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 persistent limitations in early detection combined with metastasis and therapeutic resistance maintain long-term survival rates below 45%³. Recurrence within 12-18 months is commonly observed in many patients with advanced disease⁴. These clinical realities underscore the urgent need for identifying clinically relevant diagnostic markers and therapeutic monitoring strategies. Therefore, it is valuable for identifying clinically relevant diagnostic markers.

Currently, researchers have discovered that a substantial proportion of the human genome generates long non-coding RNAs (lncRNAs)⁵. LncRNAs do not encode proteins, exist solely in RNA form, and play regulatory roles in various biological functions of cells. LncRNAs exhibit non-conservation and display highly tissue-specific expression patterns⁶. Notably, specific overexpression of lncRNAs has been observed in multiple cancer tissues⁷. The competing endogenous RNA (ceRNA) mechanism has revealed a novel mode of RNA interaction. It is well-established that microRNAs (miRNAs) can silence genes through sequence-specific interactions with 3'untranslated regions (UTR) of mRNA. Meanwhile, non-coding RNAs (ncRNAs) can competitively bind to miRNA, thereby impeding their capacity to attach to target genes and influencing gene expression⁸. The ceRNA mechanism is currently the most widely accepted hypothesis for the regulatory role of lncRNAs. The tripartite crosstalk between coding transcripts, miRNAs, and regulatory RNAs constructs intricate molecular networks that influence tumor initiation and progression⁹.

First identified in non-small cell lung cancer metastases, MALAT1 has emerged as a widely overexpressed lncRNA in numerous malignancies¹⁰. In our previous studies, we identified increased expression of MALAT1 in OvCa tissues that directly associates with OvCa staging¹¹. Here we present a protocol to genetically modulate MALAT1 expression in the epithelial OvCa (EOC) cell line SK-OV-3 and assess its impact on epithelial-mesenchymal transition (EMT)-related protein expression, migratory capacity, and invasive potential via molecular assays like qPCR, western blotting, dual-luciferase reporter assays, and functional assays such as wound healing assays and Transwell assays. The aim is to elucidate the molecular mechanism by which MALAT1 regulates EMT and metastatic dissemination in EOC through interactions with miR-200c-3p and SNAI2.

Existing methods for studying lncRNA-miRNA interactions often employ in silico prediction tools alone, which may lack experimental validation. For EMT regulation studies, traditional histological methods may not provide detailed molecular insights. By employing dual-luciferase reporter assays for experimental validation, our study overcomes these limitations, offering more reliable confirmation of predicted interactions. Furthermore, the combination of multiple functional assays, such as scratch and Transwell assays, alongside molecular techniques, enables a comprehensive understanding of EMT at both cellular and molecular levels, surpassing the depth provided by single-assay approaches.

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 its corresponding control siRNA, were obtained from commercial sources. For transfection, SK-OV-3 cells were seeded in 24-well plates at a density of 5 × 10⁴ cells/well in 500 µL of complete medium and allowed to adhere overnight. Transfections were performed using a lipid-based transfection reagent (RRID: AB_2548650) according to the manufacturer's instructions. Briefly, nucleic acids (miRNA mimics/inhibitors or siRNAs) and a lipid-based transfection reagent were diluted in reduced-serum medium separately, incubated for 5 min, then mixed together and incubated for an additional 15 min at room temperature to allow complexation. The complexes were added dropwise to the cells. After 6 h, the medium was replaced with fresh complete medium. Cells were harvested 24-48 h post-transfection for downstream analyses.

Real-time fluorescent quantitative PCR (qPCR)

Total RNA was extracted from cultured SK-OV-3 cells using the RNA extraction reagent according to the manufacturer's instructions. Briefly, cells were lysed in 1 mL of reagent per well of a 6-well plate, and RNA was isolated using a commercial RNA extraction kit following the provided protocol. RNA concentration and purity were determined using a spectrophotometer, and samples with an A₂₆₀/A₂₈₀ ratio between 1.8 and 2.0 were used for downstream applications. Reverse transcription was performed using the RT Master Mix (RRID: SCR_018597) with 500 ng of total RNA in a 20 µL reaction volume. The thermal cycling conditions were as follows: 37 °C for 15 min, followed by 85 °C for 5 s to inactivate the reverse transcriptase. The resulting cDNA was stored at −20 °C until use. Quantitative real-time PCR was conducted using a commercial miRNA qRT-PCR detection kit on a real-time PCR instrument. Each reaction was performed in a 20 µL volume containing 10 µL of 2x qPCR master mix, 1 µL of specific primers (forward and reverse, 10 µM), 2 µL of cDNA template, and 7 µL of RNase-free water. The thermal cycling conditions were: 95 °C for 10 min, followed by 40 cycles of 95 °C for 10 s and 60 °C for 30 s. Melting curve analysis was performed to verify product specificity. Expression levels were calculated using the ΔΔCt method, with GAPDH (for mRNA) or U6 (for miRNA) as internal controls. All reactions were performed in triplicate, and data are presented as mean ± SD.
NOTE: The RNA extraction reagent is a hazardous chemical. It should be handled in a fume hood as it is corrosive and may cause skin and eye irritation. Waste containing the reagent should be disposed of according to local hazardous waste disposal regulations.

Scratch assay

SK-OV-3 cells were seeded into 24-well plates at a density of 5 × 10⁴ cells/well and cultured until they reached approximately 90-100% confluency, forming a uniform monolayer. A 10 µL sterile pipette tip was then used to create a linear scratch through the cell monolayer in the center of each well. To ensure consistency, the same angle and pressure were applied during scratch formation across all wells. After scratching, PBS was added to remove detached cells with two gentle washes. Cells were then cultured in serum-free McCoy's 5A medium to minimize proliferation effects. Images of the scratch area were captured immediately (0 h) and again at 24 h and 48 h using an inverted microscope at 10x magnification. The width of the scratch at each time point was measured using ImageJ software, and the percentage of wound closure compared to the initial width used to calculate the migration rate.

Transwell assay

SK-OV-3 cells were seeded into the upper chambers of Transwell inserts (upper chamber contains 200 µL of 1% complete medium, lower chamber contains 1 mL of 5% complete medium) at a density of 5 × 10³ cells per insert, incubated at 37 °C, 5% CO₂ humidified environment for 24 h before further analysis. After incubation for 24 h, the inserts were removed and washed 3x with PBS, followed by fixation in 4% paraformaldehyde (PFA) for 15 min. After washing with PBS, cells were stained with a 1% crystal violet solution. Following staining, cells were rinsed with PBS and observed under an inverted microscope.
NOTE: Crystal violet is a biological stain and may be harmful if ingested or in contact with skin. It should be handled carefully, and waste should be disposed of properly according to local regulations.

Dual-luciferase reporter assay

Transfected cells are incubated under standard culture conditions (37 °C, 5% CO₂ humidified environment) for 48 h before lysis. After 48 h of incubation, cells were lysed and centrifuged at 5,000 × g for 5 min at 4 °C. A 50 µL aliquot of the supernatant was transferred to a 96-well plate, followed by the sequential addition of 100 µL of firefly luciferase reagent and 100 µL of Renilla luciferase reagent to each well. Firefly and Renilla luciferase activities were measured using the Dual-Luciferase Reporter Assay System on a Luminometer. Renilla luciferase activity was normalized to Firefly luciferase activity to account for variations in transfection efficiency.

Immunofluorescence assay

SK-OV-3 cells were fixed in 4% PFA for 15 min at room temperature and then washed 3x with PBS. To block non-specific binding, cells were incubated with 5% goat serum in PBS for 1 h at room temperature. Following blocking, cells were incubated overnight at 4 °C with primary antibodies against E-cadherin (RRID: AB_2833860), N-cadherin (RRID: AB_2837725), and Vimentin (RRID: AB_2835318), each diluted 1:200. The next day, cells were washed 3x with PBS and incubated with appropriate fluorophore-conjugated secondary antibodies [anti-mouse IgG-FITC (RRID: AB_2843438) and anti-rabbit IgG-FITC (RRID: AB_2843439)] diluted 1:500 for 1 h at 37 °C in the dark.
NOTE: Paraformaldehyde is toxic and a potential carcinogen. It should be handled with care, wearing appropriate personal protective equipment (PPE) such as gloves and goggles. Waste containing PFA should be disposed of as hazardous waste.

Statistical analysis

For comparisons between two groups, a t-test was used, and for multiple group comparisons, ANOVA was applied. Data are presented as mean ± SD. Statistical significance was set at *p < 0.05, **p < 0.01, and ***p < 0.001. All experiments were performed with three biological replicates (n = 3).

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