Abstract:

Objective To discuss the expression of Rh family C glycoprotein （RHCG） in the esophageal squamous cell carcinoma （ESCC） tissue and its effect on the malignant biological behavior of ESCC cells， and to clarify the value of RHCG as a diagnostic and prognostic marker for the ESCC patients. Methods A total of 143 ESCC tissue samples and 105 adjacent normal tissue samples were collected. Using immunohistochemical staining method， 141 ESCC samples were divided into two groups： RHCG low expression group （immunohistochemistry score ≤6） and RHCG high expression group （immunohistochemistry score >6）. Immunohistochemical method was used to detect the RHCG protein expression in 143 ESCC tissues and 105 normal tissues， and the relationship between the clinicopathological characteristics of the ESCC patients was analyzed. Receiver operating characteristic （ROC） curve and Kaplan-Meier survival analysis were used to evaluate the value of RHCG in diagnosis and prognosis of the ESCC patients； univariate and multivariate COX regression analysis were used to determine the independent risk factors affecting the prognosis of the ESCC patients. Gene Expression Profiling Interactive Analysis （GEPIA2） database was used to analyze the expression of RHCG mRNA in various tumor tissues. The ESCC TE-1 cells were cultured and transfected in to 6-well cell culture plates with different Lipofectamine2000∶RHCG ratios； the cells in RHCG transfection group were transfected with weights of 2.0， 2.5， and 3.0 μg for 24 and 48 h， respectively， and the cells in NC group transfected with empty vector as control. Western blotting method was used to detect the RHCG protein expression level in the TE-1 cells in various groups after transfection at different concentrations and verify the optimal transfection conditions； cell counting kit-8 （CCK-8） assay was used to detect the proliferation activities of the TE-1 cells； plate clone formation assay was used to detect the colony formation numbers of the TE-1 cells； Transwell chamber assay was used to detect the numbers of migrating TE-1 cells. Results Compared with adjacent normal tissue， the RHCG gene expression level in various cancer tissues including ESCC， glioblastoma multiforme， and head and neck squamous cell carcinoma was significantly decreased （P<0.05）. RHCG protein was mainly located on the cell membrane of normal esophageal squamous epithelial cells； the RHCG protein expression intensity in ESCC tissues was lower than that in adjacent normal esophageal tissue （χ2=109.373， P<0.001）， and the patients in RHCG low expression group had poorer differentiation than those in RHCG high expression group （P=0.041）. The area under the curve （AUC） value of RHCG for diagnosing ESCC was 0.86， with sensitivity and specificity of 95.1% and 75.0%， respectively； the Kaplan-Meier survival analysis results showed that compared with high RHCG expression group， the patients in low RHCG expression group had shorter survival time and poorer prognosis ［harard ratio（HR）=0.269，95% confidence interval（CI）： 0.113-0.639， P=0.020］； the COX regression analysis results showed that low RHCG expression could serve as an independent risk factor affecting the prognosis of ESCC ［HR=4.569， 95%CI=1.315-15.877， P=0.017）］. The Western blotting results verified that the optimal transfection condition was 3.0 μg RHCG plasmid for 48 h， at which time RHCG overexpression was optimal and RHCG protein expression level was highest. The CCK-8 assay results showed that compared with control group， the proliferation activity in RHCG overexpression group was decreased on the 4th day after cell seeding （P<0.001）. In the TE-1 cells， the colony formation number of the TE-1 cells in RHCG over-expression group was lower than that in control group （t=17.70， P<0.001）. The Transwell chamber assay results showed that compared with control group， the number of migrating cells in RHCG over-expression group was decreased （t=23.74， P<0.001）. Conclusion RHCG expression is decreased in ESCC tissues and associated with poor prognosis in ESCC patients； overexpression of RHCG can inhibit the proliferation and migration of the TE-1 cells， providing a theoretical basis for RHCG as a novel diagnostic and prognostic marker and therapeutic target for ESCC.

Key words: Rh family C glycoprotein, Esophageal squamous cell carcinoma, Diagnosis, Prognosis, Receiver operating characteristic curve