Knockdown of S100A2 inhibits the aggressiveness of endometrial cancer by activating STING pathway

Figures & data

Table 1. Information of the antibodies.

Antibody	Catalogue	Dilution
S100A2	PA5-31861	1:5000/1:4000
E-cadherin	PA5-32178	1:500
N-cadherin	PA5-17526	1:800
cGAS	PA5-56820	0.4 µg/mL
STING	PA5-26751	1:1000
p-STING	PA5-117271	1:500
Notch1	PA5-95827	1:500
HES1	PA5-28802	1:500
PTEN	PA5-20418	2 µg/mL
AKT	PA5-29169	1:1000
p-AKT	44-602G	1:1000
β-catenin	PA5-77934	1:500
GAPDH	MA5-15738-D680	1:3000
Horseradish peroxidase conjugated goat anti-rabbit IgG polyclonal secondary antibody	A27036	1:4000

Table 2. Information of the plasmids.

Oligonucleotide	Sequence
sh-S100A2	Forward 5′-GATGAGAACAGTGACCAGCAG-3′ Reverse 5′-CTGCTGGTCACTGTTCTCATC-3′
sh-NC	Forward 5′-AGATCCGTATAGTGTACCTTA-3′ Reverse 5′-TAAGGTACACTATACGGATCT-3′
S100A2	Forward 5′-GCCAAGAGGGCGACAAGTT-3′ Reverse 5′-AGGAAAACAGCATACTCCTGGA-3′
GAPDH	Forward 5′-GTCTCCTCTGACTTCAACAGCG-3′ Reverse 5′-ACCACCCTGTTGCTGTAGCCAA-3′

Table 3. Correlation between S100A2 expression and EC clinicopathological parameters.

Parameters	N	S100A2 level		p Value
		Low (N = 23)	High (N = 23)
Age				.7101
<55	9	5	4
≥55	37	18	19
Peritoneal washing				.2156
Negative	30	17	13
Positive	16	6	10
Lymph nodes (pelvic)				.0325*
Negative	29	18	11
Positive	17	5	12
Lymph nodes (aortic)				.4366
Negative	38	20	18
Positive	8	3	5
Stage				.1267
I	29	17	12
II–IV	17	6	11
Recurrence				.4747
No	36	19	17
Yes	10	4	6

* p < .05.

Figure 1. High expression of S100A2 is associated with poor prognosis of endometrial cancer. (A) PCR analysis of S100A2 mRNA expression in tissues of endometrial cancer and adjacent normal tissues, ***p < .001, n = 46. (B) The receiver operating characteristic (ROC) curve of S100A2 in endometrial cancer, AUC = 0.8911, p < .001. (C) Kaplan–Meier’s survival curve for endometrial cancer patients according to S100A2 expression in tumour lesions, p = .0185. The protein expression of S100A2 accessed by (D) immunohistochemistry method and (E) western blot, n = 3.

Figure 2. Inhibition of S100A2 suppresses aggressive behaviours of endometrial cancer cells. (A) mRNA levels of S100A2 after transfection in HEC-1 and RL95-2 cells, n = 3, ***p < .001. (B) Cell viability evaluated by CCK-8 assay, n = 3, ***p < .001. (C–F) Migrate and invasive HEC-1 and RL95-2 accessed by Transwell method, n = 3, ***p < .001. (G) Western blot analysis of E-cadherin and N-cadherin protein expression in HEC-1 and RL95-2, n = 3.

Figure 3. The representative photos of the key protein of cGAS/STING, Notch1/HES1, PTEN/AKT and beta-catenin pathways obtained by western blot, n = 3.

Figure 4. Inactivation of cGAS/STING pathway reversed the effects of S100A2 inhibition on endometrial cancer cells. (A) Cell viability evaluated by CCK-8 assay, n = 3, ***p < .001 (vs. sh-NC), ^###p < .001 (vs. sh-S100A2). (B–E) Migrate and invasive HEC-1 and RL95-2 accessed by Transwell method, n = 3, **p < .01, ***p < .001 (vs. sh-NC), ^##p < .01, ^###p < .001 (vs. sh-S100A2). (F) Western Blot analysis of E-cadherin and N-cadherin protein expression in HEC-1 and RL95-2, n = 3.

Figure 5. Knockdown of S100A2 suppressed tumour growth in vivo. (A) The photographs, (B) tumour weight and (C) tumour volume of xenograft tumours from sh-NC and sh-S100A2 groups, n = 3, ***p < .001.

Data availability statement

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.