Regulation of Epithelial-Mesenchymal Transition in Ovarian Cancer by LncRNA MALAT1 via SNAI2 and MiR-200c-3p

Summary

This protocol describes methods to evaluate the role of MALAT1 in promoting epithelial-mesenchymal transition in epithelial ovarian cancer through its regulation of the miR-200c-3p/SNAI2 axis.

Abstract

Long non-coding RNA MALAT1 regulates epithelial-mesenchymal transition (EMT) and metastasis in epithelial ovarian cancer (EOC) through a competing endogenous RNA (ceRNA) mechanism involving miRNA modulation. This study aimed to elucidate the molecular pathway by which MALAT1 influences EMT and metastatic behavior via interaction with miR-200c-3p and SNAI2. MALAT1 expression was genetically manipulated in the EOC cell line SK-OV-3 by either overexpression or knockdown. Functional effects on EMT-related protein levels, cell migration, and invasion were assessed using Western blotting, wound healing, and Transwell assays, respectively. Bioinformatics analysis identified miR-200c-3p as a common target of MALAT1 and SNAI2. The MALAT1/miR-200c-3p/SNAI2 axis was further validated by dual-luciferase reporter assays and immunofluorescence staining to confirm direct molecular interactions. Overexpression of MALAT1 enhanced SK-OV-3 cell migration by 20% and invasion by 5%, accompanied by a significant increase in SNAI2 expression (P < 0.01). Conversely, MALAT1 knockdown suppressed these phenotypes. Dual-luciferase assays confirmed that miR-200c-3p directly binds to both MALAT1 and SNAI2 (P < 0.001). miR-200c-3p overexpression reduced MALAT1-driven EMT by downregulating SNAI2 (P < 0.05), whereas restoring SNAI2 reversed the inhibitory effects of MALAT1 silencing on metastasis. This protocol demonstrates that MALAT1 promotes EMT and metastasis in EOC by functioning as a ceRNA that sequesters miR-200c-3p, leading to derepression of SNAI2. The findings provide a novel mechanistic insight and identify the MALAT1/miR-200c-3p/SNAI2 axis as a potential therapeutic target to inhibit ovarian cancer metastasis.

Introduction

Ovarian cancer (OvCa) is a highly insidious and late-diagnosed malignancy. It typically progresses asymptomatically until reaching later stages, characterized by aggressive growth primarily within the peritoneal cavity. This pathological process typically presents with ascites, reduced therapeutic responsiveness, and a poor prognosis¹. Epidemiological statistics from 2018 revealed about 295,000 newly diagnosed cases worldwide, with 185,000 mortality events, reflecting a persistent upward trajectory in disease burden². Although modern treatment protocols incorporate surgical intervention and pharmacological regimens, the ....

Protocol

Cell culture and transfection

The SK-OV-3 human OvCa cell line was cultured under standard conditions. Cells were maintained in a culture medium comprising 89% McCoy's 5A, 10% fetal bovine serum, and 1% penicillin-streptomycin solution, in a humidified incubator at 37 °C with 5% CO₂. The medium was replaced every 2-3 days, and cells were passaged upon reaching 80-90% confluence using 0.25% trypsin-EDTA. Lentiviral constructs for MALAT1 overexpression and MALAT1 knockdown (shRNA), as well as miR-200c-3p mimic, inhibitor, and negative controls, along with SNAI2-targeting siRNA and i....

Results

Migratory and invasive capacities of SK-OV-3 cells modulated by MALAT1

The experimental workflow is outlined in Figure 1. To investigate the role of MALAT1 in OvCa cell migration and invasion, we modulated its expression in SK-OV-3 cells. Upon knockdown of MALAT1, qPCR confirmed significant suppression of MALAT1 levels (p

Discussion

OvCa, the fifth leading gynecologic cancer, remains a major concern for women's health². However, most cases are identified in advanced stages, which significantly limits effective treatment options, resulting in minimal improvement in overall survival rates¹². Cancer metastasis stands out as a major contributor to patient mortality in EOC¹³. Emerging evidence implicates lncRNAs and miRNAs as pivotal regulators of EOC pathogenesis

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.22 μm PVDF membranes	Beyotime	FFP32
All-in-One miRNA qRT-PCR Detection Kit	FulenGen	QP115
Bovine serum albumin	Beyotime	ST025
Chemiluminescence substrate	Suzhou Haixing Bioscience Co., Ltd.	TCH-C335
Chemiluminescence substrate	Millipore	WBKLS0100
Dual Luciferase Reporter Gene Assay Kit	beyotime	RG027
Foetal bovine serum	Suzhou Haixing Bioscience Co., Ltd.	FBP-C550
Goat Anti-Mouse IgG FITC	Affinity	S0007
Goat Anti-Rabbit IgG FITC	Affinity	S0008
Lipofectamine 2500	Guangzhou Yingxin Biotechnology Co., Ltd.	sj-lip-2500
MALAT1 interference lentivirus	SHANGHAI GENECHEM Co., Ltd.	LV-MALAT1-sh
MALAT1 overexpression lentivirus	SHANGHAI GENECHEM Co., Ltd.	LV-MALAT1-OE
McCoy's 5A	Suzhou Haixing Bioscience Co., Ltd.	GUMD-B305
miR-200c-3p mimic	SHANGHAI GENECHEM Co., Ltd.	mic-hsa-200c-3p
Multifunctional enzyme immunoassay analyzer	Perkin Elmer	Envision 2100
Penicillin-streptomycin solution	Suzhou Haixing Bioscience Co., Ltd.	GUSA-R002
Phosphatase inhibitor cocktail	GSbioth	GS1439A
Primary antibody E-cadherin	Affinity	BF0219
Primary antibody N-cadherin	Affinity	AF5239
Primary antibody Vimentin	Affinity	AF7013
PrimeScript RT Master Mix	Takara	RR036A
Real-Time PCR Detection System	Bio-Rad	CFX96
Refrigerated Centrifuge	eppendorf	5810R
RIPA lysis buffer	GSbioth	GS504A
SDS-polyacrylamide gels	Beyotime	P0012A
Secondary antibody Goat Anti-Mouse IgG FITC	Affinity	S0007
Secondary antibody Goat Anti-Rabbit IgG FITC	Affinity	S0008
SNAI2 siRNA	Guangzhou Yingxin Biotechnology Co., Ltd.	si-hsa-snail2
StarBase database	Sun Yat-sen University	http://starbase.sysu.edu.cn
The BCA assay kit	Dingguo Biotech	BCA02
The Cell culture incubator	Heraeus	Hera cell 150
The imaging analysis system	Bio-Rad	GEL DOC 1000
The inverted microscope	Mshot	MI152-N
The RNA extraction kit	ECOTOP	EK-1328-50T
TRIzol reagent	Guangzhou Yingxin Biotechnology Co., Ltd.	T751379
Universal electrophoresis power supply	wixscientific	WIX-EP600