circFBLIM1 act as a ceRNA to promote hepatocellular cancer progression by sponging miR-346

Abstract

Backgroud: Accumulating evidences indicate that circular RNAs (circRNAs), a class of non-coding RNAs, play important roles in tumorigenesis. However, the function of circRNAs in hepatocellular cancer (HCC) is largely unknown.

Methods: We performed circRNA microarrays to identify circRNAs that are aberrantly expressed in HCC tissues. Expression levels of a significantly upregulated circRNA, circFBLIM1, was detected by quantitative real-time PCR (qRT-PCR) in HCC cell lines and tissues. Then, we examined the functions of circFBLIM1 in HCC by cell proliferation, apoptosis, invasion and mouse xenograft assay. In addition, luciferase assay and RNA immunoprecipitation (RIP) assay were used to explore the miRNA sponge function of circFBLIM1 in HCC.

Results: Microarray analysis and qRT-PCR verified a circRNA termed circFBLIM1 that was upregulated in HCC tissues and cell lines. Knockdown of circFBLIM1 inhibited proliferation, invasion and promoted apoptosis in HCC. Via luciferase reporter assays, circFBLIM1 and FBLIM1 were observed to directly bind to miR-346. Subsequent experiments showed that circFBLIM1 and FBLIM1 regulated the expression of each other by sponging miR-346.

Conclusions: Taken together, we conclude that circFBLIM1 may function as a competing endogenous RNA (ceRNA) to regulate FBLIM1 expression through sponging miR-346 to exert regulatory functions in HCC. circFBLIM1 may be a diagnostic biomarker and potential target for HCC therapy.

Keywords: Circular RNAs; Competitive endogenous RNAs; FBLIM1; Hepatocellular cancer; miR-346.

Fig. 1

circRNA expression profiles in HCC. a The cluster heat map showed the top 20 upregulated and downregulated circRNAs in HCC tissues. Red color indicates high expression level and green color indicates low expression level. b The scatter plot revealed the variation in circRNA expression between HCC tissues and matched para-carcinoma normal tissues. The values of x and y axes were the normalized signal values of the samples (log2 scaled). c-g qRT-PCR was performed to verify the expression of the top five upregulated circRNAs in HCC cell lines. All the data are shown as the mean ± S.D

Fig. 2

Knockdown of circFBLIM1 inhibits growth and invasion, and promotes apoptosis in HCC. a Cells were transfected with si-NC or si-circFBLIM1, qRT-PCR analysis demonstrated that the transfection was successful. b CCK8 assay was performed to assess cell growth. c Cell apoptosis was determined 48 h post-transfection by JC-1 staining. Green indicates apoptosis. Original magnification, × 400. d Matrigel invasion assay was performed. Representative images of invaded cells are shown in the left panel, and the results are summarized in the right panel. Original magnification, × 200. e Representative images of xenografts tumor (five mice per group) in nude mice. f Tumor volume was monitored every 4 days for 28 days. g The weights of xenograft tumors are summarized. h Representative images of HE stained lung metastatic nodules are shown. Original magnification, × 40. The number of metastatic nodules was quantified (five mice per group) in the right panel. All the data are shown as the mean ± S.D., **P < 0.01

Fig. 3

circFBLIM1 serves as a sponge for miR-346. a The levels of U6, GAPDH and circFBLIM1 were assessed by qRT-PCR in nuclear and cytoplasmic fractions. b The predicted binding sites of miR-346 within circFBLIM1 were shown. c Cells were transfected with miR-346 mimics or miR-346 LNA, qRT-PCR analysis demonstrated that the transfection was successful. d Luciferase assay of cells co-transfected with miR-346 mimics or miR-346 LNA and luciferase reporter containing circFBLIM1 (circFBLIM1 wt) or mutant construct (circFBLIM1 mut). e Cells were transfected as described, and the expression of circFBLIM1 was determined by qRT-PCR. f Cells were transfected with si-NC or si-circFBLIM1, and the expression of miR-346 was determined by qRT-PCR. U6 snRNA was used as an internal control. g MS2bp-MS2bs based RIP assay in HCC cells transfected with MS2bs-circFBLIM1, MS2bs-circFBLIM1mt, or MS2bs-Rluc. h The expression level of circFBLIM1 in 50 HCC tissues and matched para-carcinoma normal tissues was determined by qRT-PCR. i The expression level of miR-346 in the aboved tissues was determined by qRT-PCR. All the data are shown as the mean ± S.D., *P < 0.05 and **P < 0.01

Fig. 4

circFBLIM1 and FBLIM1 act as ceRNAs in HCC through regulation of miR-346. a The expression level of FBLIM1 was determined by qRT-PCR (left) and Western blot (right) in HCC cell lines. β-actin was used as a control. b The expression level of FBLIM1 in 50 HCC tissues and matched para-carcinoma normal tissues was determined by qRT-PCR. c The predicted binding sites of miR-346 within FBLIM1 were shown. d Luciferase assay of cells co-transfected with miR-346 mimics or miR-346 LNA and luciferase reporter containing FBLIM1 3′-UTR (FBLIM1 wt) or mutant construct (FBLIM1 mut). e Cells were transfected as described, and the expression of FBLIM1 was determined by qRT-PCR. f Cells were transfected with si-NC or si-FBLIM1, and the expression of miR-346 was determined by qRT-PCR. g Cells were transfected with NC, si-FBLIM1 or si-FBLIM1 + miR-346 LNA, and the expression of circFBLIM1 was determined by qRT-PCR. h Cells were transfected with NC, si-circFBLIM1 or si-circFBLIM1 + miR-346 LNA, and the expression of FBLIM1 was determined by qRT-PCR. i Cells were transfected with miR-NC, miR-346 or miR-346 + si-circFBLIM1, and CCK8 assay was performed. j Cells were transfected as described, and apoptosis assay was performed. Original magnification, × 400. All the data are shown as the mean ± S.D., *P < 0.05 and **P < 0.01