Introduction
Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer worldwide [
1]. Viral hepatitis, alcohol abuse, and nonalcoholic hepatic steatosis are the main risk factors for HCC [
2,
3]. Because of the lack of early symptoms and signs, most HCC patients are already in the advanced stage at the time of diagnosis. Despite recent achievements in HCC treatment, its diagnosis and prognosis remain challenging [
4,
5]. Therefore, seeking effective diagnosis and treatment is crucial. As reported in our previous study, plasma hsa_circ_0005397 represents a good value for HCC [
6]. However, the underlying mechanisms of HCCprogression or metastasis have not been fully elucidated.
Circular RNAs (circRNAs) are noncoding RNAs that are formed through backsplicing of linear RNA, resulting in a circular structure [
7]. Due to their stable structure, they are resistant to degradation [
8]. Furthermore, circRNAs are widely distributed in body fluids and blood, underscoring their potential as stable biomarkers [
9,
10]. Moreover, circRNAs participate in cancer pathogenesis, invasion and metastasis. For example, circ_103809 targets miR-620 to suppress proliferation and invasion in HCC [
11], and hsa_circ_104348 modulates the miR-187-3p/RTKN2 axis in hepatocellular carcinoma progression [
12]. Overall, reports indicate that circRNAs are promising biological targets for HCC diagnosis and prognosis.
Recently, knowledge of RBPs has increased substantially owing to the development of new technologies [
13]. RBPs influence RNA biology post-transcriptionally, and an increasing amount of evidence suggests that abnormal RBPs expression and function are associated with cancer metastasis [
14]. As reported, circRNAs regulate the biological activity of downstream targets by binding to RBPs [
15]. EIF4A3 is a commonly studied RBP involved in posttranscriptional regulation [
16,
17]. For example, EIF4A3 interacts with circ_0084615, promoting progression of colorectal cancer via miR-599/ONECUT2 [
18]. Additionally, EIF4A3 binds to circ_0004296, inhibiting metastasis of prostate cancer [
19]. Moreover, hsa_circ_0068631 recruits EIF4A3, which increases expression of c-Myc in breast cancer, showing an important role in cancer metastasis [
20]. As reported, hsa_circ_0005397 is a kind of circRNA originating from RHOT1 that promotes HCC by regulating the miR-326/PDK2 axis [
21]. Hsa_circ_0005397 might also be involved in other pathogeneses with various binding sites. In this study, we found a meaningful interaction between hsa_circ_0005397 and EIF4A3 in HCC progression.
Materials and methods
Clinical tissue samples
We collected 57 tumor tissues and paired adjacent tissues from HCC patients at Nantong Third People’s Hospital. Patients who received surgical treatment without any other treatment were chosen for the study. Tissues were immediately stored at -80 °C. The clinical data of all patients were collected. This study was approved by the Ethics Committee of Nantong Third Affiliated Hospital of Nantong University and were conducted in accordance with the Declaration of Helsinki. All participants signed informed consent forms in this study.
Cell culture
The normal hepatocyte cell line (LO2) and HCC cell lines (SMMC-7721, SK-Hep1, HCCLM3, BEL-7404, Huh7) were acquired from Type Culture Collection Cell Committee of the Chinese Academy of Sciences (Shanghai, China). The cells were cultured in suitable medium containing 10% fetal bovine serum (Lonsera, USA) at 37 °C with 5% CO2.
Construction of plasmids and siRNAs
SiRNAs targeting the negative control (si-NC), and hsa_circ_0005397 (si1, si2) were obtained from GenePharma (Suzhou, China). ShRNAs targeting the EIF4A3 (sh EIF4A3) and negative control (sh NC) were obtained from GenePharma (Suzhou, China). Overexpression plasmids, human hsa_circ_0005397 cDNA was synthesized and cloned into the pcDNA3.1 vector (pcDNA) and empty vector was used as the negative control (vector) which obtained from Shanghai (Gechem, China). We used lipofectamine 3000 and P3000 (Invitrogen, USA) for plasmid overexpression and lipofectamine 3000 for siRNA transfection according to the manufacturer’s instructions.
RNA preparation and qRT-PCR
Total RNA was isolated by TRIzol reagent (Invitrogen, USA) and the concentration of total RNA was detected by NanoDrop spectrophotometer (Thermo Fisher Scientific, USA). CDNA was synthesized by Synthesis Kit (Thermo Fisher Scientific, USA), and SYBGreen I Master Mix (Roche, Germany) was used for qRT-PCR. 18 S was used as an endogenous control. All data were normalized to internal control, and relative expression was calculated by the 2–ΔΔCT method. Hsa_circ_0005397 primers were purchased from Guangzhou (Geneseed, China), and other primers were purchased from Shanghai (Sangon Biotech, China). The primers used in this study were as follows: hsa_circ_0005397 (Forward: 5′-GACAAAGACAGCA GGTTCCT-3′; Reverse: 5′-CTCTGTTCTGCTTCTGAGTA-3′); EIF4A3 (Forward: 5’-CAGGGCGTGTTTTTGATATGAT-3’; Reverse: 5’-ATCAGCTTCATCCAAAACCAAC-3’; RHOT1 (Forward: 5’-CTGATTTCTGCAGGAAACACAA-3’; Reverse: 5’-GCAAAAACAGTAGCACCAAAAC-3’); and 18 S rRNA (Forward: 5’-GTAACCCGTT GAACCCCATT-3’; Reverse: 5’-CCATCCAATCGGTAGTAGCG-3). After qRT-PCR, PCR products were examined by 2% agarose gel electrophoresis. The experiments were performed at least three times independently with triplicate samples.
RNase R resistance assay
For RNase R treatment, 1 µg of total RNA was incubated with or without 0.1 µL RNase R (20 U/µL) and 1µL 10X Reaction Buffer for 0.5 h at 37 °C. After purified, qRT-PCR was used to examine the levels of hsa_circ_0005397 and RHOT1.
Western blotting
Proteins were extracted in RIPA Lysis buffer (Beyotime, China) with protease and phosphatase inhibitors. Protein lysates were separated by 10% SDS-PAGE gels and transferred to PVDF membrane (Millipore Corporation, China). After blocking in non-fat milk, membranes were incubated with primary antibody overnight at 4 ℃, and incubated with a secondary antibody for 1 h at room temperature. The primary antibody used as follows: anti-EIF4A3 (1:1000, Proteintech, China), anti-GAPDH (1:20000, Proteintech, China), HRP-conjugated goat anti-rabbit IgG (1:1000, Beyotime, China). The bands were visualized by an enhanced chemiluminescence detection system (Tanon, China).
Immunohistochemistry (IHC) examination
After dewaxed with xylene and gradient of ethanol concentration. The slices were subjected to citrate microwave heating treatment for antigen retrieval. 3% H2O2 was used to block endogenous peroxidase activity. Next, the slices were incubated with anti-EIF4A3 (1:100, Proteintech, China) overnight at 4℃ and subsequently with horseradish peroxidase-conjugated anti-mouse/rabbit secondary antibody (1:1000, Changdao Biotechnology, China) for 1 h at room temperature. After washed, the slices were stained with hematoxylin and 3,3’-diaminobenzidine (DAB, MaixinBio, China). Finally, the slices were imaged in five randomly fields. Finally, two pathologists evaluated all stained sections independently. The staining intensity was scored as 0 (no staining), 1 (weak staining), 2 (medium staining) and 3 (strong staining); the degree of staining was scored as 0 (≤ 10%), 1 (> 10-25%), 2 (> 25-50%), 3 (> 50-75%) or 4 (> 75%). Scoring was based on the product of the staining intensity score and degree score.
Nuclear and cytoplasmic extraction
The nuclear and cytoplasmic fractions of RNA were collected with extraction kits (Thermo Fisher Scientific, USA) according to the manufacturer’s instructions. After collected, the purified RNA was extracted and detected by qRT-PCR.
Actinomycin D assay
2.5 µg/mL Actinomycin D (Merck, Germany) was added to cells and cultured for 8 h. TRIzol reagent (Invitrogen, USA) was added at 0, 2, 4, 6, and 8 h to collect RNA. after collected, the purified RNA was extracted and detected by qRT-PCR.
Cell counting kit-8 (CCK-8) assay
After 48 h, transfected cells were collected, and 3,000 cells/well were seeded in 96-well plates. After incubation with CCK-8 solution (MCE, USA) for 2 h, the OD value was measured at 450 nm by using a microplate reader (Thermo Fisher Scientific, USA).
After transfection, total of 3,000 cells were added to a six-well plate for two weeks and fixed with 4% formaldehyde (Beyotime, China). After stained with 0.1% crystal violet (Sigma, USA), cell clones were counted and analyzed under a light microscope.
Transwell assay
After transfection for 48 h, 5 × 104 cells were suspended in 200µL serum-free medium and seeded into the upper chamber (Costar, USA) with or without precoated with 40 µl Matrigel (BD Biosciences, USA), and 600µL MEM media containing 20% FBS was put into bottom of chambers. After incubated for 48 h, cells were fixed with 4% paraformaldehyde for 15 min, and stained with 0.1% crystal violet (Sigma, USA) for 30 min and photographed under a microscope (Olympus, Japan). Cell counts were counted in five randomly fields, The experiments were performed at least three times independently with triplicate samples.
Cell cycle assay analysis
The DNA content test kit (Solarbio) was used to evaluate cell cycle. After transfection, the treated cells were collected and fixed in ice-cold 70% ethanol overnight. After removing the 70% ethanol, the cells were washed by PBS. Cells were resuspended with 100 µL of RNase A solution at 37 °C for 30 min, and incubated with 400 µL of PI staining solution for 30 min at 4 °C in the dark. Cell cycle was detected by flow cytometry (Bio-Rad, USA).
RNA immunoprecipitation (RIP) assay
RIP Kit (Geneseed, China) was used to perform RIP assay. A total of 1 × 107 cells were lysed, some of them was collected to acquire input RNA, while another was incubated with magnetic beads conjugated with human anti-EIF4A3 (anti-EIF4A3) or anti-immunoglobulin G (anti-IgG) antibody at 4 °C, after incubated, the purified RNA was extracted and detected by qRT-PCR.
Statistical analysis
Data, based on at least three independent experiments, are presented as the mean ± standard deviation (SD) and were analyzed by GraphPad Prism 7.0 (GraphPad Software, USA). The t test and one-way analysis were employed to analyze differences between two groups or among more than two groups. Correlation was calculated by Pearson’s correlation analyses. P values < 0.05 were considered significant.
Discussion
Despite advancements in this field [
24,
25], the survival rate of patients with HCC remains unsatisfactory due to challenges in early tumor diagnosis, as well as tumor recurrence and metastasis [
26,
27]. Although serum AFP has been widely utilized as a tumor marker for HCC diagnosis and screening, its sensitivity and specificity are not sufficiently high, necessitating further improvements [
28,
29]. Recent years, research on circular RNAs (circRNAs) has provided a novel avenue that is expected to contribute to clinical diagnosis and prognosis of HCC [
30‐
32]. Hsa_circ_0005397 was a kind of circRNA, which has been identified to play a key role in several cancers. For instance, hsa_circ_0005397 was upregulated and promoted cell proliferation and invasion in pancreatic cancer [
33]. More importantly, hsa_circ_0005397 was overexpressed in HCC tissues, which could promote hepatocellular carcinoma progression by regulating the miR-326/PDK2 axis [
21]. In this study, we also verified that hsa_circ_0005397 were markedly overexpressed in both HCC tissues and cell lines. We found that hsa_circ_0005397 had good sensitivity and specificity for diagnosis in HCC patients, and upregulated expression of hsa_circ_0005397 was significantly related to tumor size and stage. To explore the function of has_circ_0005397 in HCC cells, we designed two siRNAs targeting the back splicing junction of has_circ_0005397, the results shown that downregulated expression of has_circ_0005397 may affect cell migration, invasion and proliferation while overexpression of has_circ_0005397 shown the opposite yield, indicating the oncogenic role in HCC growth.
With the development of sequencing and other technologies, the diversity and biological functions of circRNAs are being extensively explored. CircRNAs can sponge miRNAs, bind to RBPs, and regulate gene transcription and translation to regulate tumorigenesis [
20,
34,
35]. Unfortunately, previous studies mainly focused on circRNAs serving as miRNA sponges. Actually, circRNAs could also competitively bind to and interact with RBPs [
36]. In our study, we found that EIF4A3 was a RBP of hsa_circ_0005397 from bioinformation websites. EIF4A3 plays a crucial role in post-transcriptional regulation and has been reported to interact with RNAs and act as a diagnostic marker in many cancers [
37‐
39]. Recent Studies have demonstrated that EIF4A3 binds to the upstream region to promote circMMP9 expression, showing an important role in mRNA splicing [
40]. In addition, EIF4A3 was found to bind to circZFAND6 pre-mRNA transcript upstream region, leading to the high expression of circZFAND6 in breast cancer [
41]. All these results concluded that EIF4A3 may be an important circRNA regulator and play important role in the post-transcriptional process. In our study, we found that EIF4A3 was remarkably overexpressed, and the expression of EIF4A3 was positively correlated with the expression of hsa_circ_0005397. Importantly, the expression of hsa_circ_0005397 was downregulated when depletion of EIF4A3, showing that the RBP protein EIF4A3 may affect the transcription level of hsa_circ_0005397. What’s more, the RIP assay was confirmed that EIF4A3 was able to interact with hsa_circ_0005397. Moreover, recent studies have demonstrated that EIF4A3 could regulate EMT process and facilitate tumor progression in HCC [
42]. In our study, we found that EIF4A3 inhibition could reverse the overexpression effects of hsa_circ_0005397 on HCC cell proliferation, migration and invasion, showing that hsa_circ_0005397 may regulate HCC progression through EIF4A3. Overall, these findings demonstrated that hsa_circ_0005397 promoted HCC progression and metastasis through EIF4A3. Our study provided evidence for the underlying mechanism of hsa_circ_0005397 functions in the tumorigenesis of HCC, and shed light on the potential biomarkers and therapeutic targets for HCC.
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