| BackgroundGastric cancer is one of the leading malignant diseases in China. Althoughdeclining in recent years, the incidence of gastric cancer still rank the first of allmalignancies. Many studies on the risk factors of gastric cancer have proved that thechanges of anti-oncogene, oncogene, DNA repair gene, cell adhesion molecule,Telomere/telomerase, cell cycle regulatory factor and growth factor/acceptor systemet al play certain roles in the carcinogenesis and progress of gastric cancer. Furtherstudies discovered more factors. These works made the pathogenesis of gastric cancerclear increasingly, and also provide new therapeutic targets.According to recent studies, signal pathway that controls tissue differentiationand embryo development may play a major role in the pathogenesis of carcinoma."The signal pathway that controls embryo development in vertebrate might relatewith carcinoma of mankind." This point of view offered a new direction to theresearches of pathogenesis of carcinoma.The signal pathway which was recently studied deeply shows that Sonichedgehog plays a very important role in embryo development of mankind. It has beenconfirmed that the occurrence of basal cell carcinoma, small cell lung cancer,pancreatic cancer, prickle cell carcinoma, prostatic carcinoma, carcinoma of bladder,medulloblastoma and gastric cancer of mankind have a close relationship with the abnormal activation of Sonic hedgehog signaling pathway. This signal pathwaymainly constructed by the combination (composed of secreting type signalglucoprotein Shh ligand, transmembrane protein receptor Ptch and anothertransmembrane protein Smo) and downstream transcription factor Gli protein (Gli1,Gli2,Gli3). There were two abnormal activation of Sonic hedgehog signalingpathway. 1) Ligand-independent activation: abnormal activation of intracellular signalpathway induces by loss-of-function mutation Pcth gene in cells or gain-of-functionmutation Smo gene. 2) Ligand-dependent activation: Shh ligand of paracrine isoverexpressed, which combine Ptch recipient of cytomembrane, thus activate theintracellular signal pathway.Other studies have confirmed that Shh and Ptch ligands are highly expressed inthe tissue of gastric cancer, which means the abnormal activation of Sonic hedgehogsignaling pathway is concerned with the occurrence of gastric cancer, resulting in ahypothesis that the possible pathogenesis of this abnormal activation might beligand-dependent activation: Overexpressed Shh ligands of paracrine combine Ptchrecipient of cytomembrane, thus activate the intracellular signal way, and lead tocancer. However, This hypothesis can not explain why Ptch gene which serves as anantioncogene is highly expressed in of gastric cancer.Smo protein is a transcriber in the Sonic hedgehog signaling pathway. It's anactivator that change extracellular Shh signal into intracellular Gli1 signal, thusinitiate the gene transcription in the cell nucleus. Some studies proved thegain-of-function mutation of Smo gene can induce human basal cell carcinoma. Themutation rate of Smo in familial and sporadic basal cell carcinoma is 20ï¼…. So wepresume gain-of-function of Smo gene might be concerned with the carcinogenesis ofgastric cancer, though whether Ligand-independent activation way resulted from theoverexpression of Smo gene do exist in abnormal activation of sonic hedgehog signaling pathway or not, and its contribution is not yet clear.ObjectivesTo study the contribution of Smo gene in the growth and progress of gastriccancer, and its contribution in abnormal activation of sonic hedgehog signalingpathway in gastric cancer. To find the molealar mechanin for growth of gastric cancer,and establish a theory for searching new effective target for biotherapy.Methods1. To construct a tissue microarray for gastric cancer: with a control of amatched normal gastric mucosa, then to detect the expressive level of Smo proteinand Smo mRNA in gastric cancer tissue by immunohistochemical and hybridizationin situ techniques, and to analyze the relationship of the expression level of Smo genewith pathological types, location and clinicopathological staging of gastric cancer.2. To construct a tissue microarray for gastric mucosa with atypical hyperplasiawith a control of matched normal mucosa, then to detect the expression of Smoprotein and Smo mRNA in atypical hyperplasia mucosa by immunohistochemicaland hybridization in situ techniques, and to analyze the relationship between theexpression level of Smo gene in gastric mucosal with atypical hyperplasia and theextent of atypical hyperplasia.3. Using the gastric cancer tissue microarray, to detect the expression level ofGli1 protein in gastric cancer tissue with immunohistochemical technique, then ttoanalyze the relationship with Pathological types of gastric cancer, and to combine theresults about Smo protein gained from the above, to analyze correlation of Smo andGli1 protein expression in gastric cancer.4. Using Western blot and Semi-quantitative RT-PCR techniques, to detect theexpression level of Smo protein and Smo mRNA in MGC803 gastric cancer cells, three fragments of small interfering RNA (siRNA) chemically synthesized in vitroagainst Smo mRNA were transfected into MGC 803 gastric cancer cells by positiveliposomal transfection methods, and intracellular Smo mRNA expression wasdefected by RT-PCR, which could be interfered by siRNA.Finally influences on Gli1 protein and mRNA expression, proliferation andapoptosis of gastric cancer MGC 803 cells were detected by flow cytometry. RT-PCRand MTT, after intracellular Smo mRNA being degradated.Results1. The expression rate of Smo protein in normal gastric mucosa tissue is 32ï¼…,with weak stain mainly, and that in Stomach papillary adenocarcinoma, highdifferentiated tubular adenocarcinoma, medium differentiated tubularadenocarcinoma, low differentiated adenocarcino- -ma gastric cancer tissue is 85.7ï¼…,77.8ï¼…, 88.5ï¼…, 94.4ï¼…respectively, with strong stain mainly, significantly higher thanthat of normal tissues. But that in mucoid carcinoma and signet-ring cell carcinomaare 45.5ï¼…, 14.3ï¼…respectively, mainly weak stain. No difference with that of normaltissues. Further more, Smo protein and Smo mRNA have same expression level ingastric normal mucosal and cancer tissue. The expression intensity are significantlydifferent between different clinicopathological staging of gastric cancer, it's higher instageâ…¢andâ…£than in stageâ… , and more advanced disease, more strongerexpression.2. The expression of Smo protein in mucosa with atypical hyperplasia: grade 1to 3 it is 45ï¼…, 48.4ï¼…, 57.1ï¼…respectively, mainly weak stain. Though there was aincreasing tread of Smo protein expression along with the extent of mucosal atypicalhyperplasia, there is no significant difference of expression between mucosa withatypical hyperplasia and normal mucosa. There is the same findings for Smo mRNAwith Smo protein. It's expression of Smo protein in gastric cancer tissue, was significantly higher than that in mucosa with atypical hyperplasia compareing.3. The positive expressive rate of Gli1 protein in normal gastric mucosa tissueis 20ï¼…, with weak stain mainly. And that in Stomach papillary adenocarcinoma, highdifferentiated tubular adenocarcinoma, medium differentiated tubularadenocarcinoma, low differentiated adenocarcinoma gastric cancer tissue is 100ï¼…,100ï¼…, 96.2ï¼…, 100ï¼…respectively, with strong stain mainly, significantly higher thanthat of Normal tissues. But that in mucoid carcinoma and signet-ring cell carcinoma is36.4ï¼…, 7.2ï¼…respectively, mainly weak stain, no difference with that of normaltissues, the expression of Smo and Gli1 protein are positively correlation in gastriccancer tissue, with a coefficient correlation of 0.9244. There is overexpression of Smo protein, Smo mRNA in MGC 803 cells. Weuse small RNA interference technique to silence MGC 803 cells. At 48 hours aftertransfection, the expression level of Smo mRNA in MGC 803 cells of Smo siRNA-1group decreased about 61.7ï¼…, which was the most notable reduction than othergroups. Meanwhile, the expression levels of Gli1 mRNA and Gli1 protein in thisgroup also have a notable decrease, with a 56.2ï¼…decreased of Gli1 mRNA and61.3ï¼…to Gli1 protein. After 24 hours of transfection, the cell multiplication in SmosiRNA-1 group began to decrease notably, significantly lower than controls. After 48hours of transfection, the apoptosis rate of cells in Smo siRNA-1 group was 23.6ï¼…,significantly higher than controls.Conclusions1. Smo gene is related with the occurrence of stomach papillary adenocarcino--ma, high differentiated tubular adenocarcinoma, moderate differentiated tubularadenocarcinoma, low differentiated adenocarcino- -ma gastric cancer. Theoverexpression of Smo gene might activate a certain kind of mechanisms that initiatesthe carcinogenesis of gastric cancer of the above pathological types. And the up-regulation of its expression plays a role in promoting this process.2. The overexpression of Smo gene is not an early event in the process ofgastric carcinogenesis, but a much late one.3. The mechanism of Smo gene's overexpression in carcinogenesis andprogress of gastric cancer should be: to regulate expression of Gli1 protein whichserved as downstream transcription factor, then to activate abnormally sonichedgehog signaling pathway and induce carcinogenesis.4. Growth of MGC 803 gastric cancer cells is related to the abnormalexpression of Smo gene and the abnormal activation of sonic hedgehog signalingpathway.5. It's effective and feasible to silence Smo mRNA expression in 803 gastriccancer cells by RNA interference technique. The silence of Smo mRNA's expressionin MGC 803 gastric cancer cells can down-regulate the expression of intracellularGli1 protein and Gli1 mRNA, and thereby decrease the activity of Sonic hedgehogsignaling pathway.6. Smo gene is related with MGC 803 gastric cancer cellular proliferation andapoptosis. It may play a role of control. Smo gene may serve as an oncogene and playan important role in the growth and progress of gastric cancer.7. Ligand-independent activation way may exist in Sonic hedgehog signalpathway of gastric cancer. A gain-of-function mutation of Smo gene leads to highexpression of Smo protein, thus abnormally activates Sonic hedgehog signal pathwayand results in carcinogenesis.
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