LncRNA-HNF1A-AS1 functions as a competing endogenous RNA to activate PI3K/AKT signalling pathway by sponging miR-30b-3p in gastric cancer

Accumulating evidence demonstrated that long noncoding RNAs (lncRNAs) played important regulatory roles in many cancer types. However, the role of lncRNAs in gastric cancer (GC) progression remains unclear. RT-qPCR assay was performed to detect the expression of HNF1A-AS1 in gastric cancer tissues and the non-tumourous gastric mucosa. Overexpression and RNA interference approaches were used to investigate the effects of HNF1A-AS1 on GC cells. Insight into competitive endogenous RNA (ceRNA) mechanisms was gained via bioinformatics analysis, luciferase assays and an RNA-binding protein immunoprecipitation (RIP) assay, RNA-FISH co-localisation analysis combined with microRNA (miRNA)-pulldown assay. This study displayed that revealed expression of HNF1A-AS1 was associated with positive lymph node metastasis in GC. Moreover, HNF1A-AS1 significantly promoted gastric cancer invasion, metastasis, angiogenesis and lymphangiogenesis in vitro and in vivo. In addition, HNF1A-AS1 was demonstrated to function as a ceRNA for miR-30b-3p. HNF1A-AS1 abolished the function of the miRNA-30b-3p and resulted in the derepression of its target, PIK3CD, which is a core oncogene involved in the progression of GC. This study demonstrated that HNF1A-AS1 worked as a ceRNA and promoted PI3K/AKT signalling pathway-mediated GC metastasis by sponging miR-30b-3p, offering novel insights of the metastasis mechanism in GC.


INTRODUCTION
Gastric cancer (GC) is one of the most common carcinomas and is the fourth leading cause of cancer-related deaths globally. 1 Prognoses are poor, and recurrence rates are high for the patients with GC due to tumour progression. 2 Thus, developing effective targeted therapies for successful intervention is vitally important. 3 Invasion and metastasis are essential processes for tumour and progression. However, the underlying molecular mechanisms that mediate invasion and metastasis have yet to be fully clarified.
Long noncoding RNAs (LncRNAs) are transcripts that lack proteincoding potential, and are at least 200 nucleotides in length. 4 Recently, emerging evidence has implicated that lncRNAs played important roles in multiple biological functions, including proliferation, apoptosis, metastasis, angiogenesis and lymphangiogenesis. [5][6][7] LncRNAs mediate multilevel regulation of gene expression, including transcriptional regulation by recruiting chromatin-modifying complexes and post-transcriptional regulation by directly binding to microRNAs, mRNAs or proteins. 8 Some lncRNAs were identified as competing endogenous RNAs (ceRNAs) to sponge miRNAs, thus regulating cell biological functions. 9 For example, LncRNA SNHG7, functioning as a ceRNA, enhances GALNT1-induced proliferation and metastasis via sponging miR-216b in colorectal cancer. 10 Among all known cancer-related lncRNAs, HNF1A-AS1 originally was shown to be involved in human cancer development, and was identified as a potential biomarker. 11,12 Yang et al. reported that HNF1A-AS1 was involved in oesophageal tumorigenesis by promoting cancer cell proliferation, invasion and metastasis via modulation of chromatin and nucleosome assembly as well as H19 induction. 12 Fang et al. demonstrated that HNF1A-AS1 acted as an oncogene in colon cancer in part through serving as a ceRNA to modulate miRNA-34a expression, subsequently repress miR-34a/SIRT1/p53 feedback loop and then activate canonical Wnt signalling pathway. 13 In our previous study, we found that EGR1-activated HNF1A-AS1 transcription promoted cell growth and cell cycles in GC, and that it could also increase GC cell migration abilities. 14 At present, whether HNF1A-AS1 was dysregulated in GC needs to be addressed, as well as the association between HNF1A-AS1 expression and the clinicopathological parameters of the patients with GC. In addition, since the complexity of the internal and external environments in organisms is very complex and cannot be captured by in vitro models, whether HNF1A-AS1 can promote GC metastasis in vivo was investigated. Moreover, the underlying mechanism by which HNF1A-AS1 contributes to the GC metastasis is explored.
www.nature.com/bjc Real-time quantitative RT-PCR Total RNA from gastric tissue samples and cancer cells was extracted using Trizol reagent (Invitrogen, Carlsbad, CA, USA) in accordance with the manufacturer's protocol. Total RNA was reverse-transcribed to cDNA using a Reverse Transcription Kit (Toyobo Co., Ltd., Japan). Quantitative PCR was performed using SYBR Green (Roche Applied Science, Indianapolis, IN, USA). The expression level of each specific gene was normalised to that of GAPDH. The All-in-One TM miRNA qRT-PCR Detection Kit (Genecopoeia, USA) was used for miRNA RT-qPCR. The expression level of miR-30b-3p was detected using miR-30b-3p-specific primer (Genecopoeia). U6 small nuclear RNA was used as the internal control. The relative gene expression was calculated using the 2 -ΔΔCt method.
Dual-luciferase reporter assay MKN-45 and BGC-823 cells were co-transfected with wild-or mutant-type pmiRGLO plasmid and miRNA mimics using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). Forty-eight hours after transfection, the relative luciferase activity was measured using the dual-luciferase reporter system as previously reported. 15 Western blotting The anti-PIK3CD antibody (1:500 dilution), anti-PIK3R1 antibody (1:1000 dilution), anti-AKT1 antibody (1:1000 dilution), anti-AKT2 antibody (1:300 dilution), anti-AKT3 antibody (1:500 dilution) and anti-β-actin antibody (1:1000 dilution) were incubated with polyvinylidene difluoride membranes at 4°C overnight. β-actin was used as an endogenous control. A previously described detailed procedure is available. 16 Enzyme-linked immunosorbent assay (ELISA) Cell culture supernatants of MKN-45 and BGC-823 cells that were transiently transfected with the pcDNA3.1-HNF1A-AS1 and pcDNA3.1 were collected and centrifuged at 3,000 rpm for 10 min. VEGF-C and VEGF-A secretion levels were detected via ELISA according to the manufacturer's protocol (Multi Sciences Biotech, Co., Ltd., Hangzhou, China). Migration and invasion in vitro assays of GC cells The migration and invasion abilities of GC cells were observed in both Matrigel assay-coated and uncoated Transwell chambers. A detailed procedure is described in a previous study. 17 Migration, proliferation and tube-formation assay in HUVECs and HLECs Migration, proliferation, and tube-formation assay in HUVECs and HLECs were performed as described in previous study. 17,18 RNA-binding protein immunoprecipitation (RIP) assay RNA immunoprecipitation was performed using the EZ-Magna RIP RNA-binding protein immunoprecipitation kit (Millipore, Billerica, MA, USA) and the Argonaute2 (Ago2, Millipore) antibody in accordance with the manufacturer's protocol. The cells were lysed using RIP lysis buffer. A 100-μL volume of wholecell lysate was incubated with RIP buffer containing magnetic beads conjugated with human anti-Ago2 antibody (Millipore) and normal mouse immunoglobulin G (IgG, Millipore), which served as a negative control. Samples were incubated with Proteinase K buffer and immunoprecipitated RNA was then extracted. RT-qPCR analysis, using Fos and HNF1A-AS1 primers, was performed to identify the presence of binding targets in the co-precipitated RNAs.

Mouse tumour xenograft experiments
Four-week-old male Nu/nu athymic nude mice were obtained from the National Laboratory Animal Center (Weitonglihua Biotechnology, Beijing, China) and were maintained under SPF room conditions for feeding and observation. The mice were randomly divided into four groups. MKN-45 cells were transfected with lentivirus vector LV5-GFP-HNF1A-AS1 or LV5-GFP-Negative control (GenePharma Co., Ltd., Shanghai, China). The titre of lentivirus was 1 × 10 9 TU/ml. HNF1A-AS1 was initiated by the EF-1a promoter, with the full sequence map for LV5 shown in Supplementary Fig. 1a. A total of 2 × 10 6 LV5-HNF1A-AS1 or LV5-NC cells were then injected into the lateral tail vein or the axillary fossa of each mouse (n = 6 mice per group) on 18 September 2017. To ameliorate pain to the mice throughout experimental studies, nasal anaesthesia (isoflurane) was introduced. The benefits of isoflurane anaesthesia for animals are that the anaesthesia depth was easy to control, and isoflurane did not affect body metabolism. Mice were anaesthetised using an anaesthetic machine (Kodak, Rochester, USA) with MAC 1.6% isoflurane. All experiments were performed inside a biosafety cabinet during the animal's light time cycle on the first floor of the Experimental Animal Room at Shandong University. The mice were killed by cervical dislocation, and tumour, lung, liver and kidney were isolated from the mice for further analysis. All of the animal experiments were conducted according to the Guidelines for Animal Health and Use (Ministry of Science and Technology, China, 2006). Animal experiments were approved by the Committee for Animal Protection and Utilisation of Shandong University.
Immunohistochemistry for CD-34 Immunohistochemical (IHC) analysis for CD-34 was performed to determine the amount of blood isolated from the mouse xenograft tumour tissues. Procedures were performed, and the results assessed as previously reported. 19 Statistical analysis Statistical analyses were performed using GraphPad Prism Software (GraphPad Software, Inc., La Jolla, CA, USA). Significant differences between the two groups were assessed via the Student's t test. The χ 2 test was used to analyse the association between HNF1A-AS1 expression and clinicopathological parameters. Statistical significance was considered as P < 0.05.

RESULTS
High expression of HNF1A-AS1 is correlated with lymph node metastasis in GC patients HNF1A-AS1 expression was measured in human GC tissues and non-tumourous gastric mucosa tissues. HNF1A-AS1 in gastric cancer has been found to be significantly overexpressed compared with human non-tumourous gastric mucosa tissue. Further upregulation of HNF1A-AS1 was observed in GC cases with lymph node metastasis (LNM) when compared with GC cases without LNM (Fig. 1a). The expression of HNF1A-AS1 was classified as high or low expression based on a median score. The clinicopathological assay revealed that higher HNF1A-AS1 expression in GC tissues was remarkably correlated with positive LNM (P = 0.0262), but there was no correlation between HNF1A-AS1 expression and tumour size (P = 0.2308), age (P = 0.7029), gender (P = 0.4158) or differentiation (P = 0.1384) ( Table 1). A receiver-operating characteristic (ROC) curve assay was performed to evaluate whether HNF1A-AS1 expression could be used to distinguish between patients with LNM and those without LNM. The AUC value for HNF1A-AS1, distinguishing GC cases with LNM from those without LNM, was as high as 0.7650 (Fig. 1b, 95% confidence interval (CI) = 0.6177-0.9122, P = 0.0032). These results imply that HNF1A-AS1 expression is associated with LNM and may serve as a biomarker for predicting GC LNM.
HNF1A-AS1 promotes cell migration, invasion and metastasis in GC To further explore the biological functions of HNF1A-AS1 in GC cells, GC cells were transfected with the pcDNA3.1-HNF1A-AS1 plasmid or siRNAs against HNF1A-AS1. The overexpression and knockdown efficiency of HNF1A-AS1 was detected 48 h after transfection ( Fig. 1c, d, e, f). HNF1A-AS1 significantly promoted cell migration and invasion, while HNF1A-AS1 knockdown markedly suppressed cell migration and invasion (Fig. 1g, h). MKN-45 cells transduced with LV5-HNF1A-AS1/LV5-NC expressing green fluorescent protein/GFP (Fig. 1i) were injected into the axillary fossa of mice to investigate the functional roles of HNF1A-AS1 in vivo. RT-qPCR assay showed that HNF1A-AS1 was stably overexpressed in MKN-45 cells in vivo ( Supplementary Fig. 1b). Local invasion occurred in all of the mice with LV5-HNF1A-AS1-transfected tumours. In contrast, only two of six LV5-NC mice developed local invasion; the other four mice had tumours that were wellencapsulated with non-invasive margins (Fig. 1j). Moreover, we observed that the number of CD-34-positive cells was higher in the LV5-HNF1A-AS1 group compared with the control group by IHC analysis (Fig. 1k), indicating that HNF1A-AS1 promoted angiogenesis in GC. In addition, metastatic cancer cells were observed in the lungs of mice in the HNF1A-AS1 group, which may be due to HNF1A-AS1-mediated angiogenesis in GC (Fig. 1j). In homogeneous metastatic models, mice in the LV5-HNF1A-AS1 group had a greater number of more metastatic loci in the lungs compared with mice in the LV5-NC group (14.6 ± 3.24 loci vs. 6.9 ± 1.9 loci, respectively, Fig. 1i, l). Taken together, these results suggest that HNF1A-AS1 increased the invasion, metastasis and angiogenesis of GC in vivo.
HNF1A-AS1 promotes angiogenesis of HUVECs and lymphangiogenesis of HLECs Next, we explored the role of HNF1A-AS1 in GC angiogenesis and lymphangiogenesis in vitro. The Transwell migration assay revealed that the migration activity of HUVECs dramatically increased when cultured with conditioned medium from MKN-45 and BGC-823 cells transfected with pcDNA3.1-HNF1A-AS1 (Fig. 2a). In addition, HUVEC proliferation was significantly enhanced in the HNF1A-AS1 overexpression group (Fig. 2b).
Matrigel-based capillary tube-formation assays were subsequently performed to evaluate the role of HUVECs in tube formation. Tube-formation ability was increased in the HNF1A-AS1overexpression group compared with the control group (Fig. 2c).
In addition, our results showed that HNF1A-AS1 promoted HLEC tube formation compared with the control group (Fig. 2d). The above observations indicate that HNF1A-AS1 overexpression stimulates GC angiogenesis and lymphangiogenesis in vitro.
HNF1A-AS1 regulates PIK3CD, PIK3R1 and AKT3 expression To better examine the detailed regulatory mechanism of HNF1A-AS1 in GC metastasis, the PI3K/AKT signalling pathway that was involved in cancer cell invasion, 20  Three independent experiments were performed, and data are presented as mean ± SD. h Transwell assays indicated that HNF1A-AS1 knockdown inhibited cell migration and invasion abilities in MKN-45 and BGC-823 cells (×200). Three independent experiments were performed, and data are presented as mean ± SD. i HNF1A-AS1 was overexpressed stably in MKN-45 cells, with GFP as marker gene (left, ×200). The number of lung metastatic foci in the LV5-HNF1A-AS1 group was more than that in the LV5-NC group (right). j In total, 1.5 × 10 6 MKN-45 cells transfected with LV5-HNF1A-AS1 vector or LV5-NC vector were injected into the axillary fossa of the mice. Compared with the LV5-NC group, more stroma, muscle invasion or lung metastasis was observed in the LV5-HNF1A-AS1 group. Representative lung metastasis locus was shown in IVIS system imaging (top) and H&E staining (middle, ×100). Representative stromal invasion was shown in H&E staining (bottom, ×100). k IHC assays of CD-34 showed that the blood vessel density in LV5-HNF1A-AS1 group remarkably increased than that in the LV-NC group (×100). l In total, 2.5 × 10 6 MKN-45 cells transfected with LV5-HNF1A-AS1 vector or LV5-NC vector were injected into the tail vein of the mice. The number of lung metastatic foci in the LV5-HNF1A-AS1 group was more than that in the LV5-NC group. Representative lung metastasis loci were shown in IVIS system imaging (left), bright-field gross imaging (middle, ×100) and H&E staining (right, ×100), with the quantifiable results shown in Fig. 1i (right). *P < 0.05, **P < 0.01, ***P < 0.001. was studied. PI3K/AKT signalling pathway was highly expressed and promoted GC progression. 23,24 Furthermore, PIK3CD, PIK3R1, and AKT3, rather than AKT1 and AKT2, were observed to be notably upregulated in the HNF1A-AS1 overexpression group (Fig. 2e, f, g; Supplementary Fig. 1c, d, e), and were downregulated in the HNF1A-AS1-knockdown group at both the mRNA and protein levels ( Fig. 2h; Supplementary Fig. 1f, g, h, i, j). Recent studies showed that the activation of the PI3K/AKT pathway in tumour cells increases the secretion of VEGF-A 25 and VEGF-C. 26 VEGF-A exerts a pro-angiogenic role in human cancer, 27 and VECF-C is a well-known regulator of the lymphangiogenesis. 28 Therefore, an ELISA assay was performed to demonstrate the effect of HNF1A-AS1 on VEGF-A and VEGF-C secretion. Interestingly, the VEGF-A and VEGF-C secretion levels in the supernatants isolated from MKN-45 and BGC-823 cells transfected with the HNF1A-AS1-overexpression vector were respectively significantly improved compared with control cells (Fig. 2i, j; Supplementary Fig. 1k), indicating that PI3K/AKT signalling pathway may be involved in HNF1A-AS1-induced VEGF-A and VEGF-C secretion. Next, we demonstrated the effect of a PI3K inhibitor (NVP-BKM120) on HNF1A-AS1-mediated VEGF-A and VEGF-C secretion. Our results showed that the secretion levels of VEGF-A and VEGF-C were decreased in LV5-HNF1A-AS1 GCs with NVP-BKM120 treatment (1 μM) compared with the control cells treated only with DMSO (Fig. 2k, l). A rescue experiment was also performed to confirm that HNF1A-AS1 exerts its biological functions through PI3K/ AKT signalling. Interestingly, NVP-BKM120 treatment rescued the tube formation of HUVECs and HLECs, and the migration abilities of GCs enhanced by HNF1A-AS1 (Fig. 3a, b, c). Taken together, these results indicated that the HNF1A-AS1-induced biological functions might be mediated by PI3K/AKT signalling pathway.
suggesting that miR-30b-3p interacts with HNF1A-AS1 directly and in a sequence-specific manner. Next, we explored the effects of HNF1A-AS1 with miR-30b-3pmutated binding sites on GC cell migration and invasion abilities. Our data showed that HNF1A-AS1 with miR-30b-3p-mutated binding sites exerted only a weaker effect on GC migration and invasion abilities compared with the wild-type HNF1A-AS1 (Fig. 5d, e). In addition, a rescue experiment was then performed to explore whether HNF1A-AS1 exerts biological functions through miRNA. The migration and invasion abilities were enhanced in the HNF1A-AS1-overexpression group; however, the increased migration and invasion activities promoted by HNF1A-AS1 were reversed by miR-30b-3p mimics (Fig. 5f, g), suggesting that HNF1A-AS1 exerts its tumour-oncogene effects by repressing miRNAs in GC cells. Taken together, the above results indicate that HNF1A-AS1 acts as a ceRNA with miR-30b-3p.

DISCUSSION
In this study, we showed that the expression of HNF1A-AS1 in patients with LNM was significantly higher than in patients without LNM. Moreover, ROC curve analysis revealed that HNF1A-AS1 expression could be used to distinguish between cases with LNM and those without LNM, and thus it could be used as a biomarker to predict LNM in GC. Moreover, HNF1A-AS1 enhanced migration and invasion abilities in vitro, which is consistent with previous reports, 12 indicating that HNF1A-AS1 is a crucial oncogene in GC. Furthermore, we found that HNF1A-AS1 enhanced GC invasion, metastasis and angiogenesis in vivo. In addition, HNF1A-AS1 increased HUVEC and HLEC tube formation in vitro. It is known that angiogenesis promotes the initial development of primary malignant tumours, and is closely connected with infiltration and metastasis of cancer cells, 39 and that new lymphatic vessels formed through lymphangiogenesis are responsible for cancer metastasis. 40 Thus, we concluded that HNF1A-AS1 enhanced GC metastasis maybe due to its effect on angiogenesis and lymphangiogenesis.
To further explore the regulation mechanism of HNF1A-AS1 in GC metastasis, five genes, such as PIK3CD, PIK3R1, AKT1, AKT2 and AKT3, were chosen based on RT-qPCR and western blot results. HNF1A-AS1 increased the mRNA and protein expression of PIK3CD, AKT3 and PIK3CD, rather than AKT1 and AKT2, suggesting that HNF1A-AS1 exerts its oncogenic role by upregulating PIK3CD, PIK3R1 and AKT3. Moreover, HNF1A-AS1 increased the VEGF-A and VEGF-C secretion, and both are downstream of the PI3K/AKT signalling pathway. 26 Furthermore, the PI3K inhibitor (NVP-BKM120) treatment rescued the tube formation of HUVECs and HLECs, and the migration abilities of GCs were enhanced by HNF1A-AS1, indicating that the HNF1A-AS1-induced biological functions might be mediated by PI3K/AKT signalling pathway. Many lncRNAs have recently been demonstrated to act as ceRNAs by competitively binding miRNA-responsive elements and impacting the expression levels of miRNA targets. 41 For example, LncRNA PTAR competitively binds to miR-101-3p to regulate ZEB1 expression, and thus promote EMT and invasion-metastasis in serous ovarian cancer. 42 LncRNA UICLM acts as a ceRNA by Fig. 5 MiR-30b-3p abolished the HNF1A-AS1-mediated biological effects. a Co-localisation between HNF1A-AS1 and miR-30b-3p was observed by RNA-FISH in MKN-45 and BGC-823 cells; FAM-conjugated miR-30b-3p probes and Cy3-labelled HNF1A-AS1 probes were used for staining, followed by counterstain with DAPI (×200). b, c RT-qPCR analysis of HNF1A-AS1 or PIK3CD 3′-UTR levels in the streptavidin-captured fractions from MKN-45 cells (b) and BGC-823 cell (c) lysates after transfection with biotinylated miR-30b-3p or control NC. d, e Transwell assays demonstrated that HNF1A-AS1 with miR-30b-3p-mutated binding sites exerted a weaker effect on GC migration and invasion abilities compared with wild-type HNF1A-AS1 in MKN-45 cells (d) and BGC-823 cells (e) (×100). Three independent experiments were performed, and data are presented as mean ± SD. f, g The migration (f) and invasion (g) abilities were enhanced in HNF1A-AS1-overexpression group, while miR-30b-3p mimics reversed the promotion of migration and invasion capabilities by HNF1A-AS1 (×200). Three independent experiments were performed, and data are presented as mean ± SD. h, i MiR-30b-3p significantly inhibited migration and invasion abilities in MKN-45 (h) and BGC-823 cells (i) (×100). j Migration abilities of HUVECs were dramatically inhibited in both GC cells and transfected groups (×200). k MiR-30b-3p significantly inhibited tube-formation abilities of HUVECs in both GC cells and transfected groups (×40). All the above experiments were performed in three independent experiments, and data are presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.
sponging miR-215 to regulate ZEB2 expression and promote colorectal cancer liver metastasis. 43 RNA-FISH assays showed that HNF1A-AS1 was located in both the cytoplasm and nucleus. HNF1A-AS1 was hypothesised to function as a ceRNA by upregulating the expression of PIK3CD, PIK3R1 and AKT3. Bioinformatic analyses, luciferase assays, biotin-labelled miRNA pull-down assays and RIP assays were used to explore potential interactions between HNF1A-AS1 and miRNAs, and to confirm the direct binding capabilities of the predicted miRNA-responsive elements to the full-length HNF1A-AS1 transcript. As expected, miR-30b-3p was predicted to show complementary base pairing with HNF1A-AS1 and PIK3CD or AKT3. MiR-30b-3p significantly reduced the luciferase activity of the HNF1A-AS1 WT reporter vector. In addition, RIP assays indicated that endogenous HNF1A- has-miR-30b-3p LncRNA-HNF1A-AS1 functions as a competing endogenous RNA to activate. . . H-T Liu et al.
AS1 pulldown with anti-ago2 was specifically enriched compared with the IgG antibody. RNA-FISH showed that HNF1A-AS1 and miR-30b-3p were co-localised in the cytoplasm. MiR-30b-3p pulldown assays suggested more than a tenfold enrichment of HNF1A-AS1 in the biotin-labelled miR-30b-3p-captured fraction compared with the negative control. Furthermore, miR-30b-3p abolished the HNF1A-AS1-mediated biological effects. Similarly, PIK3CD was revealed to be a target gene of miR-30b-3p. Taken together, these data indicated that HNF1A-AS1 can serve as a ceRNA to sequester miR-30b-3p, thereby protecting the target gene, PIK3CD, from repression, and ultimately promoting tumorigenesis and progression in GC.
In summary, lncRNA-HNF1A-AS1 acts as a ceRNA that activates PI3K/AKT signalling by competitively binding to miR-30b-3p and exhibits oncogenic properties in GC (Fig. 6q). Understanding the role of HNF1A-AS1 in GC could provide a potential therapeutic target for treating GC.