Invest Clin 66(4): 365 - 377, 2025 https://doi.org/10.54817/IC.v66n4a02

Corresponding author. Rong Chen. Department of Medical Laboratory, The First People’s Hospital of Shuangliu

District/West China (Airport) Hospital Sichuan University, No. 120, Chengbei Upper Street, Dongsheng Street,

Shuangliu District, Chengdu, 610200,China. Tel: +86 13736798579. Email: chenrong202431@126.com

CircRNA_104293 targets miR-497-5p to

inhibit the mTOR/STAT3 pathway and

mitigate inflammation in Crohn’s disease.

Xuerui Yin, Gaigai Huang, Chuan Zheng, Yu Zhou and Rong Chen

Department of Medical Laboratory, The First People’s Hospital of Shuangliu District/

West China (Airport) Hospital Sichuan University, Chengdu, China.

Keywords: Crohn Disease; mTOR/STAT3; Inflammation; circRNA_104293; miR-497-5p.

Abstract. Crohn’s disease (CD) is a chronic inflammatory bowel disease

driven in part by dysregulation of the mTOR/STAT3 signaling pathway, where

mTOR activates STAT3 via the PI3K/AKT cascade. Circular RNAs (circRNAs)

have recently emerged as essential regulators in inflammatory processes, al-

though their specific roles in CD remain largely unexplored. In this study, cir-

cRNA expression profiles from CD patients and healthy controls were analyzed,

revealing a significant upregulation of circRNA_104293 in CD tissues. Function-

al investigations demonstrated that knockdown of circRNA_104293 reduced in-

flammatory cytokine production and DNA damage markers, and decreased cell

apoptosis. Bioinformatic analysis and experimental validation confirmed a di-

rect interaction between circRNA_104293 and miR-497-5p. Furthermore, miR-

497-5p inhibition reversed the anti-inflammatory effects of circRNA_104293 si-

lencing. Notably, both rapamycin (an mTOR inhibitor) and miR-497-5p mimics

suppressed the mTOR/STAT3 pathway and alleviated inflammatory responses.

These findings suggest that the circRNA_104293/miR-497-5p axis contributes

to CD progression by modulating the mTOR/STAT3 pathway, highlighting its

potential as a novel therapeutic target for the treatment of Crohn’s disease.

366 Yin et al.

Investigación Clínica 66(4): 2025

CircRNA_104293 actúa sobre el miR-497-5p para inhibir

la vía mTOR/STAT3 y mitigar la inflamación en la enfermedad

de Crohn.

Invest Clin 2025; 66 (4): 365 – 377

Palabras clave: Enfermedad de Crohn; mTOR/STAT3; Inflamación; circRNA_104293;

miR-497-5p.

Resumen. La enfermedad de Crohn (CD) es una enfermedad inflamatoria

crónica del intestino cuya progresión está parcialmente mediada por la disfun-

ción de la vía de señalización mTOR/STAT3, en la que mTOR activa a STAT3

a través de la cascada PI3K/AKT. Recientemente, los ARN circulares (circR-

NAs) han surgido como reguladores clave en procesos inflamatorios, aunque

sus funciones específicas en la CD aún no están bien definidas. En este estu-

dio se analizaron los perfiles de expresión de circRNAs en pacientes con CD y

en controles sanos, y se identificó una sobreexpresión significativa de circR-

NA_104293 en tejidos de pacientes con CD. Los análisis funcionales mostraron

que la reducción de la expresión de circRNA_104293 disminuyó la producción

de citocinas inflamatorias y de marcadores de daño al ADN, así como la apopto-

sis celular. Mediante análisis bioinformático y validación experimental, se con-

firmó una interacción directa entre circRNA_104293 y miR-497-5p. Además, la

inhibición de miR-497-5p revirtió los efectos antiinflamatorios inducidos por

el silenciamiento de circRNA_104293. De manera destacada, tanto la rapami-

cina (un inhibidor de mTOR) como los miméticos de miR-497-5p suprimieron

la activación de la vía mTOR/STAT3 y redujeron las respuestas inflamatorias.

Estos hallazgos indican que el eje circRNA_104293/miR-497-5p participa en

la progresión de la CD mediante la modulación de la vía mTOR/STAT3, lo que

resalta su potencial como nuevo objetivo terapéutico para el tratamiento de la

enfermedad de Crohn.

Received: 19-06-2025 Accepted: 17-09-2025

INTRODUCTION

Crohn’s Disease (CD) is characterized

by chronic, repetitive inflammation of the

gastrointestinal tract. Its etiology has not

been fully clarified, but studies have shown

that CD is closely associated with genetic,

environmental, and immune factors, as well

as changes in the gut microbiota¹.

Patients

often present with abdominal pain, diarrhea,

weight loss, fever, and other symptoms,

which in severe cases can lead to complica-

tions such as intestinal obstruction and fis-

tula formation 1, 2. The mammalian target

of rapamycin (mTOR) belongs to the phos-

phoinositide 3-kinase (PI3K)-related kinase

family and is mainly involved in processes

such as cell growth, proliferation, metabo-

lism, and autophagy 3. Signal transducer and

activator of transcription 3 (STAT3) is a tran-

scription factor that regulates cell survival,

proliferation, and immune responses. In CD,

aberrant activation of STAT3 is strongly as-

sociated with exacerbated inflammatory

responses 4. The mTOR/STAT3 pathway in-

teracts in CD to co-regulate inflammatory

The role of CircRNA104293 in Crohn’s disease 367

Vol. 66(4): 365 - 377, 2025

reactions. For example, mTOR activation has

been found to promote STAT3 phosphoryla-

tion through the phosphoinositide 3-kinase/

protein kinase B (PI3K/AKT) signaling path-

way, thereby enhancing its transcriptional

activity 5. This interaction plays a key role

in intestinal inflammation in Crohn’s dis-

ease patients and may lead to its persistence

and aggravation. But the specific molecular

mechanism is unknown.

Circular RNAs (circRNAs) are mole-

cules with unique circular structures that

play an essential role in a variety of dis-

eases 6, 7. In neurological diseases, circRNA

is widely expressed in brain tissue and is

closely related to neurodevelopment and

signaling, showing abnormal expression in

diseases such as cerebral palsy and stroke,

and is considered a potential biomarker

of neurological diseases due to its stable

structure 8,9. In malignant tumors, some

circRNAs can adsorb microRNAs (miRNAs)

as “molecular sponges”, thereby relieving

miRNA inhibition of oncogenes and promot-

ing tumor development 10. In CD, circRNA

103765 is reported to regulate disease pro-

gression 11. At present, the study of circRNA

in CD still needs to be further expanded to

fully understand its effects on CD. MicroR-

NAs (miRNAs) are also closely associated

with the progression of various inflamma-

tory diseases 12, 13. miR-497 has been report-

ed to influence the progression of associ-

ated inflammatory diseases by modulating

lipopolysaccharide (LPS) -induced inflam-

mation in vivo 14, 15. Furthermore, miR-497

expression was significantly decreased fol-

lowing LPS treatment of RAW264.7 cells

and in a mouse colitis model 16.

Here, we performed sequencing analy-

sis of circRNA expression profiles from CD

patients to explore potential therapeutic

targets and their molecular mechanisms.

Through screening, we found circRNAs that

were up- and down-regulated in CD. Further,

qPCR was used to detect the expression level

of circRNA 104293 in CD patients, and its

mechanism of action in CD development was

studied in depth. The results of this study

are expected to provide a new treatment op-

tion for patients with CD.

METHODS

Patients and genome sequencing analysis

Between June 2021 and March 2022,

we collected the inflamed colonic tissue

samples from CD patients who underwent

surgical treatment at our institution. The

tissues were obtained from inflamed areas

of the colon, confirmed by intraoperative

findings and postoperative histopathologi-

cal examination, and non-inflamed adjacent

tissues served as control. Tissue total RNA

isolation kit (RC101-01, Vazyme, Nanjing,

China) was used for the RNA extraction,

and RNA integrity was assessed using an

Agilent 2100 Bioanalyzer (Agilent Tech-

nologies, USA). High-throughput circRNA

microarray analysis was conducted by Ak-

somics Biotechnology (Shanghai, China).

Sample processing was carried out ac-

cording to the manufacturer’s protocol.

Briefly, the Seq-Star™ rRNA removal kit

(AS-MB-001, Aksomics) was employed for

the rRNA removal, and the circRNA purifi-

cation and fluorescent cRNA transcription

were performed using Seq-Star™ RNAClean

and smallEnrich Beads (AS-MB-009) and

rtStar™ First-Strand cDNA Synthesis Kit

(AS-FS-001) from Aksomics. Labeled cRNA

concentration and specific activity were de-

termined using a NanoDrop ND-1000 spec-

trophotometer (Thermo Fisher Scientific,

USA). One microgram of labeled cRNA from

each sample was fragmented, mixed with

hybridization buffer, and loaded onto a Hu-

man circular RNA Array V2.0 (8 × 15K for-

mat, Aksomics). Hybridization was carried

out at 65°C for 17 h in an Agilent Hybridiza-

tion Oven (model G2545A). After hybridiza-

tion, the microarrays were washed and fixed

with Gene Expression Wash Buffers (Agi-

lent Technologies, USA) and scanned with

an Agilent DNA Microarray Scanner (model

G2505C).

368 Yin et al.

Investigación Clínica 66(4): 2025

Differential expression analysis

Differential expression analysis was

conducted using the DESeq2 R package.

The fold change and statistical significance

were calculated based on the log2|(fold

change)|>1 and p-adjust <0.05.

Cells culture and treatment

In this study, Fetal Human Colon (FHC)

cells (normal human colon epithelial cells)

were purchased from the Cell Bank of Shang-

hai Institute of Life Sciences. Cells were cul-

tured in RPMI 1640 medium containing 10%

fetal bovine serum (FBS) and 1% penicillin-

streptomycin-glutamine (PSG) and adapted

for 2-3 days before use in experiments. To in-

duce a general inflammatory response in vi-

tro, FHC cells were treated with 500 ng/mL

of lipopolysaccharide (LPS) and control cells

were treated with serum-free medium. Treat-

ed cells were divided into two groups: small

interfering RNA (si)-circRNA group and neg-

ative control group (siNC). Cells were trans-

fected using Lipofectamine ™ 2000 follow-

ing kit instructions and cells were collected

48 hours later for subsequent experiments.

In addition, inhibitors of miR-497-5p and its

negative control (NC) were transfected us-

ing Lipofectamine ™ 2000. The mTOR tar-

geting inhibitor rapamycin (Bioengineering

Co., A606203) was used for experiments and

cells were co-incubated with 100 ng/mL ra-

pamycin to investigate its effect.

Q-PCR assay

To determine the expression levels of

miR-497-5p and circRNA 104293, we used

qPCR. TRIZOL reagent (R0016, Beyotime,

Shanghai, China) was used to extract total

RNA and RNA was isolated through cen-

trifugation. The RNA was then dissolved in

RNase-free water. Then reverse transcrip-

tion was performed to synthesize cDNA us-

ing BeyoRT™II First Strand cDNA Synthesis

Kit (D7168S, Beyotime, Shanghai, China),

which was subsequently used as the tem-

plate for qPCR amplification following the

instructions of the SYBR Green kit (D7260,

Beyotime). The qPCR cycling conditions in-

cluded initial denaturation at 95°C for 30s,

followed by 39 cycles of 95°C for 5s, 60°C

for 30s, and a final extension at 72°C for 5

seconds. The relative expression levels of cir-

cRNA_104293 were normalized to GAPDH

as an internal control. Gene expression was

calculated using the 2-ΔΔCt method.

ELISA assay

After transfection, the cells were

washed three times with PBS. Subsequently,

0.5 ml of non-denaturing protein lysis buf-

fer was added to each well and gently mixed

to lyse the cells. The lysates were then incu-

bated on ice for 20 minutes. The superna-

tants were immediately stored at -20°C. The

tumor necrosis factor-alpha (TNF-α) ELISA

Kit (PT518), interleukin-1 beta (IL-1β)

ELISA Kit (PI305), IL-6 ELISA Kit (PI325)

and IL-8 ELISA Kit (PI641) were purchased

from the Beyotime biotechnology company

(Shanghai, China). The levels of inflamma-

tory cytokines in cell lysates were measured

using ELISA assays according to the manu-

facturer’s instructions.

Flow cytometry assay

A cell suspension of LPS-treated cells

was prepared at a density of 5 × 105/mL and

seeded into a 6-well plate. After 24 hours,

the cells were transfected with si-circRNA

104293, or si-NC, followed by an additional

24-hour incubation. Post-transfection, the

cells were harvested through digestion and

centrifugation, then washed three times

with ice-cold PBS. Apoptosis was assessed

by staining the cells with 5 μL of Annexin V-

FITC/PI. The apoptosis rate was subsequent-

ly quantified using flow cytometry. This pro-

tocol ensured consistent evaluation of cell

viability under experimental conditions.

Dual-luciferase reporter gene assay

The TargetScan online tool was em-

ployed to predict potential miRNA bind-

ing sites on circRNA_104293, specifically

identifying complementary sequences for

The role of CircRNA104293 in Crohn’s disease 369

Vol. 66(4): 365 - 377, 2025

miR-497-5p. To validate this interaction,

wild-type or mutant circRNA_104293 plas-

mids were constructed by GenePharma

(Shanghai, China). The miR-497-5p mimic

or negative control was co-transfected with

either wild-type or mutant plasmids into

FHC cells using Lipofectamine™ 2000 (In-

vitrogen, USA), with triplicate wells for each

condition. After 24 h of post-transfection,

the medium was discarded, and the cells

were washed three times with PBS. Cell lysis

was performed using 100 μL of lysis buffer

(RG132S, Beyotime) at room temperature.

Firefly and Renilla luciferase activities were

measured sequentially using a Dual-Lucifer-

ase® Reporter Assay System (E1910, Pro-

mega, USA), including LARII reagent and

Stop&Glo® reagent, according to the man-

ufacturer’s instructions. Luminescence was

detected using a Glomax Multi+ Detection

System (Promega, USA).

Immunofluorescence assay

Cells were cultured on sterile glass cov-

erslips in 24-well plates and then treated

with si-circRNA 104293 as the test and si-

NC as the control. After treatment, the cov-

erslips were washed three times with PBS

and fixed with 37 g/L formaldehyde for 15

min. Permeabilization was performed using

0.5% Triton X-100 for 10 min, followed by

blocking with 100 mL/L bovine serum al-

bumin (BSA) for one h. Primary antibodies

specific to dsDNA (ab27156, Abcam) and

ssDNA (CBL407, Millipore) were applied at

appropriate dilutions, and the samples were

incubated overnight at 4°C. The coverslips

were then washed three times with PBS and

once with distilled water, then incubated

with an FITC-conjugated secondary antibody

(A-11001, Thermo Fisher Scientific) for one

h at room temperature in the dark. Finally,

the samples were mounted with glycerol and

analyzed using fluorescence microscopy. Flu-

orescence intensity was quantified in ImageJ

by measuring the mean fluorescence per cell

and normalizing to control samples.

Western blot assay

Proteins were extracted from treated

cells 48 h post-treatment using RIPA ly-

sis buffer supplemented with protease and

phosphatase inhibitors. The protein samples

were quantified using a BCA protein assay

kit (A65453, Thermo Fisher Scientific), and

equal amounts of protein (30 μg/lane) were

separated on an SDS-PAGE gel and subse-

quently transferred to a PVDF membrane.

And then the membrane was blocked with

5% skim milk in TBST for one hour. After

blocking, the membrane was incubated over-

night at 4°C with primary antibodies tar-

geting p-mTOR (ab109268, Abcam) and p-

STAT3 (ab76315, Abcam). GAPDH (ab8245,

Abcam) was used as the loading control. Fol-

lowing three washes with TBST, the sections

were incubated with appropriate HRP-con-

jugated secondary antibodies (ab205719,

Abcam). After additional TBST washes, the

membrane was treated with a chemiluminis-

cent substrate (ab5801, Abcam), and pro-

tein bands were visualized using a Fusion

Fx5 chemiluminescence detector (Vilber).

Statistical analysis

The data were processed using SPSS

21.0 software and R software (version 4.2.0).

Differences between the two groups were

evaluated using the independent samples t-

test. A one-way ANOVA followed by the SNK-

q test was used to compare multiple groups.

A p<0.05 was statistically significant.

RESULTS

CircRNA_104293 was upregulated

in CD patients

To identify differentially expressed cir-

cRNAs between the control and CD groups,

genome-wide sequencing analysis was per-

formed. A total of 415 up-regulated and 234

down-regulated circRNAs were identified

(Fig. 1A). Among these, circRNA_103765

exhibited significantly elevated expression,

highlighting its potential as a therapeutic

370 Yin et al.

Investigación Clínica 66(4): 2025

target for CD. Further validation was con-

ducted in LPS-treated FHC cells, where cir-

cRNA_104293 showed significantly higher

expression than in controls (Fig. 1B). This

finding underscores its potential role in in-

flammatory conditions.

Si-circRNA_104293 suppressed

inflammation levels.

The transfection efficiency of si-cir-

cRNA_104293 and si-NC was evaluated

using q-PCR. Results demonstrated that

si-circRNA_104293 effectively reduced cir-

cRNA_104293 expression in LPS-treated

FHC cells, confirming successful transfec-

tion (Fig. 2A). To assess the impact of si-cir-

cRNA_104293 on inflammation, ELISA was

performed, revealing a significant decrease

in pro-inflammatory cytokines TNF-α, IL-

1β, IL-6, and IL-8 (Fig. 2B). These findings

suggest that si-circRNA_104293 effectively

attenuates inflammatory responses. Addi-

tionally, flow cytometry indicated that si-cir-

cRNA_104293 markedly inhibited apoptosis

in LPS-treated FHC cells (Fig. 2C), further

supporting its role in mitigating cellular

damage under inflammatory conditions.

CircRNA_104293 targeted miR-497-5p

TargetScan was used to identify the in-

teraction between circRNA_104293 and miR-

497-5p (Fig. 3A). A dual luciferase reporter

assay confirmed this binding relationship,

showing that the miR-497-5p mimic reduced

luciferase activity in the circRNA_104293-

WT group (Fig. 3B). This indicated that

circRNA_104293 directly targets and nega-

tively regulates miR-497-5p. Further valida-

tion using q-PCR revealed that miR-497-5p

expression was lower in the LPS-treated

group compared to controls (Fig. 3C). Ad-

ditionally, silencing circRNA_104293 led to

a notable increase in miR-497-5p levels (Fig.

3D), further supporting the regulatory role

of circRNA_104293 in suppressing miR-497-

5p expression. These findings collectively

demonstrate that circRNA_104293 targets

and downregulates miR-497-5p.

Si-circRNA_104293 reduced inflammation

LPS-treated cells were divided into

three groups: si-NC + NC-inhibitor, si-cir-

cRNA_104293 + miR-497-5p-inhibitor, and

si-circRNA_104293 + NC-inhibitor, to in-

vestigate the roles of circRNA_104293 and

Fig. 1. Elevated circRNA_104293 expressions in Crohn’s disease (CD) patients and lipopolysaccharide (LPS)-

treated FHC cells. A. Volcano plot showing differentially expressed circRNAs in CD patients and

controls based on log₂|fold change| > 1 and an FDR-adjusted p-value < 0.05. B. Quantitative (q)

PCR was used to compare the levels of circRNA_104293 in fetal human colon (FHC) cells under diffe-

rent treatments, using Student’s t-test. (**p < 0.01).

The role of CircRNA104293 in Crohn’s disease 371

Vol. 66(4): 365 - 377, 2025

miR-497-5p. The efficacy of the miR-497-5p

inhibitor was confirmed via qPCR, showing

a significant reduction in miR-497-5p levels

(Fig. 4A). Immunofluorescence assays re-

vealed that the miR-497-5p inhibitor partial-

ly reversed the effects of si-circRNA_104293,

markedly decreasing dsDNA and ssDNA

levels (Fig. 4B, C). Furthermore, si-cir-

cRNA_104293 downregulated inflammatory

cytokines, while the miR-497-5p inhibitor

partially counteracted this effect (Fig. 4D).

Apoptosis assays demonstrated that si-cir-

cRNA_104293 reduced apoptosis rates. In

contrast, the miR-497-5p inhibitor increased

them (Fig. 4E). These findings indicate that

circRNA_104293 mitigates inflammation

and apoptosis by regulating miR-497-5p.

CircRNA_104293/miR-497-5p alleviated

inflammation levels

Western blot analysis revealed that both

miR-497-5p mimic and rapamycin down-

regulated the expression of p-mTOR and p-

STAT3 compared to the NC-mimic group,

indicating suppression of the mTOR/STAT3

pathway (Fig. 5A). Immunofluorescence as-

Fig. 2. Si-circRNA_104293 attenuates inflammation in LPS-treated FHC cells. A. Transfection efficiency of

si-circRNA_104293 was confirmed by quantitative (q)PCR. B. ELISA measurements of TNF-α, IL-1β,

IL-6, and IL-8 levels across different groups. C. Flow cytometry analysis of apoptosis in lipopolysac-

charide (LPS)-treated fetal human colon (FHC) cells. (**p < 0.01 vs. si-NC group).

Fig. 3. circRNA_104293 targets miR-497-5p. A. Predicted binding site between circRNA_104293 and miR-497-

5p. B. The dual luciferase assay confirmed the interaction between circRNA_104293 and miR-497-5p.

C. qPCR analysis of miR-497-5p expression in control and lipopolysaccharide (LPS) groups. D. qPCR for

miR-497-5p levels in cells treated with si-NC or si-circRNA_104293. (**p<0.01 vs. control or NC group).

372 Yin et al.

Investigación Clínica 66(4): 2025

says showed reduced levels of ssDNA and ds-

DNA in the miR-497-5p-mimic and rapamy-

cin groups (Fig. 5B, C). Additionally, ELISA

results demonstrated decreased levels of in-

flammatory cytokines in these groups (Fig.

5D). Apoptosis analysis further confirmed

that miR-497-5p mimic and rapamycin sig-

nificantly lowered apoptosis rates compared

to the NC-mimic group (Fig. 5E).

DISCUSSION

CircRNA is more stable than linear

RNA, making it of great value for clini-

cal diagnosis and prognostic evaluation of

CD 17. Recently, some studies have shown

that circRNAs such as circRNA102610, cir-

cRNA103516, and circRNA102685 are in-

volved in the inflammatory process of CD.

However, the role of most circRNAs in CD

pathogenesis remains elusive and there-

fore requires further exploration18,20. cir-

cRNA103765 has been identified as a key

regulator of CD11 pathogenesis. In this

study, significantly increased expression of

circRNA104293 was observed in LPS-treated

FHC cells and in CD patients. These results

suggest a potential role for circRNA104293

in regulating inflammation in CD.

In IBD, TNF-α plays a critical role in

the intestinal mucosa through autocrine

and paracrine mechanisms 21. LPS produced

by the gut microbiota can directly activate

macrophages in the intestinal lamina pro-

Fig. 4. Si-circRNA_104293 reduces inflammation via miR-497-5p. A. qPCR for the miR-497-5p levels in NC-

inhibitor and miR-497-5p inhibitor. (**p < 0.01) B-C. Immunofluorescence analysis for the dsDNA and

ssDNA levels in different treatment groups. (**p <0.01, si-circRNA+NC-inhibitor vs si-NC+NC-inhibi-

tor, ## p <0.01, si-circRNA+miR-497-5p-inhibitor vs si-NC+NC-inhibitor). D. ELISA for the expres-

sion of inflammatory cytokines in different treatment groups. (**p<0.01, si-circRNA + NC-inhibitor vs

si-NC + NC-inhibitor, ## p<0.01, si-circRNA + miR-497-5p-inhibitor vs si-circRNA + NC-inhibitor). E.

Flow cytometry for the cell apoptosis in different groups. (**p<0.01, si-circRNA + NC-inhibitor vs si-NC

+ NC-inhibitor, ## p<0.01, si-circRNA + miR-497-5p-inhibitor vs si-circRNA + NC-inhibitor).

The role of CircRNA104293 in Crohn’s disease 373

Vol. 66(4): 365 - 377, 2025

pria, promote their proliferation, and induce

TNF-α release 22. It has been shown that TNF-α

and IL-1β expression are significantly abnor-

mal in mucosal biopsies from pediatric CD

patients 23. In addition, the synergistic effect

of TNF-α with interferon γ disrupts barrier

function and alters the morphological struc-

ture of intestinal epithelial cells, resulting in

increased permeability of the intestinal mu-

cosa and vascular wall, ultimately triggering

ulcer formation 24, 25. In the pathogenesis of

CD, inhibition of apoptosis leads to exces-

sive accumulation of T cells, which in turn

aggravates chronic mucosal inflammation, a

process closely related to IL-6 signaling26,27.

Notably, IL-8 expression is abnormal in the

serum and intestinal tissues of immune CD

patients, and its level not only reflects disease

severity but also serves as an independent in-

dicator of disease activity 28. In this study, we

found that si-circRNA104293 significantly re-

duced levels of inflammatory factors.

Fig. 5. circRNA_104293/miR-497-5p alleviates inflammation by inhibiting mTOR/STAT3 pathway. A. Wes-

tern blot analysis of p-mTOR and p-STAT3 expression across different groups. B-C. Immunofluores-

cence analysis of dsDNA and ssDNA levels. (**p<0.01, miR-497-5p-mimic vs NC-mimic, ## p<0.01,

rapamycin vs NC-mimic). D. ELISA measurements of TNF-α, IL-1β, IL-6, and IL-8 levels. E. Flow

cytometry assessment of cell apoptosis in different groups. (**p<0.01 vs. NC-mimic group).

374 Yin et al.

Investigación Clínica 66(4): 2025

In this study, circRNA104293 was found

to play a regulatory role by targeting and in-

hibiting miR-497-5p expression. It has been

shown that miR-497-5p can upregulate IL-6

expression, thereby inhibiting muscle cell

atrophy, and affects the inflammatory pro-

cess by participating in the NF-κB pathway

and regulating T cell function 29. The re-

sults of this work, consistently, shows that

si-circRNA104293 significantly decreased

the rate of apoptosis, whereas the miR-497-

5p inhibitor increased it. In addition, si-cir-

cRNA104293 effectively reduced the inflam-

matory response by regulating miR-497-5p,

indicating that the circRNA104293/miR-

497-5p axis plays a vital role in regulating

inflammation in CD. It was also found that

si-circRNA104293 significantly reduced ds-

DNA and ssDNA levels, while miR-497-5p in-

hibitors partially reversed this effect, further

confirming the key role of circRNA104293 in

inflammation and cell damage.

In this study, we found that p-mTOR

and p-STAT3 expression levels were signifi-

cantly decreased in miR-497-5p mimic and

rapamycin groups compared with NC mimic

groups 30. Previous studies have shown that

p-mTOR levels are increased in colonic tis-

sue of CD patients. mTOR, a key molecule

regulating cellular energy metabolism, mi-

tochondrial fusion, and glucose and lipid

metabolism, has inhibitors that can effec-

tively suppress the expression of inflamma-

tory factors 31. In addition, STAT3 acts as a

multifunctional transcription factor and can

activate inflammatory responses in T cells

through IL-6-mediated JAK/STAT3 signaling

pathway32. Blocking IL-6 signaling not only

reduces STAT3 activation but also induces

monocyte apoptosis, thereby relieving coli-

tis triggered by IL-10 deficiency 33, 34. In CD

patients, total STAT3 and phosphorylated

STAT3 levels were significantly increased in

inflammatory intestinal mucosa, and phos-

phorylated STAT3 levels were positively cor-

related with the degree of inflammatory

injury 35. The results of this study further

confirmed that circRNA104293/miR-497-5p

significantly reduced CD inflammatory lev-

els by inhibiting the mTOR/STAT3 pathway.

In summary, our findings suggest that the

circRNA_104293/miR-497-5p axis regulates

LPS-induced inflammatory responses by mod-

ulating the mTOR/STAT3 signaling pathway.

These results indicate its potential as a thera-

peutic target in inflammatory processes.

Funding

Chengdu Medical Scientific Research

Project (2021333).

Conflict of interest

The authors declare that they have no

conflict of interest regarding this study.

Ethics approval and consent to participate

This study was conducted in accordance

with the Declaration of Helsinki, and was con-

ducted with approval from the Ethics Commit-

tee of The First People’s Hospital of Shuangliu

District/West China (Airport) Hospital, Sich-

uan University. Written informed consent was

obtained from all participants.

ORCID number of authors

• Xuerui Yin (XY):

0009-0006-6698-8009

• Gaigai Huang (GH):

0009-0008-4424-1179

• Chuan Zheng (CZ):

0009-0005-4178-415X

• Yu Zhou (YZ):

0009-0007-4611-3069

• Rong Chen (RC):

0009-0005-7624-8285

Author contributions

Conceived and designed the study: XY,

RC, GH; performed the literature search and

data extraction: XY, CZ, YZ; analyzed the

data: RC; drafted the manuscript: XY, RC

The role of CircRNA104293 in Crohn’s disease 375

Vol. 66(4): 365 - 377, 2025

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