Circular RNA LPAR3 sponges microRNA-198 to facilitate esophageal cancer migration, invasion, and metastasis

Abstract

In this study, we explored expression and functions of circular RNA LPAR3 (circLPAR3) in esophageal squamous cell carcinoma (ESCC). The differential expression of circular RNAs (circRNAs) in 10 ESCC and corresponding paracarcinoma tissues was analyzed through circRNA microarray, then the candidate circRNAs were detected and verified through quantitative RT-PCR, and a novel circRNA was screened, which was circLPAR3. Circular RNA LPAR3 showed apparently high expression in ESCC tissues and cells, which was closely correlated with the clinical stage and lymph node metastasis of ESCC patients. Circular RNA LPAR3 was mainly located in the cytoplasm of ESCC cells, which was more stable than the baseline gene. Circular RNA LPAR3 upregulated MET gene expression through sponge adsorption of microRNA (miR)-198, activated the RAS/MAPK and the PI3K/Akt pathways, and promoted ESCC cell migration, invasion, and metastasis in vivo and in vitro. However, it had no effect on ESCC cell proliferation. Circular RNA LPAR3 can regulate the miR-198-MET signal axis to promote the migration, invasion, and metastasis of esophageal cancer cells, which can thereby serve as a potential diagnostic and therapeutic target of esophageal cancer.

Keywords: MET; circLPAR3; circular RNA; esophageal squamous cell carcinoma; miR-198.

FIGURE 1

Screening of target gene circular RNA LPAR3 (circLPAR3) as the biomarker of esophageal squamous cell carcinoma (ESCC) invasion and metastasis. A, The high‐throughput sequencing results of 10 pairs of ESCC and paracarcinoma tissues, the differential expression of circRNA in ESCC and paracarcinoma tissues is analyzed through heat map and hierarchical clustering analysis, and the relative expression levels of circRNA were arranged from the highest to the lowest levels, as denoted in red and green, respectively. B, The X axis in the volcano plot represents the fold change (FC); the Y axis indicates the P value. The P value at the green boundary = .05, FC = 2.0, and the red points in the plot represent the differentially expressed circRNAs. C, Scatter plot is drawn to learn the expression data distribution in the microchip, and a greater data scattering degree indicates a greater difference degree. X and Y axes indicate the signal values after standardization, in which the green line stands for the FC. In this experiment, the differential expression standards are set at FC ≥ 2.0 or ≤0.5, which refer to the region above the upper green line and the region below the lower green line in the plot, respectively. D, CircLPAR3 expression in 10 pairs of ESCC and paracarcinoma tissues verified by qRT‐PCR. E, CircLPAR3 expression in 52 pairs of ESCC tissues and matched paracarcinoma tissues detected by quantitative RT‐PCR. F, CircLPAR3 expression in ESCC‐related cell lines. **P < .01, ***P < .001

FIGURE 2

Biological characteristics of circular RNA LPAR3 (circLPAR3) in esophageal squamous cell carcinoma cells. A, CircLPAR3 origin, composition, and length. B, Sanger sequencing results of circLPAR3, in which the black arrow indicates the cyclization site. C, CircLPAR3 and linear LPAR3 mRNA expression in Kyse450 cells before and after RNase R treatment detected by quantitative RT‐PCR. D, E, RNA nuclear‐cytoplasmic separation (D) and FISH (E) experiments to understand circLPAR3 distribution in Kyse450 cells, with U6 and 18S rRNA as the positive controls of nuclear component and cytoplasmic component, respectively (scale bar = 20 µm). ***P < .001

FIGURE 3

Circular RNA LPAR3 (circLPAR3) promotes esophageal squamous cell carcinoma cell migration and invasion in vitro. A, CircLPAR3 expression levels in si‐circLPAR3‐transfected Kyse450 cells detected by quantitative (q)RT‐PCR. B, C, CircLPAR3 and LPAR3 expression levels in si‐circLPAR3‐transfected TE13 cells detected by qRT‐PCR. D, Changes in Kyse450 cell migration and invasion capacities after downregulating circLPAR3 expression detected by Transwell assay. E, CircLPAR3 expression levels in TE13 cells transfected with high circLPAR3 expression plasmid detected by qRT‐PCR. F, G, CircLPAR3 and LPAR3 expression levels in Kyse450 cells transfected with high circLPAR3 expression plasmid detected by qRT‐PCR. H, I, Expression of LPAR3 and circLPAR3 in TE13 cells after LPAR3 overexpression plasmid transfection was determined by qRT‐PCR. J, Effect of high circLPAR3 expression on TE13 cell migration and invasion capacities detected by Transwell assay. K, Proliferation of Kyse450 cells after circLPAR3 knockout for 24, 48, 72, and 96 h detected by CCK‐8 assay. L, Kyse450 cell proliferation after circLPAR3 KO detected in cell colony formation assay. **P < .01, ***P < .001

FIGURE 4

Circular RNA LPAR3 (circLPAR3) serves as a sponge of microRNA (miR)‐198. A, Schematic diagram predicting the potential binding sites of circLPAR3 with miR‐198. B, Venn diagram suggests the common downstream target genes of miR‐198 predicted in 4 databases (PicTar, miRTarBase, TargetScan, and miRBase). C, Schematic diagram presenting the binding sites of circLPAR3‐miR‐198‐MET. D, Dual‐luciferase reporter gene plasmid mutation sites of circLPAR3. E, Luciferase activities detected after cotransfection of WT circLPAR3 or mutant circLPAR3 with miR‐198 mimics or miR‐198 mimic‐negative control (NC) detected through dual‐luciferase reporter gene assay. F, G, Binding of circLPAR3 and miR‐198 with AGO2 protein in Kyse450 and TE13 cells detected through RIP assay. **P < .01, ***P < .001

FIGURE 5

Circular RNA LPAR3 (circLPAR3) adsorbs microRNA (miR)‐198 and targets MET to promote esophageal squamous cell carcinoma (ESCC) cell migration. A‐D, Effects of downregulating and upregulating miR‐198 expression on the MET gene and c‐MET protein detected through quantitative RT‐PCR and western blotting. E, Regulation of high circLPAR3 expression on c‐MET protein level detected through western blotting. F, Regulation of low circLPAR3 expression on c‐MET protein level and the phosphorylation levels of its downstream regulated proteins detected through western blotting. G, Transwell assay shows that overexpression of miR‐198 can reverse the promoting effect of high circLPAR3 expression on ESCC cell migration. H, Regulation of miR‐198 overexpression on the c‐MET protein level and the phosphorylation levels of its downstream regulated proteins induced from the high circLPAR3 expression, as detected through western blotting. *P < .05, **P < .01, ***P < .001

FIGURE 6

Circular RNA LPAR3 (circLPAR3) promotes esophageal squamous cell carcinoma lung metastasis in vivo. A, CircLPAR3 expression levels in sh‐circLPAR3‐transfected TE13 cells detected by quantitative (q)RT‐PCR. B, Representative images of the tumor nodules could be seen on the lung surface in 2 groups of mice 6 wk after the tail vein injection of TE13 cells. C, Representative images of the H&E staining results of lung metastasis pathological sections in both groups (scale bars: upper, 1000 µm; lower, 100 µm). D, Quantitative analysis on the number of lung metastases in mice of both groups. E, Representative images of immunohistochemical results of lung metastases in both groups (scale bars: upper, 1000 µm; lower, 100 µm). F, Changes in circLPAR3 and MET expression in lung metastases of both groups detected through qRT‐PCR. ***P < .001

FIGURE 7

Schematic diagram of the circular RNA LPAR3 (circLPAR3)‐microRNA (miR)‐198‐MET regulatory signal axis