The Role And Methylation Status Of Non-receptor Protein Tyrosine Phosphatase 6 In The Progression Of Esophageal Squamous Cell Carcinoma

Esophageal cancer is the sixth leading cause of cancer-related death worldwide and the incidence ranks eighth among the malignant tumors.Squamous cell carcinoma(SCC)and adenocarcinoma(AC)are the two main histological types of esophageal cancer.Esophageal squamous cell carcinoma(ESCC)is the dominant histological type of esophageal cancer in China and carries high morbidity in some areas,especially in some counties bordering Henan,Hebei,and Shanxi Provinces in northern China.Although some tumor suppressors and oncogenes have been identified to play key roles in the occurrence and development of ESCC,the exact pathogenesis of ESCC remains unclarified.The human PTPN6 gene is located on chromosome 12p13 and codes a Mr 68,000 protein of non-receptor type protein-tyrosinephosphatase.The PTPN6 gene has been considered as a candidate tumor suppressor in hematological and solid malignancies and promoter methylation may be an epigenetic mechanism in silencing its expression.However,the detailed roles of PTPN6 and its promoter methylation status in the pathogenesis of ESCC have not been fully expounded.Based on the above research background,this study aims to explore the PTPN6 expression in ESCC tissues and esophageal cancer cells,analyze the relationship between PTPN6 expression and clinic data of ESCC.Furthermore,we detect the function of CpG sites hypermethylation in the inactivation of PTPN6,and clarify the role of PTPN6 in ESCC tumorigenesis and development.Subsequently,the effect of PTPN6 gene on proliferation,migration and invasion of esophageal cancer cells is studied by overexpressing PTPN6 gene in vitro,and the function of PTPN6 on biological behavior of esophageal cancer cells is further explored.Part one The Expression and clinical significance of PTPN6 in ESCCObjectives:To detect the expression levels of PTPN6 mRNA and protein in different esophageal cancer cell lines,71 ESCC tissues,and corresponding adjacent normal tissues,and further explore the relationship between PTPN6expression and clinic data of ESCC patients.Methods:1.Human esophageal cancer cell lines TE1,TE13,Eca109,Kyse150,Kyse170,and Yes-2 were obtained from the Biological Specimen Bank of the Fourth Hospital of Hebei Medical University.Human normal esophageal epithelial cell line HEEpiC was selected as control.A total of 71 primary ESCC tissues and corresponding adjacent normal tissues were obtained from inpatients undergoing surgical treatment in the Fourth Hospital of Hebei Medical University between the years of 2008 and 2011.And the normal tissue,5cm from the edge of cancer adjacent,was used as normal control.2.The expression levels of PTPN6 mRNA and protein in different esophageal cancer cell lines and control groups were detected by real-time quantitative RT-PCR(qRT-PCR)and Western blot method respectively.3.The expression levels of PTPN6 mRNA and protein in ESCC and adjacentnormaltissueswereanalyzedbyqRT-PCRand immunohistochemistry method respectively.4.Kaplan-Meier analysis was used to test for differences in overall survival between the groups.According to the protein expression of PTPN6 in ESCC tissue,it was divided into PTPN6 positive expression group and negative expression group,and the relationship between PTPN6 expression and the overall survival of ESCC patients was investigated.Results:1.The expression of PTPN6 mRNA and protein in six esophageal cancer cells lines was significantly lower than that in the control group,especially in Eca109 and Yes-2 cells.2.PTPN6 mRNA expression in ESCC tumor tissues was significantly decreased compared to corresponding normal tissues(P<0.05).The results of immunohistochemistry showed that the positive protein expression frequency of PTPN6 in ESCC tissues was significantly lower than that in normal tissues[32.4%(23/71)vs 77.55(55/71),χ~2=29.128,P<0.05].The expression of PTPN6 mRNA and protein in ESCC tissues was associated with TNM stage,tumor pathological grade,and lymph node metastasis(P<0.05),but not with age,sex,depth of invasion and family history of upper gastrointestinal cancer(P>0.05).3.The 5-year survival rate of PTPN6 positive expression group was39.1%,whereas the 5-year survival rate of negative expression group was only18.4%(χ~2=3.391,P<0.05,Log-rank test).Part two Methylation status of PTPN6 promoter 2 in ESCC and its relationship with prognosis of ESCC patientsObjectives:To observe the changes of PTPN6 mRNA and protein expression before and after treatment with DNA methyltransferase inhibitor5-aza-2’-deoxycytidine(5-Aza-dC).Bisulfite Genomie Sequencing(BGS)method was used to detect the methylation frequency of CpG sites in the P2promoter region of PTPN6.Primers were designed according to the distribution of methylated CpG sites,and methylation-specific polymerase chain reaction(MSP)method was used to detect the methylation status of PTPN6 in ESCC.The relationship between the down-regulation of PTPN6expression and methylation was further verified.Methods:1.Three esophageal cancer cell lines with low expression of PTPN6 were cultured routinely and were treated with 5-Aza-dC at a concentration of 5micromol/L.The cells were cultured for 48 hours and the culture medium was changed every 24 hours.After that,the cells were cultured in complete medium for 24 hours.2.The expression level of PTPN6 mRNA and protein was detected in different esophageal cancer cell lines before and after 5-Aza-dC treatment by qRT-PCR and Western blot method.3.The methylation status of CpG sites in P2 promoter region of PTPN6was analyzed by bisulfite genome sequencing(BGS)in three different esophageal cancer cell lines before and after 5-Aza-dC treatment.4.The methylation status of PTPN6 was studied by methylation-specific polymerase chain reaction(MSP)method in different esophageal cancer cell lines,ESCC tissues and adjacent normal tissues.5.Kaplan-Meier analysis was used to analyze the differences in overall survival between the methylation-positive group and methylation-negative group of P2 promoter.Results:1.After treated with 5-Aza-dC,the expression level of PTPN6 mRNA and protein was significantly up-regulated in Eca109,Kyse170 and Yes-2cells.2.The CpG loci from-167bp to-326 bp were sequenced by BGS,and the methylation frequency of CpG sites in esophageal cancer cell lines before5-Aza-dC treatment was higher than that of untreated esophageal cancer cells.3.Hypermethylation was detected in Eca109,Kyse170 and Yes-2 cell lines.After treated with 5-Aza-dC,Eca109 and Yes-2 cell lines showed unmethylation status,and Kyse170 cell line showed a unmethylation band.4.The methylation frequency of PTPN6 in ESCC tumor tissues(63.4%,45/71)was significantly higher than that in corresponding normal tissues(16.9%,12/71)(P<0.05).When stratified for clinicopathologic characteristics,methylation frequency of PTPN6 was associated with TNM stage,pathological differentiation,and LN metastasis(P<0.05).The mRNA expression level of PTPN6 in the ESCC tissues with PTPN6 methylation was significantly decreased compared to the ESCC tissues with unmethylation of PTPN6(P<0.05).The protein expression of PTPN6 in the ESCC tissues with PTPN6 methylation was significantly decreased compared to the ESCC tissues with unmethylation of PTPN6(13.3%vs 65.4%,χ~2=20.386,P<0.05).5.PTPN6 promoter methylation was correlated with ESCC patients’survival.In the ESCC patients with hypermethylation of promoter P2,the5-year survival rates were 15.2%as opposed to the ESCC patients with unmethylation of promoter P2 displaying 5-year survival rates of 42.3%(χ~2=5.383,P<0.05,Log-rank test).Part three Effect of PTPN6 on biological behavior of esophageal cancer cell lines in vitroObjectives:PTPN6 overexpression plasmid pcDNA3.1-PTPN6 was constructed and transfected into esophageal cancer cell lines Eca109 and Yes-2.The effects of PTPN6 overexpression on proliferation,migration,and invasion of esophageal cancer cells were observed in vitro.Methods:1.The Eca109 and Yes-2 cell lines were transfected with PTPN6expression plasmid(pcDNA3.1-PTPN6)or the empty vector(pcDNA3.1-EV)as control.The transfection efficiency was detected by qRT-PCR method.2.The transfected cells were seeded into 96-well plates and the absorbance of each well was measured.For colony formation assay,transfected cells in logarithmic growth phase were seeded in the 6-well plates and incubated for a week.Clones were stained with 0.5%crystal violet,and the number of colonies was calculated.3.The transfected cells were seeded into 6-well plates.After transfected for 24h,scratch wounds were established by a 20μl of pipette tip.The fluorescent microscope was applied to measure the relative migration distance.4.The transfected cells in logarithmic growth phase were seeded in the upper chambers coated with Matrigel.After incubated for 24 hours,invasive cells located on the lower chamber were fixed and stained with hematoxylin.The number of cells invaded through the membrane to the lower surface was counted in five microscopic fields per filter.Results:1.In the part one,we found that the expression level of PTPN6 was lowest in Eca109 and Yes-2 cell lines.The construct containing PTPN6transcripts(pcDNA3.1-PTPN6)were transfected into them.Significant upregulation of PTPN6 was detected in pcDNA3.1-PTPN6 transfected Eca109and Yes-2 cells(P<0.05).The transfection efficiency of two cell lines was highly enough to be used for subsequent functional analyses.2.Transfection of PTPN6 led to a significant inhibition of Eca109 and Yes-2 cells proliferation detected by MTS assay,and colony formation assay further verified the results(P<0.05).3.Overexpression of PTPN6 inhibited the migration of Eca109 and Yes-2cell lines in vitro(P<0.05).4.Overexpression of PTPN6 inhibited the invasion of Eca109 and Yes-2cell lines in vitro(P<0.05).Conclusions:1.The silencing expression of PTPN6 in esophageal cancer cell lines and ESCC tissues is closely related to the occurrence and progression of esophageal cancer.The hypermethylation of P2 promoters may be one of the epigenetic mechanisms in silencing PTPN6 in ESCC.2.The protein expression and P2 promoter methylation may be independent prognostic factors in ESCC patients,and may serve as potential marker in predicting ESCC patients’survival.3.Overexpression of PTPN6 may inhibit the proliferation,migration and invasion of esophageal cancer cells in vitro.