miR‐203a‐3p.1 targets IL‐24 to modulate hepatocellular carcinoma cell growth and metastasis

Hepatocellular carcinoma (HCC) is one of the most common causes of cancer‐related death. Cytokines, including interleukin 24 (IL‐24), play an important role in HCC. IL‐24 inhibits HCC metastasis but the molecular mechanism by which this occurs is still unknown. MicroRNAs (miRNAs) are regulators of cancers including hepatocellular carcinoma (HCC). However, the role that miRNAs play in the regulation of IL‐24 in HCC is unclear. The aim of this study was to investigate the effects of regulation of IL‐24 by miR‐203a‐3p.1 on liver cancer cell proliferation and metastasis. IL‐24 mRNA and miR‐203a‐3p.1 were detected by real‐time RT‐PCR, and IL‐24 protein in the cell growth medium was measured by ELISA. A luciferase assay was used to verify that the IL‐24 gene was the target of miR‐203a‐3p.1. Cell survival ability was detected by the MTT assay and colony formation. Cell metastasis was assayed by the Transwell system. The results showed that IL‐24 could be down‐regulated by miR‐203a‐3p.1 in HCC cells and that miR‐203a‐3p.1 acted as an onco‐miRNA by targeting IL‐24. Inhibition of miR‐203a‐3p.1 in cells could lead to the reversal of HCC cell proliferation and metastasis. The study highlights a novel molecular interaction between miR‐203a‐3p.1 and IL‐24, which indicates that IL‐24 and miR‐203a‐3p.1 may constitute potential therapeutic targets for HCC.

IL-24 plays important roles in tumor suppression. miRNAs are non-coding RNAs of about 20 nucleotides in length, which are play important roles in gene regulation [18,19]. However, the regulation of IL-24 by miRNAs in HCC is unclear and the purpose of this study was to explore this question. Based on the predicted results from TARGETSCAN online, we found that miR-203a-3p.1 was one of the miRNAs that regulated IL-24 expression in HCC cells. In the present study, we show that IL-24 mRNA and protein were downregulated by miR-203a-3p.1 in HCC cells and miR-203a-3p.1 acted as an onco-miRNA by targeting IL-24. Inhibition of miR-203a-3p.1 in cells could lead to inhibiting cell growth and metastasis. These data highlight a novel molecular interaction between miR-203a-3p.1 and IL-24, which indicates that IL-24 and miR-203a-3p.1 may constitute potential therapeutic targets for HCC.

HCC tissues
Informed consent was provided by the patients, and the research procedures were approved by the Ethics Committee of Dalian Municipal Central Hospital. HCC tissues and their adjacent normal tissues as the controls were obtained from The First Affiliated Hospital (Luoyang, China).

Quantitative real-time PCR
Total RNA, including mature miRNA, was isolated from the cell lines and tissues using Trizol (Life Technologies), and the RNA was quantified by spectrophotometry (Nanodro Ò ND-1000) according to the manufacturer's instructions. Reverse transcription of the miRNA was performed using a miScript Reverse Transcription kit (Qiagen, Germantown, MD, USA) starting from 1 lg of total RNA. TaqMan miRNA assays kits (Life Technologies) were used to examine the specific miRNA expression by quantitative real-time PCR (qRT-PCR) according to the manufacturer's protocol. The qRT-PCR results, which were recorded as threshold cycle numbers (C t ), were normalized against an internal control (U6 RNA), and the comparative threshold cycle method (DDC t ) was used to determine the levels of miRNA expression. miRNA mimics and vector construction miR-203a-3p.1 (forward 5 0 -TGCTGCTAGTGGTCCTAA ACATTTCACGTTTTGGCCACTGACTGACGTGAAAT GTAGGACCACTAG-3 0 ; reverse 5 0 -CCTGCTAGTGGT CCTACATTTCACGTCAGTCAGTGGCCAAAACGTG AAATGTTTAGGACCACTAGC-3 0 ) was synthesized. Lentiviral particles were obtained using the BLOCK-iT TM PolII miR RNAi Expression Vector kit with EmGFP (Thermo Fisher Scientific, Waltham, MA, USA). In brief, a small hairpin sequence corresponding to miR-203a-3p.1 was cloned into the pLenti6/V5-DEST vector, which was then packaged into replication-incompetent lentiviral particles in HEK293FT cells by co-transfecting pLenti6/V5 plasmid with the ViraPower Packaging Mix. Viral particles were collected 48 h post-transfection in the supernatant for transfection. Several clones were generated by limiting dilutions under blasticidin selection at 10 lgÁmL À1 (Thermo Fisher Scientific). IL-24 lentivirus vector was provided by Dr L. Wang. The package of IL-24 lentivirus was the same for miR-203a-3p.1.

Luciferase assay
For measuring the effect of miR-203a-3p.1 on the 3 0 -UTR of IL-24, we generated a luciferase expression construct containing part of IL-24 3 0 -UTR. We amplified the wildtype fragment IL-24 mRNA that contained potential miR-203a-3p.1 binding sites. The PCR fragment was inserted into the pGL4 Basic Vector (Promega, Madison, WI, USA) using the KpnI/XhoI endonuclease restriction sites. Mutation of the IL-24 3 0 -UTR (Mut) was performed using a mutation kit (Stratagene, La Jolla, CA, USA). For luciferase activity assays, cells were co-transfected with 100 ng of wild-type or MUT1 and MUT2 IL-24 3 0 -UTR and 100 nM miR-203a-3p.1 or control mimics using Lipofectamine 2000. Luciferase activity was assayed using a luciferase assay kit from Promega referring to the manufacturer's protocol; after 48 h transfection, luciferase activity was measured and normalized to Renilla luciferase activity.

MTT assay
The proliferation of HCC cells was examined by 3-(4,5dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay. A quantity of 2 9 10 3 cells was seeded onto a 96-well plate, and at different time points 10 lL MTT solution (5 mgÁmL À1 , Sigma-Aldrich, St. Louis, MO, USA) was added to each well and the cells were cultured for 4 h. After the incubation, the supernatant was discarded and 150 lL dimethyl sulfoxide was added to each well until the crystals dissolved completely. The absorbance was measured using an ELISA reader.

Statistical analysis
All analyses were performed using the SPSS 16.0 statistical software package (SPSS Inc., Chicago, IL, USA) or Microsoft Excel. Groups of three were analyzed by one-way ANOVA. Student's t test was used to analyze data for two groups in the cell experiments. A non-parametric test was used to determine associations among clinicopathological variables. Differences between qualitative variables were compared with the chi-square test (Pearson's test) or Fisher's exact test. Continuous parameters were presented as the mean AE SD. Every experiment was completed independently at least three times. A P value <0.05 was considered significant.

miR-203a-3p.1 expression in clinical HCC samples
Firstly, the expression of miR-203a-3p.1 in HCC samples was examined by real-time RT-PCR. The levels of miR-203a-3p.1 were higher in HCC tissues than the normal samples ( Fig. 2A). The average levels of miR-203a-3p.1 in HCC tissues were analyzed and the result showed that the levels of miR-203a-3p.1 in the tumor tissues were higher than in the normal tissues (Fig. 2B). These results suggested that miR-203a-3p.1 may promote HCC progression.

High miR-203a-3p.1 expression in HCC cells increases cellular growth
To explore the role of miR-203a-3p.1 in cellular growth, Huh7 and Hep3B cells were transfected with miR-203a-3p.1 or the control for cell survival assays. The MTT assay was used to examine cell proliferation, and the data showed that miR-203a-3p.1 could promote cell growth in Huh7 and Hep3B cells (Fig. 3A,B). We used a colony formation assay to evaluate cell proliferation, and the results showed that miR-203a-3p.1 could significantly increase colony formation rates in Huh7 and Hep3B cell lines (Fig. 3C,D).

Discussion
The gene for IL-24 has been reported as a tumor suppressor and IL-24 plays prominent roles in various cancers such as by inhibiting tumor growth, invasion and metastasis and promoting cell death. When miR-NAs bind to the target genes, the genes' expression is down-regulated at post-transcriptional levels. But IL-24 regulation by miRNAs in cancer has lacked investigation. The present research verified that IL-24 was down-regulated in HCC and its expression, cellular survival and metastasis were regulated by miR-203a-3p.1 in HCC cells.
It was shown that IL-24 is a selective anti-cancer agent regulating endoplasmic reticulum stress. IL-24 inhibits angiogenesis of cancer by suppressing vascular endothelial growth factor, basic fibroblast growth factor, transforming growth factor, and so on. IL-24 could inhibit tumor metastasis by down-regulating genes for CD44 and matrix metallopeptidase 9 and other metastasis associated genes [13][14][15][16][17]. IL-24 is regulated by miRNAs such as miR-205 in oral cancer [11] and prostate cancer [20]. IL-24 was also found to be regulated by miR-203 but the type of miR-203 was not  mentioned [21]. In another report, it was shown that IL-24 regulates miR-221 expression in cancer cells [22]. In this study, we identified that IL-24 was regulated by miR-203a-3p.1 in HCC cells.
The recent studies verify the important regulatory roles of miRNAs in HCC cell proliferation and metastasis. Some of the miRNAs in HCC are up-regulated and others are down-regulated. In our study, we identified that miR-203a-3p.1 was up-regulated in most HCC tissues used in this study and this was related to HCC metastasis. The cellular function analysis showed that miR-203a-3p.1 promoted HCC cell proliferation, migration and invasion by targeting IL-24, which indicates miR-203a-3p.1 is an onco-miRNA in HCC. There are no reports showing the role of miR-203a-3p.1 in HCC and other diseases. A report showed that miR-205 up-regulates IL-24 in KB oral cancer [10]. More studies such as ones using miRNA array need to be carried out to investigate IL-24 regulation by miRNAs.
In summary, the data presented in this study elucidate that miR-203a-3p.1 acts as on onco-miRNA by targeting IL-24. It is possible that miR-203a-3p.1 would be a therapeutic target for HCC. We hope that the combination of IL-24 and inhibiting miR-203a-3p.1 will be a good way to treat HCC in the future.