The Expression Of Claudin-2 Protein In Human Esophageal Squamous Cell Carcinoma And Its Effect On Cell Function

BackgroundEsophageal cancer is one of the most common malignant tumors in clinical practice.The incidence of esophageal cancer ranks the 8th among malignant tumors worldwide,and the mortality ranks the 6th.Due to atypical early symptoms and easy local invasion and lymph node metastasis,the 5-year overall survival rate is less than20%.Claudin protein family is one of the tight junction molecules of epithelial cells,which is composed of 27 members.Claudin are expressed in different tissues and cells,and caludin is closely related to the occurrence and development of various malignant tumors.Exploring the expression of Claudin-2 in esophageal cancer and its function in cells will provide a new theoretical basis for revealing the occurrence and development of tumors.ObjectiveTo study the expression of Claudin-2 in esophageal squamous cell carcinoma(ESCC)and its relationship with clinicopathological features and 5-year survival rate of ESCC,and to explore the role of Claudin-2 in the proliferation,migration and invasion of ESCC cells.Method1.A total of 127 patients with ESCC who underwent surgical resection irrespective of preoperative treatment(chemotherapy,radiation therapy,or chemoradiotherapy)at Henan Cancer Hospital between February 2010 and November 2017.2.Immunohistochemical method was used to detect the expression of Claudin-2 in paraffin-embedded tissue samples of ESCC.Western blot was used to detect the expression of Claudin-2 protein in fresh tissue samples,human ESCC cells,human immortalized normal esophageal epithelial cells and cells successfully transfected with lentivirus knockdown Claudin-2.3.shRNA lentiviral vector and empty vector with low Claudin-2 expression were amplified and packaged to infect KYSE450 cells respectively.The success of cell infection was judged by observing whether the cells were infected with green fluorescence and quantitative detection of Western blot,and the infected cells were collected for follow-up experiments.4.The effect of knockdown Claudin-2 on the proliferation,invasion,migration were verified using clone formation tests,scratch tests and transwell migration assays.5.Pearson’s chi-squared test or Fisher’s exact test was used to analyze the relationship between Claudin-2 expression and clinicopathological features of ESCC patients.Kaplan-Meier method and Log-rank test were used to analyze the relationship between Cluadin-2 expression and 5-year survival time.Univariate and multivariate Cox proportional hazards regression analysis were performed to investigate the risk factors of poor prognosis for ESCC patients.One-way ANOVA was used to analyze the statistical difference of the experimental data.Result1.The expression level of Claudin-2 in ESCC tissues was higher than that in paracancerous tissues(P<0.05).Claudin-2 protein was expressed in cytoplasm and cell membrane.2.The positive expression of Claudin-2 in ESCC patients was correlated with lymph node metastasis.The 5-year survival rate of patients with positive expression of Claudin-2 was lower than that of patients with negative expression of Claudin-2.The degree of differentiation(P=0.018)and the positive expression of Claudin-2(P=0.022)were independent poor prognostic factors.3.The expression of Claudin-2 in ESCC cells was higher than in human immortalized esophageal epithelial cells.4.When the screening of puromycin was terminated after KYSE450 cells were infected by lentivirus,the green fluorescent cells were more than 80%.The expression of Claudin-2 in sh-Claudin-2 group was lower than that in empty virus group and blank control group(P<0.05).5.The number of clones formed in sh-Claudin-2 group(44.00±9.54)was lower than that in sh-NT group(167.33±6.66)and control group(167.00±3.00)(P<0.05).The percentage of wound healing in sh-Claudin-2 group(23.33±5.77)was lower than that in sh-NT group(51.33 ±8.08)and the blank control group(54.17±7.22)(P<0.05).The number of invasive cells in sh-Claudin-2 group(61.67 ±7.64)was less than that in sh-NT group(134.00±5.29)and blank control group(134.00±6.25)(P<0.05).Conclusion1.The Claudin-2 expression of ESCC is higher than that of paracancerous tissues,which is positively correlated with lymph node metastasis and negatively correlated with the five-year survival rate of ESCC patients.The degree of differentiation and the expression of Claudin-2 are independent poor prognostic factors.2.Knockdown Claudin-2 expression can inhibit the proliferation,migration and invasion of human ESCC cell.It is suggested that Claudin-2 is involved in the occurrence and development of ESCC and may become a potential prognostic index,therapeutic target and tumor marker of ESCC.