Ouabain Inhibits The Proliferation Of Esophageal Adenocarcinoma Cells Via MKK6-SOX9 Axis

ObjectiveThe objective of this work is to study Ouabain's mechanism of inhibiting the proliferation of the esophageal adenocarcinoma(EAC) cells, and the in vivo and in vitro effects of Ouabain's target gene MKK6 and downstream transcription factor SOX9 on the growth of esophageal adenocarcinoma cells.Method1. Screen 1043 kinds of medicines that can inhibit the proliferation of the esophageal adenocarcinoma cells and Ouabain was selected for study. Verity its inhabitation effect on cancer cells through cell growth curve and cell xenografts.2. Search for Ouabain's target gene in EAC cells by using RNA-seq, and MKK6 was found to be the target gene. Investigate Ouabain's effects on MKK6 gene expression and protein expression through real-time PCR and Western Blot.3. Knockdown MKK6 by lentivirus mediated sh RNA transduction, and evaluate the knockdown efficiency by real-time PCR and Western Blot. Investigate the influence of MKK6's knockdown on cancer cell growth in vitro and tumor formation in vivo.4. Identify MKK6's downstream target gene from the genes that may affect EAC cell proliferation, and lock onto the transcription factor SOX9. Investigate the impact of Ouabain treatment and MKK6 knockdown on the expression of SOX9 by real time PCR and Western Blot.5. Knock out SOX9 gene in EAC cell by CRISPR-Cas9 technology, then study the role of SOX9 in EAC by observing the change of cell growth in vitro and xenografts tumor formation in vivo.Result1. Through bioinformatic analysis, Ouabain was selected for study due to the fact that the relative cell number has a sharp drop. The cell growth curves and xenografts indicate that Ouabain can inhibit OE33 and OE19 cell growth in vitro(p value < 0.05) and tumor formation in vivo(p value < 0.05) compare to the control group.2. Ouabain affects the expression of MKK6 gene and protein. RNA sequencing result indicates that MKK6 gene expression decreased by 89% in Ouabain treated OE33 cells.Meanwhile, the western blot and real-time-PCR verified MKK6 has significant reduction in both OE33(P<0.05) and OE19 cells(P<0.05) under Ouabain treatment.3. MKK6 knockdown limits the proliferation of EAC cells. sh RNA-mediated MKK6 knockdown has a high silence efficiency(almost 90%), which can significantly limit OE33 and OE19 cell proliferation(P<0.05) and tumor formation(P<0.05).4. MKK6 regulates SOX9 expression in EAC cells: real-time PCR and Western Blot results revealed that MKK6 knockdown can reduce SOX9 m RNA expression(P<0.05)and protein expression(P<0.05) in EAC cells.5. SOX9 knockout inhibits the proliferation of EAC cells: SOX9 gene expression has been totally knockout through CRISPR-Cas9 technology. Without SOX9 expression, the cell growth of OE19 was significantly reduced ex vivo(P<0.05) and tumor formation was also reduced in the xenografts(P<0.05).Conclusion1. Ouabain inhibits the proliferation of EAC cells in vivo and in vitro.2. m RNA and protein expression level of MKK6 have decreased in Ouabain treated EAC cells and tumors.3. Reduction of MKK6 gene expression can limit EAC cell growth in vivo and in vitro.4. The expression of transcriptional factor SOX9 decreases when MKK6 gene has been knocked down in EAC cells.5. CRISPR-Cas9 mediated SOX9 knockout inhibits the proliferation of EAC cells.