#These authors contributed equally in the study
MicroRNA-145-5p (miR-145-5p) reportedly inhibits hepatocellular carcinoma (HCC) by targeting ARF6, SPATS2, CDCA3, KLF5, and NRAS, indicating that miR-145-5p plays an important role in the occurrence and development of HCC by regulating the expression of various genes. In this study, we aimed to explore novel downstream targets of miR-145-5p and elucidate the potential mechanism of miR-145-5p in HCC.
A bioinformatics analysis was performed to determine the clinical significance of miR-145-5p and alpha/beta hydrolase domain-containing protein 17C (ABHD17C) in patients with HCC. The ability of Hep3B cells to proliferate, migrate, and invade was examined after overexpression of miR-145-5p and ABHD17C or knockdown of ABHD17C. Tumorigenesis of Hep3B cells overexpressing miR-145 was detected using
miR-145-5p was downregulated in HCC tissues, and this was associated with poor prognosis in patients with HCC. Based on the bioinformatics analysis, miR-145-5p was predicted to target ABHD17C, as demonstrated by a luciferase reporter assay. ABHD17C downregulation inhibited cell viability, migration, and invasion and arrested the cell cycle. Overexpression of miR-145-5p significantly reduced the expression of ABHD17C. Moreover, ABHD17C expression was elevated in HCC tissues, which was associated with an unfavorable prognosis. Re-expressing ABHD17C into HCC cells rescued the suppressed cell viability, migration, and invasion mediated by ectopic expression of miR-145-5p. Importantly, miR-145-5p suppressed tumor growth in mice and downregulated the levels of Ki67 and ABHD17C in tumor.
miR-145-5p could attenuate HCC progression via suppressing ABHD17C.
Hepatocellular carcinoma (HCC) is a leading cause of cancer-related deaths worldwide [
As a family of small non-coding RNAs, microRNAs (miRNAs) suppress target gene expression via sequence matching, thereby modulating cellular processes [
Alpha/beta hydrolase domain-containing protein 17 (ABHD17) has been identified as a protein acyl thioesterase that selectively eliminates palmitoylation of stress-regulated exon linkers to regulate channel gating [
In this study, we confirmed the clinical significance of miR-145-5p as a deregulated miRNA in patients with HCC. In-depth
Raw transcriptome analysis data of normal liver and HCC tissues from patients with HCC were downloaded from The Cancer Genome Atlas Liver Hepatocellular Carcinoma (TCGA-LIHC) (
HEK293T and Hep3B cells were obtained from IMMOCELL (Xiamen, China) and cultured in Dulbecco’s modified Eagle’s medium (Gibco, China) containing 10% fetal bovine serum (FBS; Gibco, China) at 37°C in a humidified incubator with 5% CO2.
miR-145 (pri-miR-145-5p) or ABHD17C was amplified and inserted into a pCDH-EF1A-MCS-T2A vector to generate plasmids encoding miR-145 and ABHD17C, named miR-145 OE and ABHD17C OE, respectively. The short hairpin RNA of ABHD17C was designed and inserted into a pLKO.1-puro vector to generate plasmids encoding the short hairpin RNA of ABHD17C, namely shABHD17C. The negative control (NC) of shABHD17C was named shNC. The wild-type (WT) 3’UTR of ABHD17C (ABHD17C 3’UTR WT) and mutant 3’UTR of ABHD17C (ABHD17C 3’UTR MUT) were also PCR-amplified and ligated into the pmirGLO vector. The primers used to construct plasmids are as follows. ABHD17C forward primer: 5′-CTAGAGAATTCGGATCCATGCCCGAGCCAGGCCCCAGGATGAAC-3′, ABHD17C reverse primer: 5′-AGCTTCCATGGCTCGAGGGAATTAGGAAGTTCGTGAG-3′, pri-miR-145-5p forward primer: 5′-ATTCACGCGTGCGGCCGCCGGCGGCCTTGGCGCTGAAG-3′, pri-miR-145-5p reverse primer: 5′-TAGGGATCCGGGCCCGGGGTGGGAAGGAGGCAAATCC-3′, ABHD17C 3’UTR WT forward primer: 5′-AGCTCGCTAGCCTCGAGATGGGAATGAGAGCTGAATG-3′, ABHD17C 3’UTR WT reverse primer: 5′-CATGCCTGCAGGTCGACTCTATGCACAATGTGATTGC-3′, ABHD17C 3’UTR MUT-1 forward primer: 5′-AGTGTTTGACCTAAGCTAGTGTGGTGAAAATTC-3′, ABHD17C 3’UTR MUT-1 reverse primer: 5′-ACTAGCTTAGGTCAAACACTATCACAGCTTCAC-3′, ABHD17C 3’UTR MUT-2 forward primer: 5′-TATAAATATTTGACCTATATTCTTAAACAAAAAG-3′, ABHD17C 3’UTR MUT-2 reverse primer: 5′-ATATAGGTCAAATATTTATATGGTTACAG-3′, shABHD17C forward primer: 5′-CCGGAAGAUGUUGCAGUUGAUGCGGCTCGAGCCGCATCAACTGCAACATCTTTTTTT-3′, shABHD17C reverse primer: 5′-AATTAAAAAAAGAUGUUGCAGUUGAUGCGGCTCGAGCCGCATCAACTGCAACATCTT-3′, shNC forward primer: 5′-CCGGTTCTCCGAACGTGTCACGTCTCGAGACGTGACACGTTCGGAGAATTTTT-3′, shNC reverse primer: 5′-AATTAAAAATTCTCCGAACGTGTCACGTCTCGAGACGTGACACGTTCGGAGAA-3′.
miR-145-5p mimic, NC of miR-145-5p mimic (mimic NC), miR-145-5p inhibitor, NC of miR-145-5p (inhibitor NC) (RiboBio, Guangzhou, China), shABHD17C, shNC, and ABHD17C OE were transfected into Hep3B cells using Lipofectamine™ 2000 (11668500, Invitrogen, China) to increase the level of miR-145-5p, inhibit miR-145-5p, decrease the expression of ABHD17C, and overexpress ABHD17C, respectively. The cells were analyzed two days post-transfection. Plasmids expressing pri-miR-145-5p were transfected into HEK293T cells along with pMD2G and pspax2 plasmids. After 48 h, the lentivirus-containing supernatant was collected; lentivirus was enriched, and the titer was determined as described previously [
In the presence of 5 μg/mL polypropylene, the lentivirus was transduced into Hep3B cells at a multiplicity of infection of 10. After 48 h, the medium was replaced with fresh medium, and puromycin was added at a final concentration of 1.5 μg/mL. After 72 h, the cells were collected to analyze miR-145-5p expression.
Total RNA was isolated using the TRIzol reagent (15596018, Invitrogen, USA). The same amount of RNA was transcribed into cDNA using a Mir-X miRNA first-strand synthesis kit (638315, Takara, China) with specific primers for miRNAs or using the Superscript III First-Strand Synthesis System (18080051, Invitrogen, China) with random primers for other genes, according to the manufacturer’s instructions. qPCR was performed using Mir-X miRNA qPCR TB Green® Kit (638314, Takara, China) for miRNAs and U6, or TB Green® Fast qPCR Mix (RR430A, Takara, China) for other genes, and Thermal Cycler Dice™ Real Time System III with PC (TP970, Takara, China), according to the manufacturer’s instructions. The cDNAs of miRNA and U6 were pre-denatured at 95°C for 10 s, denatured at 95°C for 5 s, annealed, and extended at 60°C for 20 s. Denaturation, annealing, and extension were performed for 40 cycles. Finally, the melting curve was obtained. The melting curve program consisted of 95°C for 60 s, 55°C for 30 s, and 95°C for 30 s. The thermal cycling conditions for qPCR of the other genes were 95°C for 30 s, 40 cycles of 95°C for 5 s, and 60°C for 15 s, followed by melting curves. Data for miRNAs and other genes were normalized using the 2−ΔΔCq method according to U6 and 18S rRNA levels, respectively. The primers used for qPCR are as follows: U6 reverse transcription primer: 5′-AACGCTTCACGAATTTGCGT-3′, U6 forward primer: 5′-CTCGCTTCGGCAGCACA-3′, U6 reverse primer: 5′-AACGCTTCACGAATTTGCGT-3′, miR-145-5p reverse transcription primer: 5′-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACAGGGAT-3′, miR-145-5p forward primer:5′-CGGTCCAGTTTTCCCAGGA-3′, miR-145-5p reverse primer: 5′-AGTGCAGGGTCCGAGGTATT-3′, 18S rRNA forward primer: 5′-AGGCGCGCAAATTACCCAATCC-3′, 18S rRNA reverse primer: 5′-GCCCTCCAATTGTTCCTCGTTAAG-3′, ABHD17C forward primer: 5′-TACTGCTTTGATGCTTTCC-3′, ABHD17C reverse primer: 5′-ATCCTCTGTACCATGAATGA-3′, ANGPT2 forward primer: 5′-GTGGCTAATGAAGGTATT-3′, ANGPT2 reverse primer: 5′-TTATTGACTGTAGTTGGAT-3′, CTNNBIP1 forward primer: 5′-TGACCAACAGAAACCTTT-3′, CTNNBIP1 reverse primer: 5′-AATCAGACCTCTTCACATT-3′, PDGFD forward primer: 5′-GGATTAGAGGAAGCAGAA-3′, PDGFD reverse primer: 5′-TCGGACTTGAATGTGATT-3′, PODXL forward primer: 5′-AAGATAAGTGCGGCATAC-3′, PODXL reverse primer: 5′-GCTTAGTGTGAATAGTGATT-3′, SMAD3 forward primer: 5′-GCACATAATAACTTGGACCT-3′, SMAD3 reverse primer: 5′-GCTCGTAGTAGGAGATGG-3′, SMAD5 forward primer: 5′-AAGCCGTTGGATATTTGT-3′, SMAD5 reverse primer: 5′-AGGTAAGACTGGACTCTC-3′, SRGAP1 forward primer: 5′-ATCAATCTCTATGGTCTTCA-3′, SRGAP1 reverse primer: 5′-ATGGTTACTCTGGTCATC-3′.
Briefly, ice-cold RIPA buffer (P0013C, Beyotime, Shanghai, China) supplemented with phenylmethanesulfonyl fluoride (PMSF; ST506, Beyotime) was used to lyse cells or tumor tissues. Proteins (20 μg) were separated via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) after determining protein concentration using a BCA Protein Concentration Assay Kit (P0010S, Beyotime). After transferring the separated proteins onto a polyvinylidene fluoride membrane, which was then blocked with 5% skimmed milk at 26°C for 2 h, the membrane was incubated with diluted ABHD17C (PA5-61831, Thermo Fisher Scientific, USA) antibody, β-actin antibody (20536-1-AP, Proteintech, China) and GAPDH antibody (10494-1-AP, Proteintech, China) at 26°C for 2 h. Subsequently, horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (SA00001-2, Proteintech, China) and an ECL substrate (Bio-Rad, China) were used to visualize the proteins. Densiometric analysis was performed using ImageJ v1.48 (National Institutes of Health).
Briefly, 1,000 Hep3B cells exhibiting upregulated miR-145-5p and ABHD17C or downregulated ABHD17C were plated on 96-well plates. The following day, cell viability was quantified using the CyQUANT™ MTT Cell Viability Assay kit (V13154, Invitrogen, China) according to manufacturer’s instruction.
Briefly, 1 × 105 Hep3B cells, exhibiting upregulated miR-145-5p and ABHD17C or downregulated ABHD17C, were suspended in serum-free medium and then added to the upper Transwell inserts in the absence (for migration assays) or presence (for invasion assays) of Matrigel. 500 μL medium containing 10% FBS were added to the lower chamber. After incubation for 48 h, cells in the lower phase of the upper chamber were fixed with 4% paraformaldehyde and stained with 0.5% crystal violet. Migrated or invasive cells were photographed and counted under a microscope (Motic Incorporation, Ltd., China).
ABHD17C 3’UTR WT or ABHD17C 3’UTR MUT and pRL-TK vector (D2760, Beyotime) were co-transfected with miR-145-5p mimic or mimic NC into HEK293T cells. After 2 days, the cells were harvested and treated using a dual luciferase reporter assay kit (E1910, Promega, China), according to the manufacturer’s instructions. Subsequently, firefly luciferase and Renilla luciferase activities were detected using a multi-mode microplate reader (Synergy Neo2, BioTek, USA). Data were normalized by dividing firefly luciferase activity by that of Renilla luciferase.
For cell cycle analysis, Hep3B cells with upregulated miR-145-5p and ABHD17C or downregulated ABHD17C were fixed with pre-cooled 75% ethanol at 4°C for 4 h, permeated with 0.2% Triton X-100 in the presence of RNase A (Beyotime, Shanghai, China) for 30 min, and stained with propidium iodide (PI; 50 μg/mL) for 0.5 h at 26°C. Subsequently, stained cells were measured using a FACScan flow cytometer (Beckman Coulter, USA), and the resulting data were analyzed using Cell Quest software.
Experiments were performed under a project license granted by the ethics board of Fujian Medical University in compliance with the institutional guidelines for the care and use of animals. Briefly, 1 × 106 Hep3B cells stably overexpressing miR-145 were subcutaneously injected into six-week male BALB/c nude mice, procured from the Shanghai Laboratory Animal Research Center. Tumors were monitored, and all mice were sacrificed on day 37 post-injection. Obtained tumors were subjected to biological analyses, including qPCR, western blotting, and immunohistochemistry (IHC) assays.
Tumor samples were sliced, fixed, and blocked, as previously described. Subsequently, blocked slices were incubated with diluted Ki67 antibody (catalog number: 27309-1-AP; Proteintech) for 12 h at 4°C. After incubation with HRP-conjugated goat anti-rabbit IgG (catalog number: SA00001-2; Proteintech), sections were incubated with DAB for color development. Subsequently, the slices were treated with hematoxylin to stain nuclei and dehydrated. Finally, the sections were sealed with transparent resin and observed under an Olympus light microscope (BX51, Olympus Corporation, Tokyo, Japan).
Data were analyzed using GraphPad Prism 8 software (GraphPad Software, Inc., La Jolla, CA, USA). The Shapiro–Wilk test was performed to determine whether data followed a normal distribution. Levene’s test was used to ensure homogeneity of variance. The Mann–Whitney U test was performed to compare differences between two groups of nonparametric data. Survival curves were calculated using the Kaplan-Meier method, and significance was determined using the log-rank test. The Student’s
To evaluate the clinical significance of miR-145-5p, we analyzed miRNA transcriptome data from normal non-tumor (NT) and HCC tissues. Notably, miR-145-5p was significantly downregulated in HCC tissues compared with that in NT tissues (
Given that a miRNA may target a panel of mRNAs for specific suppression, the six most prevalent target prediction platforms were applied to identify actual miR-145-5p targets. Ultimately, 12 genes were identified as hits with the highest possibility of miR-145-5p interaction (
To evaluate the clinical significance of ABHD17C, the same clinical data of patients with HCC was used to analyze ABHD17C expression. In contrast to miR-145-5p expression, ABHD17C expression levels were enhanced in HCC tissue samples when compared with those in NT tissues (
To determine the role of ABHD17C in the biological processes of HCC cells, we knocked down ABHD17C in Hep3B cells. Downregulation of ABHD17C expression significantly reduced cell viability (
To determine whether ABHD17C is biologically indispensable to mediate the effect of miR-145-5p in HCC cells, ABHD17C was re-expressed in Hep3B cells by transient transfection. qPCR and western blotting confirmed that ABHD17C was efficiently re-transduced into Hep3B cells overexpressing miR-145-5p (
Finally, we used a mouse xenograft model to confirm whether
HCC, which accounts for 80% of all cases of primary liver cancer, is one of the primary causes of cancer-related deaths worldwide [
The importance of miRNAs in modulating cellular processes highlights their potential role as vital candidate biomarkers and promising therapeutic agents [
It has been reported that miR-145-5p inhibits breast cancer cell proliferation by downregulating SOX2 expression [
ABHD17C, a member of ABHD17, is highly expressed in breast cancer tissues, indicating that ABHD17C could be involved in regulating tumorigenesis and breast cancer development [
To better understand the complexity and unbiased screening of miR-145-5p targets, six platforms and a dataset of upregulated genes in patients with HCC were employed, resulting in the enrichment of eight candidate genes. Importantly, miR-145-5p could downregulate six of the eight candidate genes, confirming the robustness of our screening methodology. However, none of the miR-145-5p target genes identified in previous studies were identified in the present analysis. Therefore, it is unsurprising that one miRNA may target multiple genes for degradation. Notably, a few genes (CTNNBIP1 and SMAD3) that belong to critical cancer-driving pathways, such as transforming growth factor (TGF)-β/SMAD and Wnt/CTNNB1, were enriched, although the decreased expression mediated by miR-145-5p was not as prominent as afforded by ABHD17C. We cannot exclude the possibility that miR-145-5p may tightly control these genes, thereby fine-tuning the aforementioned cancer-related signaling and affecting HCC progression. Therefore, future studies could focus on determining the contribution of these genes to miR-145-5p-mediated negative effects on HCC cell malignancy.
In this study, we revealed the direct interaction between ABHD17C 3′UTR and miR-145-5p via dual luciferase reporter analysis. Notably, the biological role of ABHD17C has been poorly investigated. Patient-derived data revealed a negative correlation between ABHD17C expression and survival of patients with HCC. Furthermore, rescue experiments showed that miR-145-5p depended on ABHD17C to exert its tumor-inhibitory function, strengthening the importance of ABHD17C expression in promoting HCC progression. Thus, in-depth investigations should be conducted to uncover the functions of ABHD17C expression in the progression of HCC and other cancer types. The mechanistic details of how ABHD17C mediates its biological effects warrant further investigation.
There are certain limitations to this study. First, the present study mainly explored the roles of miR-145-5p and ABHD17C in HCC by upregulating the expression levels of miR-145-5p and ABHD17C, which were not further verified by inhibiting miR-145-5p. Second, we did not explore the therapeutic effects of targeting miR-145-5p in HCC, which should be explored in future investigations. Third, the possible utilization of miR-145-5p in preclinical models was not further determined using a complementary experiment, patient-derived xenograft models or organoid systems.
In the study, the tumor-suppressive effects of miR-145-5p were validated
(I) Conception and design: Linpei Wang, Xiaoqiu Ma, Wei Wang, and Shaojian Chen; (II) Administrative support: Wei Wang and Shaojian Chen; (III); Provision of study materials and patients: Linpei Wang, Youqi Chen, and Shaojian Chen; (IV) Data collection and assembly: Linpei Wang, Xiaoqiu Ma, Youqi Chen, Jiahui Zhang, Jiawei Zhang, and Wei Wang; (V) Data analysis and interpretation: Linpei Wang, Xiaoqiu Ma, and Shaojian Chen; (VI) Manuscript writing: All authors; (VII) Final approval of the manuscript: All authors.
The authors declare that all data supporting the findings of the present study are available within the article and its supplementary information.
The authors are accountable for all aspects of the work, ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. Experiments were performed under a project license granted by the Ethics Board of the Second Affiliated Hospital of Fujian Medical University (Approval No. 2020 No. 15) in compliance with the institutional guidelines for the care and use of animals.
This work was supported by the
All authors declare that there is no conflict of interest exists in the submission of this manuscript and approve the manuscript for publication.