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. 2021 Dec 14:11:773864.
doi: 10.3389/fonc.2021.773864. eCollection 2021.

LncRNA FOXP4-AS1 Promotes the Progression of Esophageal Squamous Cell Carcinoma by Interacting With MLL2/H3K4me3 to Upregulate FOXP4

Yunfeng Niu  1 , Gaoyan Wang  2 , Yan Li  1 , Wei Guo  1 , Yanli Guo  1 , Zhiming Dong  1
Affiliations

LncRNA FOXP4-AS1 Promotes the Progression of Esophageal Squamous Cell Carcinoma by Interacting With MLL2/H3K4me3 to Upregulate FOXP4

Yunfeng Niu et al. Front Oncol. .

Erratum in

Abstract

Malignant tumors are a grave threat to human health. Esophageal squamous cell carcinoma (ESCC) is a common gastrointestinal malignant tumor. China has a high incidence of ESCC, and its morbidity and mortality are higher than the global average. Increasingly, studies have shown that long noncoding RNAs (lncRNAs) play a vital function in the occurrence and development of tumors. Although the biological function of FOXP4-AS1 has been demonstrated in various tumors, the potential molecular mechanism of FOXP4-AS1 in ESCC is still poorly understood. The expression of FOXP4 and FOXP4-AS1 was detected in ESCC by quantitative real-time PCR (qRT-PCR) or SP immunohistochemistry (IHC). shRNA was used to silence gene expression. Apoptosis, cell cycle, MTS, colony formation, invasion and migration assays were employed to explore the biological functions of FOXP4 and FOXP4-AS1. The potential molecular mechanism of FOXP4-AS1 in ESCC was determined by dual-luciferase reporter, RNA immunoprecipitation (RIP) and chromatin immunoprecipitation (ChIP). Here, we demonstrated that FOXP4-AS1 was significantly increased in ESCC tissues and cell lines, associated with lymph node metastasis and TNM staging. Cell function experiments showed that FOXP4-AS1 promoted the proliferation, invasion and migration ability of ESCC cells. The expression of FOXP4-AS1 and FOXP4 in ESCC tissues was positively correlated. Further research found that FOXP4-AS1, upregulated in ESCC, promotes FOXP4 expression by enriching MLL2 and H3K4me3 in the FOXP4 promoter through a "molecular scaffold". Moreover, FOXP4, a transcription factor of β-catenin, promotes the transcription of β-catenin and ultimately leads to the malignant progression of ESCC. Finally, FOXP4-AS1 may be a new therapeutic target for ESCC.

Keywords: ESCC; FOXP4; FOXP4-AS1; MLL2; long noncoding RNA.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
FOXP4-AS1 is upregulated in ESCC and correlates with clinicopathological data. (A) The differentially expressed lncRNAs in GSE45670 by the volcano plots (log2|FC|>1). (B) Highly expressed lncRNAs in GSE45670 and GSE161533. (C) FOXP4-AS1 expression in various tumors by GEPIA dataset. (D–F) The expression of FOXP4-AS1 in ESCC tissues, different subgroups and cell lines by qRT–PCR. (G) ROC analysis shows an AUC of 0.8679 for differentiating ESCC from normal tissue. OS (H) and RFS (I) of ESCC patients according to FOXP4-AS1 by Kaplan–Meier analysis. *p < .05 and **p < .01.
Figure 2
Figure 2
FOXP4-AS1 promotes the malignant progression of ESCC. Expression of FOXP4-AS1 in transfected sh-FOXP4-AS1 (A) or pcDNA3.1-FOXP4-AS1 (B) by qRT–PCR. (C) MTS assay showing the inhibition of YES-2 proliferation ability with sh-FOXP4-AS1. (D) Increased proliferative capacity of Kyse150 with pcDNA3.1-FOXP4-AS1 by MTS assay. (E) Colony formation assay indicates that the number of colony formation in YES-2 cells was reduced after FOXP4-AS1 deregulation. (F) Improved colony formation in Kyse150 cells transfected with pcDNA3.1-FOXP4-AS1. Transwell migration (G) and invasion assays (I) showing the decreased trans-cells of YES-2 with sh-FOXP4-AS1. Transwell migration (H) and invasion assays (J) demonstrated more Kyse150 trans-cells with pcDNA3.1-FOXP4-AS1. Apoptosis (K) and cycle (L) profiles of YES-2 cells transfected with sh-FOXP4-AS1 by FCM. **p < .01.
Figure 3
Figure 3
FOXP4 is highly expressed in ESCC and increased by FOXP4-AS1 at the mRNA and protein levels. The expression of FOXP4 in ESCC tissues (A) and different subgroups (B) by qRT–PCR. (C) FOXP4 protein in ESCC by IHC (SP Left×200; Right×400). a: ESCC tissue; b normal tissue. FOXP4 mRNA (D) and protein (E) levels with FOXP4-AS1 by qRT–PCR or WB. (F) The correlation between FOXP4-AS1 and FOXP4 by qRT–PCR. Subcellular localization of FOXP4-AS1 in YES-2 (G) and Kyse150 (H), GAPDH and U6 were used as cytoplasmic and nuclear controls. *p < .05 and **p < .01.
Figure 4
Figure 4
FOXP4-AS1 upregulates FOXP4 expression through interacting with MLL2. (A) Epistatic regulatory maps of FOXP4 and FOXP4-AS1 by UCSC. (B) The binding of FOXP4-AS1 to MLL2 in Kyse150 by RIP. (C) The expression of MLL2 in ESCC by qRT–PCR. (D) The association between FOXP4-AS1 and MLL2 by qRT–PCR. FOXP4 mRNA (E, G) and protein (F, H) by coexpressing FOXP4-AS1 and MLL2. (I) The correlation between FOXP4-AS1 and MLL2 by the qRT–PCR. (J) FOXP4-AS1 increased MLL2 and H3K4me3 enrichment in the FOXP4 promoter as determined by ChIP assay. **p < .01.
Figure 5
Figure 5
FOXP4 promotes the malignant progression of ESCC. The expression of FOXP4 mRNA (A, C, D) and protein (B, E) in transfected sh-FOXP4 or pcDNA3.1-FOXP4 by qRT–PCR or WB. (F) MTS assay showing the inhibition of YES-2 proliferation ability with sh-FOXP4. (G) Increased proliferative capacity of Kyse150 with pcDNA3.1-FOXP4 by MTS assay. (H) Colony formation assay indicated that deregulated FOXP4 follows the lower colony formation in YES-2 cells. (I) Enhanced colony formation in Kyse150 cells with pcDNA3.1-FOXP4. Transwell migration (J) and invasion assays (K) show the decreased trans-cells of YES-2 with sh-FOXP4. Transwell migration (L) and invasion assay (M) demonstrated more Kyse150 trans-cells with pcDNA3.1-FOXP4. **p < .01.
Figure 6
Figure 6
FOXP4 regulates β-catenin expression as a transcription factor. The mRNA (A, B, C) and protein (D) levels of EMT‐related genes (E‐cadherin, β-catenin, and Vimentin) with sh‐FOXP4 or pcDNA3.1-FOXP4 by qRT–PCR or WB. The expression of β-catenin in ESCC (E) and different subgroups (F) by qRT–PCR. (G) The correlation between FOXP4 and β-catenin by qRT–PCR. (H) The predicted FOXP4 binding site sequence in the promoter of β-catenin. (I) The effect of FOXP4 on the luciferase activity of β-catenin by dual-luciferase reporter assay. (J) The direct interaction of FOXP4 with the β-catenin promoter by ChIP. *p < .05 and **p < .01.
Figure 7
Figure 7
Coexpression of FOXP4-AS1 and FOXP4 promotes β-catenin expression and ESCC progression. (A) The mRNA and protein levels of β-catenin with FOXP4-AS1 and FOXP4 by qRT‐PCR or WB. The effect of FOXP4-AS1 and FOXP4 coexpression on ESCC cell proliferation by MTS (B) or Colony formation (C) assay. FOXP4-AS1 and FOXP4 coexpression on ESCC cell migration (D) and invasion (E) by Transwell assay. (F) The possible molecular mechanisms for the involvement of FOXP4-AS1 in ESCC progression. **p < .01.
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