LncRNA TCF7 Promotes Epithelial Ovarian Cancer Viability, Mobility and Stemness via Regulating ITGB8

Abstract

This study aimed to investigate the carcinogenic role of long non-coding RNA T-cell factor 7 (lnc-TCF7) in epithelial ovarian cancer (EOC). Lnc-TCF7 overexpression and shRNA plasmids were transfected into SKOV3 and OVCAR3 cells, followed by measurement of cell proliferation, migration, invasion, apoptosis, stemness, and mRNA profile (via microarray). Besides, lnc-TCF7 expression was measured in tumor and adjacent tissues from 76 EOC patients. Lnc-TCF7 was upregulated in EOC cell lines; its overexpression increased cell proliferation, migration, invasion, but decreased apoptosis and promoted CD44, CD133 expressions, CD44⁺CD133⁺ cell proportion, spheres formation efficiency and drug resistance to cisplatin in SKOV3 and OVCAR3 cells. Besides, lnc-TCF7 ShRNA exhibited opposite effects comparing with its overexpression. Microarray analysis revealed 267 mRNAs were modulated by lnc-TCF7 dysregulation, among which ITGB8 was the most dysregulated one, which was validated by subsequent western blot and RT-qPCR. Furthermore, ITGB8 overexpression not only induced proliferation, migration, invasion and stemness, but also attenuated the effect of lnc-TCF7 ShRNA on these functions in SKOV3 and OVCAR3 cells. In addition, lnc-TCF7 was upregulated in tumor tissues and correlated with higher pathological grade, tumor size, International Federation of Gynecology and Obstetrics (FIGO) stage and worse overall survival in EOC patients. Conclusively, lnc-TCF7 regulates multiple oncogenic pathways, promotes proliferation, migration, invasion, stemness via upregulating ITGB8. It also correlates with advanced tumor features and poor prognosis in EOC, implying its potential as a target for EOC treatment.

Keywords: cancer stem cells; clinicopathological features; epithelial ovarian cancer; integrin β8; long non-coding RNAs T-cell factor 7; overall survival.

Figure 1

Effect of lnc-TCF7 on cell proliferation, apoptosis, migration and invasion in SKOV3 cells. (A) The expression of lnc-TCF7 in EOC cells lines and normal ovarian epithelial cells; (B) The expression of lnc-TCF7 expression after transfection; (C) Cells proliferation in SKOV3 cells; (D, E) Cells apoptosis in SKOV3 cells. (F) Western blot; (G, H) Cells migration in SKOV3 cells; (I, J) Cells invasion in SKOV3 cells. Experiments were performed in triplicates. * represented P < 0.05, ** represented P < 0.01, *** represented P < 0.001.

Figure 2

Effect of lnc-TCF7 on cell stemness in SKOV3 cells. (A, C) CD44 expression after transfection; (B, C) CD133 expression after transfection; (D, E) CD44⁺CD133⁺cell percentage after transfection; (F, G) spheres formation efficiency after transfection; (H) drug resistance to cisplatin after transfection. Experiments were performed in triplicates. * represented P < 0.05, ** represented P < 0.01.

Figure 3

Microarray analysis. Volcano plots (A), GO enrichment (B), KEGG enrichment (C) of mRNA profile between OE-TCF7 group vs. OE-NC group; Volcano plots (D), GO enrichment (E), KEGG enrichment (F) of mRNA profile between SH-TCF7 group vs. SH-NC group; Cross analysis (G), Heatmap (H), GO enrichment (I), KEGG enrichment (J) of accordant mRNA profile.

Figure 4

Validation of top 5 DEGs expressions after transfection. SNX2 (A), PCDHB8 (B), ITGB8 (C), IL1A (D), FN1 (E) mRNA expressions after transfection; and their protein expressions after transfection (F). Experiments were performed in triplicates. * represented P < 0.05, ** represented P < 0.01, *** represented P < 0.001, ns represented no significance.

Figure 5

Effect of ITGB8 overexpression on cell proliferation, apoptosis, migration and invasion in SKOV3 cells. Lnc-TCF7 expression (A), ITGB8 expression (B, C), cell proliferation (D), cell apoptosis rate and apoptotic markers (E–G), cell migration (H, I), cell invasion (J, K) after corresponding transfections. Experiments were performed in triplicates. * represented P < 0.05, ** represented P < 0.01, *** represented P < 0.001, ns represented no significance.

Figure 6

Effect of ITGB8 overexpression on cell stemness and cisplatin sensitivity in SKOV3 cells. CD44 (A), CD133 (B) mRNA expressions, their protein expressions (C), CD44⁺CD133⁺cell percentage (D), spheres formation efficiency (E) and cisplatin sensitivity (F) after corresponding transfections. Experiments were performed in triplicates. * represented P < 0.05, ** represented P < 0.01, ns represented no significance.

Figure 7

Effect of lnc-TCF7 on cell proliferation, apoptosis, migration, invasion, stemness and cisplatin sensitivity in OVCAR3 cells. Lnc-TCF7 expression (A), cell proliferation (B), cell apoptosis rate (C, D), cell migration (E, F), cell invasion (G, H), CD44 expression (I), CD133 expression (J) CD44⁺CD133⁺ cell percentage (K, L), spheres formation efficiency (M, N) and cisplatin sensitivity (O) after corresponding transfections. Experiments were performed in triplicates. * represented P < 0.05, ** represented P < 0.01, *** represented P < 0.001, ns represented no significance.

Figure 8

Effect of ITGB8 knockdown on cell proliferation, apoptosis, migration, invasion, stemness and cisplatin sensitivity in OVCAR3 cells. Lnc-TCF7 expression (A), ITGB8 expression (B), cell proliferation (C), cell apoptosis rate (D, E), cell migration (F, G), cell invasion (H, I), CD44 expression (J), CD133 expression (K) CD44⁺CD133⁺ cell percentage (L), spheres formation efficiency (M) and cisplatin sensitivity (N) after corresponding transfections. Experiments were performed in triplicates. * represented P < 0.05, ** represented P < 0.01, *** represented P < 0.001, ns represented no significance.

Figure 9

Correlation of lnc-TCF7 with ITGB8 and survival. The expression of lnc-TCF7 in tumor tissue and adjacent tissue of EOC (A); The correlation of lnc-TCF7 with OS in EOC patients (B); The correlation of lnc-TCF7 with ITGB8 expression in EOC tumor tissue (C–E); The correlation of ITGB8 with FIGO stage (F, G).