| BackgroundPeutz-Jeghers syndrome(PJS) is an autosomal dominant hereditary diseases characterized by mucocutaneous melanin deposition and multiple benign gastrointestinal hamartomatous polyps as well as not gastrointestinal multiple tumors. There is a high recurrence rate of gastrointestinal polyps after surgical resection.In earlier studies, PJS Polyps were thought to be hamartomatous polyps which had rare risk of canceration. Along with the further researeh on PJS, it is found that some PJS polyps are adenomatoid polyps which have higher risk of canceration. And it is known as hamartoma-adenoma-carcinoma sequenee, but there were not studys of its authenticity from the gene and its expression levels.PJS patients not only have a rate of cancer risk with gastrointestinal polyps, but usually with much of organs rumors outside the gastrointestinal tract such as pancreas, ovary, testis, mammary gland, uterus, lung, and so on, and the incidence of cancer in PJS families is higher than the general population. Clinical PJS mainly performance for multiple polyps in the gastrointestinal tract, abdominal symptoms with repeatedly intussusception, mainly gastrointestinal bleeding and intermittent abdominal pain, PJS polyps can occur in any part of the gastrointestinal tract, mainly in the jejunum and ileum,followed by a colon and stomach. PJS is a moderate degree of canceration of hereditary colorectal cancer syndromes, slow cancer, its mechanism is not fully elucidated, no interventions that can effect a radical cure and prevention of illness and evolution of PJS.Major causative gene of PJS has been identified as LKB1, research have reported about 30 to 67% of PJS germline mutations of the LKB1 gene, a small percentage of somatic mutations, and there is a clear family history of PJS patients may be only 10% to 20% due to de-novo LKB1 germline mutations.This genetic heterogeneity means that there is still a significant part of PJS can not be used alone LKB1 mutations to explain. Nicholas found that LKB1 mutation is not associated with PJS cancer incidence in so far the largest clinical studies (419 PJS patients), Hamid for 46 PJS genealogy research also confirmed this point of view,which was highly suggestive of other genes involved in the PJS evolution process.DNA mismatch repair(MMR) system is a kind of security system in human cells to recognize and repair DNA base mismatch, to maintain high fidelity of DNA replication. Until now a total separation from the human cell cloning to nine MMR gene, which is hMLH1, hMSH2, hMSH5, hMSH6, hMSH4, hMSH3 hPMSl, hPMS2 hMLH3 respectively, they are responsible for identifying and repair of DNA during replication gene deletions, insertions, point mutations and other errors. Mismatch repair genes (MMR) and hereditary nonpolyposis colorectal cancer (HNPCC) are closely related.It has been proved that abmormal hMLHl and hMSH2 protein expression and its function defects cause the hereditary nonpolyposis colorectal cancer (HNPCC). hMSH2 gene contains a 2727bp open reading frame, one kind of a protein consisting of 909 amino acid sequence of its translation postcoder,85% of which is in line with the corresponding area of the yeast protein.hMLHl contains 2268bp open reading frame, encoding 756 amino acid residues. Its promoter methylation can lead to the MMR deficiency, recent studies have found that the inactivation of hMLHl is associated with the occurrence and development of tumor, which is associated with about 30% of HNPCC onset.Mismatch repair gene mutations can produce defective mismatch repair proteins, mismatch repair functions can not function properly, leading to microsatellite instability or DNA replication errors,thereby increasing the instability of the cell genome, causing abnormal proliferation and differentiation of cells, prompting a variety of malignant tumors. The study found that the characteristic manifestations of hereditary non-polyposis colorectal cancer (HNPCC) for DNA mismatch repair genes (MMR genes) mutations cause DNA replication errors (RER) and instability (MSI). PJS canceration is slow, with a similar clinical and pathological features of hereditary non-polyposis colorectal cancer (HNPCC):(1) the incidence of younger age; (2) prone to enteral and parenteral multiple tumors of other organs; (3) with a high mutation significantly increased the number and growth regulation, apoptosis-related gene mutations; (4) less metastasis, easy to limitations, the better prognosis. On the basis of HNPCC high grade but low malignant phenotype,an explanation has not been fully confirmed for the the RER tumors of gene mutations frequently, it transferred invasion blocked. Thus, it is necessary to research the relationship between PJS and the MMR genes, to study the authenticity from the gene and its expression level; then the introduction of PJS polyps evolution mechanism.ObjectiveThe subject based on previous work, to detect the expression of MMR genes hMLHl and hMSH2 expression and active status in the PJS tissue, and then combined with the expression of LKB1 to explore the possible correlation between them.From a new direction to study the PJS onset and malignant transformation mechanism, aim to provide theoretical basis for PJS syndrome early diagnosis, cancer risk assessment, and its prevention; In addition, analysis of the expression of the three molecules in mRNA and protein level in the normal intestinal mucosa, colon adenoma and colon adenocarcinoma, to explore their expression in different tissues and the evolution of trends.Materials and Methods1、hMLH1±hMSH2±LKB1 mRNA detection:18 PJS polyps, colorectal adenomas, colorectal adenocarcinomas, paired non-tumor normal mucosa were collected to extract organization RNA, respectively. Using reverse transcription and the SYBR Green realtime PCR to examine hMLH1±hMSH2、LKB1 expression.2、The paraffin specimens of 25 PJ polyps is the experimental group,25 colorectal adenomas,25 colorectal adenocarcinomsa,25 paired non-tumor normal mucosa as control group. Immunohistochemical(IHC) method is used to detect LKB1、hMLH1、KMSH2 protein expression.3、Using SPSS 13.0 statistical software to deal with experimental data. If raw data consistent with parameters test conditions, one-way ANOVA was used to compared independent samples in many groups. Kruskal-Wallis test and Mann Whitney U-test were used to assess the differences of staining intensity. Bivariate correlation was used to determine whether there was a positive or negative correlation. Differences were considered significant if P<0.05. r correlation coefficient indicates the relation between two related closely.Results1.. In mRNA level hMLH、hMSH2 relative expression quantity in PJS polyps was 0.68±0.62、0.71±0.62, lower than paired non-tumor normal mucosa (0.96±0.65、 0.98±0.65), higher than colorectal adenomas (0.45±0.87、0.33±0.75), and colorectal adenocarcinomas (0.35±0.78、0.23±0.87), respectively there were significant differences among them. LKB1 relative expression quantity in PJS polyps was 2.72±1.06, higher than paired non-tumor normal mucosa (1.62±0.65), lower than colorectal adenomas (4.32±2.59) and colorectal adenocarcinomas (7.84±3.26),there was no significant difference between PJS mucosas and colorectal adenoma.2、Immunohistochemical results show that the expression of LKB1、hMLH1、 hMSH2 proteins were limited to epithelium basal layer, with subcellular location mainly in the nucleus, also visible in cell membrane and cytoplasm. The nuclear immunostaining of hMLHl and hMSH2 proteins in normal group showed the strongest expression(15/25,17/25) or very faint expression(8/25,7/25), decreasing in colorectal adenocarcinomas group. There were two different patterns of LKB1 staining in PJS samples. In one there was both membranous and cytoplasmic expression in glandular epithelial cells. In a second pattern there was loss of expression for LKB1. The polyps that showed loss of LKB1 expression also had strong of hMLHl and hMSH2 expression.The staining in adenoma and adenocarcinoma groups were related to cellular atypia.The staining of hMLHl and hMSH2 proteins in eumorphism area relatively stronger than that in atypical area, while the LKB1 dyeing opposite distribution.3、The mean rank of hMLHl protein in the normal group is 70.28, PJS group is 60.84. The staining in PJS group was significantly lower than that in normal group, difference was statistically significant(P=0.019). The mean rank of hMSH2 protein in the normal group is 73.20, PJS group is 56.76. The staining in PJS group was significantly lower than that in normal group, difference was statistically significant(P=0.009). The mean rank of LKB1 protein in the normal group is 33.13, PJS group is 41.98, difference was a statistically significant(P=0.039).The comparison among four groups (normal group, the PJ polyps group, the adenoma group and adenocarcinoma group) showed that the positive rate and immunostaining difference of three proteins were statistically significant(P<0.05). The hMLHl and hMSH2 positive rate and staining in normal group was the highest, and then was PJS group and adenoma group, which was lowest in adenocarcinoma group, but for LKB1, the positive rate and staining in normal group was lowest; the group of PJS, colonic adenoma, colonic carcinoma group in turn on the rise, and comparing two groups, the difference between PJS and adenoma group was no statistically significant(P>0.05).4、 The bivariate correlation was analyzed between LKB1 and hMLHl, LKB1 and hMSH2, hMSH2 and hMLHl. In mRNA level, the correlation coefficient is r=-0.732, (P<0.01); r=-0.834, (P<0.05); r=1.000, (P<0.05); respectively. In protein level, the correlation coefficient is r=-0.637, (P<0.05); r=-0.653,(P<0.01); r=0.967, (P<0.01); respectively. LKB1 and the expression of hMLHl and hMSH2 has significant negative correlation, respectively. hMLHl and hMSH2 have significant positive correlation, the correlation relationship between the two closely.conclusion1、The expression levels of hMLHl and hMSH2 in PJS polyps were significantly lower than that in normal group, the group of PJS, colonic adenoma, colonic carcinoma group in turn on the decline. Prompt the MMR repair the genetic mutation caused by lack of protein expression may not be in PJS cancerous process critical step but an early event.2、The expression intensity of hMLHl and hMSH2 decreased gradually with increasing degree of malignancy. But the expression intensity of LKB1 increased gradually with increasing degree of malignancy. LKB1 and the expression of hMLHl and hMSH2 has significant negative correlation, respectively. The results is suggesting that LKB1 and mismatch repair genes may exist between interaction in the course of PJS cancerization.3、The immunostaining of LKB1 in PJS polyps is diverse, which suggest the genetic heterogeneity of PJS. LKB1 loss expression of P-J polyps in MMR expression level increased slightly, speculated that LKB1 may be the regulation of the MMR. |
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