Circ_0089761 accelerates colorectal cancer metastasis and immune escape via miR-27b-3p/PD-L1 axis

Abstract

Circular RNAs have been implicated as critical regulators in the initiation and progression of colorectal cancer (CRC). This study was intended to elucidate the functional significance of the circ_0089761/miR-27b-3p/programmed cell death ligand 1 (PD-L1) axis in CRC. Our findings indicated that circ_0089761 expression was significantly elevated in CRC tissues and cell lines. Furthermore, the high expression of circ_0089761 was correlated with TNM stage and tumor size. Silencing circ_0089761 inhibited CRC cell proliferation, migration, and invasion, and increased apoptosis. Mechanistically, circ_0089761 facilitated its biological function by binding to miR-27b-3p to upregulate PD-L1 expression in CRC. Coculture experiments confirmed that low expression of circ_0089761 impeded CD8 + T cell apoptosis and depletion, activated CD8 + T cell function, and increased secretion of the immune effector cytokines IFN-γ, TNF-α, perforin, and granzyme-B. MiR-27b-3p inhibition or PD-L1 overexpression partially impeded CD8 + T cell function. The circ_0089761/miR-27b-3p/PD-L1 axis is postulated to exert pivotal functions in the mechanistic progression of CRC. Furthermore, it holds promising prospects as a feasible biomarker and therapeutic target for CRC.

Keywords: PD‐L1; circ_0089761; colorectal cancer; miR‐27b‐3p.

FIGURE 1

Circ_0089761, miR‐27b‐3p, and PD‐L1 expression in CRC. (a) RT‐qPCR was used to examined circ_0089761 in 75‐paired CRC tumor and adjacent nontumor sample. (b) Relationship between circ_0089761 expression level and overall survival in colorectal cancer patients. (c) ROC curves based on circ_0089761 levels. (d) Analysis of circ_0089761 expression in DLD‐1, HT29, SW480, and HCT116 cells. (e) RT‐qPCR was utilized to examined miR‐27b‐3p in 75‐paired CRC and adjacent nontumor sample. (f) RT‐qPCR was utilized to examined PD‐L1 in 75‐paired CRC tumor and adjacent nontumor sample. (g) The immunohistochemistry staining was conducted to measure the PD‐L1 protein from three CRC patients. Scale bars: 200 μm. *p < 0.05 versus nontumor or FHC; **p < 0.01 versus FHC; ***p < 0.001 versus FHC.

FIGURE 2

circ_0089761, miR‐27b‐3p and PD‐L1 mRNAs expression in different clinical stages. (a) RT‐qPCR was utilized to quantify circ_0089761 expression in stage I + II, III and IV. (b) RT‐qPCR was utilized to assess miR‐27b‐3p expression in stage I + II, III and IV. (c) RT‐qPCR was utilized to determine PD‐L1 mRNA expression in stage I + II, III and IV. ***p < 0.001 versus I + II stage or III stage.

FIGURE 3

Pearson's correlation assay among circ_0089761, miR‐27b‐3p, and PD‐L1 in CRC tumor and nontumor tissues. Correlations between circ_0089761 and miR‐27b‐3p in CRC (a) and nontumor tissues (b) were analyzed with Pearson's correlation coefficient. Correlations between miR‐27b‐3p and PD‐L1 in CRC (c) and nontumor tissues(d) were analyzed with Pearson's correlation coefficient. Correlations between circ_0089761 and PD‐L1 in CRC (e) and nontumor tissues (f) were analyzed with Pearson's correlation coefficient.

FIGURE 4

circ_0089761 regulated PD‐L1 in CRC cells via targeting miR‐27b‐3p. (a) The potential binding sequence between circ_0089761 and miR‐27b‐3p was predicted by starbase online software. (b) The binding relationship between circ_0089761 and miR‐27b‐3p was verified by luciferase reporter assay. (c) The potential binding sequence between miR‐27b‐3p and PD‐L1 was predicted by starbase online software. (d) The binding relationship between miR‐27b‐3p and PD‐L1 was validated by luciferase reporter assay. (e) RT‐qPCR was utilized to examine the efficiency of circ_0089761 overexpression and knockdown in HCT116 and SW480 cells. (f) RT‐qPCR was used to measure miR‐27b‐3p expression after circ_0089761 overexpression and knockdown in HCT116 and SW480 cells. (g) RT‐qPCR was utilized to measure PD‐L1 mRNA expression in HCT116 and SW480 cells after different transfection. (h) Western blotting was conducted to measure PD‐L1 protein expression in HCT116 and SW480 cells after different transfection. *p < 0.05 versus oe‐nc; **p < 0.01 versus nc mimic, oe‐nc, si‐nc, or oe‐circ; ***p < 0.001 versus nc mimic, oe‐nc, si‐nc, or oe‐circ.

FIGURE 5

circ_0089761 knockdown inhibited CRC progression via targeting miR‐27b‐3p/PD‐L1 axis. (a) CCK‐8 assay was utilized to detect cell proliferation in HCT116 and SW480 cells after different transfection. Transwell assay was utilized to detected cell migration (b) and invasion (c) ability in CRC cells after different transfection. Scale bars: 20 μm. (d) Flow cytometry was used to measure cell apoptotic rate. (e) Detection of E‐cadherin, N‐cadherin and vimentin expression in CRC cells using western blotting. *p < 0.05 versus si‐circ; **p < 0.01 versus si‐nc or si‐circ; ***p < 0.001 versus si‐nc or si‐circ.

FIGURE 6

Silencing circ_0089761 attenuated CRC tumor size in vivo. (a) Tumor volume was measured weekly. (b) Tumor images of sh‐NC or sh‐circ_0089761 group in 5th week. (c) Tumor weight of sh‐NC or sh‐circ_0089761 group was measured in 5th week. (d) Expression of circ_0089761, miR‐27b‐3p, and PD‐L1 in tumor samples was determined by RT‐qPCR. (e) Western blotting was utilized to measure the PD‐L1 protein expression in tumor samples. *p < 0.05 versus sh‐nc; **p < 0.01 versus sh‐nc; ***p < 0.001 versus sh‐nc.

FIGURE 7

Silencing circ_0089761 facilitated the proliferation of CD8+ T cells. (a) The proliferative capacity of CD8 + T cells was assessed by measuring Ki67 expression in CD8 + T cells by flow cytometry. (b) Apoptosis of CD8+ T cells was evaluated by TUNEL assay. (c–f) ELISA was applied to detect the levels of IFN‐γ, TNF‐α, perforin, and granzyme‐B in CD8+ T cell supernatants. *p < 0.05 versus si‐nc or si‐circ; **p < 0.01 versus si‐nc or si‐circ; ***p < 0.001 versus si‐nc or si‐circ.