Hsa_circ_0005397 promotes hepatocellular carcinoma progression through EIF4A3

Abstract

Purpose: The purpose of this study was to explore the expression and potential mechanism of hsa_circ_0005397 in hepatocellular carcinoma progression.

Methods: Quantitative reverse transcription-polymerase chain reaction(qRT-PCR) was used to measure the expression level of hsa_circ_0005397 and EIF4A3 from paired HCC tissues and cell lines. Western Blot (WB) and immunohistochemistry (IHC) were used to verify the protein level of EIF4A3. The specificity of primers was confirmed by agarose gel electrophoresis. Receiver Operating Characteristic (ROC) Curve was drawn to analyze diagnostic value. Actinomycin D and nuclear and cytoplasmic extraction assays were utilized to evaluate the characteristics of hsa_circ_0005397. Cell Counting kit-8 (CCK-8) and colony formation assays were performed to detect cell proliferation. Flow cytometry analysis was used to detect the cell cycle. Transwell assay was performed to determine migration and invasion ability. RNA-binding proteins (RBPs) of hsa_circ_0005397 in HCC were explored using bioinformatics websites. The relationship between hsa_circ_0005397 and Eukaryotic Translation Initiation Factor 4A3 (EIF4A3) was verified by RNA Binding Protein Immunoprecipitation (RIP) assays, correlation and rescue experiments.

Results: In this study, hsa_circ_0005397 was found to be significantly upregulated in HCC, and the good diagnostic sensitivity and specificity shown a potential diagnostic capability. Upregulated expression of hsa_circ_0005397 was significantly related to tumor size and stage. Hsa_circ_0005397 was circular structure which more stable than liner mRNA, and mostly distributed in the cytoplasm. Upregulation of hsa_circ_0005397 generally resulted in stronger proliferative ability, clonality, and metastatic potency of HCC cells; its downregulation yielded the opposite results. EIF4A3 is an RNA-binding protein of hsa_circ_0005397, which overexpressed in paired HCC tissues and cell lines. In addition, expression of hsa_circ_0005397 decreased equally when EIF4A3 was depleted. RIP assays and correlation assay estimated that EIF4A3 could interacted with hsa_circ_0005397. Knockdown of EIF4A3 could reverse hsa_circ_0005397 function in HCC progression.

Conclusions: Hsa_circ_0005397 promotes progression of hepatocellular carcinoma through EIF4A3. These research findings may provide novel clinical value for hepatocellular carcinoma.

Keywords: EIF4A3; Hepatocellular carcinoma; hsa_circ_0005397.

Fig. 1

The characteristics of hsa_circ_0005397 in hepatocellular carcinoma. (A) qRT-PCR detected the expression level of hsa_circ_0005397 in HCC tissues (n = 57). (B) The relative expression level of hsa_circ_0005397 in HCC tissues were normalized to normal tissues (n = 57). (C) Construction of an ROC curve for HCC patients based on hsa_circ_0005397 expression. (D) qRT-PCR analyzed the expression of hsa_circ_0005397 in cell lines. (*P < 0.05, **p < 0.01, ***p < 0.001). (E) The PCR product of qRT-PCR was run on 2% agarose gel, showing a single electrophoresis band of has_circ_0005397. (F) qRT-PCR analyzed relative expression after treatment with or without RNase R in BEL-7404. (G) qRT-PCR was used to measure the expression level of hsa_circ_0005397 in the nuclear and cytoplasmic. (H) qRT-PCR was used to detected the relative expression of hsa_circ_0005397 and RHOT1 mRNA in BEL-7404 cells treated with Actinomycin D at different time points

Fig. 2

Hsa_circ_0005397 affects proliferation in HCC cell lines. (A) two siRNAs target the back splicing junction of hsa_circ_0005397. (B) qRT-PCR detected the expression of hsa_circ_0005397 in BEL-7404, HCCLM3 and SK-Hep1 cells. (C) Cell proliferation was determined by CCK-8 assays in BEL-7404, HCCLM3 and SK-Hep1 cells. (D) Cell proliferation ability was assessed by Colony formation assays after knocking down of hsa_circ_0005397 in BEL-7404 cells. (E) Cell proliferation detection was measured by Colony formation assays after knocking down of hsa_circ_0005397 in HCCLM3 cells. (F) Cell proliferation ability was assessed by Colony formation assays after overexpression of hsa_circ_0005397 in SK-Hep1 cells. Colony formation rate was analyzed by colony counts. *P < 0.05, **p < 0.01, ***p < 0.001

Fig. 3

Hsa_circ_0005397 affects migration and invasion in HCC cells. (A) Transwell assays were used to detect cell migration and invasion of BEL-7404 cells transfected with siNC, si1and si2 (Magnification, 20X; Scale bars, 200 μm). (B) Cell migration and invasion abilities were determined by transwell assays in HCCLM3 cells transfected with siNC, si1 and si2 (Magnification, 20X; Scale bars, 200 μm). (C) The migratory and invasive capabilities of hsa_circ_0005397-overexpression SK-Hep1 cells (Magnification, 20X; Scale bars, 200 μm). *P < 0.05, **p < 0.01, ***p < 0.001

Fig. 4

Hsa_circ_0005397 affects the cell cycle in HCC cells. (A) The FACS analysis of the cell cycle distribution was shown in BEL-7404 cells transfected with siNC, si1and si2. (B) The FACS analysis of the cell cycle distribution was shown in HCCLM3 cells transfected with siNC, si1and si2. (C) The FACS analysis of the cell cycle distribution was shown for has_circ0005397-overexpression SK-Hep1 cells. (D) Comparison of the cell cycle distribution of has_circ0005397-knockdown BEL-7404 cells. (E) Comparison of the cell cycle distribution of has_circ0005397-knockdown HCCLM3 cells. (F) Comparison of the cell cycle distribution of has_circ0005397-overexpression SK-Hep1 cells. *P < 0.05, **p < 0.01, ***p < 0.001

Fig. 5

Hsa_circ_0005397 promotes the progression of HCC cell lines through EIF4A3. (A) Venn diagram analysis of the two databases (CircInteractome and CircFunBase) identified EIF4A3 as a candidate. (B) EIF4A3 expression in HCC tissues from TCGA datasets. (C) qRT-PCR detected the expression level of EIF4A3 in HCC tissues(n = 57). (D) The relative expression level of EIF4A3 in HCC cell lines. (E) Western Blot analysis of EIF4A3 in adjacent tissues and paired HCC tissues. (F) IHC staining of HCC tissues for EIF4A3 expression. ((magnification, 40x; scale bars, 100 μm). (G) Correlation between EIF4A3 and hsa_circ_0005397 mRNA expression in HCC tissues (n = 57). (H) qRT-PCR detected the expression of EIF4A3 in HCCLM3 cells transfected with shNC or shEIF4A3. (I) qRT-PCR analyzed the expression of hsa_circ_0005397 in HCCLM3 cells transfected with shNC or shEIF4A3. (J) RIP assay verified that EIF4A3 interacted with hsa_circ_0005397 and IgG was used as a control. (K) qRT-PCR detected the expression of hsa_circ_0005397 in HCCLM3 cells transfected with shNC + vector, shEIF4A3 + pcDNA or shEIF4A3 + pcDNA. (L) Cell proliferation ability was assessed by colony formation assays after HCCLM3 cells transfected with shNC + vector, shEIF4A3 + pcDNA or shEIF4A3 + pcDNA. Colony counts were counted to analyze colony formation rate, *P < 0.05, **p < 0.01, ***p < 0.001. (M) Cell migration and invasion abilities were determined by Transwell assays after HCCLM3 cells transfected with shNC + vector, shEIF4A3 + pcDNA or shEIF4A3 + pcDNA. (magnification, 20x; scale bars, 200 μm), *P < 0.05, **p < 0.01, ***p < 0.001