Knockdown of circ_0005615 enhances the radiosensitivity of colorectal cancer by regulating the miR-665/NOTCH1 axis

Abstract

Radiotherapy resistance is a challenge for colorectal cancer (CRC) treatment. Circular RNAs (circRNAs) play vital roles in the occurrence and development of CRC. This study aimed to investigate the role of circ_0005615 in regulating the radiosensitivity of CRC. The levels of circ_0005615, microRNA-665 (miR-665), and notch receptor 1 (NOTCH1) were detected by quantitative real-time PCR or western blot. The radiosensitivity of CRC cells was assessed by colony formation assay. Cell viability, apoptosis, and colony formation were assessed by Cell Counting Kit-8 assay, flow cytometry, and colony formation assay. Cell migration and invasion were confirmed by transwell assay and scratch assay. The binding relationship between miR-665 and circ_0005615 or NOTCH1 was verified by dual-luciferase reporter assay. Xenograft assay was used to test the effect of circ_0005615 on radiosensitivity in vivo. circ_0005615 and NOTCH1 were up-regulated, and miR-665 was down-regulated in CRC tissues and cells. Radiation decreased circ_0005615 and NOTCH1 levels and increased miR-665 level. Knockdown of circ_0005615 enhanced radiosensitivity of CRC cells. Moreover, circ_0005615 sponged miR-665 to regulate the radioresistance of CRC cells. Besides, miR-665 targeted NOTCH1 to mediate the radiosensitivity of CRC cells. Furthermore, circ_0005615 depletion increased CRC radiosensitivity in vivo. circ_0005615 silencing elevated radiosensitivity of CRC by regulating miR-665/NOTCH1 axis.

Keywords: NOTCH1; circ_0005615; circular RNAs; colorectal cancer; miR-665; radiosensitivity.

Figure 1

circ_0005615 is up-regulated in CRC tissues and cells. (a) circ_0005615 expression in normal tissues (n = 30), the less-radiosensitive group (n = 14), and the high-radiosensitive group (n = 16) was examined by qRT-PCR. (b) The level of circ_0005615 was measured in NCM460, LOVO, and SW480 cells (n = 3). (c and d) Levels of circ_0005615, GAPDH, and U6 were detected in nuclear and cytoplasmic fractions (n = 3). Data are shown as mean ± SD. *P < 0.05.

Figure 2

Depletion of circ_0005615 enhances the radiosensitivity of CRC cells. (a) Transfection efficiency of si-circ_0005615 in LOVO and SW480 cells was determined by qRT-PCR. (b and c) Colony formation assay was performed in LOVO and SW480 cells transfected with si-NC or si-circ_0005615 exposed to different doses of X-ray. (d and e) Expression of circ_0005615 was examined in LOVO and SW480 cells treated with various doses of X-ray. After LOVO and SW480 cells were exposed to 6 Gy irradiation alone or in combination with si-circ_0005615, cell viability (f), apoptosis (g), colony number (h), migration and invasion (i and j), and migratory rate (k) were detected by CCK-8 assay, flow cytometry, colony formation assay, transwell, and scratch assay. Data are shown as mean ± SD, n = 3. *P < 0.05.

Figure 3

circ_0005615 directly interacts with miR-665. (a) Putative binding site of miR-665 in circ_0005615 is shown. (b) Overexpression efficiency of miR-665 was detected using qRT-PCR (n = 3). (c and d) Luciferase activity was tested in LOVO and SW480 cells co-transfected with WT-circ_0005615 or MUT-circ_0005615 and miRNA NC or miR-665 mimic (n = 3). (e) miR-665 level was determined in CRC tissues and adjacent normal tissues (n = 30). (f) miR-665 level was examined in NCM460, LOVO, and SW480 cells (n = 3). (g) Transfection efficiency of miR-665 inhibitor was detected by qRT-PCR (n = 3). (h) miR-665 level was measured in LOVO and SW480 cells transduced with si-NC, si-circ_0005615, si-circ_0005615 + inhibitor NC, or si-circ_0005615 + miR-665 inhibitor (n = 3). (i) Expression of miR-665 was detected in LOVO and SW480 cells treated with 6 Gy irradiation (n = 3). Data are shown as mean ± SD. *P < 0.05.

Figure 4

circ_0005615 affects the radiosensitivity of CRC cells via regulating miR-665. LOVO and SW480 cells were transfected with si-NC, si-circ_0005615, si-circ_0005615 + inhibitor NC, or si-circ_0005615 + miR-665 inhibitor and then exposed to 6 Gy irradiation. Cell viability (a), apoptosis (b), colony number (c), migration and invasion (d and e), and migratory rate (f) were examined using CCK-8 assay, flow cytometry, colony formation assay, transwell, and scratch assay, respectively. Data are shown as mean ± SD, n = 3. *P < 0.05.

Figure 5

NOTCH1 is a target of miR-665. (a) Putative binding site of miR-665 in NOTCH1 3′-UTR was displayed. (b and c) Luciferase activity was detected in LOVO and SW480 cells co-transfected with WT-NOTCH1-3′-UTR or MUT-NOTCH1-3′-UTR and miRNA NC or miR-665 mimic (n = 3). (d) NOTCH1 mRNA level was examined in CRC tissues and adjacent normal tissues (n = 30). (e) NOTCH1 protein level was measured in NCM460, LOVO, and SW480 cells (n = 3). (f) NOTCH1 protein expression was determined in LOVO and SW480 cells transfected with pc-NC or pc-NOTCH1 (n = 3). (g) NOTCH1 protein level was detected in LOVO and SW480 cells transfected with miRNA NC, miR-665 mimic, miR-665 mimic + pc-NC, or miR-665 mimic + pc-NOTCH1 (n = 3). (h) NOTCH1 protein level was measured in LOVO and SW480 cells transfected with si-NC, si-circ_0005615, si-circ_0005615 + inhibitor NC, or si-circ_0005615 + miR-665 inhibitor (n = 3). (i) NOTCH1 protein level was detected in LOVO and SW480 cells treated with 6 Gy irradiation (n = 3). Data are shown as mean ± SD. *P < 0.05.

Figure 6

miR-665 mimic increases the radiosensitivity of CRC cells by targeting NOTCH1. LOVO and SW480 cells were introduced with miRNA NC, miR-665 mimic, miR-665 mimic + pc-NC, or miR-665 mimic + pc-NOTCH1 and then treated with 6 Gy irradiation. Cell viability (a), apoptosis (b), colony number (c), migration and invasion (d and e), and migratory rate (f) were detected using CCK-8 assay, flow cytometry, colony formation assay, transwell, and scratch assay, respectively. Data are shown as mean ± SD, n = 3. *P < 0.05.

Figure 7

circ_0005615 silencing increases the radiosensitivity in vivo. Transfected LOVO cells with sh-circ_0005615 or sh-NC were subcutaneously injected into the nude mice. One week after injection, the tumors of the mice were exposed to 6 Gy radiation. (a) Tumor volume was measured once a week. (b) Mice were sacrificed 4 weeks later, and the tumors were removed and weighed. (c–e) Levels of circ_0005615, miR-665, and NOTCH1 in xenograft tumors were measured by qRT-PCR or western blot assay. Data are shown as mean ± SD, n = 5. *P < 0.05.

Figure 8

Schematic diagram of the regulation of circ_0005615/miR-665/NOTCH1 signaling pathway in colorectal cancer.