Knockdown of CLDN6 inhibits cell proliferation and migration via PI3K/AKT/mTOR signaling pathway in endometrial carcinoma cell line HEC-1-B

Figures & data

Figure 1 CLDN6 expression level in EC was significantly upregulated.

Notes: (A) CLDN6 expression level of patients with EC (n=552) and controls (n=35) from the TCGA database was analyzed. (B) Relative expression levels of CLDN6 in EC tissues (n=82) relative to corresponding ANT tissues (n=82). CLDN6 expression was examined using PCR and normalized to GAPDH. (C) Relative expression levels of CLDN6 in EC cell line HEC-1-B relative to corresponding control cell. CLDN6 expression was examined using qRT-PCR and normalized to GAPDH. **P<0.001.
Abbreviations: UCEC, Uterine Corpus Endometrial Carcinoma; ANT, adjacent non-tumorous; CLDN6, claudin-6; EC, endometrial carcinoma; ESC, endometrial cells; TCGA, The Cancer Genome Atlas.

Table 1 Clinical association between CLDN6 expression and clinicopathological variables in EC patients

Characteristics	Expression of CLDN6		P-value
	Low	High
Age (years)			0.002*
<60	16	4
≥60	25	37
Histological type			≤0.0001*
Endometrioid endometrial adenocarcinoma	34	5
Mixed serous and endometrioid	3	3
Serous endometrial adenocarcinoma	4	33
Clinical stage			0.002*
I+II	29	15
III+IV	12	26
Grade			≤0.0001*
G1+G2	22	1
G3+G4	19	40

Notes: The chi-squared test was used to evaluate the correlation between gene expression and clinical features. The expression of CLDN6 gene is associated with age, histological type, clinical stage, and grade in patients with EC.

* P, statistically significant.

Abbreviations: CLDN6, claudin-6; EC, endometrial carcinoma.

Table 2 Univariate and multivariate analysis of clinical prognostic factors of EC

Variables	Univariate analysis			Multivariate analysis
	P-value	HR	95% CI	P-value	HR	95% CI
CLDN6 expression (high/low)	0.008*	3.629	1.402–9.398	0.861	0.902	0.285–2.858
Histological type	≤0.0001*	3.038	1.657–5.568	0.016*	2.624	1.194–5.764
Clinical stage (I–IV)	0.003*	4.319	1.665–11.202	0.226	1.921	0.668–5.526

Note:

* P-value is statistically significant.

Abbreviations: CLDN6, claudin-6; EC, endometrial carcinoma.

Figure 2 Kaplan–Meier OS curves based on CLDN6 expression level.

Note: The relationship between the CLDN6 expression and the survival time of EC patients was analyzed using Kaplan–Meier analysis and log-rank test.
Abbreviations: CLDN6, claudin-6; EC, endometrial carcinoma; OS, overall survival.

Figure 3 Knockdown efficiency was determined by qRT-PCR (A) and western blotting (B and C) in EC cells.

Notes: **P<0.001. Data represent the mean ± SD.
Abbreviations: CLDN6, claudin-6; EC, endometrial carcinoma.

Figure 4 Knockdown CLDN6 in EC cells significantly reduced the proliferative abilities, as determined by CCK-8 assay.

Note: *P<0.05, **P<0.001.
Abbreviations: CCK-8, cell counting kit-8; CLDN6, claudin-6; EC, endometrial carcinoma.

Figure 5 Colony-formation assays suggested that knockdown of CLDN6 significantly reduced the colony-forming ability of EC cells.

Notes: (A) Colonies in the two groups were showed under a microscope. (B) Bar chart showed the number of colonies. **P<0.001.
Abbreviations: CLDN6, claudin-6; EC, endometrial carcinoma.

Figure 6 Migratory/invasive capacity was measured using wound-healing and transwell assay.

Notes: (A) Microscopic images of HEC-1-B cells were assessed by wound-healing assay in two groups. (B) Microscopic images of migratory and invasive cell passing though the microwells of the transwell chamber were detected by transwell assay. (C) The number of migratory and invasive cells was counted. **P<0.01 vs si-con group. Data were presented as the mean ± SD of 3 independent experiments.
Abbreviation: CLDN6, claudin-6.

Figure 7 Effect of CLDN6 knockdown on activation of PI3K pathway in HEC-1B cells.

Notes: (A) The expressions of PI3K, AKT, and mTOR in EC cells 72 h after transfection examined using western blotting analysis. (B) Bars present means ± SD. The content of protein was normalized with the expression of GAPDH. **P<0.001 compared with the respective non-transfected group.
Abbreviations: CLDN6, claudin-6; EC, endometrial carcinoma.