Clinical Significance And Biological Function Of HSP47 Protein In Gastric Cancer

Gastric cancer is the third leading cause of cancer-related death in the world,and it is also one of the most common malignant tumors occurred in gastrointestinal tract.It poses a serious threat to human health and causes a heavy social burden.The exact cause of gastric cancer is still unclear,but the mainstream opinion is that the oncogenesis of gastric cancer is related to environment,infection factors,diet,gene mutations and so on.Among the factors of infection,helicobacter pylori（HP）is the significant factor to promote the occurrence and development of gastric cancer.Currently,HP is considered to be one of the most important risk factors for gastric cancer.The treatment of gastric cancer is still based on surgery combined with radiotherapy and chemotherapy.Although great advances have been achieved in surgery and adjuvant therapy in recent years,the long-term prognosis of gastric cancer patients is relatively unfavorable.The 5-year overall survival rate is far away from satisfactory due to the early metastasis of gastric cancer to remote sites.Therefore,it is of great significance to identify non-invasive biomarkers for early diagnosis and assessment of prognosis in patients with gastric cancer.Part I:The expression and prognostic value of HSP47 gene （SERPINH1）in gastric cancerObjective:This study was aimed to investigate the expression of HSP47 gene（SERPINH1）in gastric cancer and the normal tissues,and to further explore the correlation between SERPINH1 gene expression and survival among individuals with gastric cancer.Methods:Oncomine database was searched to explore the expression of SERPINH1 gene in human malignant tumor tissues.Public databases such as Oncomine,GEO and TCGA were mined to investigate the expression of SERPINH1gene in gastric cancer and the correspondingly normal tissues,and ROC curves were plotted to evaluate the diagnostic value of SERPINH1 m RNA in differentiating between gastric cancer specimens and normal tissues.Kaplan Meier plotter database was searched to identify the potential impact of SERPINH1 gene on the prognosis of gastric cancer patients,and subgroup analysis was also performed based on gender,treatment methods and status of HER2.Results:SERPINH1 m RNA was highly expressed in a variety of tumors,including gastric cancer,esophageal cancer,head and neck cancer and renal cancer.Oncomine database was further mined to explore the expression of SERPINH1m RNA in specific gastric cancer datasets.The expression levels of SERPINH1m RNA in gastric cancer tissues were statistically higher than those in the correspondingly normal gastric mucosa tissues in Cho gastric dataset,Cui gastric dataset and Chen gastric dataset from Oncomine database,TCGA STAD database and GSE 29272,GSE 54129 gastric cancer datasets from GEO database.ROC analysis showed that SERPINH1 m RNA exhibited very good performance in differentiating gastric cancerous tissues from normal gastric mucosa（AUC:0.945,95%CI:0.899-0.994 in Cho gastric dataset;AUC:0.807,95%CI:0.738-0.876 in Cui gastric dataset;AUC:0.945,95%CI:0.899-0.994 in Chen gastric dataset;AUC:0.919,95%CI:0.883-0.956 in TCGA-STAD;AUC:0.931,95%CI:0.901-0.960 in GSE29272;AUC:0.993,95%CI:0.983-1.000 in GSE54129）.We browsed the Kaplan Meier plotter database to explore the correlation between SERPINH1 gene expression and the prognosis of 876 patients with gastric cancer.We found that the overall survival time of gastric cancer patients with high expression of SERPINH1 m RNA was significantly shorter than that of gastric cancer patients with low expression of SERPINH1 m RNA.High expression of SERPINH1 m RNA was an important risk factor for poor overall survival in individuals with gastric cancer(HR=1.56,95%CI=1.31-1.85,P=2.9×10^-7).We further analyzed the correlation between SERPINH1 m RNA expression and progression free survival in individuals with gastric cancer.The progression free survival time of gastric cancer patients with high expression of SERPINH1 m RNA was significantly shorter than that of gastric cancer patients with low expression of SERPINH1 m RNA.High expression of SERPINH1m RNA was an important risk factor for worse progression free survival among individuals with gastric cancer(HR=1.73,95%CI=1.41-2.12,P=9.2×10^-8).Finally,we analyzed the prognosis of gastric cancer patients according to gender,treatment methods and status of HER2,and further confirmed that high level of SERPINH1 was closely related to poor prognosis of gastric cancer patients.Conclusion:The expression level of SERPINH1 m RNA in gastric cancer is significantly higher than that in normal gastric mucosa.SERPINH1 m RNA performs well in differentiating gastric cancer from corresponding gastric mucosa.High level of SERPINH1 m RNA is not only a risk factor for poor overall survival among gastric cancer patients,but also a risk factor for unfavorable disease-free progression prognosis.SERPINH1 is a reliable marker to differentiate gastric cancerous tissues from normal gastric mucosa,and also a promising biomarker to evaluate the prognosis of gastric cancer patients.Part II:The expression of HSP47 protein in gastric cancer and its correlation with prognosisObjective:This study was aimed to explore the correlation between HSP47protein and common clinicopathological parameters of gastric cancer,and to investigate the association between HSP47 protein expression and prognosis among individuals with gastric cancer.Methods:The Human Protein Altas database was searched to determine the expression of HSP47 protein in various malignant tumor tissues and their adjacent tissues.Patients diagnosed with gastric cancer in Department of gastrointestinal surgery and Department of Gastroenterology from Renmin Hospital of Wuhan University were recurred in this study.We successfully constructed tissue microarray based on paraffin embedded specimens from Department of Pathology.Immunohistochemistry technique was conducted to detect the expression of HSP47protein in gastric cancer tissues and corresponding adjacent tissues.Chi-square test was employed to explore the correlation between HSP47 protein expression and common clinicopathological parameters among individuals with gastric cancer.Kaplan Meier curves were adopted to evaluate the impact of HSP47 protein expression on the overall survival in individuals with gastric cancer.Multivariate Cox regression risk model was exploited to identify the potential risk factors affecting the prognosis among individuals with gastric cancer.We also collected five pairs of fresh gastric cancer tissues and their adjacent tissues after radical gastrectomy,and then extracted the total protein and detected the level of HSP47 protein via Western blot.Results:Results from the Human Protein Altas database showed that HSP47protein expression was relatively high in a variety of malignant tumors except for prostate cancer,carcinoid and lymphoma.The Human Protein Alta database included12 cases of gastric cancer tissues stained with HSP47 antibody.Two cases showed high expression of HSP47 protein,3 cases with moderate expression of HSP47protein,4 cases with low expression of HSP47 protein,and 3 cases without expression of HSP47 protein.The expression of HSP47 protein was low in 2 cases of normal gastric mucosa.Western blot revealed that the expression of HSP47 protein in5 gastric cancer tissues was statistically higher than that in corresponding adjacent gastric mucosa tissues.Immunohistochemical results demonstrated that 16 out of 48cases of gastric cancer adjacent tissues showed high expression of HSP47 protein;while 60 out of 102 cases gastric cancer tissues showed high expression of HSP47protein.Analysis with Chi-square test showed that the positive expression rate of HSP47 protein in gastric cancer tissues was significantly higher than that in corresponding gastric cancer adjacent tissues（X²=8.485,P=0.004）.We also found that HSP47 protein was closely related to various clinicopathological parameters of102 patients with gastric cancer,including T stage（P=0.015）,N stage（P<0.0001）and TNM stage（P<0.0001）.Univariate Cox regression analysis was utilized to explore the clinicopathological factors affecting overall survival of 102 cases with gastric cancer.The results showed that N stage（HR=2.822,95%CI:1.092-7.294,P=0.032）,TNM stage（HR=2.518,95%CI:1.097-5.781,P=0.029）and HSP47 protein（HR=4.954,95%CI:1.734-14.151,P=0.003）were important risk factors affecting overall survival in 102 cases of gastric cancer.We further utilized multivariate Cox regression to determine the independent risk factors affecting overall survival of 102 patients with gastric cancer.The results indicated that high expression of HSP47 protein was an independent risk factor for poor overall survival of gastric cancer patients（HR=4.054,95%CI:1.305-12.544,P=0.016）.Conclusion:The expression of HSP47 protein is up-regulated in gastric cancer compared with that in adjacent tissues.High expression of HSP47 protein is closely related to the late T stage,N stage and TNM stage.High level of HSP47 protein is an independent factor for worse overall survival among individuals with gastric cancer.Part III:HSP47 promotes the proliferation and metastasis of gastric cancer cells by up-regulating epithelial mesenchymal transitionObjective:This study was designed to explore the potential effects of silence or overexpression of HSP47 protein on the proliferation,migration and invasion of gastric cancer cells,and to investigate the potential biological pathways.In addition,this investigation was also designed to determine the inhibitory effect of small molecule inhibitor of HSP47 protein（Co1003）on gastric cancer cells.Methods:Four gastric cancer cell lines（HGC-27,MGC-803,AGS,SGC-7901）and one kind of normal gastric epithelial cell line（GES-1）were cultured.The expression of HSP47 protein in four gastric cancer cell lines as well as one kind of normal gastric epithelial cell line（GES-1）was detected by Western blot.Gastric cancer cell line with high expression of HSP47 protein was selected to knock down its expression by sh RNA.While gastric cancer cell line detected with low expression of HSP47 protein was then selected to overexpress by lentiviral plasmid.The proliferation,migration and invasion of knockdown and overexpression HSP47protein in gastric cancer cells were detected by colony formation assay,cell scratch assay and transwell assay.Flow cytometry was used to detect the apoptosis,and immunofluorescence was utilized to determine the epithelial mesenchymal transition of HSP47 protein both in HSP47 silenced and overexpressed gastric cancer cells.Western blot was used to detect the expression of E-cadherin,N-cadherin,MMP2 and MMP9 in HSP47-silenced and HSP47-overexpressed gastric cancer cells.Western blot was conducted to detect the expression of key proteins of Wnt/β-catenin signaling pathway（β-catenin,Wnt2,p-GSK-3β,GSK-3β,NF-κB P65,Slug,Snail1 and TWIST）in HSP47-silenced and HSP47-overexpressed gastric cancer cells.SGC-7901 gastric cancer cell line was intervened with different concentrations of small molecule inhibitor（Co1003）against HSP47 protein,and then the proliferation,migration and invasion of gastric cancer cells were investigated by CCK-8 assay,transwell test and scratch test.Results:HSP47 protein was highly expressed in four kinds of gastric cancer cell lines,but lowly expressed in the normal gastric epithelial cell line（GES-1）.HSP47protein was highly expressed in SGC-7901 cell line and lowly expressed in MGC-803cell line.Compared with sh NC group,the numbers of clone formation and invasion of gastric cancer cells in HSP47-silenced group were significantly decreased,and the scratch distance was significantly prolonged（P<0.05）.Compared with the control group,gastric cancer cell lines in HSP47-overexpressed group showed an increase in the number of cell clone formation,invasion and a significantly shorter scratch distance（P<0.05）.Silence of HSP47 protein expression with sh RNA significantly increased the apoptosis rate of gastric cancer cells,while overexpression of HSP47protein significantly decreased the apoptosis rate of gastric cancer cells.In TCGA gastric cancer dataset,the expression of SERPINH1 was positively correlated with MMP2(Spearman coefficient=0.55,P=3.25×e^-20)and MMP9(Spearman coefficient=0.24,P=4×e^-6),and the protein expression of MMP2 and MMP9 in gastric cancer cells significantly decreased after the knockdown of HSP47 protein.The protein expression of MMP2 and MMP9 in gastric cancer cells statistically increased after HSP47 overexpression.There was a negative correlation between SERPINH1 and E-cadherin（CDH1 gene）（Spearman coefficient=-0.12,P=0.019）,and a strong positive correlation between SERPINH1 and N-cadherin（CDH2 gene）(Spearman coefficient=0.4,P=1.31×e^-15).In addition,SERPINH1 is also correlated with the key genes of Wnt/β-catenin signaling pathway.Western blot analysis showed that silence of HSP47 protein,E-cadherin was up-regulated,N-cadherin was down-regulated,and the key proteins of Wnt/β-catenin signaling pathway were also significantly down-regulated.After HSP47 overexpression,the expression of E-cadherin was down-regulated,the expression of N-cadherin was up-regulated,and the key proteins of Wnt/β-catenin signaling pathway were also up-regulated.Different concentrations of small molecule HSP47 inhibitor（Co1003）were added to intervene gastric cancer cell line,and it was demonstrated that Co1003 inhibited the proliferation of gastric cancer cells in a concentration dependent manner.Compared with the control group,the number of migration and invasion cells in SGC-7901 gastric cancer cell group treated with Co1003 was significantly decreased.The scratch healing ability of gastric cancer cells treated with Co1003 was significantly weaker than that of the control group.Conclusion:HSP47 protein is highly expressed in gastric cancer cell lines,and promotes epithelial mesenchymal transition and metastasis of gastric cancer cells by up-regulating Wnt/β-catenin signaling pathway.In addition,small molecule inhibitor（Co1003）that specifically inhibits HSP47 protein has certain inhibitory effects on gastric cancer cells.HSP47 protein plays an important role in the occurrence and progression of gastric cancer,and HSP47 protein is a promising molecular target for the treatment of gastric cancer.