The RNA-binding protein RBM3 promotes cell proliferation in hepatocellular carcinoma by regulating circular RNA SCD-circRNA 2 production

Abstract

Background: With the development of RNA-seq technology, tens of thousands of circular RNAs (circRNAs), a novel class of RNAs, have been identified. However, little is known about circRNA formation and biogenesis in hepatocellular carcinoma (HCC).

Methods: We performed ribosomal-depleted RNA-seq profiling of HCC and para-carcinoma tissues and analyzed the expression of a hotspot circRNA derived from the 3'UTR of the stearoyl-CoA desaturase (SCD) gene, termed SCD-circRNA 2.

Findings: It was significantly upregulated in HCC and correlated with poor patient prognosis. Moreover, we observed that the production of SCD-circRNA 2 was dynamically regulated by RNA-binding protein 3 (RBM3). RBM3 overexpression was indicative of a short recurrence-free survival and poor overall survival for HCC patients. Furthermore, by modulating the RBM3 or SCD-circRNA 2 levels, we found that RBM3 promoted the HCC cell proliferation in a SCD-circRNA 2 dependent manner.

Interpretation: Herein, we report that RBM3 is crucial for the SCD-circRNA 2 formation in HCC cells, which not only provides mechanistic insights into cancer-related circRNA dysregulation but also establishes RBM3 as an oncogene with both therapeutic potential and prognostic value. FUND: This work was supported by the National Key Research and Development Program of China (2016YFC1302303), the National Natural Science Foundation of China (Grant No. 81672345 and 81,402,269). The funders did not have any roles in study design, data collection, data analysis, interpretation, writing of the report.

Keywords: Circular RNA; Hepatocellular carcinoma; Oncogene; RBM3; Stearoyl-CoA desaturase.

Fig. 1

Differential expression of circRNAs in paired HCC and adjacent non-tumor tissues. (a) CircRNAs detected in HCC (Tumor) and paired non-tumor (Peri-tumor) samples are mostly derived from the protein coding sequence. (b) Hierarchical clustering analysis of 1811 circRNAs that were differentially expressed between HCC samples (Tumor) and paired non-tumor samples (Peri-tumor) (P < .01, FDR < 0.01 and fold change >5 or < 0.2). Expression values are represented in shades of red and blue, indicating expression above and below the median expression value across all samples, respectively. (c) Scheme illustrating the production of SCD-circRNA 1 and SCD-circRNA 2. (d) The results of SCD-circRNA 1 and SCD-circRNA 2 upregulation in tumor tissues (Tumor) compared with those in adjacent non-tumor tissues (Peri-tumor) verify the RNA-seq results. Cohort 1, n = 20, Wilcoxon matched-pairs signed-rank test. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

SCD-circRNA 2 may be an independent prognostic factor for predicting recurrence-free survival (RFS) and overall survival (OS). (a and b) Kaplan-Meier analyses of correlations between SCD-circRNA 2 expression level in HCC and RFS or OS in 151 HCC patients (cohort 2) is shown. Log-rank test was used. Multivariate analysis of hazard ratios (HRs) for RFS (c) and OS (d). HRs are presented as the means (95% CI). The Cox proportional hazards model was used.

Fig. 3

Role of RBM3 in controlling circRNA formation. RBM3 depletion by shRNA lentivirus was verified by western blotting (a). qRT-PCR results indicated that downexpression of RBM3 could result in downregulated expression of SCD-circRNA 2 but not SCD-circRNA 1 in the Huh7 (b) and HepG2 (c) cell lines. RBM3 overexpression was verified by western blotting (d), resulting in the upregulated expression of SCD-circRNA 2 but not SCD-circRNA 1 in the Huh7 (e) and HepG2 (f) cell lines. Student's t-test was used. * P < .05.

Fig. 4

RBM3 is upregulated in hepatocellular carcinoma. We performed qRT-PCR test of RBM3 mRNA that included 151 HCC specimens (cohort 2) to explore the clinical significance of RBM3. (a) RBM3 mRNA is upregulated in HCC tissues (Tumor) compared with those in adjacent non-tumor tissues (Peri-tumor). n = 151. Wilcoxon matched-pairs signed-rank test. Kaplan–Meier analysis was used to compare the subgroups with RBM3 high expression (n = 82) to those with RBM3 low expression (n = 69). Remarkably, patients showing high expression of RBM3 exhibited worse recurrence-free survival (b) and overall survival (c) (P = .017 and P = .026, respectively). Log-rank test was used. (d) Relative expression of SCD-circRNA 2 was plotted against RBM3 RNA expression level in cohort 2 HCC tissues. Pearson correlation analysis was used.

Fig. 5

RBM3 promotes tumor growth in vitro and in vivo. Results of CCK8 indicated that cell counts for RBM3-overexpressing Huh7 (a) and HepG2 (c) cells were much higher than for of mock and negative control cells. The G1/G0 phase of RBM3-overexpressing Huh7 (b) and HepG2 (d) cells was much lower than in mock and negative control cells. For (a-d), data are presented as means ± SD; n = 3. Student's t-test was used. In vivo xenograft-transplanted nude mouse tumor models of human HCC growth were established with RBM3-overexpressing Huh7 cells. Tumor images are presented in (e). (f) In vivo subcutaneous tumor growth curves for RBM3-overexpressing Huh7 cells (Over-1 and Over-2) and control cells (Mock and Over-NC). (g) Weights of subcutaneous tumors derived from RBM3-overexpressing Huh7 cells (Over-1 and Over-2) and control cells (Mock and Over-NC) at four weeks after neoplasm seeding. For (f-g), data were presented as means ± SEM. Mann-Whitney U test was used. n = 5. * P < .05.

Fig. 6

RBM3 increases expression of SCD-circRNA 2 depending on the sequence of the flanking back-splicing site. Genomic fragments corresponding to SCD-circRNA 2 with different sequence of flanking back-splicing site were cloned into pZW1 plasmid (a). These plasmids were transfected into Huh7 or HepG2 cells for 48 h, and then expression of SCD-circRNA 2 was measured using qRT-PCR (b). RNA immunoprecipitation (RIP) experiments were performed using the RBM3 antibody to immunoprecipitate Huh7 (c) or HepG2 (d) cell extracts. Then, SCD 3’UTR and 18S (negative control) were detected using specific primers (Supplementary Table 2). RIP enrichment was determined as RNA associated with RBM3 immunoprecipitation relative to an input control. Data are presented as means ± SD; n = 3. Student's t-test was used. * P < .05.

Fig. 7

RBM3 activates cell proliferation through SCD-circRNA 2. EdU experiments were conducted to verify whether SCD circRNA-2 is associated with the cell cycle. SCD-circRNA 2 groups revealed high levels of DNA replication, while low replication levels were observed in siRNA groups relative to those in control (a-b). Data are presented as means ± SD; n = 3. Student's t-test was used. * P < .05. Scale bars, 100 μm. (c) The effect of RBM3 shRNA on the proliferation of HepG2 and Huh7 cell lines is reversed by SCD circRNA-2 overexpression. Scale bars, 100 μm. Statistics for quantitative data are shown in (d). Data are presented as means ± SD; n = 3. Student's t-test was used. To further demonstrated that SCD-circRNA2 was the downstream of RBM3, we established subcutaneously implanted tumor models with HepG2 cells infected with overexpression negative control lentivirus (HepG2 Over-NC), HepG2 cells infected with RBM3 overexpression lentivirus and shRNA negative control lentivirus (HepG2 Over-1 / shRNA NC), and RBM3 overexpressed HepG2 cells infected with SCD-circRNA 2 shRNA lentivirus (HepG2 Over-1 / SCD-circRNA 2 shRNA) in nude mice. Tumor images are presented in (e). Volume (f) and weights (g) of subcutaneous tumors derived from HepG2 Over-NC, HepG2 Over-1 / shRNA NC and HepG2 Over-1 / SCD-circRNA 2 shRNA cells at four weeks after neoplasm seeding. For (f-g), data were presented as means ± SEM. Mann-Whitney U test was used. All P values are defined as: *P < .05, **P < .01 and ***P < .001. (h) RBM3-overexpressing HepG2 and Huh7 cells were transfected with SCD-circRNA 2 siRNA. Forty-eight hours after transfection, cells were harvested. ERK, p38 and JNK phosphorylation was assessed by western blotting analysis with the indicated antibodies. (i) The overexpression of SCD-circRNA 2 was performed as described in Fig. 6a. The relative expression of phosphorylated ERK significantly increased after SCD-circRNA 2 overexpressing in HepG2 (up) and Huh7 (down) cells.

Supplementary Fig. 1

circRNA expression in HCC. The results of SCD-circRNA 1 and SCD-circRNA 2 upregulation in tumor tissues (Tumor) compared with those in adjacent normal liver tissues (Peri-tumor) verified the RNA-seq results. n = 151, Wilcoxon matched-pairs signed-rank test.

Supplementary Fig. 2

The characteristics of the circular RNA SCD-circRNA 2. (a) Scheme illustrating the circular RNA SCD-circRNA 2. PCR primers used to specifically clone full-length of SCD-circRNA 2 are indicated by arrows. Northern blot probes targeting SCD-circRNA 2 are indicated by blue bars. (b) Northern blot showed the expression of SCD-circRNA 2 in 4 paired HCC (Tumor) and adjacent normal liver tissues (Peri-tumor). (c) Random hexamer or oligo (dT)₁₈ primers were used in the reverse transcription experiments. The relative RNA levels in Huh7 cells were analyzed by qRT-PCR and normalized to the value using random hexamer primers. (d) The relative RNA levels in Huh7 cells were analyzed by qRT-PCR and normalized to the value detected in the mock group. (e) Sanger sequencing showing the back-spliced events (red arrow) and full-length of SCD-circRNA 2. PCR primers used to specifically clone full-length of SCD-circRNA 2 are indicated by black arrows.

Supplementary Fig. 3

Expression of SCD-circRNA 2 or SCD-circRNA 1 could not be downregulated in RBMS3 (a), IGF2BP3 (b), IGF2BP2 (c), TARBP1 (d), NOVA1 (e), NOVA2 (f), QKI (g) depleted Huh7 cells.

Supplementary Fig. 4

(a) RBM3 overexpression or downregulation could result in the upregulated or decreased expression of SCD-circRNA 2 in the HCT-15 and NCI-N87 cell lines. Data are presented as means ± SD; n = 3. Student’s t test was used. *P < 0.05. RBM3 could not regulate colorextal cancer cells HCT-15 (b) and gastric cancer cells NCI-N87 (c) proliferation. Scale bars, 100 μm.

Supplementary Fig. 5

The mRNA and protein levels of SCD were not changed in SCD-circRNA 2 upregulated or downregulated HCC cells. The overexpression of SCD-circRNA 2 was performed as described in Fig. 6a. The relative expression of SCD mRNA (a) and protein (b) did not significantly change after overexpressing. (c) The relative expression of SCD-circRNA 2 was downregulated in SCD-circRNA 2 siRNA-1 and SCD-circRNA 2 siRNA-2 transfected Huh7. qRT-PCR and western blot showed that the mRNA (c) and protein (d) level of SCD did not significantly change after silencing SCD-circRNA 2. (e) The expression level of SCD-circRNA 2 was downregulated in SCD-circRNA 2 shRNA lentivirus infected HepG2 cells. qRT-PCR and western blot showed that the mRNA (e) and protein (f) level of SCD did not significantly change after silencing SCD-circRNA 2.

Supplementary Fig. 6

RBM3 activates cell proliferation through SCD-circRNA 2. (a) S phase was recovered in RBM3-upregulated Huh7 and HepG2 cells by downregulating the expression of SCD-circRNA 2. The results of CCK8 arrays indicated that cell counts was recovered in RBM3-upregulated HepG2 (b) and Huh7(c) cells by downregulating the expression of SCD-circRNA 2. Data are presented as means ± SD; n = 3. Student’s t test was used.