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. 2024 Jun 13:11:1095-1112.
doi: 10.2147/JHC.S459633. eCollection 2024.

Circ_0007386 Promotes the Progression of Hepatocellular Carcinoma Through the miR-507/ CCNT2 Axis

Yanzhi Feng #  1   2   3   4 , Litao Liang #  1   2   3   4 , Wenbo Jia  1   2   3   4 , Jinyi Wang  1   2   3   4 , Chao Xu  1   2   3   4 , Deming Zhu  1   2   3   4 , Bin Xu  1   2   3   4 , Wenhu Zhao  1   2   3   4 , Xiangyu Ling  1   2   3   4 , Yongping Zhou  5 , Lianbao Kong #  1   2   3   4 , Wenzhou Ding #  1   2   3   4
Affiliations

Circ_0007386 Promotes the Progression of Hepatocellular Carcinoma Through the miR-507/ CCNT2 Axis

Yanzhi Feng et al. J Hepatocell Carcinoma. .

Abstract

Background: Circular RNAs (circRNAs) have been shown to play a crucial role in the initiation and development of Hepatocellular carcinoma (HCC). However, the mechanism and function of circ_0007386 in HCC are still unknown.

Methods: Circ_0007386 expression level in HCC tissues, and HCC cell lines was further analyzed by qRT-PCR. Agarose gel electrophoresis and Sanger sequencing were used to figure out the structure of circ_0007386. The involvement of circ_0007386 in HCC development was evaluated by experimental investigations conducted in both laboratory settings (in vitro) and living organisms (in vivo). RNA immunoprecipitation, Western blotting, luciferase reporter assay and fluorescence in situ hybridization (FISH) were applied for finding out the interaction among circ_0007386, miR-507 and CCNT2. To assess the connection between circ_0007386 and lenvatinib resistance, lenvatinib-resistant HCC cell lines were employed.

Results: The expression of circ_0007386 was found to increase in HCC tissues, and it was observed to be associated with a worse prognosis. Overexpression of circ_0007386 stimulated HCC cells proliferation, invasion, migration and the epithelial-mesenchymal transition (EMT) while silencing of circ_0007386 resulted in the opposite effect. Mechanistic investigations revealed that circ_0007386 acted as a competing endogenous RNA of miR-507 to prevent CCNT2 downregulation. Downregulating miR-507 or overexpressing CCNT2 could reverse phenotypic alterations that originated from inhibiting of circ_0007386. Importantly, circ_0007386 determines the resistance of hepatoma cells to lenvatinib treatment.

Conclusion: Circ_0007386 advanced HCC progression and lenvatinib resistance through the miR-507/ CCNT2 axis. Meanwhile, circ_0007386 served as a potential biomarker and therapeutic target in HCC patients.

Keywords: CCNT2; Lenvatinib; circ_0007386; hepatocellular carcinoma; miR-507.

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Conflict of interest statement

The authors report no conflicts of interest in this work.

Figures

None
Graphical abstract
Figure 1
Figure 1
Hsa_circ_0007386 is highly expressed in HCC tissues compared to other circRNAs derived from CRIM1. (A–B) The expression of 8 CRIM1-derived circRNAs in 10 pairs of HCC and para carcinoma tissues were detected by qRT-PCR. (C) The relative expression level of hsa_circ_0007386 in HCC tissues and matched para-cancer tissues (n = 80) was measured using qRT-PCR. (D) Kaplan–Meier survival curves of HCC patients with low and high hsa_circ_0007386 expression. (E) The fold change of hsa_circ_0007386 expression between HCC and paratumor tissues. (F) Expression of circ_0007386 in various cell lines. Data were all showed as mean ± SD; ns indicated no significance, *p <0 0.05, **p < 0.01, ***p < 0.001.
Figure 2
Figure 2
Confirmation of the circular structure of circ_0007386. (A) The schematic illustration depicted the circularization of CRIM1 exon 3 to 4 into circ_0000007386.(B) Sanger sequencing revealed circ_0007386’s splicing site.(C) RT-PCR and agarose gel electrophoresis confirmed the existence of circ_0007386 in HCC cell. (D–E) The expression of circ_0007386 and CRIM1 mRNA in HCC cells were detected by qRT-PCR with RNase R and actinomycin D. (F–G) The FISH assay and Cyto-plasmic separation experiment revealed that circ_0007386 is mainly situated in the cytoplasm. Data were all showed as mean ± SD; ns indicated no significance, *p <0 0.05, ***p < 0.001.
Figure 3
Figure 3
Circ_0007386 promoted the proliferation, invasion, migration and induces the EMT of HCC cells in vitro. MHCC97H was transfected with si-NC or si-circ, and Hep3B was transfected with scramble or oe-circ. (A) The knockdown and overexpression efficiency of circ_0007386 in HCC cells were determined by qRT-PCR. (B–C) CCK-8 assays and EdU assays were used to evaluate the proliferative capabilities of HCC cells. (D) Transwell assays was used to evaluate the migratory and invasive ability of HCC cells.(E) Western blot and immunofluorescence was used to detect EMT-related proteins after suppression or overexpression of circ_0007386. Data were all showed as mean ± SD; ns indicated no significance, **p < 0.01, ***p < 0.001.
Figure 4
Figure 4
Circ_0007386 promotes the proliferation and metastasis of HCC cells in vivo. (A) Images of subcutaneous xenograft tumors (6 mice per group) in nude mice. The tumor volume and average weight were measured. (B) Immunohistochemical verification of the expression of HE, Ki67, E-cadherin, N-cadherin and Vimentin in tumors tissues. (C) Circ_0007386 stable knockdown or overexpression cells were used to generate lung metastasis. Lung metastatic nodules were stained with HE. The number of metastases was calculated. Data were all showed as mean ± SD; ns indicated no significance, **p < 0.01, ***p < 0.001.
Figure 5
Figure 5
Circ_0007386 binds to miR-507 in HCC cells. (A) Venn diagram presented miRNAs of circ_0007386, based on circBank and circInteractome. (B–C) The relative expression of miR-507 in HCC tissues and cells was determined using qRT-PCR. (D) Spearman correlation analysis of circ_0007386 expression versus miR-507 expression in HCC tissues. (E) The expression of miR-507 was upregulated upon circ_0007386 knockdown while being downregulated upon circ_0007386 overexpression. (F) Pulldown assay showed that miR-507 could be pulled down by the circ_0007386 probe. (G) RIP assay showed that circ_0007386 and miR-507 could be enriched by AGO2 antibody. (H) Wild-type (WT) and mutant plasmids (MUT) of circ_0007386 were designed according to the Starbase. (I) Luciferase activity in HCC cells confirmed the binding of circ_0007386 and miR-507. Data were all showed as mean ± SD; ns indicated no significance, **p < 0.01,***p < 0.001.
Figure 6
Figure 6
CCNT2 is a direct target of miR-507 in HCC. (A) Overlapped target genes of miR-507 predicted by miRDIP, TargetScan, miRDB and miRTarBase. (B) CCNT2 expression in HCC tissues compared to matched normal tissues (n = 80). (C–D) Spearman correlation analysis of miR-507 with CCNT2, and circ_0007386 with CCNT2 in HCC tissues. (E–F) CCNT2 were tested by Western blot in HCC cells after knockdown or overexpression of mir-507 and circ_0007386. (G) Relative luciferase activity were measured after co-transfection with CCNT2 3′UTR-WT or CCNT2 3′UTR-MUT and miR-507 mimics or mimics NC. Data were all showed as mean ± SD; ns indicated no significance, ***p < 0.001.
Figure 7
Figure 7
The function of the circ_0007386/miR-507/CCNT2 axis in HCC was verified by rescue experiments. (A and B) The EdU assay and colony formation assays were used to assess the proliferative potential of various groups. (C and D) The Transwell assay and wound healing assays was used to test the capacity of HCC cells to migrate and invade in different groups. Data were all showed as mean ± SD; ns indicated no significance, **p < 0.01, ***p < 0.001.
Figure 8
Figure 8
Knockdown of circ_0007386 inhibited lenvatinib resistance of HCC cells. (A) Cells were treated with lenvatinib for 48 hours, and their cell survival curves and IC50 were calculated by CCK-8 assay. (B) The level of circ_0007386 in the two lenvatinib-resistant HCC cell lines was measured by qRT-PCR. (C) The knockdown efficiency of circ_0007386 in the two lenvatinib-resistant HCC cell lines was measured by qRT-PCR.(D–E) The CCK-8 and colony formation assays were used to assess the effect of circ_0007386 knockdown on lenvatinib resistance. Data were all showed as mean ± SD; ns indicated no significance, **p < 0.01, ***p < 0.001.
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