| Part one Expression of Smo gene in gastric cancer tissue and its clinical significanceObjective:Gastric cancer is one of the most common malignant tumors.The incidence of gastric cancer in our country is relatively high,almost twice as much as the average all over the world,which is the second largest malignant tumor only behind lung cancer.Recent research has found that the signal pathway is an important factor to induce cell carcinogenesis.The signal pathway plays an important regulatory function in the process of embryonic growth.At present,the medical community is more concerned about the research of hedgehog Sonic signal pathway,which plays an important role in the growth and development of spinal animals.The data indicated that Sonic hedgehog signal pathway's abnormal activation was related with pancreatic cancer,bladder cancer,prostate cancer,gastric cancer and other malignant diseases.The Hedgehog Sonic Signal Pathway is composed of Shh ligand(a secreted signal glycoprotein),Ptch(transmembrane protein),Smo(transmembrane protein)and Gli protein(transcription factor).In current research,there are two ways leading to the signal pathway abnormal activation:1)Non-ligand dependence,the mutation of Signaling pathway member gene:specifically Smo gene function change or Ptch gene function lossing,then activated the signal pathway;2)Ligand dependence,(ligand hyperexpression):specifically,the abnormal expression of Shh ligand,it binds to the Ptch receptor to activated the signal pathway.Clinical studies show that Shh and Ptch was strongly expression in gastric cancer cells,suggesting that the abnormal activation of sonic hedgehog pathway is an important cause of gastric cancer.Through lots of experiments,we found that ligand dependence way may be the most improtant activating factor,but as so far,there is not enough research to elucidate the high expression of Ptch gene in this activating way.Smo protein is like an information converter,which can convert Shh to Gli1 signal,activating the Sonic hedgehog signal pathway.A large number of studies have showed that,functional acquired mutations of Smo is associated with basal cell carcinoma(mutation rate is 20%).But whether the Smo gene mutation would induce gastric cancer,whether the ligand independence way is the reasion of activation are still not reached a consensus.This part is the first part of this paper,so its purpose is to explore the expression of Smo gene in gastric cancer tissue and its clinical significance.Method:Creating chip of gastric cancer cells and normal gastric tissue(85/20),relying on related technology to detect Smo protein and Smo mRNA in gastric cancer cells in the degree of expression.Analysis the lesions and the mutation site of Smo gene in gastric cancer cells,clinical and pathological stage of the relationship.Result:In 20 normal gastric tissues,the Smo protein expression rate was25%,80%of them showing weakly positive expression.However,in papillary adenocarcinoma of the stomach,the expression rate was 85.7%.In the high,medium and low differentiated tubular adenocarcinoma(9\26\18),the expression rate of Smo are 88.9%?92.3%?94.4%,the rate of strongly positive expression are 25%?25%and 35%.In mucinous adenocarcinoma,the expression rate was 54.5%,83.3%of them are weakly positive expression.And the expression rate in signet ring cell carcinoma is 28.6%(100%weakly positive expression),It can be seen that in gastric papillary adenocarcinoma,high,medium and low differentiated tubular adenocarcinoma among the expression level of Smo protein significantly much higher than normal gastric tissue.However,in mucinous adenocarcinomas?signet ring cell carcinoma and the normal gastric tissue is not much difference.In addition,whether it is normal or diseased the gastric tissue,the expression level of Smo mRNA and Smo protein are the same.The Smo expression level of different clinical stages of gastric cancer cells are not the same;in III?IV stages,The expression level of Smo protein was significantly higher than that in stage I.What needs to be emphasized is that with the development of clinical pathology stage.The expression of Smo protein showed a gradually increasing trend.Summary:There is a close relationship between the expression of the Smo and gastric adenocarcinoma especially papillary adenocarcinoma,high,medium and low differentiated tubular adenocarcinoma,Smo highly expression can induce cell canceration,its expression level is associated with the stage of tumor and severity of illness.Part two Correlation analysis of the expression of Smo protein and its downstream transcription factor Gli1 protein in gastric cancerObjective:To explore the correlation between Smo protein and its downstream transcription factor Gli1 protein expression in gastric cancer tissue.Method:Creating gastric cancer tissue microarray,relying on immunohistochemistry to detect the expression level of Gli1 protein in gastric cancer cells.Actively explore the relationship between the expression level and the type of gastric cancer.With reference to the first part of Smo protein related data,analyse the correlation of Smo and Gli1 protein expression in gastric cancer cells.Result:In normal gastric tissue,the Gli1 protein expression rate is 20%,75%of them showed a weakly positive expression.In papillary adenocarcinoma,the expression rate is 100%,28.6%of them showed a strongly positive expression;In the high,middle and low differentiation of gastric tubuloid adenocarcinoma,the expression rates are 77.8%?96.2%and100%,28.6%?28%and 61.1%of them showed a strongly positive expression;In mucinous adenocarcinoma,the expression rate was 27.3%,66.7%of them are weakly positive expression.And the expression rate in signet ring cell carcinoma is 21.4%(100%weakly positive expression).As can be seen,the Gli1 protein expression in papillary adenocarcinoma and the high,middle and low differentiation of tubular adenocarcinoma was significantly higher than in normal gastric tissue,the difference was statistically significant(?~2=67.02,P<0.001),but in mucous adenocarcinoma and signet ring cell carcinoma,there was no significant difference comparing with the expression level of Gli protein in normal gastric tissue.In our reserch,there was a significant positive correlation between Smo and Gli1 protein expression,the correlation coefficient is 0.8609.Summary:As the downstream gene of Smo,Gli1 is highly expression in papillary adenocarcinoma and the high,middle and low differentiation of tubular adenocarcinoma,its expression is consistent with Smo expression in the corresponding organization(positive correlation),The results revealed the expression status of major members of Sonic hedgehog signaling pathway in gastric cancer tissues at some level.Part three The expression of Smo protein in human gastric cancer MGC803 cells and the impact on the proliferation and apoptosis of gastric cancer MGC803 cells after small RNA interfering the expression of Smo proteinObjective:To explore the expression of Smo gene in human gastric cancer MGC803 cells and small RNA interfering with Smo gene expression and its effect on the proliferation and apoptosis of gastric cancer MGC803cells.Method:Relying on Western blot and semi quantitative RT-PCR technology to detect Smo protein and Smo mRNA expression level in gastric cancer cell MGC803.Through artificial synthesis we firstly get three pairs of specific interference RNA(siRNA),which is mainly based on Smo m RNA.Relying on positive liposomes way to transfact siRNA into MGC803 cells,take semi quantitative RT-PCR in the way to detect the moderating effect of MGC803 Cells.Then relying on in Western blot and flow cytometry to detect the Gli1 protein expression level and compare the variation,and explore its effect on the reproduction and apoptosis of MGC803.Result:Smo protein and Smo m RNA showed strongly expression in gastric cancer cell MGC803,siRNA-1 has the most significant interference comparing with other two(siRNA-1:61.7%;siRNA-2:41.2%;siRNA-3:little interference).After silencing Smo gene,the expression of Gli1 decreased(Gli1mRNA down to 56.2%,Gli1 protein down to 61.3%).Meanwhile,we found the slowing down of MGC803 cell's multiplication rate after 24 hours of transfection,the apoptosis rate rised up to 23.6%after 48 hours of transfection,so we can find that the apoptosis rate is relatively rising.Summary:Same as the previous studies,Smo gene show strongly expression in gastric cancer cell MGC803.Relying on the small RNA interference technology to silence the express of Smo mRNA,we can effectively reduce the expression level of Gli1 protein and Gli1 mRNA,On the other hand,the upstream and downstream relationship between Smo and Gli1in the signal pathway is verified again.The expression of Smo gene has close relatonship with the proliferation and apoptosis of MGC803 cells,we can slow down the proliferation rate and speed up the apoptosis rate through inhibitting its expression in gastric cancer cells.Part four The effect of Sonic Hedgehog pathway on the invasion and motor ability of gastric cancer cellsObjective:To explore the effect of Sonic Hedgehog pathway on the invasion and motor ability of gastric cancer cells.Method:After treated with different concentrations of Cyclopamine(inhibitor of Hh signal pathway),we observed the situation of cell scratch healing of MGC803 cells(24 h,48h and 72h),measured the number of cells penetrating the Transwell microventricular semi-permeable membrane and basement membrane to observe the movement and migration,at last,we tested the expression of E-cadherin using Western blot to discuss the ralationship of Sonic Hedgehog signal pathway and the infiltration and metastasis of gastric cancer.Result:Along with the increase of cyclopamine concentration(2,5,l0?M),the mobility of gastric cancer cells decreased gradually,and also,the healing ability gradually decreased.The cell healing rate decreased from 78.51±3.04%in blank group to 23.31±0.83.The average number of cells passing through the Transwell chamber decreased from 2204±47.9 in the blank group(P<0.05)to 523±16.5.We found that the relatively low expression level of E-cadherin in MGC803 cells,but it is increased when cyclopamine is used to block the signaling pathway in MGC803 cells by Western blotting.Summary:Cyclopamine can decreases the expression of E-cadherin,slows down the mobility of gastric cancer cells and reduces the ability of invasion and metastasis through inhibiting the expression of Smo and Gli1 in SHH signal pathway.Conclusion:1.The elevating of Smo gene's expression is closely related to gastric papillary adenocarcinoma and high,medium and low differentiation adenocarcinoma.The increase of its expression level is positively correlated with the stage,which may indicate the severity of the disease to some extent.2.As the downstream gene of Smo,Gli1 is highly expression in papillary adenocarcinoma and the high,middle and low differentiation of tubular adenocarcinoma,its expression is consistent with Smo expression in the corresponding organization(positive correlation),The results revealed the expression status of major members of Sonic hedgehog signaling pathway in gastric cancer tissues at some level.3.Relying on the small RNA interference technology to silence the express of Smo mRNA,we can effectively reduce the expression level of Gli1protein and Gli1 mRNA,On the other hand,the upstream and downstream relationship between Smo and Gli1 in the signal pathway is verified again.The expression of Smo gene has close relatonship with the proliferation and apoptosis of MGC803 cells,we can slow down the proliferation rate and speed up the apoptosis rate through inhibitting its expression in gastric cancer cells.4.Cyclopamine can decreases the expression of E-cadherin,slows down the mobility of gastric cancer cells and reduces the ability of invasion and metastasis through inhibiting the expression of Smo and Gli1 in SHH signal pathway.5.Smo gene and its downstream transcription factor Gli1 are highly expressed in gastric cancer,through our study,we have revealed their important clinicopathological significance in the occurrence and development of gastric cancer. |
PDF Full Text Request