared and analyzed.3. We also examined the methylation status of SFRPs family in primary colorectal tumors by methylation specific PCR (MSP). DNA of all samples were extracted and modified by sodium bisulfite treatment converting unmethylated, but not methylated. This modified DNA was used as a template for PCR. Methylation Specific PCR (MSP) was performed to observe the methylation of sFRP1, sFRP2,sFRP5 in LST,pAs and their normal tissues of patients. Frequencies of Methylation of Individual Genes in LST,PAs,and Normal Colon Samples were compared and analyzed.Bisulfite modified DNA was used as a template for MSP using primers specific for either the methylated or unmethylated sequences. The amplification products were separated on a 2% agarose gel and visualized by ethidium bromide staining and UV transillumination.4. We also examined the protein expression of SFRP1 by immunostaining in neoplastic colonic tissues. Immunoreactivity was estimated semiquantitatively.5. Fisher exact tests. Differences were considered significant if P<0.05. All significance.tests are two-tailed. All statistical tests were performed using the software SPSS3. Results1. The sFRP1 is inactivated by hypermethylation in primary CRCs.The methylation of sFRP1, sFRP2, sFRP5 in colon tissue was significantly higher in LSTs and PAs than that in normal control samples (P<0.05). There was no significant difference in the methylation of sFRP2, sFRP5 between LSTs group and Pas group, while the methylation of sFRP1 in colon samples, LSTs group was significantly higher than that in PAs group (P=0.030). Meanwhile, the expression of sFRPl mRNA in LST group was significantly lower than that in PAs group (P=0.002).. However, Protein expressions of SFRP1 were decreased significantly in LSTs compared with those in protruded adenomas (PAs) (Mann-Whitney U test, Z=-2.973, P=0.003;). SFRP1 was expressed at a very low level in LSTs. In addition, positive staining for SFRP1 was frequently observed in the adjacent morphologically normal tissue of LSTs.2. SFRPs mRNA showed a complete absence of expression in HCT116 cells. After treatment with 5-aza-dCyd for 5 days, induction of SFRPs mRNA expression occurred in HCT116 cells.4. Conclusions1. Absence or downregulation of SFRP1 expression may be a characteristic in LSTs.2. Hypermethylation of SFRP1 plays an important role in LSTs.