hsa_circ_0008305 facilitates the malignant progression of hepatocellular carcinoma by regulating AKR1C3 expression and sponging miR-379-5p

Abstract

Circular RNAs (circRNAs) are widely involved in diverse biological processes of cancers. Nonetheless, the potential function of hsa_circ_0008305 in hepatocellular carcinoma (HCC) remains largely unknown. This study aims to elucidate the role and underlying mechanism of hsa_circ_0008305 in HCC. Our findings reveal that the novel circRNA hsa_circ_0008305 (circPTK2) is significantly upregulated in HCC tissues, with its elevated expression being positively correlated with advanced tumor T stage and vascular invasion. The circular characteristics and subcellular localization of hsa_circ_0008305 was determined by RNase R treatment and RNA nucleocytoplasmic separation. Further functional assays, including CCK8, EdU, colony formation assays, scratch-healing, transwell assays, and Xenograft tumor models were conducted to explore the biological functions of circPTK2. The regulatory mechanisms of circPTK2 were elucidated through RNA sequencing, enrichment analysis, and dual luciferase reporter assay. Our findings indicate that circPTK2 is stably localized in the cytoplasm. Functionally, circPKT2 promoted the HCC cells proliferation, migration, and invasion both in vitro and vivo. Mechanistically, circPTK2 was found to positively regulates the expression of AKR1C3 by acting as a sponge for miR-379-5p. Inhibition of miR-379-5p significantly mitigates the biological effects induced by circPTK2. AKR1C3 is identified as a direct target of miR-379-5p, and silencing AKR1C3 overturns the promotion progression effects of miR-379-5p inhibitor. In conclusion, our results revealed that circPTK2 facilitates the malignant progression of HCC via sponging miR-379-5p to up-regulate AKR1C3 expression.

Keywords: AKR1C3; Hepatocellular carcinoma; Hsa_circ_0008305; Tumor progression; miR-379-5p.

Fig. 1

circPTK2 is highly expressed in HCC tissues and cells. (A) Volcano plot shows the expression profiles of circRNAs in HCC tissues from GSE97332. (B–D) circPTK2 is highly expressed in HCC samples based on GSE97332(B), GSE94508(C), GSE166678(D). (E) Expression analysis of circPTK2 in 72 paired HCC samples by qRT-PCR (Mean ± SD, n = 72, ***p < 0.001). (F) The overall distribution of the fold change (tumor tissues vs. adjacent tissues) of circPTK2 among 72 HCC patients. (G) The expression level of circPTK2 at different tumor stages(Mean ± SD, *p < 0.05). (H) The expression level of circPTK2 in samples with vascular invasion and without microvascular invasion(Mean ± SD, *p < 0.05). (I) The expression level of circPTK2 in samples within different tumor size (≤ 5 cm and > 5 cm)(Mean ± SD, ***p < 0.001). (J) circPTK2 in HCC tissues and adjacent normal tissues detected by FISH. (K) The relative expression of circPTK2 in HCC cell lines and normal live cell (Mean ± SD, n = 3, **p < 0.01, ***p < 0.001). (L) Schematic illustration displays the origination and circularization site sequence of circPTK2 according to circBase and Dideoxy sequencing. (M) Compared with liner PTK2, circPTK2 is more resistant to R Nase R treatment in HCC cells (Mean ± SD, n = 3, ns: no significance, ***p < 0.001). (N) The half-life of circPTK2 is longer than that of linear transcriptome PTK2 after actinomycin D(2ug/mL) treatment (Mean ± SD, n = 3, ***p < 0.001). (O–P) nuclear-cytoplasmic fractions (O) and FISH (P) confirmed that circPTK2 is mainly distributed in cytoplasm.

Fig. 2

circPTK2 promotes HCC cells proliferation and tumor growth in vitro and vivo. (A) Cell viability of HCC-LM3 and MHCC 97-H cells transfected with sh-circPTK2 or shRNA-NC was evaluated by CCK8 (Mean ± SD, n = 6, ** p < 0.01, ***p < 0.001). (B) Cell viability of HCC-LM3 and MHCC 97-H cells transfected with overexpression plasmids (OE-circPTK2 or OE-NC) was detected by CCK8 (Mean ± SD, n = 6, **p < 0.01, ***p < 0.001). (C) DNA replication capacity of HCC-LM3 and MHCC 97-H transfected with sh-circPTK3 or shRNA-NC examined by EdU assays (Mean ± SD, n = 3, **p < 0.01, ***p < 0.001), scale bar: 100 μm. (D) DNA replication capacity of HCC-LM3 and MHCC 97-H transfected with circPTK2 overexpression vector or negative control (Mean ± SD, n = 3, ***p < 0.001), scale bar: 100 μm. (E) Colony formation assays detected the cell growth ability of HCC-LM3 and MHCC 97-H transfected with sh-circPTK2 or shRNA-NC (Mean ± SD, n = 3, ***p < 0.001). (F) Colony formation assays analyzed the cell growth ability of HCC-LM3 and MHCC 97-H transfected with OE-circPTK2 or OE-NC (Mean ± SD, n = 3, ***p < 0.001). (G) Subcutaneous tumors using HCC-LM3 cells infected with LV-sh-circPTK2 or LV-sh-NC. (H) Tumor weight and tumor volume were measured in sh-circPTK2 and sh-NC groups(Mean ± SD, n = 5, ***p < 0.001). (I) Subcutaneous tumors using HCC-LM3 cells infected with LV-OE-circPTK2 or LV-OE-NC. (J) Tumor weight and tumor volume were measured in OE-circPTK2 and OE-NC groups (Mean ± SD, n = 5, **p < 0.01, ***p < 0.001). (K) The expression of Ki- 67 was evaluated using IHC in sh-circPTK2 and sh-NC. Scale bar: 50 μm. (L) The expression of Ki- 67 was detected using IHC in OE-circPTK2 and OE-NC. Scale bar: 50 μm.

Fig. 3

circPTK2 increases the migration and invasion capability of HCC cells. (A,B) Cell migration ability of HCC-LM3 and MHCC 97-H cells transfected with sh-circPTK2 plasmid(A), or OE-circPTK2 plasmid(B), assessed by scratch-healing assays; Mean ± SD, n = 3, **p < 0.01, ***p < 0.001, scale bar:100 μm. (C,D) The transwell assays without Matrigel evaluate the migration ability of HCC-LM3 and MHCC 97-H after circPTK2 knockdown(C) or overexpressing(D); Mean ± SD, n = 3, **p < 0.01, ***p < 0.001. (E,F) The invasion capability of HCC cells transfected with sh-circPTK2 plasmid(E), or OE-circPTK2 plasmid(F), was examined using transwell assays with Matrigel; Mean ± SD, n = 3, **p < 0.01, ***p < 0.001, vs. corresponding control plasmids.

Fig. 4

AKR1C3 can be regulated by circPTK2 and associates with poor prognosis in HCC. (A) Volcano plot displays the differential genes when silencing circPTK2 in HCC-LM3 cell via RNA sequencing. (B) A heatmap shows the top 20 down-regulated genes after circPTK2 knockdown in HCC cell. (C) Western blot analysis verifies the protein level of AKR1C3 in HCC-LM3 and MHCC 97-H transfected with sh-circPTK2 or OE-circPTK2 vectors (Mean ± SD, n = 3, ***p < 0.001). (D) Spearman correlation analysis showing the association between circPTK2 and AKR1C3 expression in HCC tissues (n = 72). (E) The expression level of AKR1C3 in HCC tissues and normal tissues based on TCGA and GTEx databases (***p < 0.001). (F) The diagnostic accuracy of AKR1C3 estimated by receiver operating characteristic (ROC) curves. (G) Expression analysis for AKR1C3 in 72 paired HCC specimens by qRT-PCR (Mean ± SD, n = 72, ***p < 0.001). (H) The overall distribution of the fold change (tumor tissues vs. adjacent tissues) of AKR1C3 among 72 patients with HCC. (I) The protein level of AKR1C3 in 8 paired tumor tissues and adjacent normal tissues measured by Western blot.(J–L) The high expression of AKR1C3 significantly associated with poor OS (J), PFI (K), and DSS(L) based on Kaplan–Meier analysis.

Fig. 5

AKR1C3 promotes HCC cells proliferation, migration and invasion. (A) The expression level of AKR1C3 in HCC cells detected by Western blot. (B) The knockdown efficacy of AKR1C3 in HCC-LM3 and MHCC 97-H after transfecting siRNA. (C) Cell viability of HCC-LM3 and MHCC 97-H cells transfected with si-AKR1C3 or si-NC was evaluated by CCK8 (Mean ± SD, n = 6, ** p < 0.01, ***p < 0.001). (D) DNA replication capacity of HCC-LM3 and MHCC 97-H transfected with si-AKR1C3 or si-NC examined by EdU assays (Mean ± SD, n = 3, **p < 0.01), scale bar: 100 μm. (E) Cell migration ability of HCC-LM3 and MHCC 97-H cells transfected with si-AKR1C3 assessed by scratch-healing assays; Mean ± SD, n = 3, ***p < 0.001, scale bar:100 μm. (F) The transwell assays without Matrigel evaluate the migration ability of HCC-LM3 and MHCC 97-H after AKR1C3 knockdown; Mean ± SD, n = 3, ***p < 0.001. (G) The invasion capability of HCC cells transfected with si-AKR1C3 was examined using transwell assays with Matrigel; Mean ± SD, n = 3, ***p < 0.001.

Fig. 6

circPTK2 promotes HCC cells proliferation, migration, and invasion via AKR1C3. (A) The protein level of AKR1C3 in HCC-LM3 and MHCC 97-H cells transfected with sh-circPTK2 plasmid alone or co-transfected with AKR1C3 overexpression vectors. (B,C) Cell viability of HCC-LM3(B) and MHCC 97-H (C) transfected with sh-circPTK2 plasmid alone or co-transfected with OE-AKR1C3 plasmids, measured by CCK8 (Mean ± SD, n = 6, ***p < 0.001). (D,E) Clone forming capacity of HCC-LM3 (D) and MHCC 97-H (E) cells co-transfected with sh-circPTK2 and OE-AKR1C3, evaluated by colony formation assays (Mean ± SD, n = 3, ***p < 0.001). (F,G) EdU assays estimated the DNA replication capacity of HCC-LM3(F) and MHCC 97-H (G) cells transfected with sh-circPTK2 plasmid alone or co-transfected with OE-AKR1C3. Mean ± SD, n = 3, ***p < 0.001, scale bar: 100 μm. (H–J) The cells migration of HCC-LM3 and MHCC 97-H co-transfected with sh-circPTK2 and OE-AKR1C3, assessed by scratch-healing experiments (H,I) and transwell migration assays (J). Mean ± SD, n = 3, **p < 0.01, ***p < 0.001, scale bar:100 μm. (K) The invasion ability of HCC-LM3 and MHCC 97-H co-transfected with sh-circPTK2 plasmid and OE-AKR1C3 vector, measured by transwell invasion assays. Mean ± SD, n = 3, **p < 0.01, ***p < 0.001.

Fig. 7

Identification of miRNA binding to circPTK2 that regulates the proliferation, migration and invasion in HCC cells. (A) RIP assays validated the combination of circPTK2 with anti-AGO2 in HCC-LM3 and MHCC 97-H cells (Mean ± SD, n = 3, ***p < 0.001). (B) Venn plot showed the miRNAs combination to AKR1C3 predicted by miRDB, Targetscan, and mirDIP. (C) miRNAs combination sites with circPTK2 forecast by miRanda v3.3a. (D) Spearman correlation analyses showing the relationships of miR-379-5p and AKR1C3 (left), and circPTK2 (right) in HCC tissues (n = 72). (E) Biotin-labeled miRNA pull-down assay confirmed the interaction of miR-379-5p with circPTK2 and AKR1C3 (Mean ± SD, n = 3, ***p < 0.001). (F) The co-location of miR-379-5p and circPTK2 in HCC cells judged by FISH. (G) Luciferase reporter assay detected the luciferase activity in HEK-293T cells co-transfected with miRNA-379-5p mimic (or mimic-NC) and circPTK2-wt (or circPTK2-mut) luciferase reporter vectors. Mean ± SD, n = 3, ***p < 0.001. (H) Cell viability of HCC-LM3 and MHCC 97-H cells transfected with sh-circPTK2 alone or co-transfected with miR-379-5p inhibitor, measured by CCK8(Mean ± SD, n = 6, ***p < 0.001). (I) EdU assays estimates the DNA replication capacity of HCC-LM3 and MHCC 97-H cells transfected with sh-circPTK2 alone or co-transfected with miR-379-5p inhibitor. Mean ± SD, n = 3, **p < 0.01, ***p < 0.001, scale bar: 100 μm. (J,K) The cells migration of HCC-LM3 and MHCC 97-H co-transfected with sh-circPTK2 and miR-379-5p inhibitor, assessed by scratch-healing experiments (J) and transwell migration assays (K). Mean ± SD, n = 3, ***p < 0.001, scale bar:100 μm. (L) The invasion ability of HCC-LM3 and MHCC 97-H transfected with sh-circPTK2 and miR-379-5p inhibitor, measured by transwell invasion assays. Mean ± SD, n = 3, ***p < 0.001.

Fig. 8

miR-379-5p targets AKR1C3 to sustain HCC progression. (A) Western blot detected the protein expression of AKR1C3 in HCC-LM3 and MHCC 97-H transfected with miR-379-5p mimic or inhibitor.(B) Schematic of AKR1C3 3′ UTR wild-type (wt) and mutant (mut) luciferase reporter vectors; Luciferase activity of HEK-293T cell co-transfected with miR-379-5p mimic and AKR1C3 3′-UTR (wt) or AKR1C3 3′-UTR (mut) vectores determined by dual-luciferase reporter assays, Mean ± SD, n = 3, ***p < 0.001. (C,D) Cell viability of HCC cells transfected with miR-379-5p inhibitor alone or co-transfected with si-AKR1C3, measured by CCK8 (Mean ± SD, n = 6, **p < 0.01, ***p < 0.001). (E,F) DNA replication capacity of HCC-LM3 (E) and MHCC 97-H (F) cells co-transfected with miR-379-5p inhibitor and si-AKR1C3, evaluated by EdU assays (Mean ± SD, n = 3, ***p < 0.001). scale bar: 100 μm. (G–I) The cells migration of HCC-LM3 and MHCC 97-H transfected with miR-379-5p inhibitor and si-AKR1C3, assessed by scratch-healing experiments (G,H) and transwell migration assays (I). Mean ± SD, n = 3, *p < 0.05, **p < 0.01, ***p < 0.001, scale bar:100 μm. (J). The invasion ability of HCC cells transfected with miR-379-5p inhibitor and si-AKR1C3, measured by transwell invasion assays. Mean ± SD, n = 3, ***p < 0.001, scale bar:100 μm. (K) Western blot analyzed the expression of AKR1C3 in HCC-LM3 and MHCC 97-H co-transfected with sh-circPTK2 and miR-379-5p inhibitor. (L) Schematic diagram shows circPTK2 promoting HCC cell proliferation, migration and invasion via miR-379-5p/AKR1C3 axis.