Objective To investigate the effects of long non-coding RNA microRNA503 host gene (LncRNA MIR503HG) on the radiosensitivity of colon cancer cells by regulating the expression of microRNA-224-5p chondroitin sulfate synthas 1 (miR-224-5pCHSY1).
Methods A retrospective analysis was conducted on 48 patients with colon cancer treated in PLA Rocket Force Characteristic Medical Center from March 2019 to January 2022. These patients were selected as tissue samples. The cohort included 28 males and 20 females, aged (57.43±5.28) years. On the basis of the lesions after radiotherapy, they were divided into the radiation resistance group (n=23) and the radiosensitive group (n=25). The expression levels of LncRNA MIR503HG and miR-224-5pCHSY1 in colon cancer tissues and cell lines (CCD841, COLO320, SW480, RKO, and HCT116) were detected by real-time quantitative polymerase chain reaction (qRT-PCR). The radiation-resistant colon cancer cell line HCT116R was constructed and divided into the overexpressing LncRNA MIR503HG (MIR503HG) and its negative control group (mimic-NC), miR-224-5pCHSY1 inhibition group (anti-miR-224-5p) and its negative control group (anti-miR-NC), overexpressing LncRNA MIR503HG+overexpressing miR-224-5pCHSY1 group (MIR503HG+miR-224-5p), and overexpressing LncRNA MIR503HG+negative control group (MIR503HG+miR-NC). The targeting effect of LncRNA MIR503HG and miR-224-5pCHSY1 was verified by dual-luciferase assay, and the cell survival fraction (SF) and apoptosis rate were detected. The data between two groups were compared using t-test.
Results Compared with the radiosensitive group, the expression of LncRNA MIR503HG in the radiation resistance group was significantly lower (1.40±0.36 vs. 0.72±0.17), and the expression of miR-224-5pCHSY1 was significantly higher (1.06±0.25 vs. 1.54±0.27) in the radioresistant group, and the differences were statistically significant (t=8.247, 6.529; both P<0.05). The relative expression levels of LncRNA MIR503HG in the CCD841, COLO320, SW480, RKO, HCT116, and HCT116R cell lines were 2.38±0.06, 1.03±0.05, 0.87±0.03, 0.86±0.02, 0.77±0.04, and 0.54±0.09, respectively. The expression of miR-224-5pCHSY1 were 0.38±0.06, 0.56±0.01, 0.59±0.02, 0.59±0.05, 0.63±0.04, and 0.82±0.06, respectively. Compared with the normal cell line CCD841, the relative expression of LncRNA MIR503HG in the colon cancer cell lines COLO320, SW480, RKO, and HCT116 decreased significantly (t=2.061, 1.665, 4.058, 6.201; all P<0.05), while the expression of miR-224-5pCHSY1 increased significantly (t=1.238, 1.930, 2.037, 1.742; all P<0.05). Compared with the HCT116 cell line, the relative expression of LncRNAMIR503HG in HCT116R significantly decreased ((0.77±0.04)% vs. (0.54±0.09)%), whereas the expression of miR-224-5pCHSY1 significantly increased ((0.63±0.04)% vs. (0.82±0.06)%)(t=1.267, 2.370; both P<0.05). When the irradiation doses were 4, 6, and 8 Gy, the SF of HCT116R cells in the MIR503HG group were significantly lower than that in the mimic-NC group ((7.25±1.11)% vs. (11.34±2.65)%, (2.85±0.58)% vs. (6.08±1.10)%, and (0.58±0.05)% vs. (3.08±0.84)%), and the differences were statistically significant( t=1.064, 1.937, 2.650; all P<0.05)). After overexpression of LncRNAMIR503HG, HCT116R sensitization enhancement ratio (SER) of the MIR503HG group was 1.399. The apoptosis rates of HCT116R cells in the mimic-NC, MIR503HG, mimic-NC+4 Gy, and MIR503HG+4 Gy groups were (8.10±0.23)%, (18.44±1.57)%, (17.33±2.35)%, and (29.83±1.89)%, respectively. Compared with the mimic-NC group, the apoptosis rates of HCT116R cells in the MIR503HG and mimic-NC+4 Gy groups were significantly higher (t=2.003, 1.475; both P<0.05). Compared with the anti-miR-NC group, the luciferase activity of MIR503HG-Wt in the anti-miR-224-5p group increased significantly (1.02±0.20 vs. 1.60±0.25), and the difference was statistically significant (t=5.366, P<0.05). After the mutation of the miR-224-5pCHSY1 and LncRNA MIR503HG binding site, there was no significant difference in MIR503HG-Mut activity between the anti-miR-224-5p group and anti-miR-NC group (0.97±0.25 vs. 1.00±0.22)(t=0.291, P>0.05). Compared with the mimic-NC group, the expression of miR-224-5pCHSY1 in the MIR503HG group was significantly lower (1.97±0.13 vs. 1.12±0.12), and the difference was statistically significant (t=3.915, P<0.05). Moreover, the expression of miR-224-5pCHSY1 in the anti-miR-224-5p group was significantly lower than that in the anti-miR-NC group (1.99±0.19 vs. 0.92±0.18)(t=2.664, P<0.05). When the irradiation doses were 4, 6, and 8 Gy, the SF of HCT116R cells in the anti-miR-224-5p group was significantly lower than that in the anti-miR-NC group ((0.59±0.08)% vs. (0.79±0.12)%, (0.39±0.06)% vs. (0.67±0.07)%, and (0.19±0.04)% vs. (0.52±0.04)%), the differences were statistically significant (t=1.281, 2.034, 2.911; all P<0.05). After inhibiting the expression of miR-224-5pCHSY1, the SER of HCT116R cells in the anti-miR-224-5p group was 1.403. The apoptosis rates of the anti-miR-NC, anti-miR-224-5p, anti-miR-NC+4 Gy, and anti-miR-224-5p+4 Gy groups were (5.08±0.78)%, (14.48±1.21)%, (13.89±1.36)%, and (23.64±1.03)%, respectively. Compared with the anti-miR-NC group, the apoptosis rates of the anti-miR-224-5p and anti-miR-NC+4 Gy groups were significantly higher (t=2.067, 1.934; both P<0.05). Compared with the anti-miR-NC+4 Gy group, the apoptosis rate of the anti-miR-224-5p+4 Gy group was significantly higher (t=4.026, P<0.05). The SFs of the mimic-NC, MIR503HG, MIR503HG+miR-NC, and MIR503HG+miR-224-5p groups were (0.82±0.17)%, (0.53±0.12)%, (0.54±0.11)%, and (0.78±0.15)%, respectively, when the irradiation dose was 4 Gy; (0.66±0.13)%, (0.38±0.09)%, (0.35±0.08)%, and (0.57±0.10)%, respectively, when the radiation dose was 6 Gy; and (0.49±0.10)%, (0.15±0.06)%, (0.13±0.05)%, and (0.43±0.11)%, respectively, when the radiation dose was 8 Gy. Compared with the mimic-NC group, the SF of HCT116R cells in the MIR503HG group decreased significantly when the irradiation dose was ≥4 Gy (t=1.609, 1.533, 1.927; all P<0.05), and the SF of HCT116R cells in the MIR503HG+miR-NC group decreased significantly (t=1.294, 1.490, 1.825; all P<0.05). Compared with the MIR503HG+miR-NC group, the SF of HCT116R cells in the MIR503HG+miR-224-5p group increased significantly when the irradiation dose was ≥ 4 Gy, and the differences were statistically significant (t=1.573, 1.204, 1.937; all P<0.05). After overexpression of miR-224-5pCHSY1, the SER of HCT116R cells in the MIR503HG+miR-224-5p group was 0.825, which was significantly lower than that of MIR503HG group. The apoptosis rates of the mimic-NC, MIR503HG, MIR503HG+miR-NC, and MIR503HG+miR-224-5p groups were (11.61±2.10)%, (24.97±0.91)%, (24.81±1.27)%, and (16.15±1.10)%, respectively. Compared with the mimic-NC group, the apoptosis rate of HCT116R cells in the MIR503HG and MIR503HG+miR-NC groups were significantly higher (t=2.304, 2.159; both P<0.05). Compared with the MIR503HG+miR-NC group, the apoptosis rate of HCT116R cells in the MIR503HG+miR-224-5p group was significantly lower (t=2.067, P<0.05).
Conclusion Overexpression of LncRNA MIR503HG can increase the radiosensitivity of colon cancer cells by inhibiting miR-224-5pCHSY1 expression.
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