Objective: To explore the effect of connexin 32 (Cx32) on the cell proliferation, migration, invasion of human hepatocellular carcinoma (HCC) cell line Huh7 and its mechanism. Methods: Firstly, bioinformatics techniqu...Objective: To explore the effect of connexin 32 (Cx32) on the cell proliferation, migration, invasion of human hepatocellular carcinoma (HCC) cell line Huh7 and its mechanism. Methods: Firstly, bioinformatics techniques were used to analyze the difference in expression of Cx32 between HCC tissues and normal liver tissues, and the relationship between Cx32 expression and important clinicopathological features of HCC was also explored. Subsequently, Cx32 expression in HCC cell lines and normal hepatic epithelial cell line was detected in vitro. Huh7 cell line with stable over⁃expression of Cx32 was further established, and the change in cell proliferation ability was measured by MTT assay, changes in migration and invasion capacities were detected by wound⁃healing assay and transwell assay, on this cell line. Finally, western blot and immunofluorescence (IF) were used to investigate the alterations of expression of epithelial⁃mesenchymal transition (EMT) markers. Results: Bioinformatics analyses showed that Cx32 mRNA and protein expression levels in HCC tissues were lower than those in normal liver tissues, and the mRNA expression level of Cx32 was negatively correlated with T stage, histological grade and clinical stage of HCC patients (all P<0.05). Results of in vitro experiments revealed Cx32 protein expression in different HCC cell lines was down⁃regulated compared to that in normal hepatic epithelial cell line LO2. Cx32 stably over⁃expressed (Cx32 OE) Huh7 cell line was successfully constructed by lentivirus infection and showed high expression of Cx32 protein in the cell line. Compared to the control group and (or) the negative control (NC) group, the Cx32 OE group exhibited decreased OD490 value, wound healing rate and invasive cell number (all P<0.05). Furthermore, an increase in the expression of epithelial marker E⁃cadherin, and a decrease in the expression of mesenchymal markers Snail and Vimentin, were observed in Cx32⁃OE Huh7 cell line. Conclusion: Cx32 is low expressed in HCC tissues and cells, while the proliferation, migration and invasion ability of Huh7 cells can be inhibited by over⁃expression of Cx32, of which the underlying mechanism may be related to the inhibition of EMT process.展开更多
AIM To clarify the mechanisms of connexin 32 (Cx32) downregulation by potential transcriptional factors (TFs) in Helicobacter pylori (H. pylori)-associated gastric carcinogenesis. METHODS Approximately 25 specimens at...AIM To clarify the mechanisms of connexin 32 (Cx32) downregulation by potential transcriptional factors (TFs) in Helicobacter pylori (H. pylori)-associated gastric carcinogenesis. METHODS Approximately 25 specimens at each developmental stage of gastric carcinogenesis [non-atrophic gastritis, chronic atrophic gastritis, intestinal metaplasia, dysplasia and gastric carcinoma (GC)] with H. pylori infection [H. pylori (+)] and 25 normal gastric mucosa (NGM) without H. pylori infection [H. pylori (-)] were collected. After transcriptional factor array analysis, the Cx32 and PBX1 expression levels of H. pylori-infected tissues from the developmental stages of GC and NGM with no H. pylori infection were measured by real-time polymerase chain reaction (RT-PCR) and Western blot analysis. Regarding H. pylori-infected animal models, the Cx32 and PBX1 mRNA expression levels and correlation between the gastric mucosa from 10 Mongolian gerbils with long-term H. pylori colonization and 10 controls were analyzed. PBX1 and Cx32 mRNA and protein levels were further studied under the H. pylori-infected condition as well as PBX1 overexpression and knockdown conditions in vitro. RESULTS Incremental PBX1 was first detected by TF microarray in H. pylori-related gastric carcinogenesis. The identical trend of PBX1 and Cx32 expression was confirmed in the developmental stages of H. pylori-related clinical specimens. The negative correlation of PBX1 and Cx32 was confirmed in H. pylori-infected Mongolian gerbils. Furthermore, decreased PBX1 expression was detected in the normal gastric epithelial cell line GES-1 with H. pylori infection. Enforced overexpression or RNAi-mediated knockdown of PBX1 contributed to the diminished or restored Cx32 expression in GES-1 and the gastric carcinoma cell line BGC823, respectively. Finally, dual-luciferase reporter assay in HEK293T cells showed that Cx32 promoter activity decreased by 30% after PBX1 vector co-transfection, indicating PBX1 as a transcriptional downregulator of Cx32 by directly binding to its promoters. ONCLUSION PBX1 is one of the determinants in the Cx32 promoter targeting site, preventing further damage of gap junction protein in H. pylori-associated gastric carcinogenesis.展开更多
基金National Natural Science Foundation of China(No.81402514)Support Program for Excellent Young Talents in Universities of Anhui Province(No.gxyq2022042)"512 Talent Cultivation Plan" of Bengbu Medical College(No.by51202208)。
文摘Objective: To explore the effect of connexin 32 (Cx32) on the cell proliferation, migration, invasion of human hepatocellular carcinoma (HCC) cell line Huh7 and its mechanism. Methods: Firstly, bioinformatics techniques were used to analyze the difference in expression of Cx32 between HCC tissues and normal liver tissues, and the relationship between Cx32 expression and important clinicopathological features of HCC was also explored. Subsequently, Cx32 expression in HCC cell lines and normal hepatic epithelial cell line was detected in vitro. Huh7 cell line with stable over⁃expression of Cx32 was further established, and the change in cell proliferation ability was measured by MTT assay, changes in migration and invasion capacities were detected by wound⁃healing assay and transwell assay, on this cell line. Finally, western blot and immunofluorescence (IF) were used to investigate the alterations of expression of epithelial⁃mesenchymal transition (EMT) markers. Results: Bioinformatics analyses showed that Cx32 mRNA and protein expression levels in HCC tissues were lower than those in normal liver tissues, and the mRNA expression level of Cx32 was negatively correlated with T stage, histological grade and clinical stage of HCC patients (all P<0.05). Results of in vitro experiments revealed Cx32 protein expression in different HCC cell lines was down⁃regulated compared to that in normal hepatic epithelial cell line LO2. Cx32 stably over⁃expressed (Cx32 OE) Huh7 cell line was successfully constructed by lentivirus infection and showed high expression of Cx32 protein in the cell line. Compared to the control group and (or) the negative control (NC) group, the Cx32 OE group exhibited decreased OD490 value, wound healing rate and invasive cell number (all P<0.05). Furthermore, an increase in the expression of epithelial marker E⁃cadherin, and a decrease in the expression of mesenchymal markers Snail and Vimentin, were observed in Cx32⁃OE Huh7 cell line. Conclusion: Cx32 is low expressed in HCC tissues and cells, while the proliferation, migration and invasion ability of Huh7 cells can be inhibited by over⁃expression of Cx32, of which the underlying mechanism may be related to the inhibition of EMT process.
基金Supported by the New Xiangya Talent Project of the Third Xiangya Hospital of Central South University,No.20150310
文摘AIM To clarify the mechanisms of connexin 32 (Cx32) downregulation by potential transcriptional factors (TFs) in Helicobacter pylori (H. pylori)-associated gastric carcinogenesis. METHODS Approximately 25 specimens at each developmental stage of gastric carcinogenesis [non-atrophic gastritis, chronic atrophic gastritis, intestinal metaplasia, dysplasia and gastric carcinoma (GC)] with H. pylori infection [H. pylori (+)] and 25 normal gastric mucosa (NGM) without H. pylori infection [H. pylori (-)] were collected. After transcriptional factor array analysis, the Cx32 and PBX1 expression levels of H. pylori-infected tissues from the developmental stages of GC and NGM with no H. pylori infection were measured by real-time polymerase chain reaction (RT-PCR) and Western blot analysis. Regarding H. pylori-infected animal models, the Cx32 and PBX1 mRNA expression levels and correlation between the gastric mucosa from 10 Mongolian gerbils with long-term H. pylori colonization and 10 controls were analyzed. PBX1 and Cx32 mRNA and protein levels were further studied under the H. pylori-infected condition as well as PBX1 overexpression and knockdown conditions in vitro. RESULTS Incremental PBX1 was first detected by TF microarray in H. pylori-related gastric carcinogenesis. The identical trend of PBX1 and Cx32 expression was confirmed in the developmental stages of H. pylori-related clinical specimens. The negative correlation of PBX1 and Cx32 was confirmed in H. pylori-infected Mongolian gerbils. Furthermore, decreased PBX1 expression was detected in the normal gastric epithelial cell line GES-1 with H. pylori infection. Enforced overexpression or RNAi-mediated knockdown of PBX1 contributed to the diminished or restored Cx32 expression in GES-1 and the gastric carcinoma cell line BGC823, respectively. Finally, dual-luciferase reporter assay in HEK293T cells showed that Cx32 promoter activity decreased by 30% after PBX1 vector co-transfection, indicating PBX1 as a transcriptional downregulator of Cx32 by directly binding to its promoters. ONCLUSION PBX1 is one of the determinants in the Cx32 promoter targeting site, preventing further damage of gap junction protein in H. pylori-associated gastric carcinogenesis.
