miR-22-Mediated Regulation of Wnt/β-Catenin Signaling by Curcumin in Retinoblastoma

Summary

This study presents a protocol for evaluating the effects of curcumin on retinoblastoma cell behavior through modulation of the Wnt/β-catenin pathway and miR-22 expression.

Abstract

Retinoblastoma (RB) is a common intraocular malignant tumor affecting infants and children, yet its precise etiology and pathogenesis remain incompletely understood. Curcumin, a bioactive polyphenol, inhibits tumor progression via microRNA-mediated modulation of the Wnt/β-catenin signaling cascade. This study aimed to clarify how curcumin mediates its antitumor effects in RB by investigating its regulation of miRNA-22 (miR-22) expression and exploring the underlying molecular mechanisms. Two validated retinoblastoma models (SO-RB50/WERI-Rb-1) were treated with curcumin at varying concentrations. To delineate miR-22's regulation of Wnt/β-catenin signaling, target cells were transduced with either a miR-22 mimic lentivirus or a non-functional control lentivirus. Xenograft tumor models were established in mice using human RB cells to observe the in vivo effects of curcumin on tumor size, miR-22 expression, and Wnt/β-catenin protein levels. Cellular proliferation, invasion, and apoptosis were assessed using the CCK-8, Transwell, and Annexin V-APC-PI dual staining assay, respectively. miR-22 levels were quantified by RT-PCR, and Wnt1 and β-catenin expression profiles were determined by Western blot analysis. Curcumin treatment resulted in decreased proliferation and invasiveness in RB cells, while enhancing apoptosis and elevating miR-22 expression. Inhibition of miR-22 diminished curcumin's effects on the Wnt/β-catenin signaling pathway. In xenograft studies, curcumin significantly reduced tumor size and enhanced miR-22 expression within the tumors, effectively suppressing Wnt/β-catenin signaling. These findings demonstrate that curcumin inhibits RB cell proliferation and invasiveness while promoting apoptosis, primarily mediated through miR-22 upregulation and subsequent inhibition of the Wnt/β-catenin pathway.

Introduction

Retinoblastoma (RB), a pediatric malignancy with high aggressiveness arising in retinal tissues, poses a substantial health threat, especially to children under the age of five. Genetic mutations, particularly those affecting the RB1 gene, are closely associated with the development of this disease¹. Considering the significant prevalence of retinoblastoma (RB) among infants and children, coupled with the fact that approximately 9000 new cases are reported annually, predominantly in low-income and developing countries, a comprehensive understanding of the disease mechanisms is imperative for developing effective therapeutic strategies and impro....

Protocol

All procedures involving animals were reviewed and approved by the Animal Ethics and Welfare Committee (AEWC) of Tianjin Eye Hospital (Approval No. NKYY-DWLL-2023-054). The reagents and the equipment used are listed in the Table of Materials.

1. Cell culture

Cells of the human retinoblastoma lines WERI-Rb-1 and SO-RB50 were cultured in high-glucose Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. Cells were seeded at an appropriate density in culture flasks and maintained at 37 °C in a humidified atmosphere containing %CO

Results

Dose-Dependent anti-proliferative and anti-invasive effects of curcumin on human retinoblastoma cells, accompanied by Wnt/β-catenin pathway deactivation
Human RB cells were treated with varying curcumin doses over a 24 h period. The findings revealed a dose-dependent decline in cell viability and invasion, coupled with an increase in apoptosis. Specifically, as the concentration of curcumin increased from 10 to 50 µM, cell proliferation decreased progressively in both SO-Rb50 and WERI-Rb-1 cell lines.......

Discussion

Curcumin, a polyphenol derived from Curcuma longa rhizomes, is renowned for its anti-inflammatory, antiangiogenic, anti-proliferative, and antioxidant properties^23,24. Additionally, it has antitumor properties, particularly its ability to induce apoptosis in malignant cells²⁵. Although prior research has emphasized the growth-suppressive effects of curcumin on RB cells^22,26,

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Annexin V-APC/PI dual staining kit	Solarbio	CA1020	quantitatively assess apoptosis in RB cells via flow cytometry
CCK-8 Assay Kit	Beyotime	C0038	measure the absorbance at 490 nm for evaluating cell proliferative capacity
CO2 incubator	Eppendorf		maintain optimal culture conditions (37°C, 5% CO2, and saturated humidity) for RB cell propagation
Curcumin	Sigma	C1386	Investigating the Anti-Tumor Effects of Curcumin on Retinoblastoma (RB)
ECL chemiluminescent substrate	Bioss	C05-07004	visualize protein bands in Western blot analysis
Fetal Bovine Serum (FBS)	VivaCell	C04001-500 C04002-500	Cell Culture
Flow cytometry	Beckman Coulter	Gallios	quantitatively analyze the apoptosis rate of cells
ImageJ Software	National Institutes of Health	version 6	Western blot band analysis,assess protein expression levels
Inverted microscope	Olympus	CKX53	observe and count invasive cells at 400× magnification in the Transwell assay
Lipofectamine 3000	Invitrogen	L3000015	Cell Transfection
Microplate reader	Thermo	K3	enzyme-linked immunosorbent assay (ELISA) quantification
miR-22 mimic Lentivirus	Hanheng Biotechnology	hsa-miR-22	establish a stably transfected cell line for animal experiments
Multiplex real-time PCR system	Bio-Rad	CFX384	perform quantitative RT-PCR (qRT-PCR) for precise gene expression analysis
Penicillin-Streptomycin (PS)	Abbkine	BMC1030	Added to cell culture media to prevent bacterial contamination
Protein Extraction Kit	Solarbio	BC3640-50T	protein isolation from cellular and tissue samples
psiCHECK-2 vector	promega	JR20110328	construct Wnt1-WT (wild-type) and Wnt1-MUT (mutant) reporter plasmids for investigating the interaction between miR-22 and Wnt1 3'UTR
PVDF membrane	Merck	ISEQ00010	protein blotting
Rabbit IgG secondary antibody	Cell Signaling Technologies	14708	Western blot detection by binding to the primary antibody
SPSS statistical software	IBM	SPSS 20.0	perform statistical analysis on the experimental data
SuperScript III First-Strand Synthesis SuperMix Kit	Invitrogen	11752-050	reverse transcription of RNA into cDNA for subsequent RT-PCR analysis
SYBR Premix Ex Taq II Kit	TaKaRa Bio	RR820A	For RT-PCR to quantitatively detect the expression of Wnt1, β-actin, and other target genes
TRIzol reagent	Invitrogen	12183-555	RNA extraction from both cellular and tissue samples
UV spectrophotometer	Beckman	DU800	determine the concentration and purity of RNA samples
Wnt1 antibody	Abcam	ab15251	Western blot analysis to detect Wnt1 protein expression
β-actin antibody	Abcam	ab8227	used as an internal reference to normalize protein loading in Western blot analysis
β-catenin antibody	Cell Signaling Technologies	37447	Western blot analysis to detect β-catenin protein expression