miR-195 enhances the radiosensitivity of colorectal cancer cells by suppressing CARM1

Figures & data

Figure 1 The expression of miR-195 and CARM1 in CRC cell lines HCT-116 and HT-29.

Notes: (A) qRT-PCR analysis of the expression levels of miR-195 in HCT-116, HT-29 cells, and HCEpiC cells. (B) The protein level of CARM1 was detected and quantified in HCT-116, HT-29, and HCEpiC cells by Western blot. *P<0.05 vs control group.
Abbreviations: qRT-PCR, quantitative reverse transcription-polymerase chain reaction; CRC, colorectal cancer.

Figure 2 miR-195 mediates radiosensitivity of CRC cells.

Notes: HCT-116 and HT-29 cells were transfected with miR-control (miR-con) or miR-195 mimics at 24 h of incubation. Survival fractions were calculated in transfected HCT-116 (A) and HT-29 cells (B) after treatment with 0, 2, 4, 6, and 8 Gy of ionizing radiation for 24 h. Cell apoptosis was detected by flow cytometry in transfected HCT-116 (C and D) and HT-29 (E and F) cells at 24 h after treatment with or without ionizing radiation (4 Gy). FITC, fluorescein isothiocyanate. The levels of apoptosis-related proteins Bax, Bcl-2, and γ-H2AX were measured by Western blot in transfected HCT-116 (G) and HT-29 cells (H) after treatment with or without ionizing radiation (4 Gy) for 24 h. *P<0.05 vs miR-control.
Abbreviations: NC, negative control; PI, propidium iodium; FITC, fluorescein isothiocyanate; CRC, colorectal cancer.

Figure 3 Effect of CARM1 knockdown on the radiosensitivity of CRC cells.

Notes: HCT-116 and HT-29 cells were transfected with si-control (si-con) and si-CARM1 at 24 h of incubation. Survival fractions were calculated in transfected HCT-116 (A) and HT-29 cells (B) after treatment with 0, 2, 4, 6, or 8 Gy of ionizing radiation for 24 h. The CARM1 level was detected using Western blot in HCT-116 (C) and HT-29 cells (D) after treatment with or without ionizing radiation (4 Gy) for 24 h. Cell apoptosis was detected by flow cytometry in transfected HCT-116 (E) and HT-29 (F) cells after treatment with or without ionizing radiation (4 Gy) for 24 h. The levels of apoptosis-related proteins Bax, Bcl-2, and γ-H2AX were determined by Western blot in transfected HCT-116 (G) and HT-29 cells (H) after treatment with or without ionizing radiation (4 Gy) for 24 h. *P<0.05 vs si-control.
Abbreviations: NC, negative control; CRC, colorectal cancer.

Figure 4 miR-195 directly targets 3′-UTR of CARM1 and regulates its expression in CRC cells.

Notes: (A) Wild type (WT) and mutant (MUT) 3′-UTR binding sites for miR-195 are shown. The mutated bases are labeled with a horizontal line. (B and C) The relative luciferase activity was measured in HCT-116 and HT-29 cells co-transfected WT or MUT CARM1 3′-UTR with miR-195 mimic or miR-control. (D and E) The relative luciferase activity was measured in HCT-116 and HT-29 cells co-transfected WT or MUT CARM1 3′-UTR with anti-miR-195 or anti-miR-control. The relative mRNA (F) and protein (G and H) level of CARM1 was detected in HCT-116 and HT-29 cells transfected with miR-195 mimics or anti-miR-195. *P<0.05 vs miR-control.
Abbreviations: NC, negative control; CRC, colorectal cancer.

Figure 5 miR-195 enhances radiosensitivity of CRC cells by regulating CARM1.

Notes: HCT-116 and HT-29 cells were transfected with miR-control or miR-195 mimics or co-transfected miR-195 mimics with pcDNA-control (pcDNA con) or pcDNA-CARM1. (A) The level of CARM1 was detected by Western blot. (B) Survival fractions were estimated in transfected HCT-116 and HT-29 cells after treatment with 0, 2, 4, 6, or 8 Gy of ionizing radiation. (C) Cell apoptosis assay of flow cytometry in transfected HCT-116 and HT-29 cells after treatment with ionizing radiation (4 Gy). Apoptosis-related proteins Bax, Bcl-2, and γ-H2AX were detected by Western blot in transfected HCT-116 (D) and HT-29 (E) cells after treatment with ionizing radiation (4 Gy). *P<0.05 vs control groups.
Abbreviations: NC, negative control; CRC, colorectal cancer.

Figure 6 miR-195 upregulation increases CRC cell radiosensitivity in vivo.

Notes: HT-29 cells overexpressing miR-195 or miR-control were subcutaneously injected into nude mice aged 4–6 weeks. Radiation was performed with 8 Gy X-rays when the tumor volume was about 100 mm³. (A) Body weight of the mouse was determined every 5 days after radiation in different groups. (B) Image of representative tumor in different groups at Day 20 after radiation. (C) Weight of tumor in different groups at Day 20 after radiation. (D) Tumor growth curve in different groups. *P<0.05 vs control groups.
Abbreviation: CRC, colorectal cancer.