CircularRNA Hsa_circ_0093335 promotes hepatocellular carcinoma progression via sponging miR‐338‐5p

Abstract Circular RNAs play an important role in the development of various malignancies, including hepatocellular carcinoma (HCC). Nevertheless, the role of Hsa_circ_0093335 (circ0093335) in HCC has not yet been explored. To investigate the biological effects and molecular mechanisms of circ0093335 on HCC. Circ0093335 expression was detected in HCC cells and clinical specimens using qRT‐PCR. The association between circ0093335 expression and HCC patients' clinical characteristics was determined using SPSS. The role of circ0093335 in HCC was estimated by overexpression and knockdown experiments in vitro and in vivo. qRT‐PCR, nucleoplasma separation assay, FISH assay, RIP, dual luciferase reporter assay and rescue assay were used to validate the regulatory effect of circ0093335 on miR‐338‐5p. The study findings showed that circ0093335 was upregulated in HCC. High circ0093335 expression was linked with the tumour‐node‐metastasis stage and microvascular tumour invasion. circ0093335 is greatly involved in HCC cell proliferation, aggressive ability and mouse tumour growth, according to many in vitro and in vivo tests. Mechanistically, circ0093335 downregulated miR‐338‐5p expression by sponging, consequently promoting HCC progression. Our research indicated that circ0093335 might be a target for HCC therapy since it promotes tumour progression by acting as a miR‐338‐5p ‘sponge’.

major interventions. 2,7Further research into the molecular elements and their signalling pathways that influence the emergence and development of HCC malignancies is needed because they may represent a therapeutic target for the disease.
Non-coding RNAs (ncRNAs) are considered key players in cancer biology and participate in gene regulation.When ncRNAs are dysregulated, they contribute to the development of cancer. 8Circular RNAs (circRNAs), a novel family of ncRNAs produced by back-splicing pre-mRNAs into stable closed-loop architecture without 5′ caps and 3′ tails, are implicated in biological processes that contribute to tumorigenesis. 9,102][13] Recent developments in research have indicated that circRNAs are dysregulated in HCC and are involved in the emergence of HCC.5][16] However, further studies are needed to fully understand how circRNAs act in HCC.BMI1, a proto-oncogene, regulates a number of physiological processes, including cell differentiation and self-renewal, and proper gene silencing, all of which are crucial for biological function. 17,18A growing body of evidence points to BMI1 as a crucial gene in carcinogenesis.3][24] Consequently, we hypothesized that cir-cRNAs derived from BMI1 are involved in the development of HCC.
Hsa_circ_0093335 (circ0093335), reported in oesophageal cancer, was formed by back-splicing of exons 6-12 of BMI1 and had an entire length of 589 bp. 25 We speculated that circ0093335 is involved in the progression of HCC and should be investigated more.
In this research, we identified and confirmed circ0093335 existence in HCC cell lines and its expression in HCC clinical specimens.We further studied the relationships between circ0093335 expression and clinical parameters.Moreover, we showed that circ0093335 promotes HCC by sponging miR-338-5p, suggesting a possible HCC therapeutic target.

| | Transfection and cell culture
Human liver cell (LO2), HCC cells (Huh7, Hep3B, MHCC97H and HCCLM3) and HEK293 cell, which was bought from the Shanghai Chinese Academy of Sciences cell bank for use in this work.At 37°C, 5% CO2, cells were grown in the appropriate medium with penicillin G, streptomycin, and fetal bovine serum (Sigma Aldrich).Sangon Biotech provided the pcDNA3.1-circ0093335,sh-circ0093335, miR-338-5p mimic and negative controls (NCs) for cell transfection.
Transfection was conducted with Lipofectamine TM 3000 (Invitrogen) under the kit manuals.
Sequence of sh-circ0093335: C CTA AT A C TT TCC AG G A TT TTT TC A A GA GAA AA A T CC TGG AAA GTATTAGGTTTTTT.

| | Patient samples
Clinical specimens were collected from 71 HCC patients at Huashan Hospital from 2013 to 2017.For the target gene expression test, these samples were stored at −80°C.All patients' clinicopathological data was gathered.The Huashan Hospital's ethical committee issued the study the stamp of approval.All patients who had signed up provided their informed consent and agreement.

| | RNA extraction and quantitative real-time polymerase chain reaction assay(qRT-PCR)
TRIzol Regent was used for total RNA extraction of patient samples and cell lines (Invitrogen).RNA quality was tested by NanoDrop 2000 (Thermo Scientific).FastQuant RT Kit or miRNA RT Kit was used for reverse transcription (TIANGEN).Super-Real PreMix Plus Kit or the miRNA qPCR Detection Kit (TIANGEN) was used to analyse target gene expression in Roche LightCycler480.GAPDH expression and U6 expression were employed as internal control, respectively.The 2−∆∆Ct method was served for statistical analysis.

| | RNase R treatment assay
RNA was mixed with RNase R Kit (Beyotime) and incubated (15 min, 37°C) for the RNase R treatment experiments.To measure the levels of RNA expression, qRT-PCR was used.
Every well-received CCK-8 reagent addition at 0, 24, 48 and 72 h, respectively, was incubated for 1 h.Subsequently, using the MRX II absorbance reader (Dynex Technologies), the absorbance (450 nm) of each well was measured.The transwell experiments examined the capacity for cell invasion and migration.Following the addition of 200 μL of cells (adjusted to 2.5 × 10 5 cells/mL) to the transwell upper chamber (Corning) with or without matrix (Corning) and 600 μL medium (10% FBS) to the lower room.The upper was swabbed after 24-48 h of incubation.The invaded and migrating cells surface were fixed (4% polyformaldehyde) and stained (0.1% crystal violet) for 15 min, respectively.With the use of an inverted microscope (Olympus), pictures were captured.

| | Plate clone formation assay
The 6-well plate (Corning) was seeded with 400 cells, and the cells were spread equally by gently shaking the plate.Every 2-3 days, cell growth was observed with an Olympus microscope (Japan), and the media was changed often.The cells were fixed (4% polyformaldehyde), stained (0.1% crystal violet) (Beyotime) and photographed 10-14 days later.

| | Haematoxylin-eosin (H&E) and immunohistochemical (IHC) staining
Mouse tumours were fixed in paraffin and divided into 5 μm slices.a blocking solution for 15 min, the slices were incubated (4°C) overnight with antibodies.The recovery processes followed the pattern already mentioned.A microscope was used to take pictures (Olympus).Double-blind reviewers assessed the staining outcomes.

| | Nuclear and cytoplasmic extraction and fluorescence in situ hybridization (FISH)
An RNA Sub-cellular Isolation Kit was used to separate the RNA into its nuclear and cytoplasmic portions (Active Motif).To gauge the levels of RNA expression, qRT-PCR was used.

| | RNA immunoprecipitation (RIP) assay
RIP kit (Merck) was used to conduct the RIP assay.After being lysed with RIP lysis solution, cells were treated with magnetic beads (4°C, 6 h) preconjugated with either an anti-AGO2 antibody or anti-IgG antibody.After that, proteinase K was used to wash and digest the magnetic beads.To analyse pure RNA, qRT-PCR was used.

| | Dual-luciferase reporter gene assays
The circ0093335 gene was cloned and inserted into the psiCHECK-2 vector along with the appropriate mutations of the miR-338-5p.HEK293 were transfected with luciferase reporter plasmids of NC, miR-338-5p mimics, wild-type vector (WT), or mutant vector (MUT) in 6-well plates using Lipofectamine3000.
The Luciferase Reporter kit was applied to determine luciferase activity (Promega).

| | Statistical analysis
Using SPSS Inc., results are shown as mean ± SD.Graph Pad Prism7 was applied to create the graphs.The statistic was determined by the one-way analysis of variance (anova) test or the Student's t-test (paired, two-tailed).

| | circ0093335 is highly expressed in HCC
The junction site of circ0093335 (derived from chromosome 10: 22615359-22,617,627) was verified using Sanger sequencing.It consists of seven consecutive exons in the BMI1 gene (Figure 1A).cDNA and gDNA templates were taken from Hep3B and HCCLM3 cells for convergent and divergent primers designed to characterize the ring structure.The PCR results were assessed by agarose gel electrophoresis.The outcomes revealed that only cDNA, not gDNA, was amplified by divergent primers for circ0093335 (Figure 1B).Moreover, the RNase R digestion experiments revealed that circ0093335 was more stable than BMI1 (Figure 1C).
Circ0093335 expression was tested in both HCC cells and LO2.
The findings showed a considerable upregulation of circ0093335 expression in HCC cells.The expression level was relatively high in HCCLM3 cells and relatively low in Hep3B cells (Figure 1D).
Circ0093335 expression levels in 71 HCC specimens were ascertained using qRT-PCR.The findings revealed that circ0093335 was significantly upregulated in the tumour (Figure 1E).Based on the medium expression, patients were categorized into two groups.The relationships between clinicopathological variables and circ0093335 expression levels were evaluated.The findings revealed that higher expression of circ0093335 correlated with advanced tumour-node-metastasis (TNM) stage and tumour microvascular invasion (MVI) (p < 0.05; Table 1).However, no statistically significant differences in age, gender, tumour size, or hepatitis B virus infection were observed.These findings indicate that circ0093335 is elevated in HCC cell lines and tissues and plays an important role in HCC oncogenesis.

| | Circ0093335 promoted HCC progression in vitro and in vivo
The impact of circ0093335 on HCC was investigated in vitro using or circ0093335-overexpressed lentiviral vectors (Hep3B-circ0093335) were injected subcutaneously to investigate the impact of circ0093335 on mouse tumour growth in vivo.The TV was markedly increased in the Hep3B-circ0093335 group compared with the Hep3B-NC group (Figure 2E).HE staining of the tumour in each group revealed that the Hep3B-circ0093335 group had more tumour features than the Hep3B-NC group (Figure 2F).
Furthermore, the immunohistochemistry assay detected the expression of the proliferative indicator Ki-67 in each group.Ki-67 expression was relatively high in the Hep3B-circ0093335 group (Figure 2F).These results prove that circ0093335 promotes HCC tumorigenesis in vivo.

| | Knockdown of Circ0093335 inhibits HCC progression in vitro and in vivo
The impact of knockdown of circ0093335 on HCC was investi- Compared to the Hep3B-NC group, the TV was decreased in the HCCLM3-sh-circ0093335 group (Figure 3E).HE staining of the tumour in each group revealed that the HCCLM3-NC group had more tumour features than the HCCLM3-sh-circ0093335 group (Figure 3F).Furthermore, the IHC assay detected the expression of the proliferative indicator Ki-67 in each group.The Ki-67 expression was relatively high in the HCCLM3-NC group (Figure 3F).

Knockdown of circ0093335 inhibited HCC tumorigenesis in vivo,
according to these findings.

| | Circ0093335 functions as a sponge of miR-338-5p
Recently, growing evidence has shown that miRNAs can modulate the actions of other ncRNAs.The probable targets of circ0093335 were predicted using a bioinformatics method to better understand the underlying mechanism (Circinteractome: https:// circi ntera ctome.nia.nih.gov/ ) (Figure 4A).The results showed that miR-338-5p has a tumour-suppressive effect on gliomas and oesophageal squamous cell carcinomas (ESCCs).MiR-338-5p is a probable target of circ0093335, and its potential binding site was predicted (Figure 4B).Then, circ0093335 WT and MUT luciferase plasmids were synthesized, which had the binding site for the putative miR-338-5p.A nuclear and cytoplasmic extraction test was performed, which showed that circ0093335 was primarily localized in the cell cytoplasm (Figure 4C).We also conducted a FISH experiment to demonstrate that the majority of circ0093335 (red) and miR-338-5p (green) are co-located in the cytoplasm of HCC cells (Figure 4D).
MiR-338-5p expression was downregulated in OE-circ0093335 cells (Figure 4E).Moreover, circ0093335 was enriched by Ago2 over IgG antibody, suggesting that it may serve as a miR-338-5p 'sponge', based on the results of RIP experiments (Figure 4F).A dual-luciferase reporter test also consistently showed that miR-338-5p could reduce the luciferase enzyme activity in the circ0093335-WT as compared with the circ0003998-MUT (Figure 4G).These findings indicated that circ0093335 directly targeted miR-338-5p.

| | DISCUSS ION
CircRNAs are a new family of ncRNAs that lack a 5′-end cap or 3′-end poly (A) tail, are widely found in plant and animal cells in a closed loop structure. 9They can exist independently of proteins, are not affected by exonucleases, and can modulate gene expression due to their specific structure. 26Recently, growing research has demonstrated the regulatory role of circRNAs in the onset and advancement of numerous malignancies, including HCC, cell cycle, cell proliferation, apoptosis and metastasis. 27Li et al. reported that the oncogenic circRNA circ0000098 facilitated HCC advancement via the miR-383/MCUR1 axis. 16Li et al. demonstrated that the miR-148a/STX3-PTEN axis was the mechanism through which circMRPS35 triggered its carcinogenic effect in HCC. 28Liu et al.
Thus, it is still worthwhile to investigate how circRNAs regulate HCC.
This study revealed that circ0093335 expression had significantly higher levels in HCC cells and tumour specimens.According to the patient's clinical data assessment, high circ0093335 expression was linked with the TNM stage and MVI.Circ0093335-overexpressed lentiviral vectors and NC lentiviral vectors were transfected into HCC cells, and cell function studies were conducted to explore the effect of circ0093335 on HCC development.These findings revealed that circ0093335 increased HCC cell proliferation and aggressive abilities in addition to mouse tumour growth.
CircRNAs are mostly found in the cytoplasm and interact with miRNAs, binding to targeted miRNAs and inhibiting their post-transcriptional regulatory functions. 10,26To further investigate the mechanism of circ0093335 in HCC, we predicted the miRNAs that bind to circ0093335 via Circinteractome (https:// circi ntera ctome.Hsa_circ_01370008 inhibits CRC progression by spongy absorption of miR-338-5p. 37In ESCC, the level of miR-338-5p in tumour tissue, serum samples and ESCC cells were significantly downregulated.
MiR-338-5p can inhibit the migration and invasion of ESCC cells.Lin et al. showed that miR-338-5p plays an anti-cancer role in ESCC and mediates the sensitivity to cisplatin by inhibiting FERMT2. 34

2. 7
| | Xenograft tumour model BALB/c female nude mice, which are 4-6 week old, were prepared at Shanghai Model Organisms Center.Each nude mouse received a cell injection (1 × 10 7 /mouse) into the right armpit.Mice were euthanized at 35 days.The transplanted tumours were taken out, photographed and the tumour volume (TV) was assessed every 7 days.
4% paraformaldehyde was used to fix the tumour tissues of mice, which were then ethanol-dried, paraffin-embedded, cut into 4 μm slices and treated with 3% H 2 O 2 solution.After being treated with F I G U R E 1 Identification and characteristics of circ0093335 in HCC.(A) Diagram showing the creation of circ0093335 and Sanger sequencing outcomes.(B) Divergent primers detected circ0093335 in cDNA.GAPDH detection was a control.►◄convergent primers, ◄►divergent primers.(C) RNase R tests to verify circ0093335 stability.(D) Circ0093335 expression in LO2 and HCC cells.(E) Circ0093335 expression in 71 HCC specimens.*p<0.05,**p<0.01,and ***p<0.001.

TA B L E 1
Association between clinical features and circ0093335 expression of hepatocellular carcinoma patients.F I G U R E 2 Circ0093335 overexpression enhanced HCC growth.(A) The proliferation of NC or OE-circ0093335 cells was tested by CCK-8 regent.(B) The colony-forming ability of NC or OE-circ0093335 cells was determined.(C, D) Transwell assays with or without matrix was used to assess the aggressive ability of NC or OE-circ0093335 cells, scale bar and 50 μm.(E) Tumours were harvested on Day 35 after subcutaneous injection (1 × 10 7 cells, n = 4).Tumour volume (TV) was recorded every 7 days for the first 35 days following the initial injection.(F) Representative images of HE staining was carried out in two groups (Hep3B-NC and Hep3B-circ0093335), and the IHC test was used to identify Ki-67 expression, scale bar, 100 μm.*p < 0.05, **p < 0.01, and ***p < 0.001.| 4085QIN et al.

CCK- 8
and colony-forming experiments.The outcomes revealed F I G U R E 2 (Continued) F I G U R E 3 Knockdown of circ0093335 inhibits HCC progression in vitro and in vivo.(A) The proliferation of negative control (NC) or sh-circ0093335 cells was tested by CCK-8 regent.(B) The colony-forming ability of NC or sh-circ0093335 cells was determined.(C,D) Transwell assays with or without matrix was used to assess the aggressive ability of NC or sh -circ0093335 cells, scale bar, 50 μm.(E) Tumours were harvested on Day 35 after subcutaneous injection (1 × 10 7 cells, n = 4).TV was recorded every 7 days for the first 35 days following the initial injection.(F) Representative images of HE staining was carried out in two groups (HCCLM3-NC and HCCLM3-sh-circ0093335), and the IHC test was used to identify Ki-67 expression, scale bar, 100 μm.*p < 0.05, **p < 0.01, and ***p < 0.001.| 4087 QIN et al. that circ0093335 overexpression markedly facilitated the growth of Hep3B and HCCLM3 (Figure 2A, B).Additionally, the impact of circ0093335 on the mobility and aggression ability of cells was investigated using transwell experiments.The outcomes revealed that circ0093335 overexpression significantly promoted Hep3B and HCCLM3 to migrate and invade (Figure 2C, D).These results prove that circ0093335 increases HCC cell proliferation and motility.Hep3B cells transfected with NC lentiviral vectors (Hep3B-NC) in vitro using CCK-8 and colony-forming experiments.The outcomes revealed that knockdown of circ0093335 markedly inhibited the growth of Hep3B and HCCLM3 (Figure3A,B).Additionally, the impact of knockdown of circ0093335 on the mobility and aggression ability of cells was investigated using transwell experiments.Knockdown of Circ0093335 significantly inhibited Hep3B and HCCLM3 to migrate and invade, according to the findings (Figure3C,D).These results prove that knockdown of circ0093335 inhibited HCC cell proliferation and motility.HCCLM3 cells transfected with NC lentiviral vectors (HCCLM3-NC) or sh-circ0093335 lentiviral vectors (HCCLM3-sh-circ0093335) were injected subcutaneously to investigate knockdown of circ0093335 impact on mice tumour growth in vivo.
nia. nih.gov/ ) and conducted literature studies on the candidate miRNAs.The human miR-338-5p gene is located on chromosome 17 and the mature miR-338-5p length is 22 nt.It was first found in colorectal cancer (CRC) research and then reported in HCC, ESCC, breast cancer, glioma and other types of cancer.It plays a variety of biological regulatory roles in various malignant tumours.[33][34][35]Xu et al. reported that miR-338-5p expression was downregulated in hypoxic CRC cell lines and that miR-338-5p inhibition was closely related to hypoxia-induced CRC resistance.Further mechanism study found that HIF-1α mediates the inhibition of miR-338-5p, interferes with the regulation of miR-338-5p on the downstream direct target IL-6, mediates the activation of STAT3/BCL2 signalling pathway and enhances CRC resistance to oxaliplatin.36However, Yang et al.found that Hsa_circ_01370008 overexpression inhibited the expression of Ki67 and PCNA and the proliferation, invasion and EMT ability of CRC cells under normoxia.Mechanism research found that