ITGA7 functions as a tumor suppressor and regulates migration and invasion in breast cancer

Abstract

Background: Breast cancer is the most common malignancy in women and the underlying mechanism of breast cancer cell metastasis is still far from uncover. Integrin subunit alpha 7 (ITGA7) is a functioning protein. It has been detected in many malignancies. But the function of ITGA7 in breast cancer is not clear. Our aim is to explore ITGA7 expression and its role in breast cancer.

Methods: Real-time PCR was performed to determine ITGA7 expression in BC tissues and normal adjacent tissues. The specific functions of ITGA7 in breast cancer cell lines (MDA-MB-231 and BT-549) transfected with small interfering RNA were determined through migration, invasion assays. Western blot assays were performed to determine the expression of c-met and vimentin.

Results: ITGA7 was down-regulated in breast cancer tissues compared to the adjacent normal tissues (T:N =7.68±27.38: 41.01± 31.47, P<0.001) and this observation was consistent with the TCGA cohort (T:N =4.51±0.45:5.40±0.61, P<0.0001). In vitro experiments showed that knocking down ITGA7 significantly inhibited the migration and invasion of the breast cancer cell lines (MDA-MB-231 and BT-549). Meanwhile, knockdown of ITGA7 promoted c-met and vimentin expression, which may induce invasion and migration.

Conclusion: ITGA7 plays an important tumorigenic function and acts as a suppress gene in breast cancer. Our findings indicate that ITGA7 was the gene associated with breast cancer.

Keywords: ITGA7; breast cancer; invasion; migration.

Figure 1

ITGA7 expression in breast cancer in validated cohort and TCGA cohort. Notes: (A) ITGA7 expression was examined by RT-qPCR in 36 paired human breast cancer tissues and adjacent noncancerous tissues (paired t-test, P<0.001). A logarithmic scale of 2^−ΔΔCt is used to represent the fold change in quantitative real-time PCR detection. (B) The TCGA cohort contained 1,100 breast tumor tissues and 113 normal tissues. RPKM is used to represent expression of ITGA7. The analysis was done using the Mann–Whitney U-test. ***P<0.001: ****P<0.0001. Abbreviations: ITGA7, integrin subunit alpha 7; RPKM, reads per kilobases per million reads; RT-qPCR, real-time quantitative PCR; TCGA, The Cancer Genome Atlas.

Figure 2

The expression level of ITGA7 in breast cancer cell lines. Notes: (A) The relative expression of ITGA7 gene (compared with the GAPDH gene) using RT-qPCR. Compared to the other cell lines, ITGA7 was expressed at a higher level in MDA-MB-231 and BT-549. (B–D) MDA-MB-231 and BT-549 cells were transfected with siRNA-1, siRNA-2, or si-NC for 48 h, and both mRNA and protein levels of ITGA7 were significantly reduced. Compared with corresponding control group, the expression of ITGA7 gene in S1 and S2 group was lower. We defined ITGA7 siRNA-1 as S1, ITGA7 siRNA-2 as S2, and ITGA7 siRNA-3 as S3. *P<0.05; **P<0.01; ***P<0.001. Abbreviations: ITGA7, integrin subunit alpha 7; NC, negative control; RT-qPCR, real-time quantitative PCR.

Figure 3

Downregulation of ITGA7 gene expression in MDA-MB-231 and BT-549 cells inhibits migration. Notes: These experiments were done at least 3 independent times. (A) In MDA-MB-231, Transwell migration assays in downregulation ITGA7 cells and their corresponding control cells. (B) In BT-549, Transwell migration assays in downregulation of ITGA7 cells and their corresponding control cells. Quantitative results of migration assays. The stained cells were then counted manually from 5 randomly selected fields and normalized with cell proliferation. **P<0.01 in comparison with the NC group using Student’s t-test. We defined ITGA7 siRNA-1 as S1 and ITGA7 siRNA-2 as S2. Abbreviations: ITGA7, integrin subunit alpha 7; NC, negative control.

Figure 4

Downregulation of ITGA7 gene expression in MDA-MB-231 and BT-549 cells inhibits invasion. Notes: These experiments were done at least 3 independent times. (A) Transwell invasion assays in downregulation ITGA7 cells and their corresponding NC cells in MDA-MB-231. (B) Transwell invasion assays in downregulation ITGA7 cells and their corresponding negative control cells in BT-549. Quantitative results of invasion assays. The stained cells were manually counted from 5 randomly selected fields and normalized with cell proliferation. ***P<0.001 in comparison with the NC group using Student’s t-test. We defined ITGA7 siRNA-1 as S1 and ITGA7 siRNA-2 as S2. Abbreviations: ITGA7, integrin subunit alpha 7; NC, negative control.

Figure 5

Downregulation of ITGA7 regulates migration and invasion via addition to c-met-regulated vimentin. Notes: These experiments were done at least 3 independent times. We defined ITGA7 siRNA-1 as S1 and ITGA7 siRNA-2 as S2. (A) The influence of ITGA7 expression on c-MET in MDA-MB-231 and BT-549 cells by Western blot. (B) The influence of ITGA7 expression on vimentin in MDA-MB-231 and BT-549 cells by Western blot. **P<0.01 and ***P<0.001 in comparison with the NC group using Student’s t-test. We defined ITGA7 siRNA-1 as S1 and ITGA7 siRNA-2 as S2. Abbreviations: ITGA7, integrin subunit alpha 7; NC, negative control.