mcr-23-0478-html.html

LRRC75A-AS1 drives the epithelial-mesenchymal transition in cervical

cancer by binding IGF2BP1 and inhibiting SYVN1-mediated NLRP3

ubiquitination

Running title:

LRRC75A-AS1 activates NLRP3/IL-1β/Smad2/3 signaling and EMT

Hongying Sui

, Caixia Shi, Zhipeng Yan, Jinyang Chen, Lin Man, Fang Wang

The department of gynecological oncology, Hunan Cancer Hospital/the Affiliated Cancer Hospital of

Xiangya School of Medicine, Central South University, Changsha 410013, P.R. China

Corresponding author: Hongying Sui

, The department of gynecological oncology, Hunan Cancer

Hospital/the Affiliated Cancer Hospital of Xiangya School of Medicine, Central South University,

Changsha 410013, P.R. China

Email: suihongying0420@163.com

Tel: +86-18975118580

Conflict of interest

The authors declare that there are no conflicts of interest.

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Abstract

Cervical cancer severely affects women’s health with increased incidence and poor survival for patients

with metastasis. Our study aims to investigate the mechanism by which lncRNA LRRC75A-AS1

regulates the epithelial-mesenchymal transition (EMT) of cervical cancer through modulating m6A and

ubiquitination modification. In this study, tumor tissues were collected from patients to analyze the

expression of LRRC75A-AS1 and SYVN1. Migratory and invasive capacities of HeLa and CaSki cells

were evaluated with wound healing and transwell assays. CCK-8 and EdU incorporation assays were

employed to examine cell proliferation.

The interaction between LRRC75A-AS1, IGF2BP1, SYVN1,

and NLRP3 was evaluated through RNA immunoprecipitation, RNA pull-down, FISH, and Co-IP

assays, respectively. MeRIP-qPCR was applied to analyze the m6A modification of SYVN1 mRNA

. A

subcutaneous tumor model of cervical cancer was established. We showed LRRC75A-AS1 was

upregulated in tumor tissues, and LRRC75A-AS1 enhanced EMT through activating NLRP3/IL-

1β/Smad2/3 signaling in cervical cancer. Furthermore, LRRC75A-AS1 inhibited SYVN1-mediated

NLRP3 ubiquitination by destabilizing SYVN1 mRNA. LRRC75A-AS1 competitively bound to

IGF2BP1 protein and subsequently impaired the m6A modification of SYVN1 mRNA and its stability.

Knockdown of LRRC75A-AS1 repressed EMT and tumor growth via inhibiting NLRP3/IL-

1β/Smad2/3 signaling in mice. In conclusion, LRRC75A-AS1 competitively binds to IGF2BP1 protein

to destabilize SYVN1 mRNA, subsequently suppresses SYVN1-mediated NLRP3 ubiquitination

degradation and activates IL-1β/Smad2/3 signaling, thus promoting EMT in cervical cancer.

Implication

LRRC75A-AS1 promotes cervical cancer progression, and this study suggests LRRC75A-AS1 as a

new therapeutic target for cervical cancer.

Keywords:

Cervical cancer; m6A modification; LRRC75A-AS1; SYVN1; NLRP3; Ubiquitination

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Introduction

Cervical cancer is a type of gynecologic malignancy that starts in the cervix and ranks No.4 in

women’s cancers (1). Globally, more than 500,000 new cases are reported each year, and the incidence

of cervical cancer in advanced stage is increasing (2, 3). The treatment for cervical cancer includes

surgery, radiation therapy, and chemotherapy, contributing to a high five-year survival rate of over 90%

for patients with early-stage (4). However, cervical cancer still causes more than 300,000 deaths each

year globally, and it remains challenging to treat distant-metastasis patients (2). Metastasis is the

primary barrier for cancer treatment that leads to cancer deaths (5). Epithelial-mesenchymal transition

(EMT) is a key process that confers cancer cells metastatic properties via enhancing migration,

invasion, and apoptosis resistance (6). Thus, uncovering the mechanism underlying the regulation of

EMT is very important for developing novel therapeutics for cervical cancer.

Emerging evidence has demonstrated various roles of long non-coding RNAs (lncRNAs) in cancer

metastasis and tumorigenesis (7). LncRNA AFAP1-AS1 was upregulated in cervical cancer and

promoted cell migration and invasion (8). Feng et al. reported that lncRNA-CTS promoted EMT and

metastasis in cervical cancer (9). LncRNA LRRC75A-AS1, also known as SNHG29, has been found to

be implicated in various cancers. Li and colleagues found that LRRC75A-AS1 promoted proliferation

and invasion in breast cancer (10). Chen et al. reported that LRRC75A-AS1 functioned as a tumor

suppressor to repress proliferation and invasion in colorectal carcinoma (11). However, the

involvement of LRRC75A-AS1 in cervical cancer needs further study.

NOD-like receptor pyrin domain containing-3 (NLRP3) is a sensor that detects intracellular stress.

Upon activation, NLRP3 recruits apoptosis-associated speck-like protein containing a caspase

recruitment domain (ASC) and pro-caspase-1 to form NLRP3 inflammasome that leads to increased

cleaved caspase-1 and subsequent secretion of pro-inflammatory cytokines IL-1β and IL-18, thus

inducing pyroptosis (12, 13). NLRP3 participates in the regulation of EMT. A previous study suggested

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that NLRP3 modulated hepatocyte EMT via IL-1β/Smad2/3 signaling (14). Moreover, NLRP3 was

required for EMT via inducing IL-1β in various cancers such as breast cancer (15), colorectal cancer

(16), and oral squamous cell carcinoma (17). NLRP3 was reported to be highly related to cervical

cancer progression, but its activity in EMT of cervical cancer remains unknown. N6-methyladenosine

(m6A) modification is the most widespread RNA modification that affects cancer progression, and

m6A-dependent regulation in cervical cancer progression has been reported recently (18-20). How

LRRC75A-AS1 affects the m6A modification in cervical cancer to induce the activation of NLRP3/IL-

1β/Smad2/3 signaling is the main objective in our study.

Insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1), a conserved single-stranded RNA-

binding protein, plays key roles in carcinogenesis and embryogenesis via regulating RNA localization,

stability or translatability (21). Intriguingly, IGF2BP1 can function as a m6A reader and facilitate the

stability of m6A-modified RNAs (22, 23). LncRNAs can interact with RNA binding proteins to

modulate mRNA stability and decay (24). He et al. reported that LINC01093 interacted with IGF2BP1

to reduce the stability of GLI1 mRNA in hepatocellular carcinoma (25). Synoviolin 1 (SYVN1) is an

E3 ubiquitin ligase that regulates downstream protein ubiquitination and degradation (26). We

predicted the potential interaction of IGF2BP1 protein and LRRC75A-AS1 or SYVN1 mRNA through

bioinformatics (Starbase), suggesting the possibility that LRRC75A-AS1 might regulate SYVN1

mRNA stability by interacting with IGF2BP1, and their interaction has never been reported. In addition,

bioinformatics analysis (Ubibrowser) also showed that SYVN1 could modulate ubiquitination

modification of NLRP3.

In this study, we hypothesized that LRRC75A-AS1 might bind to IGF2BP1 protein to reduce m6A

modification of SYVN1 mRNA and then destabilize SYVN1, thus repressing NLRP3 ubiquitination

degradation, activating IL-1β/Smad2/3 signaling and subsequently promoting EMT in cervical cancer.

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Materials and Methods

Clinical specimens

Carcinoma and para-carcinoma tissues were collected from thirty patients diagnosed with cervical

cancer via pathological examination at the Hunan Cancer Hospital/the Affiliated Cancer Hospital of

Xiangya School of Medicine, Central South University following below inclusion criteria: (1) Equal or

older than 18-year-old; (2) Patients did not receive any chemoradiotherapy; (3) No other cancers and

serious disorders such as cardiovascular and autoimmune diseases; (4) No pregnancy and nursing.

Tissues were stored at -80℃ for RNA extraction and reverse transcription and quantitative PCR (qRT-

PCR) assay. Written informed consent was obtained from each patient, and this study was conducted in

accordance with Declaration of Helsinki and approved by the Ethics Committee of Hunan Cancer

Hospital/the Affiliated Cancer Hospital of Xiangya School of Medicine, Central South University.

Cell culture and treatment

Normal primary cervical epithelial cells (NPCEC, Cat# PCS-480-011), C-33A (RRID: CVCL_1094),

HeLa (RRID: CVCL_0030), SiHa (RRID:

CVCL_0032), CaSki (RRID: CVCL_1100) and ME-180

(RRID:

CVCL_1401) cells were provided by the American Type Culture Collection (ATCC, Manassas,

VA, USA) in 2021.2 and maintained in Dulbecco’s Modified Eagle Medium (DMEM, Thermo Fisher

Scientific, Waltham, MA, USA) containing 10% fetal bovine serum (FBS, Thermo Fisher Scientific).

All cells were tested for no mycoplasma contamination and authenticated via short tandem repeat (STR)

profiling every two months. After thawing, cells at passage 4-6 were used for subsequent assays. HeLa

and CaSki cells were treated with actinomycin D (Sigma-Aldrich, St. Louis, MO, USA) at 2 μg/mL for

0, 4, 8, 12 or 24 h, and RNA was extracted for analyzing the stability of SYVN1 mRNA via qRT-PCR.

For examining NLRP3 protein stability, HeLa and CaSki cells were treated with cycloheximide (CHX,

Sigma-Aldrich) at 5 μg/mL for 0, 1, 3 or 6 h, and protein was extracted for Western blotting.

Cell transfection

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shRNAs against LRRC75A-AS1 (sh-LRRC75A-AS1), IGF2BP1 (sh-IGF2BP1), SYVN1 (sh-SYVN1),

and scramble control (sh-NC) were provided by Ribobio (Guangzhou, Guangdong, China). The coding

sequences for NLRP3 were amplified through PCR and cloned into pcDNA3.1 plasmid (OE-NLRP3,

Thermo Fisher Scientific). sh-LRRC75A-AS1 was cloned into pLKO.1 puro lentivirus vector (YouBio,

Changsha, Hunan, China) to silence LRRC75A-AS1 expression. LRRC75A-AS1 was synthesized by

Sangon Biotech (Shanghai, China) and inserted into pCMV-EGFP lentivirus vector (OE-LRRC75A-

AS1, Addgene,

Watertown, MA, USA) to achieve its overexpression. For knockdown or

overexpression of LRRC75A-AS1, lentiviral particles were packaged in 293T cells (ATCC) through

co-transfection with sh-LRRC75A-AS1 or OE-LRRC75A-AS1, and psPAX2/pMD2.G system

(Addgene). After 48 h, the lentiviral supernatants were collected, mixed with fresh complete medium

and added into cells for transfection. For NLRP3 overexpression and IGF2BP1/SYVN1 knockdown,

cells were transfected with indicated plasmids using Lipofectamine 3000 (Thermo Fisher Scientific) for

48 h.

Wound healing assay

HeLa and CaSki cells with indicated transfection were seeded and maintained to form a monolayer, and

a 200 μL pipette tip was used to create scratches on the monolayer. Subsequently, cells were

maintained for 24 h, and the scratch was observed and imaged via a microscope (Zeiss, Germany).

Image J software was used to quantify wound healing speed.

Cell counting kit-8 (CCK-8) assay

Cells with indicated transfection and control cells were seeded in 96-well plates (5

cells per well)

and incubated for 12 h. Then, the medium was removed, 100 µL of pre-warmed fresh completed

medium was added into each well. CCK-8 reagent (10 µL, Beyotime, Shanghai, China) was added into

each well, and cells were incubated for 1 h. The absorbance was measured at 450 nm.

EdU incorporation assay

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Cells with indicated transfection and control cells were seeded in 24-well plates and incubated for 12 h.

EdU was added into each well at 10 µM, and cells were incubated for additional 12 h. Subsequently,

EdU incorporation was detected via click chemistry reaction with Click-iT® EdU Alexa Fluor® 594

Imaging Kit (Thermo Fisher Scientific) following the manual. Cells were counter-stained with DAPI

(Beyotime) and mounted for imaging under a microscope (Zeiss).

Transwell assay

6.5 mm Transwell inserts (8 µm pore size, Corning, Corning, NY, USA) were coated with a mixture of

Matrigel (Corning) and complete culture medium (1:1) in 24-well plates and allowed to solidify for 1 h

in a cell incubator. Then, cells with indicated transfection were seeded to the upper chamber and

maintained for invasion. After 24 h, cells in the lower chambers were fixed, stained with crystal violet

(Sigma-Aldrich) and imaged by a microscope (Zeiss).

Co-immunoprecipitation (Co-IP) assay

Approximately 5×10

cells were collected, resuspended in IP lysis buffer (Thermo Fisher Scientific)

supplemented with protease inhibitor cocktail (Thermo Fisher Scientific) and incubated on ice for 30

min. Subsequently, cell lysates were harvested, and an anti-NLRP3 (1:1000, Abcam, Cambridge, UK;

RRID: AB_2889890) or anti-SYVN1 (1:1000, Cell Signaling Technology; RRID: AB_2798607)

antibody was mixed with the lysates and incubated overnight. Normal rabbit IgG (Abcam; RRID:

AB_2687931) was used as the negative control. The following day, Protein A/G magnetic beads

(Abcam) were added, and complexes enriched by magnetic beads were eluted with SDS buffer. The

supernatants were collected, and 20 μL of sample was loaded on 10% SDS-PAGE for gel

electrophoresis and subjected to Western blotting. The ubiquitination of NLRP3 was determined with

an anti-ubiquitin antibody (1:500, Abcam), respectively.

RNA immunoprecipitation (RIP) and MeRIP-qPCR

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HeLa and CaSki cells were harvested after trypsinization. Cell pellets were resuspended in RIP solution,

and supernatants were collected after centrifugation. The anti-SYVN1 (1:1000, Cell Signaling

Technology; RRID: AB_2798607) and anti-IGF2BP1 (1:1000, Abcam; RRID: AB_2893198)

antibodies were added into the supernatants and incubated for 6 h with rotation. Normal rabbit IgG

(1:1000, Abcam; RRID: AB_2687931) was used as the negative control. Subsequently, Protein A/G

magnetic beads were added and incubated for 2 h with rotation. Beads were washed in a magnet, and

RNA was isolated using Trizol reagent (Thermo Fisher Scientific) and subjected to qRT-PCR analysis

of LRRC75A-AS1 and SYVN1. For MeRIP-qPCR, Magna MeRIP m6A Kit was ordered from

Millipore (Billerica, MA, USA). Total RNA was extracted and fragments with a length of ~150 nt were

prepared. An anti-m6A antibody (1:1500, Abcam; RRID: AB_2916290) was mixed with RNA

fragments and incubated overnight. Magnetic beads were washed, added into samples and incubated

for 2 h. Subsequently, RNA was purified and directly subjected to qRT-PCR analysis of SYVN1

expression using iTaq Universal Probes One-Step Kit (Bio-Rad, Hercules, CA, USA) following the

manual.

RNA pull-down assay

For RNA pull-down assay, biotinylated sense and anti-sense LRRC75A-AS1 probes were synthesized

by Sangon Biotech. Magnetic RNA-Protein Pull-Down Kit was provided by Thermo Fisher Scientific,

and RNA pull-down assay was performed according to the manual. RNA-binding protein complexes

were eluted using biotin elution buffer. The supernatant was collected, and were then heated for 10 min

at 95-100°C and loaded on 10% SDS-PAGE for gel electrophoresis and subjected to Western blot

analysis.

Fluorescence in situ hybridization (FISH) combined with immunofluorescence

HeLa and CaSki cells seeded on coverslips were fixed in 4% formaldehyde solution for 20 min and

permeabilized in 0.5% Triton X-100 for 15 min. After washing, cells were immersed in proteinase K at

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15 µg/µL and pre-hybridized. Subsequently, the FITC-labeled LRRC75A-AS1 probe (50 nM) was

added, and cells were incubated with the probe overnight. Then, cells were washed and incubated with

an anti-IGF2BP1 antibody (1:500, Abcam; RRID: AB_10865721) overnight at 4°C. After washing,

cells were incubated with a secondary antibody (Abcam) for 2 h in the dark. DAPI (Beyotime) was

used to stain the nuclei, and cells were mounted and imaged by a fluorescence microscope (Zeiss).

Subcutaneous tumor model

C57BL/6 nude mice (6-week-old, male) were provided by Hunan SJA laboratory animal (Changsha,

Hunan, China) and maintained in the animal facility of Hunan Cancer Hospital/the Affiliated Cancer

Hospital of Xiangya School of Medicine, Central South University. Mice were blindly grouped into sh-

NC, sh-LRRC75A-AS1, OE-NC, and OE-LRRC75A-AS1 groups (n=7 per group). sh-NC or sh-

LRRC75A-AS1-transfected HeLa cells and OE-NC or OE-LRRC75A-AS1-transfected CaSki cells

were subcutaneously injected into the right flank (1×10

cells in 200 µL PBS/mouse). After 25 days,

mice were sacrificed, and tumors were excised. The volume and weight of tumors were measured.

Animal procedures were approved by the Ethics Committee of Hunan Cancer Hospital/the Affiliated

Cancer Hospital of Xiangya School of Medicine, Central South University.

Immunohistochemistry (IHC) staining

Tumors were fixed in 4% formaldehyde and embedded in paraffin, and sections (5 µm) were prepared.

Sections were dewaxed, rehydrated and processed for antigen retrieval in citrate buffer (pH 6.0). After

washing, sections were incubated in 0.5% Triton X-100 for 20 min and blocked. Subsequently, sections

were incubated with an anti-Ki-67 (1:100, Abcam; RRID: AB_302459) or anti-E-cadherin (1:200,

Abcam; RRID: AB_2923285) antibody for 12 h. Sections were rinsed and incubated with HRP-

conjugated secondary antibodies for 2 h. DAB (Beyotime) was added to visualize the signal. Sections

were then stained with hematoxylin (Sigma-Aldrich) and mounted for observation.

qRT-PCR assay

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Total RNA was isolated from patient tissues and mice tumor tissues, and NPCEC, C-33A, HeLa, SiHa,

CaSki and ME-180 cells. RNA was reversely transcribed into cDNA using PrimeScript RT Master Mix

(Takara, Tokyo, Japan). The expression levels of LRRC75A-AS1, E-cadherin, N-cadherin, NLRP3,

SYVN1, MARCH1, MARCH7, MARCH9 and NEDD4 were determined via quantitative PCR using

SYBR Green Master Mix (Applied Biosystems, Carlsbad, CA, USA) and normalized to GAPDH. The

−∆∆Ct

method was used to analyze their expression. Primers were provided from Sangon Biotech.

Western blotting

Tumors were homogenized and cells were lysed in lysis buffer. Lysates were centrifugated for

supernatant collection. 10% SDS-PAGE was prepared, and protein (~30 µg) was loaded for

electrophoresis and transferred to PVDF membranes (Bio-Rad). Membranes were blocked in 5% BCA

for 2 h at room temperature and incubated with anti-IGF2BP1 (1:1000, Abcam; RRID: AB_2893198),

anti-SYVN1 (1:1500,

Cell Signaling Technology; RRID: AB_2798607), anti-NLRP3 (1:1000, Abcam;

RRID: AB_2889890), anti-ASC (1:2000,

Thermo Fisher Scientific;

RRID: AB_2636363), anti-MMP-9

(1:2000, Abcam;

RRID: AB_2928971), anti-E-cadherin (1:1000, Abcam; RRID: AB_2923285), anti-

N-cadherin (1:500, Cell Signaling Technology; RRID: AB_10694647), anti-slug (1:500, Cell Signaling

Technology; RRID: AB_2239535), anti-cleaved caspase-1 (1:1000, Cell Signaling Technology;

RRID:

AB_1903916), anti-IL-1β (1:500, Cell Signaling Technology; RRID: AB_2715503), anti-p-Smad2

(1:1000, Cell Signaling Technology; RRID: AB_2798798), anti-Smad2 (1:1000, Cell Signaling

Technology; RRID: AB_10626777), anti-p-Smad3 (1:1000, Cell Signaling Technology; RRID:

AB_2193207), or anti-Smad3 (1:1000, Cell Signaling Technology; RRID: AB_2193182) antibody

overnight at 4°C. Subsequently, membranes were incubated with HRP-conjugated secondary

antibodies, and ECL substrate (Bio-Rad) was used to visualize bands. Primary and secondary

antibodies were obtained from Abcam.

Statistical analysis

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Experiments in this study were biologically repeated for at least three times, which were analyzed using

the GraphPad Prism 6.0, and results were expressed as mean ± standard deviation (SD). All the data

meet the assumption of normal distribution. The variance of two and multiple groups was analyzed

using the student’s

test and one-way analysis of variance (ANOVA) with Tukey post hoc test,

respectively.

< 0.05 was statistically significant.

< 0.05, **

< 0.01 and ***

< 0.001.

Data Availability

The data generated in this study are available upon request from the corresponding author.

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Results

LRRC75A-AS1 promoted EMT and NLRP3/IL-1β/Smad2/3 signaling in cervical cancer

To explore the role of LRRC75A-AS1 in cervical cancer, we collected carcinoma and para-carcinoma

tissues from cervical cancer patients and found that LRRC75A-AS1 was highly expressed in tumor

tissues compared to para-carcinoma tissues

(Fig. 1A

), suggesting that LRRC75A-AS1 might be

involved in cervical cancer progression. We examined LRRC75A-AS1 expression in SiHa, HeLa, C-

33A, CaSki and ME-180 cervical cancer cells, and HeLa and CaSki cells showed highest and lowest

expression of LRRC75A-AS1, respectively (

Fig. 1B

). Therefore, we chose HeLa and CaSki cells as

vitro

cell models. Knockdown of LRRC75A-AS1 led to about 70% decrease in LRRC75A-AS1

expression in HeLa cells, and its overexpression led to about 3.5-fold upregulation in CaSki cells (

Fig.

). CCK-8 and EdU incorporation assays showed that knockdown of LRRC75A-AS1 inhibited Hela

cell proliferation with around 50% decrease, but its overexpression promoted CaSki cell proliferation

with a 1.4-fold increase (

Fig. 1D

and

). Moreover, silencing of LRRC75A-AS1 suppressed about 30%

of HeLa cell migration, but overexpression of LRRC75A-AS1 promoted CaSki cell migration with a 2-

fold increase (

Fig. 1F

). Silencing of LRRC75A-AS1 led to around 70% decrease in the invasive

capacity of HeLa cells, but overexpression of LRRC75A-AS1 promoted CaSki cell invasion with about

2-fold increase (

Fig. 1G

). Furthermore, knockdown of LRRC75A-AS1 repressed the expression of

MMP-9 (~60%), N-cadherin (~40%) and slug (~70%), and promoted E-cadherin expression (~3 fold)

in HeLa cells, whereas overexpression of LRRC75A-AS1 exerted opposite effects on the expression of

these EMT-related markers in CaSki cells (

Supplementary Figure 1A

). Besides, LRRC75A-AS1

silencing reduced the expression of NLRP3 (~45%), ASC (~80%), cleaved caspase-1 (~50%), IL-1β

(~45%), p-Smad2 (~40%), and p-Smad3 (~30%) in HeLa cells, but overexpression of LRRC75A-AS1

promoted their expression levels in CaSki cells (

Supplementary Figure 1C

). Also, we confirmed that

silencing of LRRC75A-AS1 upregulated E-cadherin with about 5-fold increase while downregulated

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around 70% of N-cadherin expression at the mRNA levels, and overexpression of LRRC75A-AS1

exhibited the reverse effects (

Supplementary Figure 1B

). However, knockdown or overexpression of

LRRC75A-AS1 did not affect the mRNA level of NLRP3 (

Supplementary Figure 1B

), indicating that

LRRC75A-AS1 may regulate NLRP3 expression only at the protein level. Collectively, these results

showed that LRRC75A-AS1 enhanced EMT and NLRP3/IL-1β/Smad2/3 signaling in cervical cancer.

LRRC75A-AS1 silence repressed EMT through suppression of NLRP3/IL-1β/Smad2/3 signaling

in cervical cancer

To evaluate whether LRRC75A-AS1-mediated regulation of EMT in cervical cancer was dependent on

NLRP3, LRRC75A-AS1 was knocked down and NLRP3 was overexpressed in HeLa cells. LRRC75A-

AS1 was efficiently knocked down, and overexpression of NLRP3 did not affect its expression (

Fig.

). Silencing of LRRC75A-AS1 significantly reduced NLRP3 expression at protein level, but it was

restored by NLRP3 overexpression (

Fig. 2B

and

). LRRC75A-AS1 silencing-mediated suppression

of HeLa cell migration (

Fig. 2D

) and invasion (

Fig. 2E

) was reversed by NLRP3 overexpression. In

addition, the downregulation of MMP-9, N-cadherin and slug, and upregulation of E-cadherin in

LRRC75A-AS1-silenced cells were reversed by NLRP3 overexpression (

Fig. 2F

and

). Furthermore,

LRRC75A-AS1 silencing-mediated downregulation of ASC, cleaved caspase-1, IL-1β, p-Smad2, and

p-Smad3 expression was reversed by overexpression of NLRP3 (

Fig. 2H

). These findings suggested

that LRRC75A-AS1 facilitated EMT dependent on NLRP3.

LRRC75A-AS1 suppressed SYVN1-dependent NLRP3 ubiquitination degradation

To further investigate the mechanism by which LRRC75A-AS1 only regulates NLRP3 protein level,

we treated LRRC75A-AS1-silenced HeLa cells and LRRC75A-AS1-overexpressed CaSki cells with

CHX. Knockdown of LRRC75A-AS1 accelerated NLRP3 protein degradation in response to CHX

treatment, whereas overexpression of LRRC75A-AS1 decelerated its degradation (

Fig. 3A

). Moreover,

the ubiquitination level of NLRP3 was enhanced with about 2.5-fold increase by LRRC75A-AS1

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knockdown in MG-132-treated HeLa cells but reduced about 80% by LRRC75A-AS1 overexpression

in CaSki cells (

Fig. 3B

), suggesting that LRRC75A-AS1 repressed NLRP3 ubiquitination degradation.

As shown in

Fig. 3C

, several E3 ubiquitin ligases were predicted to be implicated in NLRP3

ubiquitination modification through Ubibrowser database. We examined the expression of top 5 E3

ubiquitin ligases and found that only SYVN1 was significantly upregulated by LRRC75A-AS1

silencing and downregulated by LRRC75A-AS1 overexpression (

Fig. 3D

). The expression levels of

MARCH9, NEDD4, MARCH1 and MARCH7 were not affected by LRRC75A-AS1 (

Fig. 3D

Furthermore, Co-IP assay showed that knockdown of LRRC75A-AS1 promoted SYVN1/NLRP3

interaction in HeLa cells and overexpression of LRRC75A-AS1 reduced their interaction in CaSki cells

(

Fig. 3E

). Besides, SYVN1 was downregulated in cervical cancer tissues (

Fig. 3F

) and cells (

Fig. 3G-

), and its expression was negatively correlated with LRRC75A-AS1 expression in cancer tissues (

Fig.

). Thus, our findings suggested that LRRC75A-AS1 inhibited NLRP3 ubiquitination degradation via

disrupting SYVN1/NLRP3 interaction in cervical cancer.

LRRC75A-AS1 reduced NLRP3 ubiquitination via destabilizing SYVN1 mRNA

Furthermore, we found that SYVN1 mRNA (

Fig. 4A

) and protein (

Fig. 4B

) levels were also

upregulated in LRRC75A-AS1-silenced HeLa cells and downregulated in LRRC75A-AS1-

overexpressed CaSki cells. Moreover, silencing of LRRC75A-AS1 improved SYVN1 mRNA stability

in HeLa cells in response to actinomycin D treatment, and overexpression of LRRC75A-AS1 promoted

SYVN1 mRNA decay (

Fig. 4C

). The increased SYVN1 expression in LRRC75A-AS1-silenced HeLa

cells was abolished by SYVN1 knockdown (

Fig. 4D

and

). In addition, increased ubiquitination level

of NLRP3 in LRRC75A-AS1-silenced HeLa cells in response to MG-132 was reversed by

simultaneous SYVN1 knockdown (

Fig. 4F

). LRRC75A-AS1 silencing-mediated enhanced

SYVN1/NLRP3 interaction was also disrupted by SYVN1 knockdown (

Fig. 4G

). In conclusion,

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LRRC75A-AS1 promoted SYVN1 mRNA decay to restrain NLRP3 ubiquitination degradation in

cervical cancer.

SYVN1 knockdown reversed the effects on EMT and NLRP3/IL-1β/Smad2/3 signaling caused by

LRRC75A-AS1 silencing

Subsequently, we determined whether LRRC75A-AS1 promoted EMT process through SYVN1. We

observed that LRRC75A-AS1 silencing-mediated suppression of HeLa cell migration (

Fig. 5A

) and

invasion (

Fig. 5B

) was abrogated by SYVN1 knockdown. Besides, reduced expression of MMP-9, N-

cadherin, slug, and increased E-cadherin expression in LRRC75A-AS1-silenced HeLa cells were

reversed by silencing of SYVN1 (

Fig. 5C

and

). Furthermore, LRRC75A-AS1 silencing-mediated

decreased expression of ASC, cleaved caspase-1, IL-1β, p-Smad2, and p-Smad3 was upregulated by

silencing of SYVN1 (

Fig. 5E

). These observations showed that LRRC75A-AS1 promoted EMT and

NLRP3/IL-1β/Smad2/3 signaling through modulating SYVN1.

LRRC75A-AS1 competitively bound to IGF2BP1 to destabilize SYVN1 mRNA via reducing its

m6A modification

To clarify the regulatory relationship between LRRC75A-AS1 and SYVN1, RIP method was used and

results showed no direct interaction between SYVN1 protein and LRRC75A-AS1 (

Fig. 6A

). IGF2BP1,

a m6A modification reader, was predicted to interact with LRRC75A-AS1 and SYVN1 mRNA (

Fig.

). Furthermore, RIP assay verified that both LRRC75A-AS1 and SYVN1 mRNA could be

efficiently enriched by IGF2BP1 protein (

Fig. 6C

), and co-localization of LRRC75A-AS1 and

IGF2BP1 protein in cytoplasm was confirmed by FISH method (

Fig. 6D

). The accumulation of m6A at

SYVN1 mRNA was significantly reduced by knockdown of IGF2BP1 in HeLa and CaSki cells (

Fig.

). Subsequently, LRRC75A-AS1-mediated enrichment of IGF2BP1 was reduced by LRRC75A-AS1

knockdown but enhanced by LRRC75A-AS1 overexpression through RNA pull-down assay (

Fig. 6F

Silencing of LRRC75A-AS1 raised the IGF2BP1 protein-mediated enrichment of SYVN1 mRNA in

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HeLa cells, whereas overexpression of LRRC75A-AS1 reduced its enrichment in CaSki cells (

Fig. 6G

These data implied that LRRC75A-AS1 competitively bound to IGF2BP1 protein and subsequently

reduced the m6A modification of SYVN1 mRNA, thus promoting SYVN1 mRNA decay in cervical

cancer.

LRRC75A-AS1 promoted tumor growth and EMT through NLRP3/IL-1β/Smad2/3 signaling in

vivo

To evaluate the function of LRRC75A-AS1 in cervical cancer progression

in vivo

, mice were

subcutaneously injected with sh-LRRC75A-AS1-transfected HeLa cells or OE-LRRC75A-AS1-

transfected CaSki cells. As expected, knockdown of LRRC75A-AS1 significantly caused about 70%

decrease in tumor growth, and overexpression of LRRC75A-AS1 exerted opposite results with about 2-

fold increase (

Fig. 7A-C

). Additionally, knockdown of LRRC75A-AS1 inhibited Ki-67 expression and

enhanced E-cadherin expression, overexpression of LRRC75A-AS1 upregulated Ki-67 but reduced E-

cadherin expression in tumors (

Fig. 7D

and

). SYVN1 was upregulated with about 5-fold increase in

tumors formed by LRRC75A-AS1-silenced HeLa cells and downregulated around 80% in tumors

formed by LRRC75A-AS1-overexpressed CaSki cells (

Fig. 7F

). In addition, silencing of LRRC75A-

AS1 reduced the expression of NLRP3 (~85%), ASC (~50%), cleaved caspase-1 (~80%), IL-1β

(~40%), p-Smad2 (~60%), and p-Smad3 (~50%), whereas overexpression of LRRC75A-AS1 increased

their expression levels in tumors (

Fig. 7G

). In conclusion, LRRC75A-AS1 promoted tumor growth and

EMT by activating NLRP3/IL-1β/Smad2/3 signaling.

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Discussion

Cervical cancer is a serious global health issue that affects women, especially in developing countries

(27). Although the overall survival rate of cervical cancer is high and its incidence declines in the past

several decades, the morbidity and mortality still remain high (28, 29). Here, we firstly reported a novel

mechanism of LRRC75A-AS1 in regulation of EMT in cervical cancer through modulating m6A

modification and ubiquitination degradation. Specifically, we confirm that LRRC75A-AS1

competitively binds to m6A modification reader IGF2BP1 to destabilize SYVN1 mRNA, which

decreases SYVN1-mediated NLRP3 ubiquitination and activates IL-1β/Smad2/3 signaling, thus

promoting EMT in cervical cancer (

Supplementary Figure 2

Emerging lncRNAs are implicated in EMT and metastasis of cervical cancer (30). NEAT1, an

oncogenic lncRNA, enhances EMT by inhibiting miR-361 expression in cervical cancer (31). LncRNA

SFTA1P promotes the proliferation, invasion, and metastasis in cervical cancer via interacting with

PTBP1 to enhance TPM4 mRNA degradation (32). As a novel lncRNA, LRRC75A-AS1 was found to

exert both oncogenic and anti-tumor activities in various cancers such as breast cancer and colorectal

carcinoma (10, 11). Pang et al. reported that LRRC75A-AS1 inhibited proliferation and enhanced

apoptosis in multiple myeloma by sequestering miR-199b-5p and releasing PDCD4 (33). Han and

colleagues found that LRRC75A-AS1 enhanced cell proliferation and EMT through the miR-223-

3p/CTNND1 axis in glioblastoma (34). Therefore, the oncogenic or anti-tumor activity of LRRC75A-

AS1 may be attributed to or shaped by diverse tumor microenvironments of various cancers (10, 21).

However, the activity of LRRC75A-AS1 in cervical cancer needs further study. Strikingly, we reported

increased LRRC75A-AS1 expression in cervical cancer tissues, and the upregulation of LRRC75A-

AS1 significantly promoted cervical cancer cell proliferation, migration, invasion and EMT, and tumor

growth in mice, demonstrating the notion that LRRC75A-AS1 acted as an oncogene to facilitate the

progression of cervical cancer.

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LncRNAs exert functions through various mechanisms, such as competing endogenous RNAs

(ceRNAs), chromatin interaction and remodeling, and interaction with RNA binding proteins (35-37).

Mostly, m6A readers, such as the RNA binding protein IGF2BP1, are major mediators for m6A-

involved molecular mechanisms and biological activities by regulating mRNA stability (38). In our

study, we found that IGF2BP1 directly bound to SYVN1 mRNA and knockdown of IGF2BP1

significantly suppressed m6A modification of SYVN1 mRNA in cervical cancer cells. LRRC75A-AS1

could competitively bind to IGF2BP1 protein, thus repressing the m6A modification and the stability of

SYVN1 mRNA. For the first time, our findings suggest a novel molecular mechanism involving the

interaction between LRRC75A-AS1, IGF2BP1 and SYVN1 mRNA in cervical cancer.

Growing evidence has suggested that lncRNAs regulate protein stability through the ubiquitin-

proteasome system (39). For instance, lncRNA HEPFAL promoted SLC7A11 ubiquitination to impair

its stability (40). In our study, the ubiquitination of NLRP3 was suppressed by LRRC75A-AS1

overexpression but upregulated by LRRC75A-AS1 silencing in cervical cancer cells. E3 ubiquitin-

protein ligases have been identified as NLRP3 suppressors (41). TRIM31 promoted proteasomal

degradation of NLRP3 to attenuate NLRP3 inflammasome activation (42). Spop enhanced the

ubiquitination and degradation of NLRP3 by directly interacting with NLRP3 (43). We firstly proved

E3 ubiquitin ligase SYVN1 modulated the ubiquitination degradation of NLRP3 in cervical cancer.

However, clinical samples and

in vivo

assays involved in this study are very limited. To further explore

its significance in clinical application, more patients and

in vivo

assays should be involved in future

studies. Further direct evaluation of metastasis to the lung, liver and lymph nodes will be employed via

establishing the mouse model of tumor metastasis through tail vein injection, beyond just IHC staining

of E-cadherin according to reviewer’s comment. The inverse correlation between LRRC75A-AS1 and

SYVN1 expression in cancer tissues is fairly weak although statistically significant. We have searched

numerous public datasets and consulted several experts, but found no suitable available expression

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datasets showing the negative correlation. However, besides Fig. 3I, results in Fig. 4A-C also proved

their negative correlation between LRRC75A-AS1 and SYVN1 expression in cervical cancer. In

addition, the interplay between LRRC75A-AS1 and other regulators of m6A modification including

methyltransferases (writers), demethylase (erasers) and methylation recognition proteins (readers)

needs further exploration.

Collectively, we firstly demonstrated that LRRC75A-AS1 promoted EMT of cervical cancer via

impairing m6A modification of SYVN1 mRNA and its stability, subsequently suppressing NLRP3

ubiquitination and activating IL-1β/Smad2/3 signaling. Our findings provide novel molecular insight

for regulating EMT in cervical cancer and suggest potential therapeutic targets such as LRRC75A-AS1

SYVN1, and NLPR3.

Acknowledgements

This work was supported by National Cancer Center Climbing Fund (NCC2018B66), Key R&D

projects of the Hunan Cancer Hospital Research Climbing Plan (YF2020003) and General Guidance

Topic of Hunan Provincial Health Commission (202205015239).

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Figure legends

Figure 1. LRRC75A-AS1 promoted proliferation, migration and invasion in cervical cancer.

(A) qRT-PCR analysis of LRRC75A-AS1 in normal and tumor tissues from cervical cancer patients

(n=30). (B) LRRC75A-AS1 expression in SiHa, HeLa, C-33A, CaSki and ME-180 cells was

determined by qRT-PCR. HeLa cells were transfected with sh-NC or sh-LRRC75A-AS1, and CaSki

cells were transfected with OE-NC or OE-LRRC75A-AS1 vector for 48 h. (C) qRT-PCR analysis of

LRRC75A-AS1 level in HeLa and CaSki cells. (D) Cell proliferation was analyzed via CCK-8 assay.

(E) EdU incorporation (red) was examined to evaluate cell proliferation. The nuclei (blue) were stained

with DAPI. Scale bar, 100 μm. (F) Cell migration was determined with wound healing assay. (G) Cell

invasion was determined with transwell assay. Cell experiments in this study were biologically

repeated for at least three times. *

< 0.05, **

< 0.01 and ***

< 0.001.

Figure 2.

LRRC75A-AS1 silence repressed EMT through suppression of NLRP3/IL-1β/Smad2/3

signaling in cervical cancer

HeLa cells were grouped with control, sh-NC, sh-LRRC75A-AS1, sh-LRRC75A-AS1+OE-NC or sh-

LRRC75A-AS1+OE-NLRP3. (A-B) qRT-PCR analysis of LRRC75A-AS1 and NLRP3 levels in HeLa

cells. (C) NLRP3 protein level was detected via Western blotting in HeLa cells. (D-E) Migration and

invasion were determined with wound healing and transwell assays in HeLa cells. (F) MMP-9, E-

cadherin, N-cadherin, and slug levels were detected via Western blotting in HeLa cells. (G) qRT-PCR

analysis of E-cadherin and N-cadherin in HeLa cells. (H) ASC, cleaved caspase-1, IL-1β, p-Smad2,

Smad2, p-Smad3, and Smad3 protein levels were detected via Western blotting. Cell experiments in

this study were biologically repeated for at least three times. *

< 0.05, **

< 0.01 and ***

< 0.001.

Figure 3. LRRC75A-AS1 suppressed SYVN1-dependent NLRP3 ubiquitination degradation.

HeLa cells were transfected with sh-NC or sh-LRRC75A-AS1, and CaSki cells were transfected with

OE-NC or OE-LRRC75A-AS1 vector for 48 h. (A) NLRP3 protein abundance in response to CHX

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treatment (0, 1, 3 or 6 h) was detected via Western blotting. (B) NLRP3 ubiquitination level was

determined via Co-IP method. (C)

E3 ubiquitin ligases were predicted to be implicated in NLRP3

ubiquitination modification from Ubibrowser database. (D) MARCH9, NEED4, MARCH1, MARCH7,

and SYVN1 expression levels were examined via qRT-PCR. (E) The NLRP3/SYVN1 interaction was

evaluated through Co-IP assay. (F-H) The expression levels of SYVN1 in normal and tumor tissues

(n=30) and cells were analyzed by qRT-PCR and Western blotting. (I) Correlation analysis of

LRRC75A-AS1 and SYVN1 expression in tumor tissues (n=30)

. Cell experiments in this study were

biologically repeated for at least three times. *

< 0.05, **

< 0.01 and ***

< 0.001.

Figure 4. LRRC75A-AS1 reduced NLRP3 ubiquitination via destabilizing SYVN1 mRNA.

HeLa cells were transfected with sh-NC or sh-LRRC75A-AS1, and CaSki cells were transfected with

OE-NC or OE-LRRC75A-AS1 vector for 48 h. (A-B) SYVN1 mRNA and protein levels were detected

via

qRT-PCR

and Western blotting. (C) SYVN1 mRNA stability analysis by

qRT-PCR

in response to

actinomycin D (2 μg/mL) treatment. HeLa cells were grouped with control, sh-NC, sh-LRRC75A-AS1,

sh-LRRC75A-AS1+sh-NC or sh-LRRC75A-AS1+sh-SYVN1. (D-E) qRT-PCR and Western blot

analysis of SYVN1 expression in HeLa cells. (F) NLRP3 ubiquitination level was determined via Co-

IP method. (G)

The NLRP3/SYVN1 interaction was evaluated through Co-IP assay

. Cell experiments

in this study were biologically repeated for at least three times. *

< 0.05, **

< 0.01 and ***

0.001.

Figure 5. SYVN1 knockdown reversed the effects on EMT and NLRP3/IL-1β/Smad2/3 signaling

caused by LRRC75A-AS1 silencing.

HeLa cells were grouped with sh-NC, sh-LRRC75A-AS1, sh-LRRC75A-AS1+sh-NC, or sh-

LRRC75A-AS1+sh-SYVN1. (A-B) Migration and invasion were determined with wound healing and

transwell assays in HeLa cells. (C) MMP-9, E-cadherin, N-cadherin, and slug levels were detected via

Western blotting in HeLa cells. (D) qRT-PCR analysis of E-cadherin and N-cadherin in HeLa cells. (E)

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ASC, cleaved caspase-1, IL-1β, p-Smad2, Smad2, p-Smad3, and Smad3 protein levels were detected

via Western blotting. Cell experiments in this study were biologically repeated for at least three times.

< 0.05, **

< 0.01 and ***

< 0.001.

Figure 6. LRRC75A-AS1 competitively bound to IGF2BP1 to destabilize SYVN1 mRNA via

reducing its m6A modification.

(A)

The LRRC75A-AS1/SYVN1 protein interaction was determined with RIP assay. (B) IGF2BP1

protein was predicted to interact with LRRC75A-AS1 and SYVN1 mRNA from Starbase database. (C)

The interaction of IGF2BP1 with LRRC75A-AS1 or SYVN1 mRNA was confirmed by RIP assay. (D)

FISH combined with immunofluorescence assay showed co-localization of LRRC75A-AS1 (green) and

IGF2BP1 (red) in cytoplasm. The nuclei were stained with DAPI (blue). Scale bar, 20 µm. (E) The

m6A modification of SYVN1 mRNA was determined after IGF2BP1 knockdown with MeRIP-qPCR

assay.

HeLa cells were transfected with sh-NC or sh-LRRC75A-AS1, and CaSki cells were transfected

with OE-NC or OE-LRRC75A-AS1.

(F) RNA pull-down assay for determining the LRRC75A-

AS1/IGF2BP1 protein interaction. (G) RIP assay for analyzing the IGF2BP1/SYVN1 mRNA

interaction under LRRC75A-AS1 expression intervention

. Cell experiments in this study were

biologically repeated for at least three times. *

< 0.05, **

< 0.01 and ***

< 0.001.

Figure 7. LRRC75A-AS1 promoted tumor growth and EMT through NLRP3/IL-1β/Smad2/3

signaling in vivo.

Mice were subcutaneously injected with sh-LRRC75A-AS1-transfected HeLa cells or OE-LRRC75A-

AS1-transfected CaSki cells. 7 mice were included in each group. (A)

Tumor images in each group. (B-

Tumor volume and weight

in each group

. (D-E) IHC staining of Ki-67 and E-cadherin in tumors. (F)

qRT-PCR analysis of LRRC75A-AS1 and SYVN1 expression

in tumors

. (G) NLRP3, ASC, cleaved

caspase-1, IL-1β, p-Smad2, Smad2, p-Smad3, and Smad3 were detected via Western blotting. *

0.05, **

< 0.01 and ***

< 0.001.

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