miR-889-3p targeting BMPR2 promotes the development of retinoblastoma via JNK/MAPK/ERK signaling

MicroRNAs (miRNAs) are vital regulators of tumor pathogenesis, including that of retinoblastoma (Rb). This study investigated the functions and mechanisms of action of miR-889-3p in Rb. BMPR2 and miR-889-3p levels were assessed by quantitative reverse transcription PCR (qRT-PCR) or western blotting. Through several cell function tests, the effects of miR-889-3p and BMPR2 on cell proliferation, migration, and JNK/MAPK/ERK signaling were evaluated. The interaction between miR-889-3p and BMPR2 was investigated using a luciferase reporter assay. In vivo tumor development was investigated using a xenograft test. The association between miR-889-3p and BMPR2 expression was identified using Pearson’s correlation analysis. miR-889-3p was increased in Rb cells, and miR-889-3p knockdown inhibited Rb cell proliferation, migration, and phosphorylation of c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase (MAPK), and ERK1/2 in vitro, as well as tumor growth in vivo. Further, they were inversely associated in Rb tissues and miR-889-3p may directly attached to the 3′-UTR of BMPR2 mRNA. Finally, the inhibition of BMPR2 inverted the negative effects of the miR-889-3p inhibitor on migration, proliferation, and activation of JNK, p38 MAPK, and ERK1/2 in Rb cells. Our results indicate that miR-889-3p, which targets BMPR2 and promotes Rb growth by controlling the JNK/MAPK/ERK pathway, is an oncogene in Rb. These results suggested that the miR-889-3p/BMPR2 axis may be a new therapeutic target for Rb.


Quantitative RT-PCR (qRT-PCR)
Following the manufacturer's instructions, total RNA was extracted from tissues and cells using TRIzol reagent (catalog number: 15596-026, Invitrogen).cDNA was reverse-transcribed using the PrimeScript RT Reagent kit (Catalog number: RR037A) from Takara (Dalian, China).For qRT-PCR, a SYBR Green PCR kit (Catalog number: RR820A; Takara) was used.Each gene of interest was amplified using a specific set of primers.Data were calculated using the relative quantitation (2 −ΔΔCt ) technique, and each experiment was performed at least thrice.Real-time PCR primer sequences are listed in Table 1.

Cell counting Kit-8 assay (CCK8)
Cells were placed in 96-well plates at a density of 3 × 10 3 cells per well in 100 µL of RPMI 1640 medium supplemented with 10% FBS to transfect the oligo fragments into the cells.Then, CCK-8 reagent (10 µL, Catalog number: C0037, Beyotime, Shanghai, China) was supplemented to each well and the 96-well plate was placed in 5% CO 2 at 37 °C for 1 h.Microplate Readers (Molecular Devices, Sunnyvale, CA, USA) were used to measure the OD 450 values.Each experiment was performed at least thrice.

Scratch test
Scratching experiments were performed to quantify the cell migration.Trypsinized cells were seeded onto 6-well plates with three parallel wells per group and cultivated until 90% confluence.The cells were then maintained in the FBS-free media while having vertical scratches made with a 100 µL tip.Under an inverted microscope, the cells were examined at 0 h and 24 h, and the migration rate was calculated using the formula (0 h scratch width-24 h scratch width)/0 h scratch width × 100%.

RNA pull-down assay
An RNA pull-down kit (Catalog number: KT103-01, Guangzhou Saicheng Biotechnology Co., LTD, Guangzhou, China) was used to perform the assay.Biotinylated miR-889-3p (Bio-miR-889-3p) and its negative control (bio-NC) were purchased from GenePharma Co., Ltd.(Shanghai, China) and transfected into HXO-RB44 and SO-RB50 cells.After 48 h of transfection, cells were collected and treated with lysis buffer.The lysate was incubated with streptavidin beads pretreated with RNase-free Bovine Serum Albumin (BSA) and yeast tRNA.
After incubation for 2 h, the RNA complex was eluted and isolated to detect the relative enrichment of PLCB1, MEF2C, and BMPR2 using qRT-PCR.

Luciferase reporter gene assays
Luciferase reporter gene assays were conducted using a Dual-luciferase Reporter Assay System (Catalog number: E1910, Promega, Madison, WI, USA).Brief, the fragments of BMPR2 3′-UTR featuring a miR-889-3p wild type (WT) or a mutated (MUT) binding site was cloned into a pmirGLO reporter vector (Catalog number: E1330, Promega), which were known as pmirGLO-BMPR2-WT or pmirGLO-BMPR2-MUT, respectively.They were then transfected using Lipofectamine 2000 into SO-RB50 and HXO-RB44 cells, which also co-expressed the miR-NC or miR-889-3p mimic.Forty-eight hours after transfection, the activity of firefly luciferase in each well was compared to that of Renilla luciferase.

Xenograft assay
Ten male BALB/c nude mice were obtained from the Southern Medical University Laboratory Animal Centre at the age of six weeks and housed in a pathogen-free environment.The weight of the mice ranged from 18 to 20 g.The hospital Ethics Committee approved all animal experiments.Mice were randomly assigned to antagonist miR-889-3p and antagonist NC groups (n = 5 per group) prior to injection.To create animal models, HXO-RB44 cells were microinjected into the left armpit of mice, together with Antago miR-889-3p or Antago-NC obtained from GenePharma.Tumor development was tracked beginning on the fourth day, and the mice were weighed every 4 d.The formula used to determine the tumor size was volume (m 3 ) = (length × width 2 ) × 0.5.After 28 d, all mice were euthanized by CO 2 inhalation and the tumor nodules were removed and weighed.The Animal Experimental Ethics Committee of Wuhan No. 1 Hospital approved the mouse experiments, and all animal experiments were performed in accordance with the ARRIVE guidelines.

Statistical analysis
The GraphPad Prism 8.3.0 was used to analyze the data, and the results were given as mean ± SD.One-way ANOVA followed by Tukey's post hoc test was used to assess differences across several groups, and t-tests were used to analyze differences between two groups.The expression levels of miR-889-3p and BMPR2 were compared using Pearson's correlation analysis.Statistical significance was set at P < 0.05.

Ethical approval
The medical ethics committee of Wuhan No. 1 Hospital approved this study (Wuhan, China).The availability of tissue sampling in experiment is in strict compliance and was in standardized on ethical ground by declaration of Helsiniki.Each patient signed a document requesting their informed permission.The animal tests were conducted out in Wuhan, all animal methods were carried out in accordance with ARRIVE principles and were authorized by the Animal Care Centre of Wuhan No. 1 Hospital.

Participant agreement
Each patient completed an informed consent form in writing.

MiR-889-3p modulates JNK/MAPK/ERK signaling in vitro to encourage Rb cell migration and proliferation
To determine whether miR-889-3p significantly contributes to illness, we first employed qRT-PCR to estimate the trend in miR-889-3p expression levels in Rb cells or tissues.Rb tissues, including SO-RB50, Y79, and HXO-RB44 www.nature.com/scientificreports/Rb cells, expressed miR-889-3p at higher levels than matched nearby normal tissues or the ARPE-19 normal human retinal epithelial cell line (Fig. 1A,B).The biological function of miR-889-3p was determined using loss-of-function studies because HXO-RB44 and SO-RB50 cell lines expressed miR-889-3p at higher levels.The downregulated expression of miR-889-3p was confirmed by qRT-PCR (Fig. 1C).The miR-889-3p inhibitor dramatically reduced the viability of HXO-RB44 and SO-RB50 cells, according to the CCK-8 assay (Fig. 1D).Scratch testing revealed that HXO-RB44 and SO-RB50 cells treated with the miR-889-3p inhibitor migrated considerably less than those in the NC group (Fig. 1E).Additionally, we assessed the key proteins of the JNK/MAPK/ERK signaling pathway by western blotting and found that JNK, p38 MAPK, and ERK1/2 were less phosphorylated when miR-889-3p was downregulated (Fig. 1F).These findings indicate that miR-889-3p modulates JNK/MAPK/ ERK signaling in vitro to promote the proliferation and migration of Rb cells.

Directly targeting BMPR2 by miR889-3p
We predicted a possible target for the miR-889-3p molecule using the miRNA target analysis tool miRDB and found downregulated genes in Rb samples using an mRNA microarray GSE97508 from GEO DataSets (screening criteria: adj.P < 0.05 and logFC < − 2) to gain a better understanding of the molecular process behind the capacity of miR-889-3p to promote Rb progression.Venny 2.1 analysis showed that 93 genes overlapped with miRDB and GSE97508 (Fig. 3A).We then uploaded these 93 genes to STRING for Gene Ontology (GO) enrichment, which showed that PLCB1, MEF2C, and BMPR2 were correlated with the regulation of cell migration (Fig. 3B).
After performing the RNA pull-down assay, only BMPR2 was pulled down by miR-889-3p in R cells (Fig. 3C).
Figure 3D shows that miR-889-3p potentially targets BMPR2 mRNA.To further validate BMPR2 as a target of miR-889-3p, we performed luciferase reporter gene assays using luciferase production plasmids expressing either WT or MUT BMPR2.According to these findings, miR-889-3p mimic transfection in HXO-RB44 and SO-RB50 cells caused the BMPR2-WT construct, but not the BMPR2-MUT construct, to decrease luciferase activity (Fig. 3E).We verified that BMPR2 is a downstream target of miR-889-3p before examining its expression in Rb tissues and cells.The results demonstrated that BMPR2 mRNA levels in Rb tissues or cells, such as SO-RB50 and HXO-RB44, were considerably lower than those in matched normal tissues (Fig. 3F) or in the normal human retinal epithelial cell line, ARPE-19 (Fig. 3G).Furthermore, miR-889-3p expression was inversely associated with BMPR2 expression (Fig. 3H).These findings indicate that miR-889-3p specifically targets BMPR2.

Knockdown of BMPR2 blocked the miR-889-3p inhibitory impacts on activation, cell proliferation, and migration of JNK/MAPK/ERK signaling in Rb cells
We performed rescue trials to investigate whether miR-889-3p exerted its biological function by regulating BMPR2 expression.The protein expression of BMPR2 was found to be lower in SO-RB50 and HXO-RB44 cells transfected with si-BMPR2, indicating that the BMPR2 interference fragment was valid.Compared to inhibitor-NC, BMPR2 expression at the protein level was elevated in HXO-RB44 and SO-RB50 cells transfected with the miR-889-3p inhibitor (Fig. 4A).As expected, cell viability (Fig. 4B), migration (Fig. 4C), and protein phosphorylation of JNK, p38 MAPK, and ERK1/2 (Fig. 4D) were diminished in SO-RB50 and HXO-RB44 cells transfected with the miR-889-3p inhibitor compared to inhibitor-NC, whereas si-BMPR2 had the opposite biological functions of the miR-889-3p inhibitor.Additionally, the effects of the miR-889-3p inhibitor were reduced by cotransfection with si-BMPR2 and miR-889-3p inhibitors.These findings suggest that miR-889-3p promotes Rb migration, activation, and proliferation in JNK/MAPK/ERK signaling by negatively regulating BMPR2 expression.

Discussion
Rb is the most dangerous intraocular tumor of the retina in children, and its incidence is increasing worldwide 23 .
According to a growing body of evidence, miRNAs are essential players in the pathophysiology of Rb 10 .As a result, miRNAs are considered potent molecular indicators for both diagnosis and prognosis as well as treatment targets in Rb.Here, we found that miR-889-3p was upregulated in Rb and that silencing miR-889-3p suppressed the malignancy of Rb cells by targeting BMPR2 to activate the JNK/MAPK/ERK pathway.Previous studies have demonstrated that miR-889-3p functions in a variety of solid tumors.For instance, in cervical cancer, decreased miR-889-3p expression promoted cell proliferation, motility, and invasion while suppressing apoptosis and radiosensitivity 12,13 .However, according to another study 16 , miR-889-3p functions as an oncogene in osteosarcoma 16 .Furthermore, miR-889-3p promoted the invasive capacity of malignant peripheral nerve sheath tumors 15 .The function of miR-889-3p in Rb currently remains unknown.In the current study, we found that Rb tissues and cell lines exhibited significantly higher levels of miR-889-3p.MiR-889-3p can be knocked down to inhibit tumor growth, migration, and proliferation.Our findings are in accordance with those of previous research on malignant peripheral nerve sheath tumors and osteosarcoma, suggesting that miR-889-3p is an oncogene in Rb.
There is growing evidence that miRNAs may bind to target genes' 3-UTRs and change their expression 6 .MiR-889-3p has been shown to affect multiple genes involved in the tumorigenesis of several malignancies [13][14][15][16] .Here, we discovered that miR-889-new 3p's target gene was BMPR2.A crucial component of BMP signaling is BMPR2, a member of the BMP receptor family of transmembrane serine/threonine kinases 24 .Recently, abnormal BMPR2 expression was identified in lung cancer 17 , pancreatic ductal adenocarcinoma 18 , and osteosarcoma 19 .The JNK/MAPK/ERK signaling pathway is a key pathway related to multiple human diseases, including osteoarthritis 22 , gastric cancer 25 , and obesity 26 .In cancer, the activation of the JNK/MAPK/ERK signaling pathway by PAGE4 promotes prostate cancer cell survival 27 .According to the previous study from Xiao et al. 22 , BMPR2 overexpression inhibits the activation of the JNK/MAPK/ERK signaling pathway in osteoarthritis.In our study, the downregulation of BMPR2 enhanced the phosphorylation levels of JNK, p38 MAPK, and ERK1/2 in Rb cells, suggesting that BMPR2 knockdown activated the JNK/MAPK/ERK signaling pathway in Rb cells.Our results are in agreement with those reported by Xiao et al. in osteoarthritis.
Despite these limitations, we determined the role and regulation mechanism in Rb cells of miR-889-3p.The number of clinical samples was insufficient to determine the clinical value of miR-889-3p; therefore, more clinical samples should be collected in the future to determine the function of miR-889-3p.Additionally, the upstream role of miR-889-3p in Rb has not yet been investigated.In the future, we will investigate the important regulators that improve the miR-889-3p regulatory network in Rb.

Figure 2 .
Figure 2. In vivo, miR-889-3p supports the formation of retinoblastoma (Rb) tumors.(A) The tumor samples from the nude mice given Antago-NC or Antago-miR-889-3p (Antago-miR) injections are shown here.(B) The curve of tumor volume from the nude mice injected with Antago-NC or Antago-miR, whereas **P < 0.001 versus NC. (C) The tumor weight of the nude mice was measured on the 28th day after injection with Antago-NC or Antago-miR, **P < 0.001 versus NC.