Abstract:

Objective To discuss the expression and biological function of gap junction protein β2 （GJB2） in the lung adenocarcinoma （LUAD） A549 cells， and to procide the basis for the treatment of LUAD. Methods The TIMER database was used to analyze the differential expression of GJB2 gene in various tumors； the GEPIA database was used to detect the mRNA expression of GJB2 in LUAD and analyze its correlation with different clinical stages of LUAD； the Kaplan-Meier plotter database was used to analyze the correlation between GJB2 protein and the prognosis of the LUAD patients. A total of 111 pairs of cancer tissues and adjacent normal lung tissues of LUAD patients were collected， and immunohistochemical staining was used to observe the expression of GJB2 protein in LUAD tissues and adjacent normal lung tissues. The human LUAD A549 cells were cultured in vitro and divided into GJB2 overexpression group （OE-GJB2 group， transfected with GJB2 overexpression plasmid） and its negative control group （OE-NC group， transfected with GJB2 empty vector plasmid）， small interfering RNA （siRNA）-GJB2 group （si-GJB2 group， transfected with GJB2-siRNA） and its negative control group （si-NC group， transfected with control siRNA）. Western blotting method was used to verify the transfection efficiency of the cells； cell couting kit-8 （CCK-8） method was used to detect the proliferation activities of the A549 cells in various groups； 5-ethynyl-2'-deoxyuridine （EdU） staining was used to detect the positive expression rates of the A549 cells in various groups； colony formation assay was used to detect the number of colony formation of the A549 cells in various groups； cell scratch assay was used to detect the migration rate of the A549 cells in various groups； Transwell chamber assay was used to detect the number of the invasion A549 cells in various groups； Western blotting method was used to detect the expression levels of GJB2 protein and epithelial-mesenchymal transition （EMT）-related proteins in the A549 cells in various groups. Results The RT-PCR results showed that GJB2 gene was abnormally expressed in various tumors； compared with adjacent normal lung tissue， the mRNA expression level of GJB2 in cancer tissue of LUAD patients was significantly increased （P<0.05）， and the high expression of GJB2 was correlated with the clinicopathological stage of LUAD （P<0.05）. The Kaplan-Meier plotter results showed that compared with patients with low GJB2 expression， the overall survival of the patients with high GJB2 expression was significantly decreased ［hazard ratio （HR）=1.71， 95% CI： 1.34-2.18， P<0.05］. GJB2 protein was negatively expressed in adjacent normal alveolar and bronchial epithelial cells； compared with adjacent normal lung tissue， the expression of GJB2 protein in cancer tissue of LUAD patients was significantly enhanced. In LUAD， the positive expression of GJB2 protein was significantly associated with lymphnode metastasis （P<0.05） but not with gender （P=0.626）， age （P=0.639）， or TNM stage （P=0.837） （P>0.05）. The CCK-8 results showed that compared with OE-NC group， the proliferation activity of the A549 cells in OE-GJB2 group was significantly increased （P<0.05 or P<0.01）； compared with si-NC group， the proliferation activity of the A549 cells in si-GJB2 group was significantly decreased （P<0.05 or P<0.01）. The EdU staining results showed that compared with OE-NC group， the positive expression rate of EdU in the A549 cells in OE-GJB2 group was significantly increased （P<0.01）； compared with si-NC group， the positive expression rate of EdU in the A549 cells in si-GJB2 group was significantly decreased （P<0.01）. The colony formation assay results showed that compared with OE-NC group， the number of colony formation of the A549 cells in OE-GJB2 group was significantly increased （P<0.01）； compared with si-NC group， the number of colony formation of the A549 cells in si-GJB2 group was significantly decreased （P<0.01）. The Transwell chamber assay results showed that compared with OE-NC group， the number of invasion A549 cells in OE-GJB2 group was significantly increased （P<0.01）； compared with si-NC group， the number of invasion A549 cells in si-GJB2 group was significantly decreased （P<0.01）. The cell scratch assay results showed that 24 h after scratching， compared with OE-NC group， the migration rate of the A549 cells in OE-GJB2 group was significantly increased （P<0.01）； compared with si-NC group， the migration rate of the A549 cells in si-GJB2 group was significantly decreased （P<0.01）. The Western blotting results showed that compared with OE-NC group， the expression levels of GJB2 and N-cadherin proteins in the A549 cells in OE-GJB2 group were significantly increased （P<0.01）， while the expression level of E-cadherin protein was significantly decreased （P<0.05）； compared with si-NC group， the expression levels of GJB2 and N-cadherin proteins in the A549 cells in si-GJB2 group were significantly decreased （P<0.01）， while the expression level of E-cadherin protein was significantly increased （P<0.01）. Conclusion GJB2 is highly expressed in cancer tissue of LUAD patients and is associated with the poor prognosis in LUAD patients. GJB2 promotes the proliferation， invasion， migration， and EMT of the A549 cells.

Key words: Gap junction protein β2, Lung adenocarcinoma, Cell proliferation, Cell invasion, Cell migration, Epithelial-mesenchymal transition