circTNFRSF21, a newly identified circular RNA promotes endometrial carcinoma pathogenesis through regulating miR-1227-MAPK13/ATF2 axis

Abstract

Background: Circular RNA is a type of non-coding RNA with great potential in regulating gene expression and associated with disease progression. However, the role of circular RNA in endometrial carcinoma (EC) remains largely unknown.

Results: In this study, we found that circTNFRSF21 was highly expressed in EC cells and tumor tissues. In vitro and in vivo results showed that circTNFRSF21 was linked to increased EC cell growth and EC xenografts formation in nude mice. Mechanically, we showed that circTNFRSF21 acts as a sponge of miR-1227 in EC cells to rescue MAPK13/ATF2 signaling pathway activity.

Conclusions: Our studies suggested that in the EC, circTNFRSF21 promotes EC formation through downregulating miR-1227 expression and activating MAPK13/ATF2 signaling pathway. These findings provide strong evidence that circTNFRSF21-miR-1227-MAPK13/ATF2 axis is a promising target for EC treatment.

Methods: qRT-PCR was used to detect circTNFRSF21expression in EC patients and EC cell lines. Cell growth, cell colony formation, cell apoptosis, cell cycle progression, and in vivo tumor formation assays were used to evaluate the roles of circTNFRSF21 in EC. Western blot, luciferase assay, RNA pull-down, siRNA knockdown, and CRISPR gene knock out assays were applied to study the mechanisms through which circTNFRSF21 regulates EC formation.

Keywords: ATF2; MAPK13; circular RNA; endometrial carcinoma; miR-1227.

Figure 1

Detection of circ_TNFRSF21 expression in HEC1-A. (A) Diagram of circ_TNFRSF21. (B) PCR results of TNFRSF21 exon 6 or circ_TNFRSF21 from cDNA or genomic DNA (gDNA) (C) Detection of circ_TNFRSF21 and TNFRSF21 exon 6 expression before and after RNase A treatment. (D) Pull down circ_TNFRSF21 with AGO2 or EIF4A, IgG was used as negative control. *P<0.05, **P<0.01, ***P<0.001, data are represented as mean +/- SEM.

Figure 2

circTNFRSF21 inhibits TNFRSF21 expression. (A) siRNA targeting the junction of the covalently joined 3' and 5' ends of circ_TNFRSF21 was shown. F1/R1 and F2/R2 were used to amplify TNFRSF21 mRNA. (B) Detection of circ_TNFRSF21 expression in HEC1-A after silencing circ_TNFRSF21. TNFRSF21 mRNA (C) and protein level (D) after silencing circ_TNFRSF21 in HEC1-A. Overexpression of circ_TNFRSF21 (E) significantly decreased TNFRSF21 transcription (F) and protein synthesis (G). *P<0.05, **P<0.01, ***P<0.001. Data are represented as mean +/- SEM.

Figure 3

circTNFRSF21 promotes EC cell growth, proliferation and in vivo tumor formation. (A) circTNFRSF21 expression in EC patient tumor tissues and adjacent normal tissues. (B) circTNFRSF21 expression in EC cell lines and HEuEC cells. Cell growth (C) and cell cycle (D) detection after overexpressing circTNFRSF21 in HEuEC cells. (E) Cell growth after silencing circTNFRSF21 in HEC1-A and Ishikawa cells. (F) Cell colony formation after silencing circTNFRSF21 in HEC1-A and Ishikawa cells. Statistical results of colony number were shown on the right. (G) Cell cycle and (H, I) Cell apoptosis after silencing circTNFRSF21 in HEC1-A and Ishikawa cells. Etoposide induced cell apoptosis was used as positive control. (J) Image of tumors derived from si-circTNFRSF21 and control Ishikawa cells in nude mice (n=6). (K) Tumor weights and (L) growth curve of tumors from the 2 groups. *P<0.05, **P<0.01, ***P<0.001. Data are represented as mean +/- SEM.

Figure 4

Knock down or overexpress TNFRSF21 does not affect EC cell growth. (A) Western blot results of TNFRSF21 after knocking down TNFRSF21 in HEC1-A and Ishikawa cells. GAPDH was used as an internal control. Cell growth (B) and cell cycle (C) detection after knocking down TNFRSF21 in HEC1-A and Ishikawa cells. (D) Western blot results of TNFRSF21 after overexpressing TNFRSF21 in HEC1-A and Ishikawa cells. (E) Cell growth after overexpressing TNFRSF21 in HEC1-A and Ishikawa cells. Data are represented as mean +/- SEM.

Figure 5

circTNFRSF21 acts as a sponge of miR-1227 in vivo. (A) Diagram of circ_TNFRSF21 and miR-1227 interactions (B) Pull down miR-1227 with biotin labeled WT circTNFRSF21 or miR-1227 binding sites mutated circ_TNFRSF21 from HEC1-A cell lysis. Detection of miR-1227 expression after overexpressing (C) or knocking down (D) circTNFRSF21 in HEC1-A and Ishikawa cells. (E) qRT-PCR detect miR-1227 expression in EC cell lines and HEuEC. (F) miR-1227 expression in in EC patient tumor tissues and adjacent normal tissues. (G) Cell growth after overexpressing miR-1227 in HEC1-A and Ishikawa cells. (H) Cell growth after silencing miR-1227 in HEC1-A and Ishikawa cells. (I, J) Cell apoptosis detection after silencing miR-1227 in HEC1-A and Ishikawa cells. *P<0.05, **P<0.01, ***P<0.001. Data are represented as mean +/- SEM.

Figure 6

miR-1227 targets MAPK13 and inhibits EC cell proliferation. (A) Predicted MAPK13 3’UTR-miR-1227 interaction. Detection of MAPK13 mRNA transcription (B) and protein level (C) after overexpressing miR-1227 mimic in HEC1-A and Ishikawa cells. (D) Luciferase assay detects WT or miR-1227 binding sites mutated MAPK13 3’UTR activity in the presence or absence of miR-1227. (E) Western blot detects MAPK13 expression in EC cell lines and HEuEC. (F) Western blot results of MAPK13 after knocking down MAPK13 in HEC1-A and Ishikawa cells. GAPDH was used as internal control. (G) CellTiter-Glo detect cell viability after knocking down MAPK13 in HEC1-A and Ishikawa cells, cell viability was tested 5 days after transduction. *P<0.05, **P<0.01, ***P<0.001. Data are represented as mean +/- SEM.

Figure 7

circTNFRSF21 rescues MAPK13-ATF2 signaling pathway by acting as miR-1227 sponge in EC cells. Detection of MAPK13 transcription (A, B) and protein level (C). (D, E) Luciferase assay detect MAPK13 3’UTR activity. (F, G) Cell growth detection after knocking down MAPK13 in the presence or absence or circTNFRSF21. (H) Western blot detects MAPK13, ATF2, phosphorylated ATF2 expression. GAPDH was used as internal control. (I) Cell growth detection after knocking down MAPK13 in the presence or absence or ATF2. (J) Western blot detects MAPK13, ATF2 expression, GAPDH was used as internal control. *P<0.05, **P<0.01, ***P<0.001. Data are represented as mean +/- SEM.