microRNA‐1914, which is regulated by lncRNA DUXAP10, inhibits cell proliferation by targeting the GPR39‐mediated PI3K/AKT/mTOR pathway in HCC

Abstract Increasing studies have confirmed that abnormally expressed microRNAs (miRNAs) take part in the carcinogenesis as well as the aggravation of hepatocellular carcinoma (HCC). However, little information is currently available about miR‐1914 in HCC. Here, we first confirmed that miR‐1914 inhibition in HCC cell lines and tumour specimens correlates with tumour size and histological grade. In a series of functional experiments, miR‐1914 inhibited tumour proliferation and colony formation, resulting in cell cycle arrest and increased apoptosis. Moreover, miR‐1914 mediated its functional effects by directly targeting GPR39 in HCC cells, leading to PI3K/AKT/mTOR repression. Restoring GPR39 expression incompletely counteracted the physiological roles of miR‐1914 in HCC cells. In addition, down‐regulation of AKT phosphorylation inhibited the effects of miR‐1914 in HCC. Furthermore, the overexpression of lncRNA DUXAP10 negatively correlated with the expression of miR‐1914 in HCC; thus, lncRNA DUXAP10 regulated miR‐1914 expression and modulated the GPR39/PI3K/AKT‐mediated cellular behaviours. In summary, the present study demonstrated for the first time that lncRNA DUXAP10–regulated miR‐1914 plays a functional role in inhibiting HCC progression by targeting GPR39‐mediated PI3K/AKT/mTOR pathway, and this miRNA represents a novel therapeutic target for patients with HCC.


| INTRODUC TI ON
Hepatocellular carcinoma (HCC) remains one of the most common malignancies and is the main cause of cancer-related deaths worldwide. 1 Despite the remarkable advancement in therapeutic strategies, 2,3 the prognosis remains unsatisfactory because only 10%-37% of patients are suitable candidates for surgery because of late-stage tumours or hepatic functional failure. 4 Thus, further exploration of the mechanisms underlying HCC progression may help provide novel therapeutic targets.
As a set of evolutionarily conserved small non-coding RNAs, mi-croRNAs (miRNAs) have been identified as post-transcriptional gene expression regulators that act via combining with complementary sequences within 3′-untranslated regions (UTRs) of corresponding protein-coding genes, eventually leading to mRNA degradation or translation inhibition. 5,6 Mounting studies have indicated that miRNAs are vital in diverse biological processes in HCC, including cell proliferation, cell cycle progression, apoptosis, differentiation and metastasis, because they can regulate tumour suppressors or oncogenes. 7,8 Therefore, specific miRNAs are known to be potential therapeutic, diagnostic and prognostic biological markers in HCC. 9 miR-1914, a newly identified cancer-related microRNA, is aberrantly expressed and has become a predictor of cancer patients' survival. 10-12 miR-1914 facilitates cancer progression by repressing the expression of nuclear factor IX in colorectal cancer (CRC), and plasma miR-1914 suppresses chemoresistance in CRC patients. [13][14][15] Moreover, miR-1914 is dysregulated in stomach cancer. However, the expression status and specific biological effects of miR-1914, as well as the underlying molecular mechanism, require further exploration in HCC.
Herein, we revealed for the first time that the level of miR-1914 was decreased in HCC and correlated with adverse prognostic features, confirming its critical role in HCC progression and potential as a target for treating HCC.

| Clinical samples and cell culture
In total, 134 HCC specimens and corresponding neighbouring non-

| Immunohistochemical staining
Immunohistochemical staining (IHC) was conducted using an SABC kit (Maxim, Fuzhou, China) according to the manufacturer's instructions. In summary, HCC tissue sections were incubated with GPR39 (1:300, Abcam, USA) overnight at 4°C and then flushed with PBS and re-incubated with biotinylated secondary antibodies (Goldenbridge, Zhongshan, China) according to the SP-IHC assay instructions. The experiment was performed according to our previously reported protocol. 18

| Western blot analysis
Proteins were separated by SDS-PAGE and transferred to PVDF membranes. The experiment was performed as previously described. 7
F I G U R E 1 miR-1914 is reduced in Hepatocellular carcinoma (HCC). Comparing the aberrant expression level of miR-1914 among (A) HCC specimens versus tumour-adjacent tissues, and (B) HCC cell lines versus LO2 cells; HCC tissues arising from different histological grade (C) and tumour size (D). *P < .05, **P < .01

| Cell proliferation, cell cycle and apoptosis detection
The procedures for EdU, colony formation, cell cycle and apoptosis analyses were performed as reported previously. 7

| Luciferase reporter assay
The 3′-UTR binding sequence of GPR39 and the associated mu-

| Statistical analysis
To avoid systemic errors, each experiment was repeated more than three times. The results are displayed as the mean ± standard deviation. Student's t test or one-way ANOVA (one-way analysis of variance) followed by LSD post hoc test was conducted to compare the differences between two groups or more than two groups, respectively, with SPSS (SPSS 18.0; SPSS Inc, Chicago, IL, USA). A P value <.05 was regarded as statistically significant.

| The expression level of miR-1914 was decreased in HCC and correlated with adverse prognostic features
To assess the expression level of miR-1914 in HCC tissues, we selected 50 pairs of tissues randomly. The expression level of miR-1914 was markedly lower in the HCC samples than in the corresponding adjacent non-tumour tissues (P < .05, Figure 1A). Likewise, the expression of miR-1914 was obviously lower in the HCC cell lines than in the physiological liver cell line L02 (P < .05, Figure 1B). We se-  Figure 1C) and large tumour size (≥5 cm, P < .05, Figure 1D).

| miR-1914 inhibits cell proliferation, cell cycle and colony formation and promotes apoptosis in HCC cells
To determine the efficacy of miR-1914 in HCC, a series of functional experiments were conducted using a lentivirus system to stably overexpress miR-1914 in MHCC-97L cells and knock down miR-1914 in Hep3B cells (Figure 2A, P < .05).
The results of EdU assays revealed that the overexpression of miR-1914 remarkably suppressed the proliferation of MHCC-97L cells (P < .05, Figure 2B). Moreover, the ectopic expression of miR-1914 significantly inhibited the number of cell colonies, as confirmed by the colony formation assay (P < .05, Figure 2C).

| GPR39 is recognized as a direct target gene of miR-1914
Target algorithms (TargetScan, miRDB and StarBase v2.0) were utilized to uncover potential targets of miR-1914. GPR39 was the predicted target ( Figure 4A). To verify this, we examined the mRNA and protein levels of GPR39 by qRT-PCR and WB assays and demonstrated that miR-1914 significantly inversely regulated GPR39 expression (P < .05, Figure 4B,C). Similarly, a dual-luciferase reporter assay showed that enhanced miR-1914 expression effectively suppressed the relative luciferase activity of wild-type (wt) GPR39 but not that of mutant (mt) GPR39 (P < .05, Figure 4D).
Moreover, in clinical samples, we discovered that the expression of GPR39 was dramatically lower in high miR-1914 expression tumours than in low miR-1914 expression tumours (P < .05, Figure 4E,F). Additionally, we determined a dramatically negative association between miR-1914 and GPR39 mRNA by Spearman's analysis in HCC samples (P < .05, Figure 4G). Furthermore, the expression of GPR39 in xenograft tissues was analysed, and the data indicated that GPR39 expression was higher in the miR-1914 knockdown group than in the miR-1914 overexpression group (P < .05, Figure 4H).

| Characterization of GPR39 status in HCC
To investigate the level of expression and the potential role of GPR39 in HCC tissues, we conducted qRT-PCR and WB assays, and the results showed that GPR39 levels were much lower in adjacent non-tumour tissues than in HCC tissues (P < .05, Figure 5A,B).
Moreover, we established stable GPR39 overexpression or knockdown cells (P < .05, Figure 5C) by lentiviral transduction. EdU assays showed that GPR39 overexpression significantly promoted the proliferation of Hep3B cells (P < .05, Figure 5D). The ability of colony formation confirmed that GPR39 up-regulation significantly increased the number of cell colonies (P < .05, Figure 5E).
In addition, GPR39 knockdown in miR-1914-silenced Hep3B cells partly inhibited the effects of anti-miR-1914 on tumour cell proliferation, colony formation, cell cycle progression and apoptosis (P < .05, Figure 6A-F). Thus, the data showed that GPR39 was a functional target of miR-1914 in HCC.

| miR-1914 and GPR39 expression in HCC indicates the prognosis
To confirm the clinical use of miR-1914 and GPR39 in HCC, we investigate their correlation with clinical features. As shown in Table 1, miR-1914 down-regulation was dramatically related to large tumour size (≥5 cm; P = .011), high histological grade and increased GPR39 levels were associated with the shortest OS and DFS (P < .05, Figure 7E,F).

| AKT phosphorylation plays a crucial role in PI3K/AKT/mTOR signalling mediated the biological effects of miR-1914 in HCC
To explore the molecular mechanisms that mediate the efficacy of miR-1914, we examined PI3K/AKT/mTOR signalling using a WB assay. As shown in Figure 8A

| miR-1914 is negatively modulated by lncRNA DUXAP10 in HCC cells
To better elucidate the underlying mechanism by which miR-1914 was depressed in HCC, we predicted the potential targets using Starbase v2.0 and discovered that lncRNA DUXAP10 is a molecular sponge that may regulate miR-1914. First, we used the GEO database to show that DUXAP10 was obviously higher in HCC samples than in normal liver tissues, and we confirmed that DUXAP10 was higher in HCC tissues than in neighbouring non-tumour tissues ( Figure 9A, P < .05).

| D ISCUSS I ON
To date, accumulative evidence has validated that abnormal miRNA expression is pivotal in HCC carcinogenesis and progression. 22  Previous studies confirmed that miRNAs serve as prognostic biomarkers and valid pharmaceutical targets for HCC. 25,26 In the present study, we corroborate that miR-1914 impedes cell prolifer-

ACK N OWLED G EM ENTS
This study was supported by grants from National Natural Science

CO N FLI C T O F I NTE R E S T
No conflicts of interest exist.

AUTH O R CO NTR I B UTI O N S
WY and ZL conceived and designed the experiments; LS, LW, TC, BY and YW performed the experiments; QL analysed the data; and LS, WY and ZL wrote the paper. All authors read and approved the final manuscript.

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.