| BackgroundPJS is anautosomal dominant genetic diseases, characterised by mucocutaneous hyperpigmentation and multiple benign gastrointestinal hamartomatous polyps. The relative incidence of PJS is approximately1/120000-1/8300births. In earlier studies, PJS polyps were thought to be hamartomatous or hyperplastic polyps which had rare risk of canceration. Along with the further study on PJS, researchers have found that PJS patients have an increased risk of developing a variety of neoplasms, including both benign and malignant tumors, particularly of the colon, breast, pancreas, testis, uterus, cervix and ovary. It is estimated that the incidence of cancer among these patients is15.2fold higher than in the general public.The cumulative risk at the age of65is up to93%.The gene responsible for PJS has been identified as LKB1.30to70percent of PJS patients show germline mutations in LKB1gene, with a smaller proportion of individuals presenting with somatic mutations. Only10-20%of patients with family history of PJS may be caused due to de novo germline mutation.of LKB1. The heterogeneity of PJS suggests that there is still considerable cases can not be explained by LKB1mutations alone. Nicholas found that LKB1mutations was not related to the incidence of cancer in the largest-ever clinical research(419PJS patients),which was highly suggestive of other genes or signaling pathways involved in this complicated process. The emerging view is that tumours originate in the defect of stem cell behavior. Meanwhile, normal asymmetric division is dysregulated, stem cells may generate2daughter cells with stem cell features that may accumulate and form a neoplasm such as polyp. Some signaling pathways, such as the Notch, Hh and Wnt signaling pathways, that regulate stem cell renewal may also be involved in tumorigenesis.Many studies for the function of HH signaling pathway in tumors have shown that abnormality in this important pathway get involved in at least25percent tumors. The development of the gastrointestinal tract derive from endoderm.As significant endoderm signaling, HH pathway has been confirmed closely related to digestive tract tumors.Three HH homologues have been identified in mammals, including Sonic hedgehog(SHH), Indian hedgehog(IHH), and Desert hedgehog (DHH). Among them, SHH is the hot point of research. An unregulated progenitor cell proliferation induced via abnormal SHH protein expression or pathway activation has been suggested to play a role in all cancers of the gastrointestinal tract.The misregulation of sonic hedgehog signaling has also been implicated in several forms of precancerous lesions, such as Barrett’s esophagus, gastric metaplasias, ulcerative colitis and Crohn’s disease.In addition, SHH activation does not seem to be correlated with either pTNM/UICC stage, location of the neoplasm in the colon, or tumor size, which might support the concept that SHH dysregulation is an early event in colon cancer carcinogenesis, already present in polyps.Recent study found that LKB1functions as a component of HH pathways and confirmed that LKB1influenced the response to HH by controlling the length of the primary cilium and that LKB1loss resulted in the dampening of HH signaling. Undoubtedly, this viewpoint provide a new approach to explore the formation and canceration mechanism of PJ polyps. LKB1and SHH/GLI1signal paahway possibly play an important role in PJS hamartoma’s development process.ObjectiveTo detect the expression pattern of SHH, the important ligand in HH pathway, and downstream nuclear transcription factor GLI1in mRNA and protein level in PJS hamartoma and their potential clinical significance. To explore the correlation of LKB1and SHH/GLI1signaling pathway. Aim at exploring PJS pathogenesis from a new angle. To detect the expression of LKB1, SHH, GLI1in mRNA and protein level in normal mucous membrane, colorectal adenoma, colorectal adenocarcinoma. To explore their expression and variation tendency in different tissues. To make sure whether these molecules involve in the evolution of colorectal tumors.Materials and Methods1、LKB1、SHH、GLI1mRNA detection:14PJS polyps,14colorectal adenomas,14colorectal adenocarcinomas,14paired non-tumor normal mucosa were collected to extract organization RNA.Using reverse transcription and the SYBR Green realtime PCR to examine LKB1、SHH、GLI1expression.2、The paraffin specimens of20PJ polyps is the experimental group,25colorectal adenomas,25colorectal adenocarcinomsa,20paired non-tumor normal mucosa as control group. Immunohistochemical(IHC) method is used to detect LKB1, SHH,GLI1protein expression.3、Using SPSS13.0statistical 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. Partial correlation was used to determine whether there was a positive or negative correlation. Differences were considered significant if P<0.05.Results1、In mRNA level LKB1relative expression quantity in PJS polyps was2.65±1.54, there was no significant difference between PJS mucosas and colorectal adenoma(4.57±3.48). There was significant difference between PJS mucosas and colorectal adenocarcinomas(8.42±3.92). SHH、GLI1relative expression quantity in PJS polyps were2.64±1.59、3.27±2.11, the expression levels of in PJS polyps were significantly higher than that in normal group(1.18±0.7、0.98±0.51),but lower compared to that in colorectal adenoma and colorectal adenocarcinomas(4.74±2.32、4.63±3.11).There was no significant difference between PJS mucosas and colorectal adenoma.2、The expression of LKB1、SHH、GLI1proteins were limited to epithelium layer, with subcellular location in cell membrane and cytoplasm. The nuclear immunostaining of GLI1protein in colorectal adenocarcinomas group was increased. SHH and GLI1proteins in normal group showed no expression(17/20,18/20) or very faint expression(3/20,2/20). No nuclear immunostaining was detected.There were two different patterns of LKB1staining 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 LKB1expression also showed faint or loss of SHH and GLI1expression.The staining in adenoma and adenocarcinomas was related to cellular atypia.The staining of three proteins in eumorphism area was weaker than that in atypical area.3> The mean rank of LKB1protein in the normal group is18.68, PJS group is22.33. The difference was no statistically significant(P=0.305). The mean rank of SHH protein in the normal group is16.88, PJS group is24.13. The staining in PJS group was significantly higher than that in normal group, difference was statistically significant(P=0.018). The mean rank of GLI1protein in the normal group is17.05, PJS group is23.95. The staining in PJS group was significantly higher than that in normal group, difference was statistically significant(P=0.017).The comparision 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 positive rate and staining in normal group was lowest, and then was PJS group and adenoma group, which was highest in adenocarcinoma group.The difference between PJS group and adenoma group was no statistically significant.4, The partial correlation was analyzed between LKB1and SHH、SHH and GLI1、LKB and GLI1.In mRNA level, the correlation coefficient is r=0.457,(P<0.01)、r=0.314,(P<0.05)、r=0.225,(P>0.05), respectively. In protein level, the correlation coefficient is r=0.224,(P<0.05)、r=0.640,(P<0.01)、r=0.363,(P<0.01), respectively. There was significant positive correlation between two of them in protein level.conclusion1、The biallelic inactivation of LKB1is not widespread incidents in PJS polyps. Most of these PJP polyps contain wild-type allele.The immunostaining of LKB1in PJS polyps is diverse, which suggest the genetic heterogeneity of PJS.2、The expression levels of SHH and GLI1in PJS polyps were significantly higher than that in normal group.The results is suggesting that SHH dependent activation of the HH pathway may play an important role in PJS.2、The expression intensity of LKB1、SHH、GLI1increased gradually with increasing degree of malignancy. There was significant positive correlation between two of them. The results is suggesting that the abnormality of SHH-GLI1signaling pathway may be an early event in the course of PJS cancerization and may be regulated by LKB1. |
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