MicroRNA-361-3p regulates retinoblastoma cell proliferation and stemness by targeting hedgehog signaling

Abstract

Retinoblastoma (RB) is the most common type of intraocular malignancy in children. During RB oncogenesis, sonic hedgehog (SHH) is commonly differentially expressed. Additionally, microRNAs (miRs) are known to serve crucial roles as oncogenes or tumor suppressors. Specifically, miR-361-3p has been revealed to serve a vital role in cutaneous squamous cell carcinoma, cervical cancer, prostate cancer, colorectal cancer, gastric cancer, hepatocellular carcinoma, breast cancer and lung cancer. However, the role of miR-361-3p in RB and the potential molecular mechanisms involved remain unknown. Therefore, the current study aimed to determine the involvement of miR-361-3p in the development of RB by targeting SHH signaling. In the present study, miR-361-3p expression levels in RB tissue and serum samples obtained from 10 patients with RB, normal retinal tissue and serum samples obtained from 10 healthy controls, and two human RB cell lines (Y79 and Weri-Rb-1) were determined using reverse transcription-quantitative polymerase chain reaction. In addition, a cell counting kit-8 assay, a cell transfection assay, a MTT assay, western blotting, a tumor sphere formation assay and a luciferase assay were used to assess the expression, function and molecular mechanism of miR-361-3p in human RB. It was demonstrated that miR-361-3p was significantly downregulated in RB tissues, RB serum and RB cell lines compared with normal retinal tissues and normal serum. The ectopic expression of miR-361-3p decreased RB cell proliferation and stemness. Furthermore, GLI1 and GLI3 were verified as potential direct targets of miR-361-3p. miR-361-3p was also revealed to exhibit a negative correlation with GLI1/3 expression in RB samples. Taken together, the results indicate that miR-361-3p functions as a tumor suppressor in the carcinogenesis and progression of RB by targeting SHH signaling. Thus, miR-361-3p should be further assessed as a potential therapeutic target for RB treatment.

Keywords: glioma-associated oncogene homologue transcription factor 1; glioma-associated oncogene homologue transcription factor 3; hedgehog signaling; metastasis; microRNA-361-3p; proliferation; retinoblastoma.

Figure 1.

Expression of miR-361-3p is downregulated in the serum and tissue of patients with RB. (A) Reverse transcription-quantitative polymerase chain reaction analysis of miR-361-3p expression in the serum of 10 patients with RB and 10 healthy participants. (B) miR-361-3p expression in RB samples and normal tissue obtained from patients with RB. ***P<0.001 vs. normal serum or normal tissues. miR, microRNA; RB, retinoblastoma.

Figure 2.

miR-361-3p inhibits RB cell proliferation. (A) RT-qPCR analysis of miR-361-3p expression in Y79 and Weri-Rb-1 RB cells following transfection with miR-361-3p or control miR for 48 h. (B) An MTT assay was performed in Y79 and Weri-Rb-1 RB cells that exhibited miR-361-3p overexpression. (C) Relative cell proliferation was determined using a CCK-8 assay. (D) RT-qPCR analysis of miR-361-3p inhibitor expression in Y79 and Weri-Rb-1 RB cells following transfection with a miR-361-3p inhibitor or control miR for 48 h. (E) An MTT assay was performed following miR-361-3p inhibition. (F) Relative cell growth was assessed using a CCK-8 assay following miR-361-3p inhibition. *P<0.05 and **P<0.01 vs. control miR. miR, microRNA; RB, retinoblastoma; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; CCK-8, cell counting kit-8; in, inhibitor.

Figure 3.

miR-361-3p overexpression reduces RB cell stemness. (A) Reverse transcription-quantitative polymerase chain reaction and (B) western blotting of the expression level of stem cell markers (CD133, SOX-2 and Nanog) in Y79 and Weri-Rb-1 RB cells transfected with miR-361-3p. (C) A tumor sphere assay was performed in Y79 and Weri-Rb-1 RB cells transfected with miR-361-3p (magnification, ×200). *P<0.05 vs. control miR. miR, microRNA; RB, retinoblastoma; CD, cluster of differentiation; SOX-2, SRY box-2.

Figure 4.

miR-361-3p inhibition promotes RB cell stemness. (A) Reverse transcription-quantitative polymerase chain reaction analysis and (B) western blotting of stem cell marker (CD133, SOX-2 and Nanog) expression in Y79 and Weri-Rb-1 RB cells transfected with a miR-361-3p inhibitor. (C) A tumor sphere assay was performed in Y79 and Weri-Rb-1 RB cells transfected with a miR-361-3p inhibitor (magnification, ×200). *P<0.05 vs. control miR. miR, microRNA; RB, retinoblastoma; CD, cluster of differentiation; SOX-2, SRY box-2; in, inhibition.

Figure 5.

miR-361-3p directly targets GLI1 and GLI3. (A) A graphical representation of miR-361-3p binding sites in wild-type GLI1 and GLI3 3′-UTRs. (B) A luciferase assay was performed in Y79 and Weri-Rb-1 RB cells transfected with miR-361-3p and GLI1 or GLI3 3′UTR luciferase reporter plasmids. (C) Western blotting was performed to assess GLI1 and GLI3 expression in Y79 and Weri-Rb-1 RB cells transfected with miR-361-3p. *P<0.05 vs. control miR. miR, microRNA; RB, retinoblastoma; GLI, glioma-associated oncogene homologue; UTR, untranslated region.

Figure 6.

Negative correlation between miR-361-3p and GLI1/3 in RB samples. A Pearson's correlation analysis was performed between miR-361-3p and GLI1/3 in 10 RB tissues GLI1, P<0.05, r=−0.407; GLI1, P<0.05, r=−0.494. miR, microRNA; GLI, glioma-associated oncogene homologue; RB, retinoblastoma.