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Abstract

BACKGROUND:

Recently, the incidence and mortality of retinoblastoma (RB) have gradually increased. Many studies support the pivotal role of microRNAs (miRNAs) in the pathogenesis of RB. Alternation of microRNA-98 (miR-98) expression has been detected in several cancers, excluding RB. This study was designed to assess the regulatory mechanisms of miR-98 in human RB.

METHODS:

RT-qPCR and Western blot analysis were used to detect miR-98 and HMGA2 expression. The effects of miR-98 were explored using the CCK-8 and Transwell assays. Dual-luciferase reporter assay was performed to confirm the relationship between miR-98 and HMGA2.

RESULTS:

In RB, downregulation of miR-98 was identified. Upregulation of miR-98 inhibited proliferation, invasion and migration of RB cells. Further, HMGA2 was confirmed as a direct target gene of miR-98. And knockdown of HMGA2 suppressed the progression of RB. Moreover, upregulation of HMGA2 reversed the suppressive effects in the development of RB. In addition, miR-98 also showed suppressive effect on EMT and Wnt/ $\beta$ -catenin pathway.

CONCLUSION:

MiR-98 targets HMGA2 to act as a tumor suppressor in RB by mediating Wnt/ $\beta$ -catenin pathway.

Keywords

miR-98 retinoblastoma HMGA2 wnt/β-catenin pathway

1. Introduction

Retinoblastoma (RB) is a malignant tumor derived from photoreceptor precursor cells. RB usually occurs in children under 3 years old and has a family genetic predisposition [13]. RB is the most common intraocular malignancy in infants and children and is rare in adults [18]. Moreover, RB is prone to intracranial and distant metastasis, often endangering children’s lives. For families with a genetic history of RB, genetic testing and genetic counseling can reduce the risk of birth in children with RB [4]. In addition, the prognosis of RB patients is related to many factors, such as tumor size and location, and tumor differentiation [25]. To make matters worse, there are no effective preventive measures for RB. Therefore, early detection, early diagnosis and early treatment are the keys to improving the cure rate and reducing the mortality rate.

Previous studies have demonstrated that microRNAs (miRNAs) are involved in the pathogenesis of human cancers by suppressing the expression of target genes [3]. In RB, different functions of miRNAs as oncogenes or tumor suppressors have been reported. For example, overexpression of miR-497 repressed cell proliferation, invasion and migration in RB by targeting VEGFA [14]. In contrast, miR-125b promoted RB growth through suppressing DRAM2 [2]. Recently, the function of miR-98 in different human cancers has drawn our attention. Yang et al. reported that miR-98 inhibited proliferation and invasion of non-small cell lung cancer (NSCLC) cells through regulating PAK1 [37]. Moreover, low miR-98 expression has been identified as a poor prognostic biomarker for patients with NSCLC [30]. In addition, the inhibitory effect of miR-98 has also been found in hepatocellular carcinoma [15], pancreatic ductal adenocarcinoma [8] and glioma [31]. However, the role of miR-98 is unclear and needs to be investigated in RB.

As a member of the high-mobility group protein family, HMGA2 (high-mobility group A protein 2) is a chromatin remodeling factor that binds to AT-rich regions in DNA [9]. Upregulation of HMGA2 was identified in NSCLC [19] and breast cancer [22]. Moreover, increased expression of HMGA2 was related to a malignant phenotype in pancreatic exocrine tissue [1]. As an oncogene, HMGA2 is a driving factor for tumor metastasis [20]. Moreover, upregulation of HMGA2 promoted metastasis of colorectal cancer and affected survival [33]. More importantly, HMGA2 has been found to be involved in different signaling pathways. The Wnt/ $\beta$ -catenin signaling pathway was proposed to induce HMGA2 and proliferation in metastatic triple-negative breast cancer [34]. It was also demonstrated that miR-337 suppressed the progression of hepatocellular carcinoma through targeting HMGA2 and modulating PI3K/AKT and Wnt/ $\beta$ -catenin signaling pathways [6]. However, the role and underlying molecular mechanism of HMGA2 remain elusive in RB.

In the present study, we are focusing on exploring whether miR-98/HMGA2 axis can regulate cell proliferation, invasion, migration and epithelial – mesenchymal transition (EMT) in RB through mediating the Wnt/ $\beta$ -catenin pathway. This study will provide new insights into miR-98-mediated cell survival and metastasis mechanisms in RB.

2. Materials and methods

2.1 Clinical tissues

The RB tissues and normal retina tissues (located $\geqslant$ 3 cm away from the tumor) were acquired from 68 patients in the Yankuang New Journey General Hospital. The RB patients participated in the study did not receive any treatment before surgery. The retinoblastoma tissues were all pathologically diagnosed and classified by $\geqslant$ 2 pathologists. All patients provided written informed consents, and this experiment was approved by the Institutional Ethics Committee of Yankuang New Journey General Hospital based on Ethical Rules for Biomedical Research Related with Human (Trial) which issued by the national health and family planning commission and Helsinki Declaration.

Figure 1.

MiR-98 expression was decreased in RB tissues. (A) The expressions of miR-98 in RB tissues. (B) Low miR-98 expression was related to shorter overall survival of RB patients. * $P<$ 0.05, ** $P<$ 0.01.

2.2 Cell lines culture

The normal human retinal pigment epithelial cell ARPE-19 and WERI-Rb-1, Y79 cell lines were used for this experiment. These cell lines were obtained from ATCC (Manassas, VA, USA). In RPMI-1640 medium (Gibco, Grand Island, NY, USA), the cells were then seeded with 10% heat-inactivated fetal bovine serum (FBS), 100 U/l penicillin, and 100 U/l streptomycin. The cell lines were incubated in a humidified atmosphere of 95% air and 5% CO ${}_{2}$ at 37 ${}^{\circ}$ C.

2.3 Cell transfection

MiR-98 mimics or inhibitor and negative control (NC) were acquired from Genepharma Co., Ltd (Shanghai, China). HMGA2 siRNA and negative control siRNA were also purchased from Genepharma (Shanghai, China). They were separately transferred to Y79 cells by using Lipofectamine 2000 (Invitrogen, Carlsbad, USA).

2.4 Quantitative reverse transcription PCR (qRTPCR)

Total RNA was extracted in RB tissues using TRIzol ${}^{{\@setsize{\scriptsize}{8pt}{\viipt}{\@viipt}\textregistered}}$ reagent (Invitrogen, Thermo Fisher Scientifc, Inc.). The cDNA was reverse transcribed by the First-Strand cDNA Synthesis Kit (Promega, Madison, WI, USA). We performed qRT-PCR on ABI Prizm 7300 Sequence Detection System by using SYBR Premix Ex Taq mix (Takara Biotechnology, Co., Ltd.). U6 or GAPDH was used as control for miR-98 or HMGA2. And their expressions were calculated using the 2 ${}^{-\vartriangle\vartriangle ct}$ method [17].

2.5 Cell counting kit-8 (CCK-8) assay

The CCK-8 assay was used to assess cell viability in RB. First, 3 $\times$ 10 ${}^{3}$ Y79 transfected cells were put in 96 well plates. After transfection, 10 $\mu$ l of CCK-8 reagent was added to each well for 2 h at 37 ${}^{\circ}$ C (Dojindo, Kumamoto, Japan) at 24, 48, 72 and 96 h according to the manufacturer’s instructions. Cell viability was measured using a microplate reader (Bio-Rad, USA) at an absorbance of 450 nm [5].

2.6 Transwell assay

Transwell chambers (Millipore) were used to examine cell migration and invasion. First, 5 $\times$ 10 ${}^{3}$ Y79 cells with miR-98 mimics or inhibitor were put in the upper chamber for 24 h at 37 ${}^{\circ}$ C, while the lower chamber was filled with 10% FBS to induce migration of Y79 cells. In addition, cells were put in the upper chamber with Matrigel (BD) for the invasion assay. Finally, migrated or invasive cells were stained with 0.5% crystal violet. An inverted microscope (Olympus) was used to obtain cell images [8].

2.7 Dual luciferase reporter assay

The relationship between miR-98 and HMGA2 was verified by dual luciferase reporter assay (Promega Corporation, Madison, WI, USA). The DNA oligonucleotides containing the wild-type 3’-UTR of HMGA2 or the mutant 3’-UTR of HMGA2 were synthesized and cloned into the pmirGLO dual-luciferase miRNA expression vector (Promega Corporation, WI, USA). Next, Y79 cells were co-transfected with the miR-98 mimics and above vectors (Promega Corporation). After 48 h, the firefly and renilla luciferase activities were measured by a dual-luciferase reporter assay system (Promega, Madison, WI, USA) [36].

2.8 Western blot analysis

Protein samples were extracted using RIPA lysis buffer (50 mM Tris (pH 7.4), 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate). Next, proteins were separated by 10% SDS-PAGE and incubated with 5% skim milk in PVDF membranes at room temperature. Membranes were then incubated with HMGA2, GAPDH, Wnt/ $\beta$ -catenin pathway ( $\beta$ -catenin, p- $\beta$ -catenin) and EMT markers (E-cadherin, N-cadherin, vimentin) primary antibodies overnight at 4 ${}^{\circ}$ C. After washing, they were incubated with corresponding secondary antibodies for another 2 h at room temperature. Finally, protein expression was measured by ECL (Pierce Biotechnology, USA) [36].

2.9 Statistical analysis

Differences between groups were analyzed by SPSS 19.0 using one-way ANOVA with Bonferroni post hoc test. The correlation between miR-98 and clinical features of RB patients was calculated by Chi-squared test. Overall survival rates and survival differences were detected by univariate Kaplan-Meier method with log-rank test. Data were expressed as mean $\pm$ SD. Significant differences were defined as $P<$ 0.05.

Table 1
Relationship between miR-98 expression and their clinic-patholo- gical characteristics of RB patients

Characteristics	Cases	miR-98		$P$ -value
		High	Low
Age (years)				0.29
$\geqslant$ 24	35	15	20
$<$ 24	33	12	21
Gender				0.12
Male	37	14	23
Female	31	13	18
Tumor size (mm)				0.004*
$\leqslant$ 10	41	18	23
$>$ 10	27	9	18
Lymph nodes metastasis				0.008*
No	42	15	27
Yes	26	12	14
TNM classification				0.016*
T1 $+$ T2	48	20	28
T3 $+$ T4	20	7	13
Differentiation				0.014*
Well $+$ moderate	45	16	29
Poor $+$ undifferentiated	23	11	12
Tumor location				0.08
Unilateral	44	18	26
Bilateral	24	9	15

Statistical analyses were performed by the $\chi$ 2 test. * $P<$ 0.05 was considered significant.

Figure 2.

MiR-98 overexpression inhibited cell proliferation, migration and invasion in RB. (A) The miR-98 expression was detected in WERI-Rb-1, Y79 and ARPE-19 cell lines. (B) MiR-98 expression regulated by miR-98 mimics or inhibitor was examined in Y79 cells. (C, D) MiR-98 mimics or inhibitor regulated the cell proliferation. (E, F) Cell migration and invasion were regulated by miR-98 mimics or inhibitor in RB. * $P<$ 0.05, ** $P<$ 0.01.

Figure 3.

HMGA2 was a direct target gene of miR-98. (A) HMGA2 had binding sites with miR-98. (B) Luciferase reporter assay (C) MiR-98 had negative correlation with HMGA2. (D, E) The expression of HMGA2 were observed in Y79 cells containing miR-98 mimics or inhibitor. ** $P<$ 0.01.

Figure 4.

The knockdown of HMGA2 inhibited the development of RB. (A) The expressions of HMGA2 in RB tissues. (B) The HMGA2 expression in WERI-Rb-1, Y79 and ARPE-19 cell lines. (C) The expression of HMGA2 was examined in Y79 cells with HMGA2 siRNA. (D) HMGA2 siRNA regulated the cell proliferation in Y79 cells. (E, F) Cell migration and invasion analysis in cells containing HMGA2 siRNA. ** $P<$ 0.01.

Figure 5.

MiR-98 inhibited the progression of RB through targeting HMGA2. (A) The expression of HMGA2 was measured in Y79 cells with HMGA2 vector and miR-98 mimics. (B) The cell proliferation was measured in Y79 cells with HMGA2 vector and miR-98 mimics. (C, D) The cell migration and invasion in Y79 cells with HMGA2 vector and miR-98 mimics. ** $P<$ 0.01.

Figure 6.

MiR-98 regulated the tumorigenesis of RB through blocking Wnt/ $\beta$ -catenin pathway. Western blot analysis of E-cadherin, N-cadherin, Vimentin, HMGA2, $\beta$ -catenin and p- $\beta$ -catenin in Y79 cells contained miR-98 mimics or inhibitor.

3. Results

3.1 MiR-98 was downregulated in RB tissues

First, the alternation of miR-98 expression was observed in RB tissues by qRT-PCR assay. A lower miR-98 expression was detected in RB tissues than in normal tissues ( $P<$ 0.01, Fig. 1A). Next, the cases were divided into a high miR-98 expression group and a low expression group based on the median value of miR-98 expression levels in RB patients as a cutoff point. The correlation between miR-98 and clinical features of RB patients was then analyzed. Low miR-98 expression was found to be closely correlated with tumor size ( $P=$ 0.004), differentiation ( $P=$ 0.014), lymph nodes metastasis ( $P=$ 0.008) and TNM classification ( $P=$ 0.016, Table 1). In addition, RB patients with low miR-98 expression had shorter overall survival, indicating that low expression of miR-98 predicted poor prognosis in RB patients ( $P=$ 0.025, Fig. 1B). The findings indicated that miR-98 was an indicator of tumorigenesis and prognosis of RB patients.

3.2 Upregulation of miR-98 inhibited cell proliferation, migration and invasion in RB

Next, expression of miR-98 was detected in WERI-Rb-1, Y79 and ARPE-19 cell lines. MiR-98 was also downregulated in WERI-Rb-1 and Y79 cells compared to ARPE-19 cells ( $P<$ 0.01, Fig. 2A). MiR-98 mimics or inhibitor was then transfected into Y79 cells to explore its role in RB. We found that the expression level of miR-98 was enhanced by miR-98 mimics and decreased by miR-98 inhibitor ( $P<$ 0.01, Fig. 2B). Functionally, CCK-8 assay showed that upregulation of miR-98 suppressed cell proliferation in Y79 cells ( $P<$ 0.01, Fig. 2C). In contrast, silencing of miR-98 promoted cell proliferation in RB ( $P<$ 0.05, Fig. 2D). Furthermore, overexpression of miR-98 suppressed cell migration in Y79 cells, whereas knockdown of miR-98 promoted cell migration in RB ( $P<$ 0.01, Fig. 2E). Similarly, the same effect of miR-98 on cell invasion was also identified ( $P<$ 0.01, Fig. 2F). The results showed that miR-98 had an inhibitory effect on the pathogenesis of RB.

3.3 HMGA2 was a direct target gene of miR-98.

Further, theoretical target genes of miR-98 were search from TargetScan (http://www.targetscan.org). HMGA2 was predicted to be a target gene having binding sites to miR-98 (Fig. 3A). This prediction was verified by luciferase reporter assay. The results indicated that miR-98 mimics inhibited the luciferase activity of Wt-HMGA2 ( $P<$ 0.01, Fig. 3B). However, the luciferase activity of Mut-HMGA2 was not affected by miR-98 mimics. Moreover, miR-98 was found to be negatively correlated with HMGA2 expression in RB tissues ( $P<$ 0.0001, R ${}^{2}=$ 0.5709; Fig. 3C). Next, expression of HMGA2 regulated by miR-98 mimics or inhibitor was observed in Y79 cells. Consistently, HMGA2 expression was reduced by miR-98 mimics ( $P<$ 0.01, Fig. 3D) and was promoted by miR-98 inhibitor ( $P<$ 0.01, Fig. 3E). Based on these results, HMGA2 was confirmed as a direct target of miR-98, and a negative correlation between miR-98 and HMGA2 was detected.

3.4 The knockdown of HMGA2 inhibited the development of RB

Subsequently, HMGA2 expression was observed in RB. Higher expression of HMGA2 was identified in RB tissues than in normal tissues ( $P<$ 0.01, Fig. 4A). Similarly, upregulation of HMGA2 was also identified in the WERI-Rb-1 and Y79 cell lines ( $P<$ 0.01, Fig. 4B). To further confirm whether HMGA2 is involved in the progression of RB, Y79 cells were transfected with HMGA2 siRNA. We found that HMGA2 siRNA significantly reduced its expression in Y79 cells ( $P<$ 0.01, Fig. 4C). CCK-8 assay showed that knockdown of HMGA2 inhibited cell proliferation in Y79 cells ( $P<$ 0.01, Fig. 4D). Inhibition of cell migration and invasion induced by HMGA2 was also detected in RB (P $<$ 0.01, Fig. 4E and F). These findings implied that HMGA2 was upregulated in RB, and knockdown of HMGA2 inhibited the development of RB.

3.5 MiR-98 inhibited the progression of RB through targeting HMGA2

MiR-98 mimics and HMGA2 vector were then co-transfected into Y79 cells to determine their interaction. The downregulation of HMGA2 induced by miR-98 mimics was recovered by HMGA2 vector in Y79 cells ( $P<$ 0.01, Fig. 5A). Similarly, the HMGA2 vector also impaired the inhibitory effect of miR-98 on cell proliferation in Y79 cells ( $P<$ 0.01, Fig. 5B). Furthermore, overexpression of HMGA2 also blocked the inhibition of cell invasion and migration induced by miR-98 in RB ( $P<$ 0.01, Fig. 5C and D). In conclusion, miR-98 inhibited proliferation, migration and invasion of RB cells by targeting HMGA2.

3.6 MiR-98 regulated the tumorigenesis of RB through blocking Wnt/ $\beta$ -catenin pathway

Finally, the effects of miR-98 on EMT and Wnt/ $\beta$ -catenin pathway were investigated to further explore its molecular mechanism in RB. We found that miR-98 regulated the expression of EMT makers. The results suggested that overexpression of miR-98 promoted E-cadherin expression and suppressed N-cadherin and Vimentin expressions (Fig. 6). In contrast, downregulation of miR-98 had opposite effects on EMT makers (E-cadherin, N-cadherin and Vimentin), as shown in Fig. 6. Therefore, we considered that miR-98 inhibited EMT in Y79 cells to regulate cell metastasis. In addition, HMGA2 has been reported to be involved in the Wnt/ $\beta$ -catenin signaling pathway. To elucidate whether miR-98 is involved in the Wnt/ $\beta$ -catenin pathway, $\beta$ -catenin and p- $\beta$ -catenin expressions regulated by miR-98 mimics and inhibitor was examined in Y79 cells. We found that upregulation of miR-98 reduced p- $\beta$ -catenin expression (Fig. 6) in Y79 cells, and knockdown of miR-98 enhanced p- $\beta$ -catenin expression (Fig. 6). The expression of $\beta$ -catenin was not affected by miR-98 mimics and inhibitor. Overall, miR-98 inhibited cell survival and metastasis through blocking EMT and Wnt/ $\beta$ -catenin pathway in RB.

4. Discussion

RB is highly malignant, affecting 8,000 children each year, which usually leads to intracranial metastasis [26]. The pathogenesis of RB is a complex process associated with many miRNAs and genes, such as miR-183/LRP6 [29], miR-22/ENO1 [16] and miR-145/ADAM19 [27]. In particular, it had been reported that miR-98 reduced the HMGA2 expression and promoted osteogenic differentiation of mesenchymal stem cells [10]. In head and neck squamous cell carcinoma, HMGA2 had been identified as a target gene of miR-98 [12]. Therefore, the aim of this study was to investigate the role of miR-98 in RB. MiR-98 regulated proliferation, invasion and migration of RB cells by modulating HMGA2 in an attempt to identify new targets for RB therapy.

This study demonstrated the downregulation of miR-98 and upregulation of HMGA2 in RB. HMGA2 was confirmed to be a direct target gene of miR-98. It had been reported that miR-98 negatively regulated HMGA2 expression [35]. A negative correlation between miR-98 and HMGA2 was also identified in RB. Furthermore, low miR-98 expression was related to a shorter overall survival rate in RB patients. Similarly, Wang et al. revealed that low miR-98 expression was associated with poor prognosis in NSCLC patients [30]. In addition, high expression of HMGA2 was identified in RB and miR-98 was negatively correlated with HMGA2 in RB tissues. Similarly, upregulation of HMGA2 was also detected in colorectal cancer, triple-negative breast cancer and osteosarcoma, which was negatively regulated by some miRNAs [7, 11, 28]. Previous studies have further supported our results that miR-98 could directly target HMGA2.

Functionally, overexpression of miR-98 inhibited cell proliferation, invasion and migration in RB by targeting HMGA2. Similarly, miR-98 was reported to inhibit migration, invasion and proliferation of NSCLC cells through targeting ITGB3 [21]. In this study, EMT makers (E-cadherin, N-cadherin and Vimentin) associated with cell metastasis were regulated by miR-98 in RB. We found that miR-98 blocked EMT to inhibit cell metastasis in RB. It was also demonstrated that miR-200c suppressed the migration of RB cells by reversing EMT [23]. Similarly, miR-98 inhibited migration, invasion, proliferation and EMT of HCC cells [39]. In addition, knockdown of HMGA2 inhibited cell proliferation, migration and invasion in RB. Shi et al. proposed that silencing of HMGA2 promoted apoptosis and inhibited cell migration and invasion in prostate cancer, consistent with our study [24]. Further, miR-98 was found to regulate tumorigenesis of RB through blocking the Wnt/ $\beta$ -catenin pathway. Zhang et al. also reported that miR-98 suppressed the proliferation of hepatocellular carcinoma cells by regulating the Wnt/ $\beta$ -catenin pathway [38]. Moreover, miR-98 repressed breast cancer cell proliferation, invasion, and migration by targeting HMGA2 [32], which was parallel to our results.

5. Conclusion

This study preliminarily demonstrated that miR-98 inhibited the development of RB by targeting HMGA2 and mediating the Wnt/ $\beta$ -catenin pathway. It provides strong evidence for exploring effective diagnostic biomarkers of RB. However, further investigation is needed to verify the findings of this study.

Footnotes

Conflict of interest

The authors declare that they have no competing interests.

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MicroRNA-98 targets HMGA2 to inhibit the development of retinoblastoma through mediating Wnt/ β -catenin pathway

Abstract

BACKGROUND:

METHODS:

RESULTS:

CONCLUSION:

Keywords

1. Introduction

2. Materials and methods

2.1 Clinical tissues

2.3 Cell transfection

2.4 Quantitative reverse transcription PCR (qRTPCR)

2.5 Cell counting kit-8 (CCK-8) assay

2.6 Transwell assay

2.7 Dual luciferase reporter assay

2.8 Western blot analysis

2.9 Statistical analysis

Table 1 Relationship between miR-98 expression and their clinic-patholo- gical characteristics of RB patients

3.1 MiR-98 was downregulated in RB tissues

3.2 Upregulation of miR-98 inhibited cell proliferation, migration and invasion in RB

3.3 HMGA2 was a direct target gene of miR-98.

3.4 The knockdown of HMGA2 inhibited the development of RB

3.5 MiR-98 inhibited the progression of RB through targeting HMGA2

3.6 MiR-98 regulated the tumorigenesis of RB through blocking Wnt/ β -catenin pathway

4. Discussion

5. Conclusion

Footnotes

Conflict of interest

References

Table 1
Relationship between miR-98 expression and their clinic-patholo- gical characteristics of RB patients

3.6 MiR-98 regulated the tumorigenesis of RB through blocking Wnt/ $\beta$ -catenin pathway