Overexpression of KRAS enhanced the stemness of esophageal cancer cells inhibited by overexpression of circ0043898

Abstract

Background: Our previous study revealed that circ0043898 is downregulated in esophageal cancer (EC), and its overexpression attenuates the progression of EC. The objective of this article is to explore whether circ0043898 inhibits tumor progression by inhibiting cancer stem cells (CSCs) in EC.

Methods: PCDH-circ0043898 plasmid was transfected into EC cells, and the effect of overexpression was verified by qRT-PCR. Immunofluorescence, flow cytometry, and stem cell spheroidization were used to detect CSCs phenotype changes. RNA sequencing was adopted to identify downstream regulatory genes and p-PI3K, PI3K, and KRAS expressions were validated by western blot. To investigate whether KRAS functions downstream of circ0043898 in regulating cancer stemness, we co-transfected EC cells with both KRAS and circ0043898 plasmids and examined CSCs phenotypes.

Results: circ0043898 was overexpressed in the EC cells, and reduced stem cell markers (CD44 and CD133) and the number of stem cell spheroidization. In addition, overexpression of circ0043898 changed many genes expression, including reduced p-PI3K, PI3K, and KRAS expressions. Moreover, overexpression of KRAS attenuated the effect of overexpressed circ0043898 on CSCs phenotype.

Conclusions: Overexpression of circ0043898 reduced CSCs markers and the number of stem cell spheroidization. However, the overexpression of KRAS attenuated the inhibition effect of overexpressed circ0043898 on CSCs marker and the number of stem cell spheroidization. These findings identify a potential therapeutic target for the EC.

Keywords: CircRNAs; Esophageal cancer; KRAS; Stemness; circ0043898.

Fig. 1

Overexpression of circ0043898 reduced stem cell markers and weakened the ability of stem cells to form spheres in EC cells. EC cells (ECA109, Kyse-520) were transfected with PCDH empty plasmid (named PCDH-NC group) and PCDH-circ0043898 plasmid (named PCDH-circ0043898 group), respectively. After 24 h, samples were collected for qRT-PCR to detect circ0043898 and the observation of stem cell sphere formation ability. After 48 h, samples were collected for IF, flow cytometry, and Western blot to detect stemness marker genes CD44 and CD133. (A) qRT-PCR detection of circ0043898 expression. (B) IF detection of CD44 and CD133 expressions. (C) Flow cytometry detection of CD44⁺CD133⁺ double-positive cell population. (D) Western blot analysis of CD44 and CD133 protein expression following circ0043898 overexpression. Blots were cut prior to antibody incubation. (E) Representative images of stem cell spheres in EC cells. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 2

RNA sequencing reveals gene expression changes and functional enrichment following circ0043898 overexpression in EC cells. ECA109 and Kyse-520 cells were transfected with empty PCDH plasmid (named the PCDH-NC group) and PCDH-circ0043898 plasmid (named the PCDH-circ0043898 group), respectively. Samples were collected for RNA sequencing 24 h post-transfection. (A) Venn diagram showing DEGs in Kyse-520 and ECA109 cells following circ0043898 overexpression. (B) Heatmap of DEGs in Kyse-520 and ECA109 cells. (C) Top 15 GO functional enrichment analysis of 309 commonly DEGs in Kyse-520 and ECA109 cells. (D) Top 15 KEGG pathway enrichment analysis of 309 commonly DEGs. (E) qRT-PCR detection of KRAS and PI3K mRNA levels in EC cells following circ0043898 overexpression. (F) Western blot analysis of KRAS, PI3K, and p-PI3K protein expression in EC cells following circ0043898 overexpression. Blots were cut prior to antibody incubation. ***P < 0.001

Fig. 3

Overexpression of KRAS reversed the stemness inhibition induced by circ0043898 in EC cells. ECA109 and Kyse-520 cells were transfected with PCDH-NC (PCDH-NC group), PCDH-circ0043898 (PCDH-circ0043898 group), or co-transfected with PCDH-circ0043898 and PCDH-KRAS (PCDH-circ0043898 + KRAS group). After 24 h of transfection, samples were collected for qRT-PCR analysis and stem cell spheroid formation assays. After 48 h of transfection, IF and flow cytometry were performed to assess stemness markers CD44 and CD133. (A) qRT-PCR analysis of circ0043898, KRAS, and PI3K mRNA expression. (B) IF detection of CD44 and CD133 protein expression. (C) Flow cytometry analysis of CD44⁺CD133⁺ double-positive cell ratios. (D) qRT-PCR analysis confirming KRAS overexpression. (E) Western blot detection of KRAS expression in each group. Blots were cut prior to antibody incubation. (F) Representative images of stem cell spheroid formation in each group. *P < 0.05, **P < 0.01, ***P < 0.001