β-catenin axis

Accepted Manuscript CircRNA circ_0067934 promotes tumor growth and metastasis in hepatocellular carcinoma through regulation of miR-1324/FZD5/Wnt/β-catenin axis Qian Zhu, Guiyu Lu, Zihua Luo, Fenfang Gui, Jinghua Wu, Dongwei Zhang, Yong Ni PII:

S0006-291X(18)30350-4

DOI:

10.1016/j.bbrc.2018.02.119

Reference:

YBBRC 39484

To appear in:

Biochemical and Biophysical Research Communications

Received Date: 8 February 2018 Accepted Date: 13 February 2018

Please cite this article as: Q. Zhu, G. Lu, Z. Luo, F. Gui, J. Wu, D. Zhang, Y. Ni, CircRNA circ_0067934 promotes tumor growth and metastasis in hepatocellular carcinoma through regulation of miR-1324/ FZD5/Wnt/β-catenin axis, Biochemical and Biophysical Research Communications (2018), doi: 10.1016/ j.bbrc.2018.02.119. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT CircRNA circ_0067934 promotes tumor growth and metastasis in hepatocellular carcinoma through regulation of miR-1324/FZD5/Wnt/β β-catenin axis Qian Zhu1, Guiyu Lu2, Zihua Luo1, Fenfang Gui1, Jinghua Wu1, Dongwei Zhang1, Yong Ni2,# 1

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Department of Gastroenterology, Shenzhen Longhua District Central Hospital, Shenzhen

518110, China 2

Department of General Surgical, the Second People’s Hospital of Shenzhen, Shenzhen

518110, China #

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Corresponding authors: Yong Ni, Department of General Surgical, the Second People’s

Hospital of Shenzhen, No. 3002, Sungang West Road, Shenzhen 518110, China. E-mail:

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[email protected]

Running title: circ_0067934 promotes hepatocellular carcinoma growth and

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metastasis

ACCEPTED MANUSCRIPT Abstract Recently, increasing evidences demonstrate that circular RNAs (circRNAs) exert very important functions in the progression of human cancers. However, the functions and molecular mechanism of circ_0067934 in hepatocellular carcinoma (HCC) are largely

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unknown. In the present study, we found that the expression of circ_0067934 was significantly upregulated in HCC tissues and cell lines compared to adjacent normal tissues. Furthermore, we showed that circ_0067934 knockdown remarkably suppressed

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the proliferation, migration and invasion of Hep3B and HuH7 cells while inducing their apoptosis. In terms of mechanism, we found that circ_0067934 directly suppressed

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miR-1324, which targeted the 3′-UTR of FZD5 mRNA and subsequently downregulated the Wnt/β-catenin signaling pathway in HCC. Through rescue experiments, we demonstrated that circ_0067934 enhanced the proliferation, migration and invasion of HCC cells by the inhibition of miR-1324 and concomitant activation of FZD5/Wnt/β-catenin signaling pathway. In summary, the circ_0067934/miR-1324/FZD5/β-catenin signaling

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axis might serve as a promising therapeutic target for HCC intervention.

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Key words: circ_0067934; proliferation; invasion; miR-1324; hepatocellular carcinoma

ACCEPTED MANUSCRIPT 1. Introduction Hepatocellular carcinoma (HCC) is one of the most common and aggressive cancers around the world [1]. HCC is the second most leading cause of cancer-related deaths among men, especially in China [2]. Although some methods, such as resection,

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transplantation or ablation, are effective for early HCC, most patients were diagnosed at advanced stages with tumor metastasis [3], which greatly contributes to patients’ poor outcomes. Therefore, novel biomarkers for the early diagnosis of HCC are urgently required. Furthermore, investigating the mechanism of HCC development and

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progression is of vital significance.

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In recent years, the functions of circular RNAs (circRNAs) have attracted large amounts of attentions in the field of biology. CircRNAs belong to noncoding RNA family and ubiquitously exist in mammalian cells [4]. Their expression is high cell-type-specific, tissue-specific and developmental-specific. Emerging studies indicate that circRNAs regulate gene expression by interacting with microRANs (miRNAs) or other molecules [5].

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Abnormal expression of circRNAs is usually observed in various cancers [6]. By regulating cell proliferation, apoptosis and migration, circRNAs are involved in the progression of cancers, such as colorectal cancer [7], cholangiocarcinoma [8] and bladder cancer [9].Some reports imply that circRNAs might be novel biomarkers for cancer diagnosis or

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prognosis [10, 11]. As a result, understanding the functional mechanism of circRNAs will

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benefit cancer therapy.

Previous study indicated that circ_0067934 promoted the proliferation of esophageal

squamous cell carcinoma cells [12]. Whether circ_0067934 regulates HCC development requires investigation. In this study, we first found that the expression of circ_0067934 was upregulated in HCC tissues and cell lines. Circ_0067934 knockdown in Hep3B and HuH7 cells significantly suppressed cell proliferation, migration an invasion. We identified circ_0067934 as a sponge for miR-1324, which directly targeted the 3′-UTR of FZD5 mRNA and downregulated the Wnt/β-catenin signaling pathway in HCC. In collection, our results demonstrated the key role of circ_0067934 in HCC progression. Therefore, the circ_0067934/miR-1324/FZD5/Wnt/β-catenin axis might be a promising therapeutic target

ACCEPTED MANUSCRIPT for HCC treatment. 2. Materials and methods 2.1. Patient samples and cell lines

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The total of 49 HCC patients, who underwent surgeries at the Second People’s Hospital of Shenzhen, were included in this study. The adjacent normal tissues were obtained from 1 cm away from the edge of the HCC; and there were no obvious tumor cells. The diagnosis of HCC was confirmed by histological examination. Staging was

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determined by the BCLC staging system [13] and American Joint Committee on Cancer criteria [14]. Patients with HCC who had prior treatment of their tumor or history of other

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solid tumors were excluded in this study. This study was approved by the Human Research Ethics Committee from the Second People’s Hospital of Shenzhen. Informed consent was obtained from all patients.

HCC cell lines (BEL7402, Hep3B, HuH7 and MHCC97-L) and human normal hepatic cell line HL-7702 were cultured with RPMI 1640 Medium (Life Technologies, Carlsbad, CA,

2.2. MiRNA transfection

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USA) containing 10% fetal bovine serum in a humidified atmosphere of 5% CO2.

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The HCC cells were seeded at six-well plates and then transfected at 24 h with specific siRNA (100 nM) or control siRNA (100 nM) using Lipofectamine RNAi MAX

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according to the manufacturer’s protocol (Invitrogen) as described before [12]. We used the

following

siRNA

sequences

5′-UGUUGAUUGGGAUAUGUUAUU-3′;

for

hsa_circ_0067934:

sense: antisense:

5′-UAACAUAUCCCAAUCAACAUU-3′. 2.3. Cell proliferation assay Cell proliferation was detected by using the Cell Counting Kit-8 (7 sea biotech, Shanghai, China). Cells were grown in 96-well plates with 1×104 cells per well and incubated at 37 °C in 5% CO2 until cell confluence reached 70%. After 48 h of plasmid transfection, cells were incubated for an additional 24, 48, 72 and 96 h. A volume of 10 µL

ACCEPTED MANUSCRIPT of CCK8 solution was added into each well. The absorbance at 450 nm was measured with the SUNRISE Microplate Reader (Tecan, Switzerland). 2.4. Flow cytometric analysis We harvested transfected cells after 24 h. The Hep3B and Huh7 cells were stained

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with Annexin V and PI using an Annexin V-FITC/PI apoptosis detection kit (BD Biosciences) before analysis by flow cytometry (FACScan; BD Biosciences). The cell cycle analysis was performed by staining cells with propidium iodide using the Cycle TEST

analyzer.

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2.5. In vitro cell migration and cell invasion assays

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PLUS DNA Reagent Kit (BD Biosciences). The cells were then analyzed using a FACScan

Twenty-four-well plates containing transwell chambers with 8-µm pore size polycarbonate membrane inserts (Corning Incorporated, Corning, NY, USA) were used for cell migration and invasion assays. For cell invasion assays, the top side of the membrane was coated with Matrigel (BD Biosciences), and 1×105 cells in serum-free DMEM or RPMI

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1640 medium were seeded in each chamber. DMEM or RPMI 1640 containing 10% FBS was added to each well under the chamber. For cell migration assays, 5×104 cells in serum-free DMEM or RPMI 1640 medium were seeded in each well on Matrigel-free

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chamber. After 24 h of incubation, cotton swabs were used to remove the cells inside the upper chamber, while the cells on the other side of the membrane were fixed and stained

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with 0.5% crystal violet solution. Five random fields were counted for each well. 2.6. Reverse transcription and quantitative real-time PCR (qRT-PCR) The RNA was extracted from tissues or cultured cells with Trizol reagent according to

the manufacturer’s protocol (Life Technologies, Scotland, UK). Then, 500 ng of total RNA was reverse transcribed in a final volume of 10 µl using random primers and standard conditions with the Prime Script RT Master Mix. Then, we performed quantitative real-time polymerase chain reaction (qRT-PCR) using the SYBR Select Master Mix (Applied Biosystems) with 0.5 µl complementary DNA (cDNA) on the ABI7300 system (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s instructions. We used

ACCEPTED MANUSCRIPT GAPDH, β-actin, and U6 as internal controls and tested hsa_circ_0067934 and FZD5 expression levels by qRT-PCR. 2.7. Luciferase reporter assay Hep3B and HuH7 cells were seeded into a 24-well plate. Cells were co-transfected

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with wild-type or mutated FZD5 reporter plasmids, and with miR-1324 mimics or controls. Luciferase assays were conducted 24 h after transfection using the Dual Luciferase Reporter Assay System (Promega, WI, USA).

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2.8. In vivo tumor growth and metastasis assays

For tumor growth in vivo, 5×106 siCirc_0067934 or control Hep3B cells were

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subcutaneously injected into the flanks of 6-week-old BALB/c nude mice from HFK Biosciences. Tumor volumes were measured at indicative time points. And tumor weights were determined 5 weeks post injection. For tumor metastasis in vivo, we intravenously injected 5×105 siCirc_0067934 or control Hep3B cells into lateral tail veins of recipient nude mice. 4 weeks later, we analyzed lung metastases. Animal experiments were

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performed in accordance with relevant guidelines and regulations of the Institutional Animal Care and Use Committees at the Second People’s Hospital of Shenzhen.

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2.9. Statistical analysis

All data are shown as mean ± standard deviation (SD). Statistical significance was

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determined using Student's t-test by SPSS 13.0 and Graphpad Prism 6. P< 0.05 was considered statistically significant. 3. Results

3.1. Expression patterns of circ_0067934 in HCC In an attempt to explore the role of circ_0067934 in HCC, we first analyzed its expression in 49 pairs of HCC tissues and adjacent normal tissues by qRT-PCR. The results indicated that circ_0067934 was highly expressed in HCC tissues compared with adjacent normal tissues (Fig. 1A). In addition, in situ hybridization also showed that circ_0067934 expression was upregulated in HCC samples (Fig. 1B). To further determine

ACCEPTED MANUSCRIPT the correlation between circ_0067934 expression and HCC severity, we examined the expression of circ_0067934 in different stages of HCC samples. As shown in Fig. 1C, circ_0067934 expression was positively correlated with HCC TNM stage. Moreover, we found that circ_0067934 displayed the highest expression level in advanced HCC

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samples (Fig. 1D). Besides, qRT-PCR analysis also indicated that circ_0067934 was overexpressed in HCC cell lines compared to HL7702 cells (Fig. 1E). Then the Kaplan-Meier analysis was used to evaluate patient survival rates relative to circ_0067934 expression levels. The results indicated that higher expression of circ_0067934 in HCC

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patients correlated with poorer prognosis (Fig. 1F). Taken together, these data indicated circ_0067934 was overexpressed in HCC cells and positively correlated with HCC

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severity.

3.2. Knockdown of circ_0067934 repressed cell proliferation, migration and invasion while inducing cell apoptosis in HCC

To further investigate the role of circ_0067934 in HCC, we knocked down

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circ_0067934 in Hep3B and HuH7 cells (Fig. 2A). Through CCK8 assays, we found that circ_0067934 silence significantly inhibited the proliferation of Hep3B and HuH7 cells (Fig. 2B). Furthermore, fluorescence-activated cell sorting (FACS) analysis showed that circ_0067934 knockdown led to fewer cells engaged in cell cycle (Fig. 2C). On the

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contrary, we measured the effect of circ_0067934 on cell apoptosis and found that circ_0067934 depletion resulted in more deaths of Hep3B and HuH7 cells (Fig. 2D).

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Additionally, using transwell assays, we demonstrated that circ_0067934 significantly reduced the migration and invasion of Hep3B and HuH7 cells in vitro (Fig. 2E and F). To further determine the effect of circ_0067934 on HCC in vivo, we performed tumor growth and metastasis assays in vivo. Every other one week post injection, we measured the tumor volumes and found that circ_0067934 knockdown significantly delayed tumor growth (Fig. 2G). After 5 weeks, we sacrificed all recipient nude mice and found that the tumor tissues from sicirc_0067934 group were smaller (Fig. 2H and I). In addition, the results of tumor metastasis assays in vivo showed that circ_0067934 silence reduced the numbers of metastatic nodules in the lung (Fig. 2J). Taken together, these data suggested

ACCEPTED MANUSCRIPT circ_0067934 knockdown suppressed HCC cell proliferation and metastasis in vitro and in vivo. 3.3. Circ_0067934 activated Wnt/β-catenin pathway via regulation of miR-1324/FZD5 axis We decided to investigate the molecular mechanism by which circ_0067934 exerted

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its function in HCC. Previous studies demonstrated that circRNAs could regulate the expression of miRNAs [15]. We looked for potential miRNAs that could be regulated by circ_0067934. Results indicated that miR-1324 was a potential miRNA target for

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circ_0067934. There is a putative binding site for miR-1324 in circ_0067934 (Fig. 3A). MiRNAs are widely acknowledged as regulators of gene expression by targeting the

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3′-UTR region of mRNAs [16]. We also searched the downstream target molecular. We found that FZD5, a positive regulator of Wnt/β-catenin pathway, is a potential target gene of miR-1324. There is also a putative binding site of miR-1324 in the 3′-UTR of FZD5 (Fig. 3B). To verify their interaction, we performed luciferase activity reporter assays and found that miR-1324 mimics inhibited the luciferase activity of Hep3B and HuH7 cells transduced

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with wide type circ_0067934 or FZD5 sequence, while mutations of the putative miR-1324 binding sites in circ_0067934 or FZD5 abolished this effect (Fig. 3C and D). Moreover, we found that knockdown of circ_0067934 significantly promoted the levels of miR-1324 in Hep3B and HuH7 cells (Fig. 3E). Consistently, overexpression of miR-1324 suppressed

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the mRNA level of FZD5 in Hep3B and HuH7 cells (Fig. 3F). FZD5 has been reported to positively activate the Wnt/β-catenin signaling pathway [17, 18], which contributes to the

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development and progression of HCC [19]. Thus, we explored the effect of circ_0067934 and miR-1324 on Wnt/β-catenin signaling pathway by western blot. The results indicated that circ_0067934 knockdown or miR-1324 overexpression significantly suppressed the protein levels of FZD5, nucleus β-catenin and Cyclin D1 in Hep3B and HuH7 cells (Fig. 3G), which suggested that circ_0067934 activated Wnt/β-catenin signaling by inhibition of miR-1324 in HCC. 3.4. Overexpression of FZD5 or inhibition of miR-1324 reversed the effects of circ_0067934 depletion

ACCEPTED MANUSCRIPT To further determine whether circ_0067934-mediated effects on HCC depends on miR-1324 and FZD5, we inhibited miR-1324 or restored FZD5 expression in Hep3B and HuH7 cells transduced with sicirc_0067934. We checked the expression levels of miR-1324 and FZD5 in indicated cell lines by qRT-PCR (Fig. 4A and B). As shown in Fig.

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4B, inhibition of miR-1324 or restoration of FZD5 rescued the expression level of FZD5 in circ_0067934-depleted Hep3B and HuH7 cells. Then we performed CCK8, apoptosis detection and transwell assays. We found that inhibition of miR-1324 significantly promoted the proliferation, migration and invasion of circ_0067934-depleted Hep3B and

expression

also

led

to

enhanced

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HuH7 cells while reduced their apoptosis (Fig. 4C-F). Similarly, restoration of FZD5 proliferation,

migration

and

invasion

of

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circ_0067934-depleted Hep3B and HuH7 cells while decreased apoptosis (Fig. 4C-F). Collectively, our data indicated that the circ_0067934/miR-1324/FZD5/Wnt/β-catenin signaling pathway axis was involved in the regulation of HCC progression. 4. Discussion

As one of the most common and malignant cancers, HCC contributes to large

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amounts of cancer-related deaths worldwide [2]. Despite some advances made on the therapeutic methods for HCC, the outcomes of patients are still very poor. The mechanisms of hepatocarcinogenesis remain largely unknown. And there is an urgent

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requirement to look for novel diagnostic biomarkers and therapeutic targets for HCC treatment. In the recent years, emerging reports indicated that circRNAs exerted

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important functions and were closely correlated with a diversity of human cancers, such as HCC [20]. Increasing evidences indicated that circRNAs might serve as biomarkers for tumor diagnosis or prognosis [4, 21]. Nevertheless, the functions of most circRNAs have not been defined in cancers, including HCC. In this study, we identified circ_0067934 as an oncogene in HCC and demonstrated its functional mechanism. More and more evidences indicated that circRNAs could regulate cell proliferation, migration, invasion and apoptosis in various cancers. For instance, Chen et al. reported that circRNA_100782 regulates pancreatic carcinoma proliferation through the IL6-STAT3 pathway [22]. Xie et al. reported that circ_0074362 levels were significantly

ACCEPTED MANUSCRIPT downregulated in gastric cancer tissues and regulated gastric cancer initiation [23]. In addition, Luo et al. showed that knockdown of hsa_circ_0000064 with siRNA dramatically attenuated the proliferation, blocked cell cycle progression, and promoted cell apoptosis in lung [24]. Previous study demonstrated that Circular RNA circ_0067934 is upregulated in

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esophageal squamous cell carcinoma and promoted proliferation [12]. However, the role of circ_0067934 in HCC remains elusive. In the present study, we found that circ_0067934 was significantly upregulated in HCC tissues and cell lines compared to normal tissues or cells. Furthermore, we found that the expression of circ_0067934 was

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positively correlated with TNM stage and HCC progression. And higher expression of circ_0067934 in HCC patients is linked to poorer prognosis. To investigate the role of

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circ_0067934 in HCC cells, we conducted CCK8, apoptosis detection and transwell assays with circ_0067934-silenced Hep3B and HuH7 cells. The results indicated that circ_0067934 knockdown significantly inhibited the cell proliferation, migration and invasion while dramatically induced apoptosis.

An accumulation of evidence demonstrated that miRNAs played an essential role in

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the regulation of tumor development and progression, such as in pancreatic cancer [25], melanoma [26], breast cancer [27] and HCC [28]. And circRNAs have been showed to regulate gene expression through association with miRNAs [15]. Therefore, we explored

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whether circ_0067934 regulated HCC progression through targeting miRNAs. Using bioinformatics analysis and a luciferase activity reporter assay, we found and verified that

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circ_0067934 directly associated with miR-1324 in HCC cells. To date, the function of miR-1324 is totally unknown. To explore the role of miR-1324 and its downstream molecular mechanism, we also analyzed its target gene and proved that miR-1324 targeted FZD5 in HCC cells. FDZ5 is a co-receptor of Wnt and could promote activation of Wnt/β-catenin signaling pathway. To determine whether circ_0067934 promoted HCC progression by eventually activating Wnt/β-catenin signaling pathway, we analyzed the activation of Wnt/β-catenin pathway. Using western blot, we demonstrated that both circ_0067934 knockdown and miR-1324 overexpression downregulated FZD5 expression and impaired the activation of Wnt/β-catenin pathway. To further verify FZD5 or miR-1324 is indispensible for the effects of circ_0067934,

ACCEPTED MANUSCRIPT we inhibited miR-1324 or overexpressed FZD5 in circ_0067934-depleted Hep3B and HuH7 cells. Using CCK8, apoptosis detection and transwell assays, we demonstrated that either inhibition of miR-1324 or restoration of FZD5 reversed the effected of circ_0067934 knockdown on HCC cells. In conclusion, our study revealed for the first time that

axis. Conflicts of interest

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The authors declare no competing financial interests.

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circ_0067934 promoted HCC progression by regulating a miR-1324/FZD5/Wnt/β-catenin

Abbreviations

Regions. Acknowledgements

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CircRNAs, Circular RNAs; HCC, Hepatocellular Carcinoma; 3′-UTR, 3′-Untranslated

This work was supported by the Shenzhen Scientific Innovation Committee Fund

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Tang, microRNA-874 suppresses tumor proliferation and metastasis in hepatocellular carcinoma by

ACCEPTED MANUSCRIPT FIGURE LEGENDS Figure 1. Expression patterns of circ_0067934 in HCC. (A) The expression of circ_0067934 was analyzed in 49 pairs of HCC tissues and adjacent normal tissues by qRT-PCR. (B) Circ_0067934 expression was determined by in situ hybridization in paired

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HCC samples. (C) Relative expression of circ_0067934 in different TNM stages of HCC tissues. (D) Relative expression of circ_0067934 in adjacent normal tissues, early HCC (eHCC) and advanced HCC (aHCC) by qRT-PCR. (E) Relative expression of circ_0067934 in HCC cell lines. (F) This panel showed the Kaplan–Meier survival curve

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relative to circ_0067934 expression levels. All data are representative of three independent experiments and expressed as the mean ± SD. *p < 0.05, **p < 0.01 and ***p

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< 0.001.

Figure 2. Knockdown of circ_0067934 repressed cell proliferation, migration and invasion while inducing cell apoptosis in HCC. (A) Relative expression of circ_0067934 in Hep3B and HuH7 cells transfected with sicirc_0067934 or control. (B) CCK8 assay showed that

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circ_0067934 knockdown suppressed the proliferation of Hep3B and HuH7 cells. (C) FACS asnalysis indicated that circ_0067934 knockdown suppressed cell cycle progression. (D) Cell cycle was determined in Hep3B and HuH7 cells by FACS. (E and F) Transwell assay indicated that circ_0067934 silence significantly inhibited the migration

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and invasion of Hep3B and HuH7 cells. (G) Tumor volumes were measured every other one week. (H) Photo of tumor tissues. At the endpoint of experiments, mice were

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sacrificed and tumor tissues were separated. (I) Weights of tumor tissues in H were determined. (J) Quantification of the number of metastatic nodules in the lung. All data are representative of three independent experiments and expressed as the mean ± SD. *p < 0.05 and **p < 0.01. Figure

3.

Circ_0067934

activated

Wnt/β-catenin

pathway

via

regulation

of

miR-1324/FZD5 axis. (A and B) A diagram showed the binding sites of miR-1324 in circ_0067934 and FZD5. (C and D) Dual-luciferase reporter assay indicated that co-transfection with miR-1324 significantly reduced the luciferase activity of the reporter containing wild-type circ_0067934 or FZD5 in Hep3B and HuH7 cells. (E) qRT-PCR

ACCEPTED MANUSCRIPT analysis indicated that circ_0067934 knockdown promoted the expression of miR-1324 in Hep3B and HuH7 cells. (F) Overexpression of miR-1324 significantly inhibited the mRNA level of FZD5 in Hep3B and HuH7 cells. (G) Knockdown of circ_0067934 or overexpression of miR-1324 suppressed the protein levels of FZD5, nucleus β-catenin

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and Cyclin D1 in Hep3B and HuH7 cells. All data are representative of three independent experiments and expressed as the mean ± SD. *p < 0.05, **p < 0.01 and ***p < 0.001.

Figure 4. Overexpression of FZD5 or inhibition of miR-1324 reversed the effects of circ_0067934 depletion. (A and B) The expression of miR-1324 and FZD5 was

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determined by qRT-PCR in indicated cell lines. (C) Cell proliferation was measured by CCK8 assay. (D) Cell apoptosis was determined by FACS analysis. (E and F) Transwell

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assay was used to detect the migration and invasion of Hep3B and HuH7 cells. All data are representative of three independent experiments and expressed as the mean ± SD. *p

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< 0.05 and **p < 0.01.

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ACCEPTED MANUSCRIPT Dear Editor, All authors declare no competing financial interests.

Sincerely,

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Yong Ni, Department of General Surgical, the Second People’s Hospital of Shenzhen, No. 3002,

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Sungang West Road, Shenzhen 518110, China. E-mail: [email protected]