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. 2023 Feb 13:2023:8143581.
doi: 10.1155/2023/8143581. eCollection 2023.

Transcription Activation of Rab8A by PEA3 Augments Progression of Esophagus Cancer by Activating the Wnt/ β-Catenin Signaling Pathway

Shusheng Cai  1 , Xiumei Liu  2 , Weiran Gao  3 , Lianhua Ye  3 , Qiming Zhong  3 , Xin Zhang  3
Affiliations

Transcription Activation of Rab8A by PEA3 Augments Progression of Esophagus Cancer by Activating the Wnt/ β-Catenin Signaling Pathway

Shusheng Cai et al. Dis Markers. .

Abstract

Background: Rab8A has been reported as an oncogenic gene in breast and cervical cancer. However, the function and molecular mechanism of Rab8A in esophagus cancer has not been reported.

Methods: Rab8A expression was detected by qPCR and western blotting assays, small interference RNA (siRNA) was applied to reduce Rab8A expression, and the biological behaviors of esophagus cancer cells were estimated by cell counting kit-8, colony formation, and transwell and western blotting assays. The transcriptional factor of Rab8A was verified by dual-luciferase assay and chromatin immunoprecipitation assay. The protein expression of key genes in the Wnt/β-catenin signaling pathway was determined by western blotting assay. M435-1279 was used to suppress the Wnt/β-catenin signaling pathway.

Results: A significant increase of Rab8A expression has been found in esophagus cancer cells. Knockdown of Rab8A suppressed the viability, colony formation, migration, and invasion abilities of esophagus cancer cells and induced apoptosis. PEA3 transcriptionally regulated Rab8A expression and promoted the viability, colony formation, migration, and invasion abilities of esophagus cancer cells and blocked apoptosis, which were diminished by si-Rab8A transfection. Additionally, the expression levels of key genes related to the Wnt/β-catenin signaling pathway were strengthened by PEA3 overexpression, which were reduced by si-Rab8A transfection. M435-1279 treatment significantly reduced the viability and colony formation of esophagus cancer cells.

Conclusions: The data showed that Rab8A was transcriptionally regulated by PEA3 and promoted the malignant behaviors of esophagus cancer cells by activating the Wnt/β-catenin pathway. The above results indicated that Rab8A may be considered as a promising biomarker for diagnosis and precision treatment in esophagus cancer.

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Conflict of interest statement

The authors declare that they have no conflicts of interest.

Figures

Figure 1
Figure 1
Rab8A was highly expressed in esophageal cancer cell lines, and siRab8A transfection significantly reduced Rab8A. (a–c) Results from qPCR and western blotting assays showed that Rab8A was overexpressed in KYSE150, TE-1, and Eca109 cells. (d, e) siRab8A-1 or siRab8A-2 transfection obviously reduced Rab8A expression in KYSE150 and Eca109 cells. ∗∗P < 0.01 vs. control.
Figure 2
Figure 2
siRab8A transfection affected the growth, apoptosis, migration, and invasion abilities of KYSE150 and Eca109 cells. (a, b) The OD values of KYSE150 and Eca109 cells were obviously reduced after siRab8A-1 or siRab8A-2 transfection. (c, d) The number of cell clones in KYSE150 and Eca109 cells was obviously reduced after siRab8A-1 or siRab8A-2 transfection. (e–h) siRab8A-1 or siRab8A-2 transfection obviously reduced Bcl-2 and increased BAK and active caspase-3 in KYSE150 and Eca109 cells. (i–l) The number of cell migration and invasion in KYSE150 and Eca109 cells was obviously reduced after siRab8A-1 or siRab8A-2 transfection. ∗∗P < 0.01 vs. control.
Figure 3
Figure 3
PEA3 was regarded as a transcriptional factor of Rab8A and positively regulated Rab8A expression. (a) In the WT-Rab8A group, overexpression of PEA3 significantly increased the luciferase activity. (b, c) After si-NC or si-PEA3 treatment, ChIP assay was carried out using control IgG or Rab8A antibody. Then, qPCR was performed to analyze Rab8A enrichment. (d–g) In KYSE150 and Eca109 cells, siPEA3 transfection reduced the protein levels of PEA3 and Rab8A; PEA3-OE transfection increased the protein levels of PEA3 and Rab8A. (h, i) Data from western blotting assays showed that Rab8A and PEA3 were highly expressed in esophageal cancer tissues. ∗∗P < 0.01 vs. normal. T: tumor, N: normal.
Figure 4
Figure 4
The biological effects of PEA3-OE on KYSE150 and Eca109 cells were counteracted by si-Rab8A. (a–d) The promoting effects of PEA3-OE on OD values and cell clone numbers in KYSE150 and Eca109 cells were reversed by si-Rab8A treatment. (e–h) PEA3-OE treatment increased Bcl-2 expression and decreased Bak and active caspase-3 expression in KYSE150 and Eca109 cells, whereas the phenomena were reversed by the addition of si-Rab8A. (i–l) The increased invasion and migration abilities of KYSE150 and Eca109 cells caused by PEA3-OE were inhibited by the administration of si-Rab8A. ∗∗P < 0.01 vs. control; #P < 0.05 and ##P < 0.01 vs. PEA3-OE.
Figure 5
Figure 5
The Wnt/β-catenin signaling pathway was involved in the regulation of PEA3/Rab8A on KYSE150 and Eca109 cell behaviors. (a–d) Protein levels of c-MYC, β-catenin, and CyclinD1 in KYSE150 and Eca109 cells were detected by western blotting after control, PEA3-OE, or PEA3-OE+si-Rab8A treatment, respectively. ∗∗P < 0.01 vs. control; ##P < 0.01 vs. PEA3-OE. (e–h) Protein levels of c-MYC, β-catenin, and CyclinD1 in KYSE150 and Eca109 cells were detected by western blotting after control, siRab8A, inhibitor, or siRab8A+inhibitor treatment, respectively. (i, j) The OD values in KYSE150 and Eca109 cells were detected by CCK-8 assay after control, siRab8A, inhibitor, or siRab8A+inhibitor treatment, respectively. (k, l) The cell clone number in KYSE150 and Eca109 cells was detected by colony formation assay after control, siRab8A, inhibitor, or siRab8A+inhibitor treatment, respectively. ∗∗P < 0.01 vs. control; ##P < 0.01 vs. siRab8A.
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