B7-H1 Expression Is Associated with Poor Prognosis in Colorectal Carcinoma and Regulates the Proliferation and Invasion of HCT116 Colorectal Cancer Cells

<div><p>Background And Objective</p><p>The investigation concerning the B7-H1 expression in colorectal cancer cells is at an early stage. It is unclear whether B7-H1 expression may have diagnostic or prognostic value in colorectal carcinoma. Additionally, how B7-H1 is associated with the clinical features of colorectal carcinoma is not known. In order to investigate the relationship between B7-H1 and colorectal cancer, we analyzed B7-H1 expression and its effect in clinical specimens and HCT116 cells.</p> <p>Methods</p><p>Paraffin-embedded specimens from 143 eligible patients were used to investigate the expression of CD274 by immunohistochemistry. We also examined whether B7-H1 itself may be related to cell proliferation, apoptosis, migration and invasion in colon cancer HCT116 cells.</p> <p>Results</p><p>Our results show that B7-H1 was highly expressed in colorectal carcinoma and was significantly associated with cell differentiation status and TNM (Tumor Node Metastasis) stage. Patients with positive B7-H1 expression showed a trend of shorter survival time. Using multivariate analysis, we demonstrate that positive B7-H1 expression is an independent predictor of colorectal carcinoma prognosis. Our results indicate that B7-H1 silencing with siRNA inhibits cell proliferation, migration and invasion. Furthermore, cell apoptosis was also increased by B7-H1 inhibition.</p> <p>Conclusions</p><p>Positive B7-H1 expression is an independent predictor for colorectal carcinoma prognosis. Moreover, knockdown of B7-H1 can inhibit cell proliferation, migration and invasion.</p> </div

Effective knockdown of B7-H1 by siRNA in HCT116 cells.

<p>(A) RT-qPCR analysis to show the B7-H1 mRNA level. Parental or HCT116 cells transfected with scrambled siRNA or siRNA targeting B7-H1 for 48 h were harvested and RT-qPCR was performed; (B) Western blot analysis to detect the B7-H1 protein level. Parental or HCT116 cells transfected with scrambled siRNA or siRNA targeting B7-H1 for 48 h were harvested and cell lysates were prepared and used for Western blot; (C) Flow cytometric analysis and mean channel fluorescence to show the B7-H1 expression on cell membrane. Parental or HCT116 cells transfected with scrambled siRNA or siRNA targeting B7-H1 for 48 h were harvested and cell surface staining was performed before flow cytometric analysis. Data were presented as means ± SD, *P<0.05 versus the si-scramble group.</p

Effect of B7-H1 knockdown on cell proliferation and apoptosis in HCT 116 cells.

<p>(A) MTT analysis to detect cell proliferation. Parental or HCT116 cells were transfected with scrambled siRNA or siRNA targeting B7-H1 for 48 h were seeded in 96-well plates and cell proliferation was detected by MTT. Data were presented as means ± SD, *P<0.05 versus the si-scramble group. (B) Flow cytometric analysis to detect cell apoptosis. Parental or HCT116 cells transfected with scrambled siRNA or siRNA targeting B7-H1 for 48 h were collected and stained with Annexin-V-FITC and PI before flow cytometric analysis. Data were presented as means ± SD, *P<0.05 versus the si-scramble group.</p

Effect of B7-H1 knockdown on cell migration and invasion in HCT116 cells.

<p>(A) Boyden chamber assay to detect cell migration. Parental or HCT116 cells transfected with scrambled siRNA or siRNA targeting B7-H1 for 48 h were seeded in Boyden chambers without Matrigel-coated membrane, and after another 48 h, migrated cells were stained and counted under a microscope (magnification×10). Representative images were shown. (B) Number of migrated cells shown in A. Data was shown as means ± SD from five fields. *P<0.05 versus the si-scramble group. (C) Boyden chamber assay to detect cell invasion. Parental or HCT116 cells transfected with scrambled siRNA or siRNA targeting B7-H1 for 48 h were seeded in modified Boyden chambers with Matrigel-coated membrane, and after another 24 h, invasive cells that moved through the Matrigel membrane were stained and counted under a microscope (magnification×10). Representative images were shown. (D) Number of invasive cells shown in C. Data was shown as means ± SD from five fields. *P<0.05 versus the si-scramble group.</p