Role Of Hedgehog Signaling Pathway In Gastric Tumorigenesis

Background:Gastric cancer is a public healthy problem in the whole world with high incidence and mortality. Nearly one half of gastric cancer patients in global occurred in our country. More and more youth are being suffered from it in recent years. Unclear pathogenesis and lack of specific targeted drugs result in poor therapeutic effect. The exact molecular mechanism of the signaling pathways involved in gastric cancer is one of important measures for improving therapeutic effect and developing targeted therapy drugs.Hedgehog signaling pathway plays an important role in embryonic development, maintenance of the dynamic balance of cell proliferation and apoptosis, process of tissue regeneration and tumorigenesis. Many studies showed that the abnormal activation of hedgehog signaling pathway may relate to gastric cancer, but the detailed mechanism is so far unclear. There is no systematic report about how Hh signaling changes in the classic process of gastric tumorigenesis, which includes chronic superficial gastritis, atrophy gastritis, intestinal metaplasia, dysplasia and gastric cancer. Therefore, we try to investigate the variation of expression and molecular mechanism of Hh signaling in the gastric tumorigenesis in vivo and vitro.Objective:1. To investigate the significance of the core components of Hh signaling pathway, including:Shh, Smo, SuFu, Gli1, Gli2, Gli3 and downstream CyclinDl expression in gastric carcinoma and precancerous lesion in the gastric tumorigenesis process.2. To identify the relationships between Hh signaling and CyclinD1 expression, gastric mucosa cell proliferation and apoptosis, clinical stage of gastric cancer, and the possible mechanism of Hh signaling in gastric cancer.3. To evaluate the effects of Hh signaling pathway inhibitor cyclopamine on proliferation and expression of Glil and CyclinDl in gastric cancer cell, and further study the molecular mechanism of Hh signaling in the gastric tumorigenesis in vitro.MethodsClinical specimens A total of 186 cases of gastric mucosa lesions with clinical data retrieved from endoscopic biopsy and surgery were available for the study in the First Affiliated Hospital of Nanchang University from Jan.2007 to Sep.2009. The gastric mucosa lesions included chronic superficial gastritis(CSG) 44 cases, metaphastic atrophy gastritis (MAG) 44 cases, dysplasia (Dys) 48 cases and gastric cancer (GC) 50 cases, which were verified by HE dye. Expression of the core components of Hh signaling pathway, including:Shh, Smo, SuFu, Gli1, Gli2, Gli3 and CyclinD1 were assayed by immunohistochemistry, besides the expression of PCNA. TdT-mediated dUTP nick end labeling (TUNEL) was performed to detect cell apoptosis.In vitroCell morphology of gastric cancer cell lines MKN28 was observed under inverted microscope, the inhibition rates of cell growth were detected by MTT assay, and the expression of GLI1 and CyclinDl were determined by western blot after treated with cyclopamine for 24h,48h and 72h.Results:Clinical specimens(1) The expression of Shh in Dys group was the highest, which was the lowest in MAG group, Shh expression in MAG group was significantly lower than in CSG group (P=0.003<0.01), Shh expression in GC group was also lower than CSG group (P=0.035<0.05).(2) Obvious difference of Smo expression level was observed in the process of gastric tumorigenesis. The expression of Smo was low in GC group. There was no significant difference between GC and CSG group (P=0.051>0.05), but much lower in GC group than MAG and Dys group (P<0.001).(3) SuFu expression decreased gradually during the process of gastric tumorigenesis, GC group had a significant lower level than the other groups (P<0.001).(4) Gli1 was expressed abnormally and nuclear staining positive rates were higher in gastric cancer. The expression of Gli 1 protein in GC group was significantly higher than CSG group (P=0.001<0.01), whereas the difference was no significance compared with the other three groups (P>0.05).The nuclear staining positive rates of Glil expression in CSG, MAG, Dys and GC group were 4.5%,6.8%,8.3%and 32.0% (P<0.01). There was a closely correlation between Glil protein expression and differentiation degree, infiltration depth, TNM staging and node metastasis (P<0.05).(5) The expression of Gli2 in GC group was lower than that in CSG group (P=0.001<0.05), which had no significant difference compared with the other two groups (P>0.05).(6) Expression of Gli3 protein in GC group was lower compared with the other three groups (P<0.001 or 0.05).(7) The level of CyclinD1 expression increased gradually during the process of gastric tumorigenesis, which was higher in GC group than that in CSG and MAG group(P<0.O1), but had no significant difference with Dys group (P=0.245>0.05).(8) PCNA expressed higher in GC group than that in CSG and MAG group (P<0.05), but had no significant difference with Dys group (P=0.472>0.05).(9)Apoptosis index was extremely lower in GC(4.08±1.41) and Dys group(6.14±1.56) than that in CSG (12.54±4.23) and MAG group(21.37±6.27) (P<0.01).(10) Positive correlation was found between expression of Gli1 and CyclinD1 (R=0.214, P=0.003<0.01), so did PCNA (R=0.145, P=0.048), whereas negative correlation between Glil expression and cell apoptosis(R=0.278, P=0.002<0.01).In vitro(1) The proliferation of MKN28 was dose-and time-dependently inhibited by cyclopamine.(2) The expression of CyclinDl decreased significantly while inhibiting Hh signaling pathway, but the total expression of Glil was not significantly down-regulated.Conclusions:1. Hh signaling pathway was activated aberrantly in the process of gastric mucosal malignant transformation.2. Aberrant activation of Hh signaling had a closely correlation with differentiation degree, infiltration depth, TNM staging and node metastasis in gastric cancer.3. CyclinDl was down-regulated and the proliferation of MKN28 was inhibited by cyclopamine.