Mechanism of miR-200b-3p-induced FOSL2 inhibition of endometrial cancer cell proliferation and metastasis

doi:10.21203/rs.3.rs-4866332/v1

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Mechanism of miR-200b-3p-induced FOSL2 inhibition of endometrial cancer cell proliferation and metastasis

https://doi.org/10.21203/rs.3.rs-4866332/v1

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Objective The purpose of this study was to investigate how miR-200b-3p inhibits the proliferation and metastasis of endometrial cancer cells by inducing the expression of FOSL2 in the AP1 transcription family.

Methods Endometrial cancer cell line HEC-1-A was divided into 12 groups: NC-mimic ( transfected with negative control NC mimic ), miR-200b-3p mimic ( transfected with miR-200b-3p mimic ), NC-suppress ( transfected with negative control NC inhibit ), miR-200b-3p inhibit group ( transfected with miR-200b-3p inhibit ), si-NC ( transfected with negative control Si-NC ), Si-FOSL2 ( transfected with Si-FOSL2 ), oe-NC ( transfected with negative control oe-NC ), oe-FOSL2 group ( oe-FOSL2 ), MiR-200b-3p mimic + oe-NC group ( co-transfected with miR-200b-3p mimic and oe-NC ), miR-200b-3p mimic + oe-FOSL2 group ( co-transfected with miR-200b-3p mimic and oe-FOSL2 ), miR-200b-3p inhibit + si-NC group ( co-transfected with miR-200b-3p inhibit and si-NC ), miR-200b-3p inhibit + si-FOSL2 group ( co-transfected with miR-200b-3p inhibit and si-FOSL2 ). Real-time fluorescence quantitative PCR, Western blot, CCK-8 assay, scratch test and Transwell assay were used to detect the expression of miR-200b-3p mRNA, FOSL2 mRNA and protein, cell proliferation, migration and invasion.

Results In endometrial cancer cell lines, the expression of miR-200b-3p was significantly down-regulated (P<0.05), while the expression of FOSssL2 was significantly up-regulated (P<0.05). Compared with NC-mimic group, the expression of FOSL2, N-cadherin and Vimentin in miR-200b-3p mimic group was significantly decreased (P<0.05), and the expression of E-cadherin was significantly increased (P<0.05). The cell proliferation, migration rate and the number of transmembrane cells were significantly decreased (P<0.05). Compared with the miR-200b-3p mimic + oe-NC group, the expression of FOSL2, N-cadherin and Vimentin in miR-200b-3p mimic + oe-FOSL2 was significantly increased (P<0.05), the expression level of E-cadherin was significantly decreased (P<0.05), and the cell proliferation, migration rate and the number of transmembrane cells were significantly increased (P<0.05). Compared with NC-inhibit group, the expression of FOSL2, N-cadherin and Vimentin in miR-200b-3p inhibit group was significantly increased (P<0.05), and the expression of E-cadherin was significantly decreased (P<0.05). The cell proliferation, migration rate and the number of transmembrane cells were significantly increased (P<0.05). Compared with the miR-200b-3p inhibit + si-NC group, the expression of FOSL2, N-cadherin and Vimentin in miR-200b-3p inhibit + si-FOSL2 was significantly decreased (P<0.05), and the expression of E-cadherin was significantly increased (P<0.05); the cell proliferation, migration rate and the number of transmembrane cells were significantly decreased (P<0.05)

Conclusion The expression of miR-200b-3p in endometrial cancer cells is down-regulated, which can inhibit the proliferation, migration and invasion of endometrial cancer cells by regulating the EMT process, and its mechanism is related to its targeted negative regulation of FOSL2 expression.

Biological sciences/Cancer

Health sciences/Biomarkers

Health sciences/Medical research

miR-200b-3p

FOSL2

Endometrial cancer

Cell proliferation

Cell metastasis

EMT

Endometrial cancer (EC) is a malignant tumor that occurs in the epithelial lining of the uterus in the female reproductive system, with adenocarcinoma being the most common, and its incidence is steadily increasing worldwide.[1] Early treatment of EC results in a relatively good prognosis, but patients with advanced or recurrent EC have a poorer clinical prognosis and a significantly lower survival rate, with 5-year survival rates of 44% and 37% in patients with pelvic and extra-pelvic metastases, respectively.[2, 3] As the only gynecologic malignancy with an increased mortality rate,[4] there is an urgent need for further exploration of the mechanisms of its development and progression. MicroRNAs (miRNAs) is a class of small non-coding RNA (ncRNA), usually composed of 19–24 nucleotides, that can directly affect or regulate intracellular signaling pathways and indirectly influence the expression of tumor-associated proteins, thereby exerting antitumor effects or carcinogenesis.[5] A variety of miRNA have been found to be closely related to endometrial cancer development and progression.[6–8] Among them, miR-200b can affect cancer progression by regulating the EMT process in endometrial cancer cells.[9] miR-200b-3p, which belongs to the miR-200b family, has also been found to inhibit the proliferation, invasion, and migration ability of a variety of tumors by inhibiting the expression of downstream mRNA.[10] FOSL2, a member of the activator protein-1 (AP1) transcriptional family, is widely expressed in tissues, and is able to regulate growth, development, and immune responses, which play key roles in physiological and pathological processes.[11] Some studies have found that FOSL2 can act as a downstream target protein of miRNAs to regulate epithelial mesenchymal transition (EMT), metastasis, and other mediators of malignant tumor progression.[12, 13] Although numerous studies have confirmed that miR-200b-3p and FOSL2 play important roles in the progression of some cancers, the roles of both in the development of endometrial cancer have not been clarified. Therefore, the aim of this study is to investigate the role of miR-200b-3p through its regulation of FOSL2 and its effect on the proliferation and metastasis of endometrial cancer cells, and to reveal its underlying molecular mechanisms.

1.1 Cells and Primary Reagents

Human endometrial cancer (HEC-1-A, RL95-2, Ishikawa) and human normal endometrial mesenchymal hEM15A are purchased from the Shanghai Cell Bank, Chinese Academy of Sciences. 0.25% trypsin, trypsin, DMEM medium, RPMI 1640 medium, and RIPA lysate are purchased from Promega, USA. TRIzol (Invitrogen, USA). miR-200b-3p inhibitor, FOSL2 inhibitor, and β-actin are purchased from Thermo Fisher Scientific, USA. N-cadherin inhibitor, Vimentin inhibitor and human E-cadherin inhibitor are purchased from CST, USA. miR-200b-3p mimic, miR-200b-3p inhibitor, si-FOSL2, oe-FOSL2 and the corresponding negative control are purchased from Ribobio, China. wild-type FOSL2 3'UTR (FOSL2-WT), mutant FOSL2 3'-UTR (FOSL2-WT) report vectors are purchased from Vigenebio, Shandong, China.The dual-luciferase reporter gene kit is purchased from Solarbio, USA. The CCK-8 reagent and the cell cycle kit are purchased from Beyotime Biotech Inc, Shanghai, China. Transwell chamber is purchased from Thermo, USA.

1.2 Cell culture and cell transfection

Endometrial cancer cell lines HEC-1-A, RL95-2, Ishikawa and human normal endometrial mesenchymal stromal cell line hEM15A were cultured in DMEM medium containing 10% fetal bovine serum (FBS) at 37℃ with 5% CO2 incubator. The HEC-1-A cell line was divided into 12 groups, and 3 replicate wells were set up in each group: NC-mimic (transfected with negative control NC mimic), miR-200b-3p mimic (transfected with miR-200b-3p mimic), NC-inhibit (transfected with negative control NC inhibit), miR-200b-3p inhibit group (transfected with miR-200b-3p inhibit), si-NC (transfected with negative control Si-NC), Si-FOSL2 (transfected with Si-FOSL2), oe-NC (transfected with negative control oe-NC), oe-FOSL2 group (oe-FOSL2), miR-200b-3p mimic + oe-NC group (co-transfected with miR-200b-3p mimic and oe-NC), miR-200b-3p mimic + oe-FOSL2 group (co-transfected with miR-200b-3p mimic and oe-FOSL2), miR-200b-3p inhibit + si-NC group (co-transfected with miR-200b-3p inhibit and si-NC), miR-200b-3p inhibit + si-FOSL2 group (co-transfected with miR-200b-3p inhibit and si-FOSL2). The miR-200b-3p mimic, miR-200b-3p inhibitor, si-FOSL2, oe-FOSL2, and the corresponding negative control were individually or co-transfected into HEC-1-A cells using the Lipofectamine 2000 kit according to the instructions. 48h later, the transfection efficiency was verified by real-time fluorescence quantitative PCR and Western blot to verify the transfection efficiency.

1.3 Real-time fluorescence quantitative PCR

Total RNA was extracted using TRIzol reagent, and the RNA concentration and OD260/OD280 ratio were detected using a NanoDrop ultraviolet spectrophotometer. 10 µg of RNA was taken and reverse transcribed into cDNA, and RT-qPCR was performed using SYBR Green Mix (Thermo Fisher Scientific, USA). β-actin was used as an internal reference, and the relative gene expression was calculated using the -ΔΔCT method. Three parallels were set in each group.

1.4 Western blot

Total protein was extracted using RIPA protein lysate containing 100× Cocktail and 100× PMSF, and protein concentration was determined by BCA protein assay kit. Proteins were electrophoretic transfered to PVDF membranes by 10% SDS-PAGE gel. After the transfer was completed, the membrane was closed with 5% skimmed milk for 1h at room temperature and incubated with the corresponding primary antibody at 4°C overnight. The membrane was washed with TBST buffer and incubated with the corresponding secondary antibody at room temperature for 1h. ECL reagent and chemiluminescence detection kit were used for blot analysis, β-actin was used as the internal control, and Image J v1.8 was used for blot analysis.

1.5 Bioinformatics analysis

Starbase(http://starbase.sysu.edu.cn/index.php) was used to predict the potential binding site between miR-200b-3p and FOSL2 3'-UTR.

1.6 Dual luciferase test

HEC-1A cells were co-transfected with Lipofectamine 2000(miR-200b-3p mimic /NC mimic), wild-type FOSL2 3'UTR (FOSL2-WT), mutant FOSL2 3'-UTR (FOSL2-MUT). After 48h of transfection, luciferase activity was measured using Dual-Lucy Assay Kit.

1.7 Cell proliferation assay

CCK-8 was used to detect the proliferative activity of cells in each group. HEC-1A cells in logarithmic growth phase were prepared into a cell suspension of 5×10⁴ cells/mL, and 100 µL of the cell suspension was inoculated into 96-well plates and cultured at 37℃ with 5% CO2 for 2 to 3h. Subsequently, 10µL of CCK-8 reagent was added to each well, and the culture was continued for 2h. Absorbance values at 450nm were detected, and the data were recorded and the cell proliferation curves were plotted.

1.8 Cell scratch assay

Cell migration was detected using the cell scratch assay. EC cells were inoculated in 6-well plates at a density of 1×10⁵ cells/well, and when the cell fusion reached more than 80%, a 200 µL tip was used to scratch vertically at the bottom of the well plates to form cell scratches. After completion of the scratch, the cells were rinsed with PBS to remove the suspended cells. Photographs were taken at 0h and 12h after scratching with a 10x field of view. The area of the scratch at each time point was analyzed using ImageJ software, and the mobility rate was calculated.

1.9 Transwell Detection

Cell invasion was detected using the Transwell assay. HEC-1A cells were inoculated at a density of 1×10⁵ cells/well in the upper chamber of a small chamber containing 200 µL of serum-free medium, while 500 µL of medium containing 10% fetal bovine serum was added to the lower chamber. After 24 hours of incubation at 37°C with 5% CO2, cells remaining on the upper surface of the filter were removed, and cells migrating to the lower chamber were fixed using methanol and stained with crystal violet. Subsequently, five fields of view were randomly selected under a light microscope (10x objective), and the average cell number was calculated.

1.10 Statistical analysis

GraphPad Prism statistical software is applied to analyze the data and plot. Data in this study are expressed as mean ± SD. t-test, one-way ANOVA and SNK-q test were used for comparison of measurement data. P < 0.05 is considered as significant statistically difference.

2.1 miR-200b-3p mimic and FOSL2 expression in EC cells

The expression of miR-200b-3p is significantly down-regulated (P < 0.05) in EC cell lines compared to human normal endometrial cells. The lowest expression is found in HEC-1-A, which is selected for subsequent overexpression experiments. mRNA and protein expression of FOSL2 is significantly increased (P < 0.05) in EC cell lines, as shown in Fig. 1 and Table 1.

Table 1

miR-200b-3p mRNA and protein expression in EC cell lines (n = 3, x ± s)
Groups	miR-200b-3p	FOSL2 mRNA	FOSL2
hEM15A	1.01 ± 0.02	0.49 ± 0.02	0.58 ± 0.03
HEC-1-A	0.31 ± 0.04	1.24 ± 0.04	1.26 ± 0.11
RL95-2	0.49 ± 0.04	1.18 ± 0.12	1.13 ± 0.08
Ishikawa	0.61 ± 0.02	0.67 ± 0.06	0.74 ± 0.03
F	792.900	248.940	181.759
P	P < 0.001	P < 0.001	P < 0.001

2.2 FOSL2 is a downstream target gene of miR-200b-3p

The existence of potential binding site of miR-200b-3p with FOSL2 3'-UTR is predicted using Starbase(Fig. 2A).Compared with the NC-mimic group, the expression of miR-200b-3p in the miR-200b-3p mimic group was significantly increased (P < 0.001). Compared with the NC- inhibit group, and, miR-200b-3p expression in the miR-200b-3p inhibit group was significantly decreased (P < 0.001) (Table 2). Compared with the Si-NC group, FOSL2 mRNA and protein expression in the Si-FOSL2 group were significantly decreased (P < 0.001). Compared with the oe-NC group, the FOSL2 mRNA and protein expression in the oe-FOSL2 group were significantly increased (P < 0.001) (Fig. 2B and Table 3 ). Compared with the NC-mimic group, FOSL2 mRNA and protein expression in the miR-200b-3p mimic group were significantly decreased (P < 0.001). Compared with the miR-200b-3p mimic + oe-NC group, FOSL2 mRNA and protein expression in the miR-200b-3p mimic + oe-FOSL2 group were significantly increased (P < 0.001). Compared with the NC-inhibit, FOSL2 mRNA and protein expression in the miR-200b-3p inhibit group were significantly increased (P < 0.001). Compared with the miR-200b-3p inhibit + si-NC group, FOSL2 mRNA and protein expression in the miR-200b-3p inhibit + si- FOSL2 group were significantly decreased(P < 0.001), (Table 4 and Fig. 2C). As a result of dual luciferase assay, Compared with the NC-mimic group, FOSL2-WT dual luciferase activity in the miR-200b-3p mimic group was significantly reduced (P < 0.001). Compared with the NC-inhibit group, FOSL2-WT dual luciferase activity in the miR-200b-3p inhibit group was significantly higher(P < 0.001),( Table 5).

Table 2

miR-200b-3p mRNA expression in each group of cells (n = 3, x ± s)
Groups		miR-200b-3p
NC-mimic		1.01 ± 0.02
miR-200b-3p mimic		0.45 ± 0.02
t		34.293
P		< 0.001
NC-inhibit		0.99 ± 0.02
miR-200b-3p inhibit		2.59 ± 0.08
t		33.607
P		< 0.001

Table 3

Expression of FOSL2 mRNA and protein in each group of cells (n = 3, x ± s)
Groups	FOSL2 mRNA		FOSL2
si-NC	1.02 ± 0.02		1.01 ± 0.01
si-FOSL2	0.39 ± 0.02		0.31 ± 0.02
t	38.579	54.222
P	< 0.001	< 0.001
oe-NC	0.99 ± 0.02	1.02 ± 0.03
oe-FOSL2	2.54 ± 0.08	2.64 ± 0.12
t	32.557	23.105
P	< 0.001	< 0.001

Table 4

Expression of FOSL2 mRNA and protein in each group of cells (n = 3, x ± s)
Groups	FOSL2 mRNA	FOSL2
NC-mimic	1.01 ± 0.01	0.99 ± 0.01
miR-200b-3p mimic	0.45 ± 0.02	0.39 ± 0.02
t	43.377	46.476
P	< 0.001	< 0.001
miR-200b-3p mimic + oe-NC	0.42 ± 0.03	0.40 ± 0.02
miR-200b-3p mimic + oe-FOSL2	0.89 ± 0.03	0.82 ± 0.02
t	19.188	20.176
P	< 0.001	< 0.001
NC-inhibit	0.99 ± 0.02	1.00 ± 0.02
miR-200b-3p inhibit	2.44 ± 0.03	2.40 ± 0.02
t	69.656	85.732
P	< 0.001	< 0.001
miR-200b-3p inhibit + si-NC	1.41 ± 0.03	1.38 ± 0.03
miR-200b-3p inhibit + si-FOSL2	2.11 ± 0.02	2.09 ± 0.05
t	33.627	21.090
P	< 0.001	< 0.001

Table 5

Dual luciferase assay (n = 3, x ± s)
Groups	FOSL2-WT	FOSL2-MUT
NC-mimic	1.01 ± 0.09	1.00 ± 0.11
miR-200b-3p mimic	0.49 ± 0.05	0.99 ± 0.08
t	8.748	0.127
P	0.001	0.905
NC-inhibit	0.99 ± 0.12	1.01 ± 0.08
miR-200b-3p inhibit	2.15 ± 0.08	1.00 ± 0.09
t	13.931	0.144
P	< 0.001	0.893

2.3 Effect of miR-200b-3p on endometrial cancer cell proliferation, migration and invasion

Compared with the NC-mimic group, showed a significant decreased the cell proliferation activity, cell migration rate, and the number of membrane-penetrating cells in the miR-200b-3p mimic group were significantly decreased (P < 0.05), the mRNA and protein expression levels of the epithelial markers, N-cadherin and Vimentin were significantly decreased (P < 0.05), and the mRNA and protein expression levels of mesenchymal marker were significantly increased (P < 0.05). Compared with the miR-200b-3p mimic + oe-NC group, cell proliferation activity, cell migration rate, and number of membrane-penetrating cells in the miR-200b-3p mimic + oe-FOSL2 group were significantly increased (P < 0.05), the mRNA and protein expression levels of N-cadherin and Vimentin were significantly increased (P < 0.05), and the mRNA and protein expression levels of E -cadherin's mRNA and protein expression levels were significantly decreased (P < 0.05). Compared with the NC-inhibit group, the cell proliferation activity, cell migration rate, and number of membrane-penetrating cells in the miR-200b-3p inhibit group were significantly increased (P < 0.05), the mRNA and protein expression levels of N-cadherin and Vimentin were significantly increased (P < 0.05), and the mRNA and protein expression levels of E-cadherin were significantly decreased (P < 0.05). Compared with the miR-200b-3p inhibit + si-NC group, the cell proliferative activity, cell migration rate, and the number of membrane-penetrating cells in the miR-200b-3p inhibit + si-FOSL2 group were significantly decreased (P < 0.05), mRNA and protein expression levels of N-cadherin and Vimentin were significant decreased (P < 0.05), and the mRNA and protein expression levels of E-cadherin were significant increased (P < 0.05), (Fig. 3, Table 6 and Table 7).

Table 6

Proliferative activity of each group of cells detected by CCK-8 assay (n = 3, x ± s)
Groups	OD (450 nm)
Groups	0 d	1 d	2 d	3 d	4 d
NC-mimic	0.35 ± 0.06	0.51 ± 0.05	1.10 ± 0.05	1.53 ± 0.04	1.90 ± 0.03
miR-200b-3p mimic	0.34 ± 0.04	0.40 ± 0.05	0.43 ± 0.04	0.53 ± 0.05	0.61 ± 0.04
t	0.600	2.694	18.124	27.050	44.687
P	0.581	0.054	< 0.001	< 0.001	< 0.001
miR-200b-3p mimic + oe-NC	0.31 ± 0.05	0.39 ± 0.04	0.42 ± 0.05	0.51 ± 0.03	0.59 ± 0.04
miR-200b-3p mimic + oe-FOSL2	0.34 ± 0.03	0.49 ± 0.02	0.82 ± 0.03	1.01 ± 0.05	1.35 ± 0.06
t	0.891	3.873	11.882	14.852	18.255
P	0.423	0.018	< 0.001	< 0.001	< 0.001
NC-inhibit	0.37 ± 0.08	0.52 ± 0.04	1.08 ± 0.04	1.47 ± 0.08	1.91 ± 0.04
miR-200b-3p inhibit	0.36 ± 0.03	0.69 ± 0.05	1.36 ± 0.05	1.71 ± 0.09	2.13 ± 0.08
t	0.203	4.599	10.046	3.452	4.260
P	0.849	0.010	0.002	0.026	0.13
miR-200b-3p inhibit + si-NC	0.36 ± 0.03	0.66 ± 0.04	1.31 ± 0.04	1.76 ± 0.03	2.14 ± 0.06
miR-200b-3p inhibit + si-FOSL2	0.37 ± 0.02	0.55 ± 0.03	1.13 ± 0.05	1.53 ± 0.04	2.01 ± 0.03
t	0.480	4.260	4.869	7.967	3.357
P	0.656	0.019	0.008	0.001	0.028

Table 7

Comparison of cell migration, invasion and EMT-related protein expression in each group (n = 3, x ± s)
Groups	Scratch repair rate (%)	Numbers of cellpermeating septum(cells)	N-cadherin	Vimentin	E-cadherin
NC-mimic	51.31 ± 2.87	79.89 ± 7.03	1.02 ± 0.03	1.02 ± 0.01	0.80 ± 0.02
miR-200b-3p mimic	29.84 ± 3.21	36.97 ± 6.49	0.42 ± 0.03	0.51 ± 0.03	1.17 ± 0.02
t	8.636	7.770	24.495	27.934	22.658
P	< 0.001	0.001	< 0.001	< 0.001	< 0.001
miR-200b-3p mimic + oe-NC	32.46 ± 3.11	40.59 ± 8.03	0.45 ± 0.04	0.47 ± 0.03	1.10 ± 0.04
miR-200b-3p mimic + oe-FOSL2	42.89 ± 3.98	64.28 ± 6.32	0.91 ± 0.03	0.83 ± 0.04	0.94 ± 0.03
t	3.577	4.015	15.935	12.471	5.543
P	0.023	0.016	< 0.001	< 0.001	< 0.001
NC-inhibit	49.79 ± 4.11	85.31 ± 4.86	1.02 ± 0.02	1.03 ± 0.01	0.89 ± 0.02
miR-200b-3p inhibit	71.16 ± 3.15	121.40 ± 9.35	1.40 ± 0.04	1.44 ± 0.03	0.38 ± 0.03
t	3.148	5.932	14.717	22.457	24.500
P	0.002	0.004	< 0.001	< 0.001	< 0.001
miR-200b-3p inhibit + si-NC	76.59 ± 2.62	122.59 ± 6.83	1.42 ± 0.05	1.42 ± 0.03	0.40 ± 0.02
miR-200b-3p inhibit + si-FOSL2	60.97 ± 3.28	105.18 ± 7.12	1.23 ± 0.03	1.29 ± 0.04	0.73 ± 0.03
t	6.466	3.056	5.644	4.503	15.853
P	0.003	0.038	0.001	0.011	< 0.001

miRNA is an endogenous non-coding RNA that exhibit a high degree of sequence and functional conservation, and affect downstream mRNA expression through post-transcriptional regulatory mechanisms. This regulatory process involves the specific binding of miRNA to the 3' untranslated region (3'UTR) of mRNA, which in turn affects mRNA stability, translational efficiency, or directly leads to the degradation of mRNAs, thereby regulating a variety of cancer-related processes, and playing an important role in the occurrence and development of many cancers, with great potential for diagnostics and therapy. miR-200b- 3p can perform different functions depending on the tumor type. In most cases, exhibits tumor suppressor effects that are down-regulated in cancerous tissues and cells. For example, Li et al.[14] found that miR-200b-3p expression in breast cancer tissues and cell lines was significantly lower than that in normal breast tissues and mammary epithelial cells. Xia et al.[15] identified that miR-200b-3p expression was downregulated in metastatic prostate cancer. In the present study, down regulation of miR-200b-3p expression in endometrial cancer cells was also confirmed. Recent studies have shown that miR-200b can inhibit the EMT process in endometrial cancer cells by promoting the expression of NKD2, which in turn regulates their invasive and migratory abilities.[9] Hong et al.[16]also demonstrated that miR-200b-3p can inhibit the migration and invasion of breast cancer cells by regulating the expression of ezrin-radixin-moesin protein. In addition miR-200b-3p can also act on genes such as microfiber-associated glycoprotein 2 (MAGP2), Mothers Against Decapentaplegic Homolog 2 (SMAD2) and high mobility group box 3 (HMGB3) to inhibit the life processes of tumor proliferation, apoptosis, invasion, and migration by silencing their mRNA expression. [10] In this study, overexpression of miR-200b-3p significantly inhibited the ability of EC cells for proliferation, migration and invasion, while inhibition of miR-200b-3p significantly promoted the ability of EC cells for proliferation, migration and invasion. Therefore, it is hypothesized that miR-200b-3p might be involved in EC progression as a tumor suppressor gene.

FOSL2, a member of the Fos family and part of the AP-1 transcriptional complex, has been studied to be significantly up-regulated in expression in ovarian cancer tissues and to regulate ovarian cancer cell development by mediating the formation of inflammatory vesicles. [17] In this study, it is also found that FOSL2 expression is significantly increased in endometrial cancer cells, meaning that it may be involved in endometrial cancer progression as an oncogene. Some studies have found that FOSL2 can also act as a downstream target gene of miRNAs to affect cancer proliferation, migration and invasion, such as being targeted and inhibited by miR-143-3p to affect the proliferation and migration of myeloma,[18] and it can also promote the progression of colorectal cancer under the upstream regulation of miR-619-5p.[19] In this study, it is found that FOSL2 mRNA contains a binding site to miR-200b-3p by Starbase, therefore, FOSL2 is likely to be a potential target gene of miR-200b-3p, and this speculation is verified in this study by employing dual luciferase. Meanwhile, it is also observed that miR-200b-3p overexpression could inhibit the transcription and translation of FOSL2 in EC cells, and miR-200b-3p knockdown could have the opposite effect, further confirming that FOSL2 is one of the targets of miR-200b-3p, and that miR-200b-3p could negatively regulate FOSL2 expression in EC cells. In addition, the inhibitory effects of miR-200b-3p overexpression on the proliferation, migration and invasion of EC cells were able to be abrogated by FOSL2 overexpression, and the promotional effects of miR-200b-3p knockdown on the malignant phenotype of EC cells are able to be reversed by FOSL2 silencing. Thus, miR-200b-3p could inhibit the proliferation, migration and invasion of EC cells by targeting and suppressing FOSL2 expression.

Tumor development is a complex process involving multiple factors, links and regulation, and abnormal expression in any link may lead to changes in protein expression, which in turn affects the value-added, invasive and migratory abilities of tumor cells and biological behaviors such as epithelial-mesenchymal transition (EMT), and ultimately has a significant impact on tumorigenesis, metastasis and prognosis. Epithelial-mesenchymal transition (EMT) refers to the loss of epithelial cells' original polarity and intercellular adhesion, and the acquisition of the ability to migrate and invade the stroma,[20] which is involved in the invasion and metastasis of a wide range of tumors, including EC.[21, 22]EMT is characterized by a decrease in epithelial cell markers E-cadherin and β-catenin, and mesenchymal cell markers N- cadherin and Vimentin increase.[23] Some studies found that miR-200b-3p could inhibit EMT and thus tumor progression through Zinc finger E-box binding homology box 1/2 (ZEB1/2).[10] In triple-negative breast cancer, miR-200b-3p can target the Rho GDP Dissociation Inhibitor (RHOGDI) signaling pathway, thereby increasing CDH1 expression and EMT inhibition.[24] Xie et al. [25] also found that prostate leucine zipper PrLZ could further inhibit the expression of miR-200 family members by activating TGF-β1/p-smad2, thereby negatively regulating ZEB1 expression and causing EMT in prostate cancer. Yin et al.[26] found that FOSL2 promoted EMT, invasion and migration of non-small cell lung cancer cells by binding to the SNAI2 promoter to facilitate SNAI2 transcription. Some studies have confirmed that the miR-200 family and FOSL2 were involved in the regulation of EMT, however, whether miR-200b-3p regulates the involvement of FOSL2 in the EMT process of EC cells was unknown. In this study, miR-200b-3p overexpression is found to significantly inhibit the expression of N-cadherin and Vimentin and promote the expression of E-cadherin, meaning an inhibitory effect of miR-200b-3p on the EMT process in EC cells, and this effect could be abrogated by FOSL2 overexpression. Thus, the miR-200b-3p/FOSL2 axis may inhibit EC cell migration and invasion by inhibiting the EMT process.

In summary, the expression of miR-200b-3p in endometrial cancer cells was down-regulated.

It inhibits the proliferation, migration and invasion of endometrial cancer cells by regulating the EMT process, and its mechanism is related to its targeted negative regulation of FOSL2 expression. Targeting the miR-200b-3p/FOSL2 axis may become a new idea and a new target for the treatment of endometrial cancer. Future studies still need to further explore the specific mechanism of miR-200b-3p and FOSL2 in the occurrence and progression of endometrial cancer in animal models, as well as their interaction with other signaling pathways.

· Ethics approval and consent to participate

This study was approved by the Tianjin Fifth Central Hospital Ethics Committee, I confirmed that informed consent was obtained from all patients and their families, I confirmed all methods were carried out in accordance withHelsinki declaration.

· Consent for publication

All authors consent for publication

· Availability of data and materials

All data generated or analysed during this study are included in this published article

· Competing interests

not applicable

· Funding

Binhai New Area Health CommitteeTechnology projects

Funding number :2023BWKQ003

Author Contribution

Contributions: (I) Conception and design: Lijie He; (II) Administrative support: Zhe Han; (III) Provision of study materials or patients: Lijie He; (IV) Data analysis and interpretation: Jing Wang; (V) Manuscript writing: All authors; (VI) Final approval of manuscript: All authors.# Lijie He and Jing Wang these two authors contribute equally to this paper.

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No competing interests reported.

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Mechanism of miR-200b-3p-induced FOSL2 inhibition of endometrial cancer cell proliferation and metastasis

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Abstract

Figures

Introduction

Material and Methods

1.1 Cells and Primary Reagents

1.2 Cell culture and cell transfection

1.3 Real-time fluorescence quantitative PCR

1.4 Western blot

1.5 Bioinformatics analysis

1.6 Dual luciferase test

1.7 Cell proliferation assay

1.8 Cell scratch assay

1.9 Transwell Detection

1.10 Statistical analysis

Results

2.1 miR-200b-3p mimic and FOSL2 expression in EC cells

2.2 FOSL2 is a downstream target gene of miR-200b-3p

2.3 Effect of miR-200b-3p on endometrial cancer cell proliferation, migration and invasion

Discussion

Declarations

Author Contribution

References

Additional Declarations

Supplementary Files

Status:

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