LncRNA ASB16-AS1 Promotes Growth And Invasion Of Hepatocellular Carcinoma Through Regulating miR-1827/FZD4 Axis And Activating Wnt/β-Catenin Pathway.

BACKGROUND: To date, although several long noncoding RNAs (lncRNAs) are reported to regulate hepatocellular carcinoma (HCC) development, their relationship still remains elusive. ASB16-AS1 is a poorly researched novel lncRNA. We aimed to investigate its function in HCC progression.
METHODS: qRT-PCR and in situ hybridization (ISH) were used to analyze ASB16-AS1 expression in HCC tissues. CCK8, Edu incorporation and colony formation were used to determine cell proliferation. Transwell assay was used to examine migration and invasion. Luciferase reporter assay was used to analyze the interactions among ASB16-AS1, miR-1827 and FZD4.
RESULTS: Bioinformatics analysis identified ASB16-AS1 was overexpressed in HCC tissues, which was further validated by qRT-PCR and in situ hybridization (ISH). Besides, ASB16-AS1 was demonstrated to be a potential indicator for HCC prognosis. Functional studies showed ASB16-AS1 knockdown attenuated proliferation, migration and invasion of HCC cells. Mechanistically, ASB16-AS1 directly interacted with miR-1827 and promoted FZD4 expression by sponging miR-1827. Overexpressed FZD4 eventually activated Wnt/β-catenin pathway and contributed to HCC progression.
CONCLUSION: Our work is the first to identify ASB16-AS1 as an oncogene that enhances HCC progression by modulating miR-1827/FZD4/Wnt/β-catenin pathways.

Introduction

Recent study has shown hepatocellular carcinoma (HCC) becomes one of the biggest problems for human health worldwide.1 Every year, about 800,000 cases are diagnosed as HCC and it causes over 850,000 deaths per year.2 As a major type of liver cancer, its pathogenesis still remains unclear.1 Although several therapeutic methods developed, including surgery and radiotherapy, in past years, the outcomes of HCC are rather poor.3 The five-year survival rate in HCC patients is only about 30%.4 Hence, it is urgently required to investigate the in-depth mechanisms of HCC development.

Along with the advance of RNA-sequencing and bioinformatics methods, increasing long noncoding RNAs (lncRNAs) have been discovered in human transcriptome.5 LncRNA could be defined by over 200 nucleotides in length and no potential for coding protein.6 Various studies show lncRNA is involved in many pathological processes, including tumorigenesis.7 Dysregulated lncRNA expression is associated with aberrant proliferation, metastasis or differentiation of tumor cells.8 For example, upregulation of lncRNA EPB41L4A-AS2 suppresses HCC progression via increasing FOXL1 expression.9 LncRNA MCM3AP-AS1 enhances HCC cell proliferation and metastasis through modulating miR-194-5p/FOXA1 signaling.10 LincRNA-p21 aberrant downregulation in squamous cell cancer leads to tumor aggravation via STAT3 pathway.11 Obviously, lncRNAs play critical roles in cancer. And it is important to determine how lncRNA correlates with tumorigenesis.

ASB16-AS1 is a poorly characterized lncRNA. A recent study indicates that ASB16-AS1 promotes glioma development.12 Its role in other cancers remains uncovered. Here, we discovered ASB16-AS1 was aberrantly upregulated in HCC using bioinformatics screening. We validated it and further revealed that ASB16-AS1 upregulation suggested a poor prognosis. Knockdown of ASB16-AS1 led to decreased proliferation and metastasis. Mechanistically, ASB16-AS1 was demonstrated to sponge miR-1827, leading to upregulation of FZD4 and activation of Wnt/β-catenin signaling. In conclusion, we uncovered a novel mechanism of lncRNA ASB16-AS1/miR-1827/FZD4/Wnt/β-catenin signaling in HCC progression.

Materials And Methods

Human Samples

Fifty-one HCC samples and their adjacent normal controls were from The Second Affiliated Hospital of Jilin University. All tissues were stored at liquid nitrogen. Our study was approved by the Ethics Committee of The Second Affiliated Hospital of Jilin University. Our study was conducted in accordance with the Declaration of Helsinki. Written informed consent was obtained from patients.

Cell Culture

HCC cell lines and LO2 cells were obtained from the Type Culture Collection of the Chinese Academy of Sciences. Cell lines were cultured using DMEM (HyClone, Logan, UT, USA) containing 10% FBS (Hyclone) at 37°C in humidified incubator with 5% CO2.

To obtain stable knockdown cell lines, shRNAs against ASB16-AS1 (5ʹ-GGTTCTGAATCATTCAGTT-3ʹ) were cloned into GV112 vector and transfected into cells using Lipofectamine 2000 (Invitrogen) at the concentration of 2 μg per well, followed by selection using puromycin for 2 weeks.

qRT-PCR

RNA isolation along with qRT-PCR was completed using TRIzol reagent (Invitrogen) based on a previous work.13 GAPDH and U6 were the normalized controls, respectively. Relative expression was determined according to the 2−ΔΔCt method.

CCK8 Assay

HCC cells were plated into 96-well plates and cultured for described time. Then, CCK8 solution was added and cell viability was determined by measuring absorbance at 450 nm.

Ethynyldeoxyuridine (EdU) Analysis

EdU assay was completed following a previous work.14

Transwell Assay

Transwell assay was used to measure migration and invasion according to a previous work.14

Dual-Luciferase Reporter Assay

The binding site in ASB16-AS1 with miR-1827 was predicted using miRDB. And the binding site in FZD4 with miR-1827 was analyzed using TargetScan7.1. Then, the sequences of ASB16-AS1 and FZD4 3ʹ-UTR containing the wild-type (WT) or mutant (MUT) binding site with miR-1827 were inserted into pGL3 vector (Promega). Then, reporters and miR-1827 mimic or control were transfected into Huh7 cells. Forty-eight hours later, the luciferase activity was measured by using the Dual-Luciferase Reporter Assay System (Promega Corporation).

Statistical Analysis

Statistical analysis was conducted by GraphPad Prism 6 software (GraphPad Software, Inc., La Jolla, CA, USA). Results were presented as mean ± SD. Student’s t-test or one-way ANOVA was used for statistical analysis. p<0.05 was considered as statistically significant.

Results

Upregulated ASB16-AS1 Correlates With Poor Prognosis In HCC

To determine the correlation of lncRNAs with HCC, we performed bioinformatics analysis using Cancer RNA-seq Nexus tool (). We identified that ASB16-AS1 was an upregulated lncRNA in HCC tissues (Figure 1A and B) according to this database. Another data in GEPIA database also validated this phenomenon (Figure 1C). Besides, qRT-PCR showed ASB16-AS1 was also upregulated in 51 HCC tissues (Figure 1D), which was further confirmed by in situ hybridization (Figure 1E). Moreover, ASB16-AS1 level was increased in advanced stages of tumor samples (Figure 1F). Then, HCC tissues were grouped into two subgroups based on ASB16-AS1 median expression value. Kaplan-Meier analysis indicated ASB16-AS1 overexpression correlated with low survival rate (Figure 1G). Notably, the data in GEPIA database also showed ASB16-AS1 upregulation predicted low disease-free survival rate (Figure 1H). Thus, ASB16-AS1 is upregulated in HCC and may be a prognostic marker.

Figure 1

Upregulated ASB16-AS1 correlates with poor prognosis in HCC. (A) Analysis of differentially expressed lncRNAs between HCC and control tissues using Cancer RNA-seq Nexus tool (). (B) ASB16-AS1 transcripts were indicated according to Cancer RNA-seq Nexus tool. (C) ASB16-AS1 expression was analyzed using GEPIA tool (). (D) qRT-PCR analysis of ASB16-AS1 expression in 51 pairs of HCC and adjacent normal tissues. (E) In situ hybridization (ISH) for ASB16-AS1 expression in HCC and paired normal tissues. (F) Expression of ASB16-AS1 in different stages of HCC tissues. (G) Overall survival rate was analyzed based on ASB16-AS1 expression. (H) Disease-free survival rate was analyzed in HCC patients using GEPIA tool according to ASB16-AS1 expression. *p<0.05.

Effects Of ASB16-AS1 Knockdown On HCC Cells

To investigate the role of ASB16-AS1, we measured its expression in HCC cell lines. ASB16-AS1 was upregulated in tumor cell lines (Figure 2A). We then chose Huh7 and Hep3B cells for further experiments. Through shRNA, ASB16-AS1 expression was significantly decreased in these cell lines (Figure 2B). CCK8 assay showed ASB16-AS1 downregulation impaired the proliferation of Huh7 and Hep3B cells (Figure 2C). EdU incorporation and colony formation assays also achieved similar trend (Figure 2D and E). Next, cell migration and invasion was assessed by Transwell assay. Results showed that ASB16-AS1 knockdown reduced migration and invasion of Huh7 and Hep3B cells (Figure 2F and G). Hence, ASB16-AS1 is an oncogene in HCC.

Figure 2

Effects of ASB16-AS1 knockdown on HCC cells. (A) Expression patterns of ASB16-AS1 in HCC cell lines. (B) shRNA-caused knockdown of ASB16-AS1 in Huh7 and Hep3B cells was validated by qRT-PCR. (C–E) Cell proliferation was evaluated by CCK8, EdU incorporation, and colony formation assays. (F and G) Transwell assay for analysis of cell migration and invasion. *p<0.05.

ASB16-AS1 Regulates miR-1827/FZD4/Wnt/β-Catenin Pathway

The molecular mechanism was then investigated. Through bioinformatics analyses, we identified ASB16-AS1 might sponge miR-1827 and miR-1827 may target FZD4. To demonstrate it, luciferase reporter vectors were constructed (Figure 3A). Luciferase reporter assay indicated that luciferase activity of ASB16-AS1-WT or FZD4-WT reporter was reduced by miR-1827 mimic in Huh7 cells (Figure 3B and C). Furthermore, biotin-labeled miR-1827 precipitated ASB16-AS1 in HCC cells (Figure 3D) while RNA immunoprecipitation (RIP) assay suggests miR-1827 associated with FZD4 mRNA (Figure 3E). Thus, miR-1827 directly interacted with ASB16-AS1 and targeted FZD4. Furthermore, ASB16-AS1 knockdown promoted miR-1827 expression (Figure 3F). Besides, miR-1827 mimic or ASB16-AS1 knockdown led to decreased expression of FZD4 (Figure 3G). Notably, miR-1827 inhibitor could rescue the effect of ASB16-AS1 knockdown (Figure 3G), suggesting ASB16-AS1 sponges miR-1827 to upregulate FZD4. FZD4 is an activator of Wnt/β-catenin pathway in colorectal cancer.15 No evidence suggests a correlation between FZD4 and Wnt/β-catenin pathway in HCC. Thus, we speculated whether FZD4 also activates Wnt/β-catenin pathway in HCC. Interestingly, knockdown of ASB16-AS1 suppressed expression of MYC, CCND1, CCND2 and SOX4 (the classical target genes of Wnt/β-catenin pathway) in Huh7 cells (Figure 3H). However, FZD4 overexpression rescued it (Figure 3H), indicating ASB16-AS1 activates Wnt/β-catenin pathway depending on FZD4. Moreover, bioinformatics analysis suggested FZD4 was upregulated in HCC tissues and qRT-PCR further validated it (Figure 3I and J), implying FZD4 as an oncogene in HCC.

Figure 3

ASB16-AS1 regulates miR-1827/FZD4/Wnt/β-catenin pathway. (A) Strategies for construction of wild-type (WT) or mutant (MUT) ASB16-AS1and FZD4 luciferase reporter vectors. (B and C) Luciferase reporter assay using ASB16-AS1and FZD4 luciferase reporter vectors in Huh7 cells. (D) Pulldown assay indicated miR-1827 interacted with ASB16-AS1. (E) RIP assay showed miR-1827 interacted with FZD4 mRNA in Huh7 cells. (F) ASB16-AS1 knockdown increased miR-1827 level. (G) Expression of FZD4 was tested after transfection with indicated vectors. (H) Target genes (CCND1, CCND2, MYC and SOX4) of Wnt/β-catenin pathway were analyzed by qRT-PCR in Huh7 cells. (I) FZD4 expression was analyzed using UALCAN tool (). (J) Relative expression of FZD4 was analyzed by qRT-PCR in HCC tissues. *p<0.05.

FZD4 Rescues The Effects Of ASB16-AS1 Knockdown

Finally, to further determine whether FZD4 is critical for ASB16-AS1 function, rescue assays were conducted. As presented by CCK8, EdU incorporation and colony formation assays, FZD4 overexpression rescued the effects of ASB16-AS1 knockdown on proliferation (Figure 4A–C). Similarly, FZD4 restoration also promoted migration and invasion in HCC cells with ASB16-AS1 knockdown (Figure 4D and E). Summarily, ASB16-AS1 promotes HCC progression by regulating miR-1827/FZD4 and activating Wnt/β-catenin pathway.

Figure 4

FZD4 rescues the effects of ASB16-AS1 knockdown. (A–C) Cell proliferation was measured by using CCK8, EdU incorporation and colony formation assays. (D and E) Transwell assay was performed to determine migration and invasion. *p<0.05.

Discussion

As a most common tumor, HCC causes large amounts of deaths.16 Finding effective biomarkers and therapeutic targets for HCC is a pivot task. In this study, we identified ASB16-AS1 is upregulated in HCC samples. Upregulation of ASB16-AS1 indicated low survival rate. Moreover, ASB16-AS1 knockdown suppressed proliferation, migration and invasion of HCC cells.

Recently, the function of lncRNAs in tumor has been widely explored. Growing evidences show lncRNAs exert important roles in the development and progression of various cancers, including HCC.17–19 For example, lncRNA LINC00461 is upregulated in HCC and promotes cancer cell proliferation and invasion through miR-149-5p/LRIG2 pathway.20 LncRNA PVT1 regulates HCC progression via promoting autophagy.21 Overexpression of lncRNA LASP1-AS significantly promotes HCC growth and invasion by increasing expression of LASP1.22 ASB16-AS1 is a rarely investigated novel lncRNA. Only a recent study showed that ASB16-AS1 promotes progression of glioma.12 Here, we validated ASB16-AS1 was markedly upregulated in HCC tissues by bioinformatics approach and qRT-PCR detection. Furthermore, we found ASB16-AS1 expression was correlated with HCC prognosis. Then CCK8, EdU and colony formation assays demonstrated ASB16-AS1 knockdown suppressed HCC proliferation, migration and invasion. Thus, our data support ASB16-AS1 as an oncogene in HCC.

Mechanistically, lncRNAs have been reported to regulate gene expression by interacting with proteins or miRNAs.14,23 Large amounts of studies support that lncRNAs are competing endogenous RNAs (ceRNAs) for miRNAs in tumor.9,20,21 For example, lncRNA KCNQ1OT1 promotes colon cancer chemoresistance through sponging miR-34a and upregulating ATG4B.24 LncRNA MALAT1 regulates stemness of colorectal cancer cells by interacting with miR-20b-5p to promote Oct4 expression.25 In addition, lncRNA DGCR5 inhibits HCC cell proliferation and invasion via sponging miR-346 and activating KLF14.26 However, how ASB16-AS1 exerts roles remains unclear. In our study, after bioinformatics prediction, we identified ASB16-AS1 may interact with miR-1827. Through luciferase reporter assay and pulldown, their direct interaction was confirmed. Moreover, miR-1827 expression was upregulated after ASB16-AS1 knockdown. Thus, ASB16-AS1 inhibited the activity of miR-1827.

Next, FZD4 was validated to be targeted by miR-1827. And results showed that FZD4 expression was suppressed by miR-1827 or knockdown of ASB16-AS1. Thus, we found that ASB16-AS1 inhibited miR-1827 to upregulate FZD4 expression. FZD4 is an important activator of Wnt/β-catenin pathway while activation of Wnt/β-catenin pathway promotes HCC progression.15,27 However, whether FZD4 activates Wnt/β-catenin pathway is unknown. Our results showed that ASB16-AS1 increased activation of Wnt/β-catenin pathway depending on FZD4 expression in HCC. Up to date, the function of FZD4 in HCC is uncovered. We found FZD4 expression is elevated in HCC tissues, implying an oncogenic role. Thus, rescue assays were performed. We showed that FZD4 restoration effectively rescued the effects of ASB16-AS1 knockdown. Thus, our data revealed ASB16-AS1 regulates miR-1827/FZD4/Wnt/β-catenin signaling to initiate HCC tumorigenesis.

Collectively, ASB16-AS1 is upregulated in HCC and predicts poor prognosis. ASB16-AS1 overexpression promotes HCC development through regulating miR-1827/FZD4 axis and activating Wnt/β-catenin pathway. Our research provides a new mechanism regulating HCC tumorigenesis. In our study, some limitations existed. For example, in vivo animal assays should be performed to test the function of ASB16-AS1/miR-1827/FZD4 axis.