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

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

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 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

Screening for cell lines coexpressing both EPR-1 and Survivin.

<p>(A) The expressions of EPR-1 and Survivin in cell lines were analyzed by Western blotting using β-actin for standardization. (B) The expressions of EPR-1 and Survivin in HEK293 cell line were confirmed by RT-PCR using EPR-1 and Survivin specific cDNA primers.</p

Capture of RISC by both s-2′ FU, 2′ FC oligonucleotides and as-2′ FU, 2′ FC oligonucleotides.

<p>(A) Sequences of the s/as-2′ FU, 2′ FC oligonucleotides and siRNAs. (B) Analysis of RISC capture by Western blotting using anti-TRBP (<i>top</i>) and anti-Ago2 (<i>bottom</i>) antibodies. (C) Schematic representation of TRBP construct with a Flag tag. The numbers indicate amino acid loci. After cotransfecting the HEK293 cells with siRNA1 (targeting both EPR-1 and Survivin) or siRNA-caspase9 (targeting caspase-9) and TRBP, the active RISCs were captured by beads preincubated with 2′ FU, 2′ FC oligonucleotides. Then, the proteins associated with the siRNA-2′ FU, 2′ FC oligonucleotides were recovered and analyzed by Western blotting using an anti-Flag antibody. (D) The sequences of FITC modified siRNA1. After transfecting the HEK293 cells with FITC-siRNA1, the active RISCs were captured by beads preincubated with s/as-2′ FU, 2′ FC oligonucleotides and analyzed by MultisKan.</p

The function of siRNAs targeting the complementary region of EPR-1 and Survivin.

<p>(A) Sequences of siRNAs. The sense strands of siRNAs were designed against the mRNA of the Survivin. The predicted melting free energies of the 4 terminal base pairs of each siRNA are indicated. Throughout this paper, antisense siRNA strands have been presented in black and sense siRNAs strands in red. (B, D) Twenty-four hours after HEK293 cells were transfected with siRNA, the degradation of EPR-1 and Survivin was analyzed by RT-PCR and qRT-PCR using β-actin for standardization. (C) Forty-eight hours after HEK293 cells were transfected with siRNA, the expressions of EPR-1 and Survivin were analyzed by Western blotting using β-actin for standardization.</p

2′ FC, 2′ FU modification of the sense or antisense strand of siRNA does not affect the selective property of the guide strand.

<p>(A) The sequences of unmodified and modified siRNAs. as/s-2′ F indicates that both the sense and antisense strands of siRNA1 were modified with 2′ FC, 2′ FU, while in s-2′ F or as-2′ F, only the sense or antisense strands was modified with 2′ FC, 2′ FU, respectively. (B, D) Twenty-four hours after HEK293 cells were transfected with unmodified and modified siRNA1, the expressions of EPR-1 and Survivin were analyzed by RT-PCR and qRT-PCR using β-actin for standardization. (C) Forty-eight hours after HEK293 cells were transfected with unmodified and modified siRNA1, the expressions of EPR-1 and Survivin were analyzed by Western blotting using β-actin for standardization.</p

The direction of Dcr processing does not alter the selective property of the guide strand.

<p>(A) The sequences of shRNAs. The core 19 nt were obtained from siRNA1 and siRNA4 as mentioned above. The predicted melting free energies of the 4 terminal base pairs of the sense and antisense strands are similar to those of siRNA1. While in siRNA4, the energy difference between the 5′ end of sense and antisense is approximately −1.4 kal/mol. shRNA-R and shRNA-L permit processing specifically from either the 5′ or 3′ end of the incipient siRNA1 or siRNA4. (B) Twenty-four hours after HEK293 cells were transfected with shRNAs, the expressions of EPR-1 and Survivin were analyzed by RT-PCR using β-actin for standardization. (C) Forty-eight hours after HEK293 cells were transfected with shRNA, the expressions of EPR-1 and Survivin were analyzed by Western blotting using β-actin for standardization. (D) The expressions of EPR-1 and Survivin in single cell clones expanded from the siRNA1-ShRNA-R and siRNA4-ShRNA-R were analyzed by RT-PCR using β-actin for standardization. (E) The expressions of EPR-1 and Survivin in single cell clones expanded from the siRNA1-ShRNA-R and siRNA4-ShRNA-R were analyzed by Western blotting using β-actin for standardization.</p

Terminal, single-nucleotide mismatches do not alter the function of siRNA in cultured mammalian cells.

<p>(A) The sequences of siRNA and mismatched siRNA. The predicted melting free energies of the 4 terminal base pairs of sense and antisense strands are similar to those of completely matched siRNA1. The first nucleotide of 5′ end of the sense and antisense strands were mismatched individually (siRNA1-5′m and siRNA1-3′m). (B, D) Twenty-four hours after HEK293 cells were transfected with siRNA1 and mismatched siRNA1, the degradation of EPR-1 and Survivin was analyzed by RT-PCR and qRT-PCR using β-actin for standardization. (C) Forty-eight hours after HEK293 cells were transfected with siRNA1 and mismatched siRNA1, the expressions of EPR-1 and Survivin were analyzed by Western blotting using β-actin for standardization.</p