Effect of integrin α7 on cell proliferation, invasion, apoptosis and the PI3K/AKT pathway, and its association with clinicopathological features in endometrial cancer

Abstract

Targeting integrin α7 (ITGA7) suppresses malignant progression of several types of cancer, including tongue squamous cell carcinoma, hepatocellular carcinoma and non-small cell lung cancer, while the effect of its knockdown on cell function and its association with clinicopathological features in endometrial cancer (EC) is unclear. The present study aimed to investigate this issue. ITGA7 was knocked down by short-interfering (si)RNA in Ishikawa and RL95-2 cells followed by western blotting and reverse transcription-quantitative PCR assays. Subsequently, cell proliferation, apoptosis, invasion and expression levels of PI3K, phosphorylated (p-) PI3K, AKT and p-AKT were determined using Cell Counting Kit-8, TUNEL, Transwell assays and western blotting. Moreover, ITGA7 in tumor and adjacent tissues from 50 patients with endometrial cancer was detected using immunohistochemical assay. ITGA7 expression was increased in EC cell lines (HEC-1A, RL95-2, Ishikawa and KLE) compared with telomerase-immortalized human endometrial stromal cells (THESCs). In both Ishikawa and RL95-2 cells, three ITGA7 siRNAs all demonstrated good efficiency on ITGA7 knockdown, amongst which the one with the highest efficiency was selected for the following experiments. ITGA7 knockdown reduced cell proliferation and invasion, while inducing apoptosis; moreover, it suppressed p-PI3K/PI3K and p-AKT/AKT ratios. In patients with EC, ITGA7 expression was increased in tumor tissues compared with adjacent tissues, and its lower tumor expression was associated with myometrial invasion (<1/2), non-lymphovascular invasion and decreased FIGO stage. In conclusion, ITGA7 knockdown repressed proliferation, invasion and the PI3K/AKT pathway while inducing apoptosis in EC cell lines, and its insufficiency was associated with less advanced tumor features in EC patients. These results indicated that ITGA7 may be a potential target for the treatment of EC.

Keywords: PI3K/AKT pathway; cell function; clinicopathological features; endometrial cancer; integrin α7.

Figure 1.

ITGA7 expression in THESCs and EC cell lines. (A) ITGA7 mRNA level; (B) Representative images of ITGA7 protein levels as detected by western blot analysis; (C) Grayscale analyses of quantified blots of ITGA7 protein level. *P<0.05, **P<0.01 and ***P<0.001 compared with THESCs. ITGA7, integrin α7; EC, endometrial cancer; THESCs, telomerase-immortalized human endometrial stromal cells.

Figure 2.

ITGA7 expression after transfection in Ishikawa and RL95-2 cells. (A) ITGA7 mRNA level after transfection in Ishikawa cells. (B) Representative images of ITGA7 protein level detection by western blot after transfection in Ishikawa cells. (C) Grayscale analyses of blots of quantified ITGA7 protein levels after transfection in Ishikawa cells. (D) ITGA7 mRNA level after transfection in RL95-2 cells. (E) Representative images of ITGA7 protein level detection by western blot after transfection in RL95-2 cells. (F) Grayscale analyses of blots of quantified ITGA7 protein levels after transfection in RL95-2 cells. *P<0.05, **P<0.01 and ***P<0.001. ITGA7, integrin α7; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; si-, short interfering; NC, negative control.

Figure 3.

Proliferation and apoptosis after transfection in Ishikawa and RL95-2 cells. (A) Proliferation after transfection in Ishikawa cells. (B) TUNEL-positive rate after 48 h of transfection in Ishikawa cells (magnification, ×200). (C) Representative images of TUNEL analysis after transfection in Ishikawa cells. (D) Proliferation after transfection in RL95-2 cells; (E) TUNEL-positive rate after transfection in RL95-2 cells. (F) Representative images of TUNEL analysis after 48 h of transfection in RL95-2 cells (magnification, ×200). *P<0.05 and **P<0.01. ITGA7, integrin α7; NC, negative control; NS, not significant; si-, short interfering; OD, optical density.

Figure 4.

Invasion after transfection in Ishikawa and RL95-2 cells. (A) Representative images of invasion assay after transfection in Ishikawa cells (magnification, ×200). (B) Invasive cell number after transfection in Ishikawa cells. (C) Representative images of invasion assay after transfection in RL95-2 cells (magnification, ×200). (D) Invasive cell number after transfection in RL95-2 cells. *P<0.05 and **P<0.01. ITGA7, integrin α7; NC, negative control; si-, short interfering.

Figure 5.

PI3K/AKT pathway after transfection in Ishikawa and RL95-2 cells. (A) Representative images of PI3K, p-PI3K, AKT, p-AKT detection by western blot analysis after transfection. (B) Grayscale analyses of blots of PI3K, p-PI3K, AKT, p-AKT levels after transfection. **P<0.01 and ***P<0.001. ITGA7, integrin α7; NC, negative control; PI3K, phosphatidylinositol 3-kinase; AKT, protein kinase B; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; si-, short interfering; p-, phosphorylated.

Figure 6.

ITGA7 expression and its association with clinical characteristics in patients with EC. (A) Comparison of ITGA7 IHC score in tumor and adjacent tissues. Comparison of ITGA7 IHC score (B) in patients with myometrial invasion ≥1/2 vs. those with myometrial invasion <1/2, (C) in patients with cervical invasion vs. those without that, (D) in patients with lymphovascular invasion vs. those without that and (E) among patients with different FIGO stage. All images were captured with ×200 of magnification. *P<0.05, **P<0.01 and ***P<0.001. ITGA7, integrin α7; EC, endometrial cancer; IHC, immunohistochemical; FIGO, International Federation of Gynecology and Obstetrics.