Function And Mechanism Study Of Protocadherin 7(PCDH7) In Gastric Cancer

Objective:Gastric cancer is one of the most common gastrointestinal cancer,with a high rates of cancer-related mortality in all kinds of cancers worldwide.China has a high incidence of gastric cancer,the annual morbidity and mortality rates are more than two times of the world average.Due to the lack of specific clinical presentations or radiographic features,the majority of early gastric cancer patients are delayed diagnosis.Despite the considerable improvement in cancer diagnosis and comprehensive therapy,patients with advanced gastric cancer still have poor prognosis.As is well-known that invasion and metastasis are the great challenge of curing malignant tumors.Invasion and metastasis are relatively later event in the progression of gastric cancer.The mechanism of invasion and metastasis is related to plenty of genes and signaling pathways between cancer cells and the host cells,cancer cells and extracellular matrixcomponents.The earliest metastasis mechanism was put forward by Liotta,it has three step "adhesion","degradation","metastasis".Epithelial-mesenchymal transition(EMT)is a process that epithelial cells lose polarity,become mesenchymal cells and acquire the ability of migration and invasion.EMT is involved in a number of molecular markers.Cadherins are calcium-dependent transmembrane glycoproteins,which form a large family of cell-cell adhesion molecules.Cadherins families include the classic cadherins(type ? and type ? cadherin),desmosomal cadherins,Flamingo(CELSR)cadherins,fat-like cadherins and protocadherins.The protocadherins was the largest subfamily,which was first introduced by Shintaro Suzuki in 1993.They are divided into two groups(the clustered and non-clustered PCDH families)based on their genomic structure.Although it has been widely reported in nervous system development,its functions in tumors are not fully understood.PCDH7 belongs to the protocadherins and locates at human chromosome 4p15,which was first introduced in gastric cancer cells.Among the human tissues,the expression of PCDH7 is predominant in the brain and heart,which was also called BH-PCDH.PCDH7 encodes an integral membrane protein that is believed to function in cell-cell recognition and adhesion like the classic cadherins,but the adhesion is weaker.It has been widely reported in nervous system development.Recent researches suggested that PCDH7 can participate in the development of human tumor.However,no evidence has been found in its potential role in gastric cancer.The aim of this study was to detecte the expression of PCDH7 in gastric cancer and gastric mucosa tissues,analysis function of gastric cancer cells in vitro.Further,investigate the association between PCDH7 expression and the clinicopathological characteristics and the regulatory mechanism of gastric cance cells.Methods:In this study,PCDH7 expression was detected in gastric cancer tissues and nontumorous gastric mucosa tissues by RT-qPCR and immunohistochemistry.Fresh specimens of 47 cases gastric cancer tissues and 25 cases noncancerous gastric mucosal tissues for RT-qPCR were obtained and stored in liquid nitrogen.Total RNA were extracted from fresh tissues,then,converted to cDNA.The mRNA expression level was analyzed by RT-qPCR.Tissue samples for immunohistochemistry,including 119 cases of gastric cancer tissues and 75 cases of noncancerous gastric mucosa tissues,were collected from Department of Pathology,Qilu Hospital of Shandong University.The association of PCDH7 expression with age,gender,tumor size,Lauren's classification,TNM stage,lymph node metastases and distal metastasis were analyzed.Furthermore,univariate and multivariate analyses were carried out through Cox proportional hazard model.Moreover,the overall survival(OS)and disease-free survival(DFS)were also analyze.Gastric cancer cells were transfected with siPCDH7 or NC by X-treme GENE transfection reagent according to the manufacturer's recommendations.Cells were cultured and collected after 48h.The knockdown efficiency of siRNA was detected by RT-qPCR.MTS and transwell assays were performed to assess the effect of PCDH7 on proliferation,migration and invasion in gastric cancer cells.The methods of RT-qPCR were used to detect the mRNA expression of E-cadherin,N-cadherin,?-catenin,Vimentin,Twist,Snail,Zebl and Zeb2 in Gastric cancer cells.To further confirm this result,western blot was used to check the protein expression of E-cadherin,?-catenin,Vimentin and Snail.Results:1.The expression of PCDH7 was downregulated in human gastric cancer tissues.Compared with noncancerous gastric mucosal tissues,PCDH7 mRNA were down-regulated in gastric cancer tissues(p<0.0001).Furthermore,the expression of PCDH7 protein was analyzed by immunohistochemistry.In gastric cancer tissues,PCDH7 mostly presented negative immunoreactivity and the positive rate of PCDH7 was just only 32.8%(39/119).In contrast,the positive rate of PCDH7 was high in noncancerous gastric mucosa.PCDH7 positive was detected in all of the normal gastric tissue(20/20,100%)and 35 cases of intraepithelial neoplasia gastric tissue(35/55,63.6%).In addition,our result showed that PCDH7 expression in gastric cancer tissues with lymph node metastasis(LNM)was predominantly lower than that in gastric cancer tissues without LNM(P<0.001).PCDH7 expression was stepwise down-regulated from normal tissue trough intraepithelial neoplasia tissue to gastric cancer tissues(all p<0.05).In the next,receiver operator characteristic(ROC)curves were constructed to evaluate the diagnostic value of PCDH7 in gastric cancer.Between gastric cancer and noncancerous groups,the area under the curve(AUC)was 0.938,suggesting that PCDH7 expression could be used to discriminate these two groups(p=0.000).Moreover,the ROC curves indicated that the PCDH7 expression could discriminate the case with LNM from those without LNM with an AUC of 0.695(p=0.001).2.Relationship between PCDH7 expression and clinicopathological parameters in gastric cancer.PCDH7 expression was significantly negatively associated with Lauren's classification(P =0.0005),lymph node metastases(P=0.0002)and TNM stage(P=0.0221).However,there was no statistically significant difference between PCDH7 expression and age(P = 0.3009),gender(P = 0.2835),tumor size(P = 0.4465)or distal metastasis(P = 0.2219).3.Low expression of PCDH7 correlates with poor prognosis.Survival analysis demonstrated that patients with low expression of PCDH7 had a poorer overall survival(OS)and disease-free survival(DFS)than those who had high PCDH7 expression(P = 0.0490 and P = 0.0380 respectively).The univariate analysis uncovered that PCDH7 expression,depth of invasion,lymph node metastasis,distal metastasis and tumor stage were prognostic factors for OS and DFS of gastric cancer patients(P<0.05).Multivariate analysis suggested that distal metastasis and tumor stage were independent prognostic predictors for OS and DFS(P<0.05).However,our study revealed that PCDH7 expression was not an independent prognostic factor for OS and DFS of patients with gastric cancer.Despite that,it still favors PCDH7 as a protective factor(OS,HR = 0.842;DFS,HR =0.668)for patient survival.To sum up,our results demonstrated that low expression of PCDH7 was significantly associated with poor outcome.4.Cell functional experiments in vitro.Cell proliferate assay demonstrated that knockdown of PCDH7 could not influence cell proliferation obviously compared with negative controls in two GC cell lines(all P>0.05).BGC 823 and MKN 45 cells with siPCDH7 transfection displayed a significantly increased migration and invasion capability compared with the cells transfected with negative control.5.PCDH7 knockdown influenced the expression of E-cadherin.The mRNA expression level of E-cadherin was decreased in BGC 823 and MKN 45 cells after transfected with siPCDH7,whereas there was no effect on the expression of N-cadherin,?-catenin,Vimentin,Twist,Snail,Zebl and Zeb2.To further confirm this result,western blot was performed.In this result,we observed that PCDH7 knockdown could inhibit the protein expression of E-cadherin and have no effect on that of p-catenin,Vimentin and Snail in BGC 823 cells.And similar results were obtained in MKN 45 cells.Conclusions:Our data indicated that PCDH7 play an important role in the invasion and metastasis of gastric cancer,and PCDH7 could suppress cell migration and invasion through E-cadherin inhibition.PCDH7 can serve as a novel biomarker for diagnostic and prognosis in patients with gastric cancer.