LncRNA TUG1 functions as a ceRNA for miR‐1‐3p to promote cell proliferation in hepatic carcinogenesis

Abstract Background Hepatocellular carcinoma (HCC) is characterised by high malignancy, metastasis and recurrence, but the specific mechanism that drives these outcomes is unclear. Recent studies have shown that long noncoding RNAs (lncRNAs) can regulate the proliferation and apoptosis of hepatic cells. Methods We searched for lncRNAs and microRNAs (miRNAs), which can regulate IGF1 expression, through a bioinformatics website, and predicted that lncRNA taurine‐upregulated gene 1 (TUG1) would have multiple targets for miR‐1‐3p binding, meaning that lncRNA TUG1 played an adsorption role. A double luciferase assay was used to verify the targeting relationship between lncRNA TUG1 and miR‐1‐3p. Western blotting and qPCR were used to verify the targeting relationship between miR‐1‐3p and IGF1, and qPCR was used to verify the regulatory relationship between the lncRNA TUG1‐miR‐1‐3p‐IGF1 axis. CCK‐8 was used to detect the growth activity of miRNA‐transfected L‐O2 cells, and flow cytometry was used to detect cell cycle changes and apoptosis. Result The proliferation cycle of L‐O2 cells transfected with miR‐1‐3p mimics was significantly slowed. Flow cytometry showed that the proliferation of L‐O2 cells was slowed, and the apoptosis rate was increased. In contrast, when L‐O2 cells were transfected with miR‐1‐3p inhibitor, the expression of IGF1 was significantly upregulated, and the cell proliferation cycle was significantly accelerated. Flow cytometry showed that the cell proliferation rate was accelerated, and the apoptosis rate was reduced. Conclusion LncRNA TUG1 can adsorb miR‐1‐3p as a competitive endogenous RNA (ceRNA) to promote the expression of IGF1 and promote cell proliferation in hepatic carcinogenesis.

5-year survival rate of those with liver cancer is no more than 25%, and the prognosis is very poor. These outcomes are related to the characteristics of high malignancy, recurrence and metastasis of liver cancer, 4,5 so the initial mechanisms of liver cancer are worth exploring. Liver cancer is a polygenic disease. Different genes are involved in different signalling pathways in the development of liver cancer. 6 Therefore, identifying the key targets for targeted treatment has become a new strategy for cancer prevention and treatment.
Noncoding RNA (ncRNA) refers to RNA that is not translated into polypeptides. Small ncRNAs (sncRNAs) are shorter than 200 nt, and long ncRNAs (lncRNAs) are longer than 200 nt. Although these ncRNAs are not translated into proteins, they can regulate protein expression. LncRNAs can also affect gene expression 7 through chromosome modification, transcriptional regulation and post-transcriptional regulation. Taurine-regulated gene 1 (TUG1) is a lncRNA expressed mainly in the retina and brain and is very important for the normal development of photoreceptors in the retina. 8 Recent studies have found that lncRNA TUG1 is upregulated in a variety of cancers, including B-cell malignancy, oesophageal squamous cell carcinoma, ovarian cancer, liver cancer and osteosarcoma. [9][10][11][12] In these malignant tumours, knockout of TUG1 can inhibit tumour cell proliferation, migration and clone formation. However, the specific mechanism by which lncRNA TUG1 affects cell proliferation and apoptosis remains unknown.
Insulin-like growth factor 1 (IGF1) is an important growthstimulating factor that is mainly synthesised and secreted by hepatocytes. IGF1 regulates cell proliferation by activating the downstream cell signal regulatory protein serine/threonine kinase Akt, and its main receptor IGF1-R is also increased in liver cancer. 13 Bioinformatics studies have found that microRNA-1 (miRNA-1) can target and regulate the expression of IGF1. MiR-1-3p has a sequence antisense to IGF1 mRNA and can silence the expression of the IGF1 gene through interaction with IGF1 mRNA. LncRNA TUG1 may promote IGF1 expression by inhibiting the silencing effect of miR-1-3p on IGF1 expression. Therefore, we aimed to explore the relationship between lncRNA TUG1, miR-1-3p and IGF1 and their influence mechanism on the proliferation and apoptosis of liver cells to provide new insights into the prevention and treatment of liver cancer.

| Prediction of downstream targets of lncRNA TUG1
The interaction between RNA molecules is mediated by miRNA and is based on base complementary pairing. Therefore, the target downstream of TUG1 can be predicted according to relevant software. MiRcode and lncRNABase are bioscience software programmes used to predict the interaction between lncRNAs and miRNAs. Through the above bioinformatics software, we found that there are multiple seed regions of miR-1-3p in the TUG1 sequence, and IGF1 is an important downstream target gene of miR-1-3p.

| Cell culture
The human hepatic cell line L-O2 was obtained from Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. The cell lines were cultured in DMEM (Lonza, Walkersville, MD, USA) with 10% foetal bovine serum.

| Luciferase assay
For the luciferase assay, 293T cells were seeded at 50,000 cells per well in a 24-well plate. After 24 h, the cells in each well were transfected with a mixture of 0.5 µg of luciferase constructs and 0.5 µl of miR-1-3p precursor mimics or control mimics plasmid. At 48 h after transfection, the cells were lysed, and the luciferase activity was measured on a luminometer using a dual-luciferase reporter assay system (Promega, Shanghai, China) according to the manufacturer's instructions. The luciferase activity was normalised to firefly luciferase activity. The experiments were performed in triplicate and independently repeated at least three times.

| Western blot assay
Total protein was extracted from lysed cells. SDS-PAGE (10%) was used to fractionate 20 µg of the protein samples, which were subsequently transferred to polyvinylidene fluoride membranes. The membranes were blocked with 5% skim milk in TBST for 1 h and incubated with primary antibodies overnight at 4°C. After washing, the membranes were incubated with the secondary antibodies for 1 h at room temperature. Protein signalling was detected using an enhanced chemiluminescence detection system (Tanon, Shanghai, China). The primary antibody was diluted with the newly prepared blocking solution. The dilution ratios of β-actin (Solarbio, Beijing, China) and IGF1 (Abcam, Shanghai, China) antibodies were 1:5000 and 1:1000, respectively. The secondary antibodies corresponded to each protein primary antibody (mouse secondary antibody 1:3000, rabbit secondary antibody 1:3000).

| Quantitative real-time PCR assay
Total RNA was extracted from transfected target cells by TRIzol, and the concentration of extracted RNA was determined by UV analysis. RNA was reverse transcribed into cDNA. To determine the best primer annealing temperature and template amount, an RT-PCR pre-experiment was carried out; the PCR mix was prepared and centrifuged based on an Axygen kit. The above samples were placed into an IQ5 fluorescence quantitative PCR instrument and the SYBR green fluorescence quantitative PCR method was used to analyse the expression of each gene. The design of the miRNA reverse transcription primers and qPCR primers is shown in Table 1.

| CCK-8 assay
Endothelial progenitor cells (EPCs) were seeded into 96-well plates at a density of 1 × 10 3 cells/well, and 100 μl of endothelial growth medium (EGM-2) was added to each well. At 24, 48, 72, 96 and 120 h after inoculation, 10 μl of CCK-8 reagent was added to each well, and the plate was incubated at 37°C for 2 h. The absorbance at 450 nm (630 nm as a reference) was measured using a microplate reader.

| Flow cytometry
The transfected L-O2 cells in each group were collected, fixed with 70% ethanol, centrifuged at 1000 r/min to remove ethanol and rinsed once with PBS. 300-500 µl PI solution containing 10 µg/ml RNase was then added to resuspend the cells. After incubation in the dark for 30 min, the cell cycle and apoptosis were detected by a BD FACSAria cell sorter (Beckton Dickinson, San Jose, CA, USA).

| Statistical analysis
The SPSS 19.0 software (SPSS, Chicago, IL, USA) was used for statistical analysis. All values are presented as the mean ± standard deviation (SD) of three or more independent replicates. 9 The standard t-test was performed to compare the differences. *p < 0.05 and **p < 0.01 indicate significant differences.

| IGF1 is an important downstream target gene of lncRNA TUG1
The downstream targets of lncRNA TUG1 were predicted by using miRcode and lncRNABase. The results showed that there were multiple seed regions of miR-1-3p in the TUG1 sequence and that IGF1 was an important downstream target gene of miR-1-3p. Therefore, we constructed the lncRNA TUG1-miR-1-3p-IGF1 regulatory network.

| Luciferase reporter assays showed the binding of lncRNA TUG1 with miR-1-3p
To verify the binding of lncRNA TUG1 to miR-1-3p, we constructed 3´-UTR mutant and wild-type lncRNA TUG1 luciferase reporter gene vectors. The luciferase reporter gene detection results showed that miR-1-3p mimics could significantly reduce the luciferase activity of the wild-type luciferase reporter gene but could not reduce the luciferase activity of the mutant luciferase reporter gene ( Figure 1A).

| LncRNA TUG1 regulates the expression of miR-1-3p and IGF1
The above results confirmed the targeting relationship between lncRNA TUG1 and miR-1-3p. After transient transfection of lnc-TUG1 siRNA into L-O2 cells, the gene expression levels of miR-1-3p and IGF1 were detected by qPCR. The results showed that in L-O2 cells, with the downregulation of lncTUG1, the expression of TA B L E 1 Primer sequences for miRNA reverse transcription and qPCR

| Inhibition of miR-1-3p can increase the expression of lncRNA TUG1 and IGF1
From the above results, it can be seen that lncRNA TUG1 can regulate the expression of miR-1-3p and IGF1. After transfecting miR-1-3p inhibitor and mimics into L-O2 cells, we found that the expression level of miR-1-3p in the mimics group was significantly higher than that in the mimics NC group and that the expression level of miR-1-3p in the inhibitor group was significantly lower than that in the inhibitor NC group (Figure 2A). We further compared the expression changes in downstream genes; after miR-1mimics were transfected into L-O2 cells, the expression level of IGF1 was significantly downregulated with the increase in miR-1-3p, and TUG1 expression was downregulated ( Figure 2B,D). After the miR-1 inhibitor was transfected into L-O2 cells, compared to that in the inhibitor NC group, the expression level of IGF1 was significantly upregulated in the inhibitor group with the decrease in the miR-1-3p level, and TUG1 expression was upregulated ( Figure 2C,E).
We further detected the protein expression of miR-1-3p inhibitor-transfected L-O2 cells with western blotting (WB). The expression level of IGF1 in the inhibitor group was significantly higher than that in the inhibitor NC group, and the expression level of IGF1 in the mimics group was significantly lower than that in the mimics NC group (Figure 2F,G).

| Biological function of lncRNA TUG1 in hepatic cells
Data from Figures 1 and 2 verify the regulatory relationship of the lncRNA TUG1-miR-1-3p-IGF1 axis. After transfecting miR-1-3p mimics and inhibitors into hepatic cells (L-O2 cells), we detected the cell growth activity with CCK-8. What stood out in Figure 3A was that the proliferation of hepatic cells in the mimics group was slower than that in the mimics NC group, and the proliferation of hepatic cells in the inhibitor group was higher than that in the inhibitor NC group. We

| DISCUSS ION
LncRNA TUG1 is a lncRNA with 6.7 KB nucleotides. One interesting finding is that LncRNAs can act as ceRNAs to reduce the concentration of miRNAs, resulting in the inhibition of miRNA function in cells. 14,15 At present, the mechanism of ceRNAs is the most discussed mechanism in lncRNA TUG1. Li J et al. 16 reported that lncRNA TUG1 adsorbs miR-132 as a ceRNA to regulate the Hedgehog pathway to promote hepatoma cell proliferation and inhibit apoptosis. In oral squamous cell carcinoma cells, it was found that lncRNA TUG1 promotes cell proliferation and invasion by inhibiting cell mir219 or inducing the expression of FMNL2. 17 In bladder cancer cells, lncRNA TUG1 increases ZEB2 expression by inhibiting miR-142 and inhibits the Wnt/β-catenin pathway to promote cell proliferation and inhibit apoptosis. 18 For non-small-cell lung cancer, lncRNA TUG1 can enhance the sensitivity of cancer cells to chemotherapeutic drugs by interfering with miR-221-dependent PTEN inhibition. 19 In addition, Gao et al. 20  analysis, lncRNA TUG1 has multiple targets with a high binding fraction that can bind to miR-1-3p, that is, lncRNA TUG1 plays an adsorption role. Therefore, plasmids lncTUG1 gene 3′-UTR(WT-1) and lncTUG1 gene 3′-UTR (mu-1) were designed to transfect 293T cells.
The results showed that for the psiCHECK2-TUG1-3'UTR-WT transfection group, the Rluc/Luc fluorescence ratio of the mimics miR-1-3p transfection group was lower than that of the mimics NC group; for the psiCHECK2-TUG1-3'UTR-MU transfection group, there was no significant difference in the Rluc/Luc fluorescence ratio between the mimics miR-1-3p transfection group and the mimics NC groups.
Therefore, we believe that there is a targeted relationship between the lncRNA TUG1 gene and miR-1-3p. respectively. After verifying the role of the lncRNA TUG1-miR-1-3p-IGF1 regulatory axis, CCK-8 was further used to detect the cell growth state, and flow cytometry was used to detect the cell cycle and apoptosis rate. Compared to those in the mimics NC group, the cell proliferation cycle was significantly slowed down and the apoptosis rate was increased in the miR-1 mimics group. However, the cell proliferation cycle was significantly accelerated, and the apoptosis rate was decreased in the miR-1 inhibitor group, compared to those in the inhibitor NC group. Based on the above experimental results, this study demonstrated the regulatory axis of lncRNA TUG1-miR-1-3p-IGF1, verified its regulatory effect on the proliferation and apoptosis of hepatic cells, and provided a new target for and insight into the prevention and treatment of hepatoma. However, due to funding and time constraints, this study only explored the interaction between lncRNA TUG1, miR-1-3p and IGF1 at the cellular level and did not verify this ceRNA mechanism at the animal level. This project will be included in our next research plan.

CO N FLI C T O F I NTE R E S T
The authors declare no conflicts.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.