MicroRNA-532 inhibits cell growth and metastasis in retinoblastoma by targeting MDM4 CURRENT STATUS:

Background Now, numerous microRNAs (miRNAs) are found to exert effect in retinoblastoma (RB). This research mainly focused on the function of miR-532 in RB, which has not been investigated. Methods RT-qPCR and Western blot analysis were used to measure expressions of miR-532 and genes. Transwell, CCK-8 and luciferase reporter assays were applied to explore functions of miR-532 and MDM4 in RB. Results The expression of miR-532 was reduced in RB. Furthermore, overexpression of miR-532 restrained RB cell survival and metastasis and induced apoptosis. In addition, miR-532 directly targets MDM4. Moreover, downregulation of MDM4 blocked the progression of RB. And upregulation of MDM4 reversed the anti-tumor effect of miR-532 in RB.


Introduction
Retinoblastoma (RB) is the most common intraocular malignancy in infants and young children. Twothirds of RB patients are under 3 years of age, and less than 5% of those over 5 years old [1]. As a genetic abnormal disease, RB has a certain genetic predisposition. Approximately 60% of RB patients are monogenic non-genetic, and 40% are caused by genetic inheritance of a parent who is sick or carrying a disease gene [2]. Nowadays, RB is one of the most cured cancers. 95-98% of children with RB can recover, and more than 90% of patients can survive to adulthood. Although the survive rate of China's patients is slightly lower, the current 5-year survival rate can reach 80-85% [3]. However, RB is prone to intracranial and distant metastasis, often endangering the lives of children [4]. Therefore, early detection, diagnosis and treatment are the key to keeping your child's eyes and vision and improving the cure rate.
MicroRNAs (miRNAs) inhibits expressions of target genes mainly by binding to the target mRNA, or by causing the mRNA to degrade or hinder its translation [5]. In the meantime, the dysregulation of 3 miRNAs was also identified in RB. For example, miR-101 and miR-497 were downregulated in RB, while miR-221 and miR-222 were upregulated in RB [6][7][8]. Functionally, miR-138-5p acted as a tumor suppressor via targeting PDK1 in human RB [9]. Inversely, miR-498 promoted cell proliferation and inhibited cell apoptosis in RB by directly targeting CCPG1 [10]. Now, the different roles of miR-532 in human cancers aroused our attention. First, upregulation of miR-532 was observed in colorectal cancer [11]. Moreover, miR-532-3p promoted hepatocellular carcinoma progression by targeting PTPRT [12]. However, downregulating of miR-532 was detected in epithelial ovarian cancer [13].
Furthermore, miR-532 inhibited cell proliferation and invasion by acting as direct regulators of hTERT in ovarian cancer [14]. In addition, Venkatesan et al. found that miR-532 was downregulated in RB [15]. But the function of miR-532 has not been illustrated in RB and need to be explored.
MDM4 (Mdm4 p53 binding protein homolog) is well-known to be involved in p53 pathway [16]. The tumor suppressor p53 has been widely identified in human cancers [17]. Furthermore, MDM4 promoter attenuated the p53 tumor suppressor pathway and accelerated tumor formation in humans [18]. Moreover, it was found that MDM4 in combination with p53 contributed to breast cancer susceptibility [19]. Zhou et al. reported that MDM4 accelerated the tumorigenesis of esophageal squamous cell carcinoma [20]. In addition, MDM4 expression was regulated by multiple microRNAs in prostate cancer, including miR-191-5p and miR-887 [21]. Mandke et al. suggested that miR-34a modulated MDM4 expression via a target site in the open reading frame [22]. Moreover, miR-1307 regulated cisplatin resistance by targeting MDM4 in breast cancer [23]. However, the interaction between MDM4 and miR-532 was not investigated in RB. Therefore, their relationship was verified in this study. Furthermore, the potential functions of miR-532 and MDM4 was explored in RB. This research will provide a potential therapeutic target against RB.

Clinical tissues
Twenty-two RB patients with informed consents in Weifang People's Hospital were participated in this study. Moreover, patients with RB did not receive any treatment except for surgery. The permission of this research was acquired from the Institutional Ethics Committee of Weifang People's Hospital.

RNA isolation and RT-qPCR
Total RNA isolation was performed using TRIZOL reagent (Invitrogen, USA). And cDNA solution was obtained using microRNA reverse transcription kit (Takara, Dalian, China). RT-qPCR assay was performing using SYBR miRNA detection assays (Takara) based on the manufacturer's instruction. U6 or GAPDH was used as the control of miR-532 or MDM4, which were quantified with the 2 −△△cq method.

Western blot analysis
RIPA lysis buffer (Beyotime) was used to obtain protein samples. Next, 10% SDS-PAGE was used to separate protein. Protein samples were transferred to PVDF membranes. Blocked with 5% non-fat milk, protein samples were incubated overnight at 4℃ with E-cadherin, N-cadherin, vimentin, MDM4 and GAPDH primary antibodies (Abcam, Shanghai, China). Afterwards, secondary antibodies (Abcam, USA) were added to incubate protein samples for 1 hour. ECL kit (Beyotime) was used to assess protein bands.

Transwell assay
First, diluted Matrigel was added to the upper chamber for cell invasion. Cell migration assay was performed without Matrigel. After 30 min, WERI-Rb-1 cell suspension (3×10 3 cells/well) was added to the Transwell upper chamber, and RPMI-1640 medium (10% FBS) was added to 24-well plates in lower chamber. After 24 h, the moved cells were stained with 0.1% crystal violet. Observation and photographing were performed using a light microscope.

Statistical analysis
Data was shown as mean ± SD and analyzed by SPSS 17.0 or Graphpad Prism 6. Differences between groups were analyzed using one-way analysis of variance with Tukey's post hoc test. Differences were considered as significant at P < 0.05.

Results
The expression of miR-532 was reduced in RB.
To explore the dysregulation of miR-532, its expression was firstly detected in RB. We found that the expression of miR-532 was reduced in RB tissues compared with adjacent tissues (P < 0.01, Fig. 1A).
Consistently, miR-532 was also downregulated in WERI-Rb-1 cells contrast to ARPE-19 cells (P < 0.01, Fig. 1B). The above results showed that miR-532 was indeed abnormally expressed in RB. Next, WERI-Rb-1 cells with miR-532 mimics or inhibitor were prepared to explore its role in RB. RT-qPCR indicated that miR-532 expression was elevated by its mimics and declined by its inhibitor (P < 0.01, Fig. 1C).
Then, gain-loss functional experiment was performed to explore the function of miR-532 in RB.
Overexpression of miR-532 restrained the development of RB.
Downregulation of MDM4 blocked the progression of RB.
Furthermore, the reversal effect of MDM4 on Bcl-2/Bax expression was detected in WERI-Rb-1 cells with miR-532 mimics (Fig. 5C). In addition, the inhibitory effect of miR-532 on cell migration and invasion was also weakened by MDM4 vector (Fig. 5D, 5E). Based on these results, we considered that upregulation of MDM4 reversed the anti-tumor effect of miR-532 in RB.

Discussion
Many literatures now indicate that the dysregulation of many miRNAs is associated with the formation and development of RB tumors. Many of these miRNAs have been reported to exert an inhibitory effect in RB, providing a potential therapeutic method for RB [24,25]. In this study, miR-532 expression was reduced in RB. Furthermore, overexpression of miR-532 restrained RB cell viability and metastasis and induced apoptosis. In addition, MDM4 was confirmed to be a direct target gene of miR-532. Moreover, downregulation of MDM4 blocked the progression of RB. And upregulation of MDM4 reversed the anti-tumor effect of miR-532 in RB. Therefore, we considered that miR-532 acted as a tumor inhibitor in RB via binding to MDM4. Consistent with our results, decreased expression of miR-532 was also detected in ovarian cancer and hepatocellular carcinoma [26,27]. In addition, downregulation of miR-532 was found to promote the proliferation and invasion of bladder cancer cells [28]. Song et al. also reported that loss of miR-532 promoted cell proliferation and metastasis by in hepatocellular carcinoma [29]. Here, cell proliferation, migration and invasion were found to be inhibited by overexpression of miR-532 in RB.
However, upregulation of miR-532 was identified in breast cancer and gastric cancer [30,31].
Furthermore, miR-532 functioned as an oncogenic microRNA in human gastric cancer [32]. These findings are contrary to our results. Contradictory findings suggest that miR-532 expression patterns show tissue specificity in human cancers.
Previous studies revealed that miR-532 was involved in tumorigenesis through regulating target genes, such as ETS1 and Rab3IP [33,34]. In current research, miR-532 was found to directly target 8 MDM4 and inversely regulated its expression in RB. Upregulation of MDM4 has been identified in leukemia [35]. The p53 inhibitor MDM4 has been reported to be involved in tumorigenesis [36].
Moreover, it has been reported that MDM4 is an oncogene and cancer therapy [37]. Here, MDM4 was upregulated in RB and repressed cell viability and metastasis in RB, which was the same as previous studies. And upregulation of MDM4 reversed the anti-tumor effect of miR-532 in RB. The interaction between MDM4 and other miRNAs has been identified in some human cancers. For example, miR-128 induced pancreas cancer cell apoptosis by targeting MDM4 [38]. Yan et al. also showed that miR-1205 functioned as a tumor suppressor by disconnecting MDM4 in non-small cell lung cancer [39].
Consistent with the above studies, miR-532 restrained cell proliferation and metastasis in RB by suppressing MDM4 expression.

Conclusion
Briefly, this study proposed that miR-532 was an anti-tumor miRNA in RB through inhibiting survival and metastasis and inducing apoptosis. Moreover, MDM4 was confirmed as a direct target of miR-532.
This research highlights the critical role of miR-532/MDM4 axis in RB progression.

Acknowledgements
Not applicable.

Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Authors' contributions
CL designed the study and drafted the manuscript. YN and CW were responsible for the collection and analysis of the experimental data. TJ, QR and JX revised the manuscript critically for important intellectual content. All authors read and approved the final manuscript.

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The study was approved by Ethical Committee of Qilu Hospital of Shandong University (Qingdao) and conducted in accordance with the ethical standards.

Consent for publication
Not applicable. The interaction between miR-532 and MDM4 was involved in RB progression. (A) MDM4 expression was observed in WERI-Rb-1 cells containing miR-532 mimics and MDM4 vector.