LncRNA MIR503HG通过调控miR-224-5pCHSY1表达对结肠癌细胞放射敏感性的影响

Study on the effects of LncRNA MIR503HG on radiosensitivity of colon cancer cells by regulating the expression of miR-224-5pCHSY1

目的探讨长链非编码RNA微小RNA-503宿主基因(LncRNA MIR503HG)通过调控miRNA-224-5p硫酸软骨素合酶1 (miR-224-5pCHSY1 )的表达对结肠癌细胞放射敏感性的影响。方法选取2019年3月至2022年1月火箭军特色医学中心收治的48例结肠癌患者为组织样本获取对象, 进行回顾性分析, 其中男性28例、女性20例, 年龄(57.43±5.28)岁。根据放疗后的病灶情况将患者分为放射抵抗组(23例)和放射敏感组(25例)。采用实时定量聚合酶链式反应(qRT-PCR)检测2组患者结肠癌组织及各细胞株(CCD841、COLO320、SW480、RKO、HCT116)中LncRNA MIR503HG、miR-224-5pCHSY1的表达情况;构建放射抵抗的结肠癌细胞株HCT116R, 将HCT116R细胞株分为过表达LncRNA MIR503HG(MIR503HG)组及其阴性对照组(mimiC-NC)、miR-224-5pCHSY1抑制组(anti-miR-224-5p)及其阴性对照组(anti-miR-NC)、过表达LncRNA MIR503HG+过表达miR-224-5pCHSY1组(MIR503HG+miR-224-5p)、过表达LncRNA MIR503HG+阴性对照组(MIR503HG+miR-NC);通过双荧光素酶报告验证LncRNA MIR503HG与miR-224-5pCHSY1的靶向作用;检测各组细胞的存活分数(SF)和凋亡率。两组间数据的比较使用t检验。结果与放射敏感组相比, 放射抵抗组结肠癌组织中LncRNA MIR503HG的相对表达量明显降低(1.40±0.36对0.72±0.17), miR-224-5pCHSY1的相对表达量明显升高(1.06±0.25对1.54±0.27 ), 且差异均有统计学意义(t=8.247、6.529, 均P<0.05)。CCD841、COLO320、SW480、RKO、HCT116及HCT116R各细胞株LncRNA MIR503HG的相对表达量分别为2.38±0.06、1.03±0.05、0.87±0.03、0.86±0.02、0.77±0.04、0.54±0.09, miR-224-5pCHSY1的相对表达量分别为0.38±0.06、0.56±0.01、0.59±0.02、0.59±0.05、0.63±0.04、0.82± 0.06, 与正常细胞株CCD841相比, 结肠癌细胞株COLO320、SW480、RKO、HCT116的LncRNA MIR503HG相对表达量均明显降低(t=2.061、1.665、4.058、6.201, 均P<0.05), miR-224-5pCHSY1的相对表达量均明显增加(t=1.238、1.930、2.037、1.742, 均P<0.05 )。与HCT116细胞株相比, HCT116R的LncRNA MIR503HG相对表达量明显降低[(0.77±0.04)对(0.54±0.09)], miR-224-5pCHSY1的相对表达量明显增加[(0.63±0.04)对(0.82±0.06)], 差异均有统计学意义(t=1.267、2.370, 均P<0.05 )。在照射剂量分别为4、6、8 Gy时, MIR503HG组的HCT116R细胞SF明显低于mimic-NC组[(7.25±1.11)%对(11.34±2.65)%、(2.85±0.58)%对(6.08±1.10)%、(0.58±0.05 )%对(3.08±0.84)%], 且差异均有统计学意义(t=1.064、1.937、2.650, 均P<0.05 )。过表达LncRNA MIR503HG后, MIR503HG组HCT116R的放射增敏比(SER)为1.399。mimic-NC组、MIR503HG组、mimic-NC+4 Gy组、MIR503HG+4 Gy组的细胞凋亡率分别为(8.10± 0.23)%、(18.44±1.57)%、(17.33±2.35)%、(29.83±1.89)%, 与mimic-NC组相比, MIR503HG组、mimic-NC+4 Gy组HCT116R细胞的凋亡率均明显升高, 且差异均有统计学意义(t=2.003、1.475, 均P<0.05 )。与anti-miR-NC组相比, anti-miR-224-5p组MIR503HG-Wt的荧光素酶活性明显增加(1.02±0.20对1.60±0.25 ), 且差异有统计学意义(t=5.366, P<0.05);miR-224-5pCHSY1与LncRNA MIR503HG结合位点突变后, 与anti-miR-NC组相比, anti-miR-224-5p组MIR503HG-Mut的活性无明显变化(0.97±0.25对1.00±0.22), 2组间的差异无统计学意义(t=0.291, P> 0.05 )。与mimic-NC组相比, MIR503HG组miR-224-5pCHSY1的表达水平明显降低(1.97±0.13对1.12±0.12), 差异有统计学意义(t=3.915, P<0.05)。与anti-miR-NC组相比, anti-miR-224-5p组中miR-224-5pCHSY1的表达水平明显降低(1.99±0.19对0.92±0.18 ), 差异有统计学意义(t=2.664 , P<0.05 )。在照射剂量分别为4、6、8 Gy时, anti-miR-224-5p组HCT116R细胞的SF均明显低于anti-miR-NC组[(0.59±0.08 )%对(0.79±0.12)%、(0.39±0.06 )%对(0.67±0.07)%、(0.19±0.04 )%对(0.52±0.04)%], 且差异均有统计学意义(t=1.281、2.034、2.911, 均P<0.05)。抑制表达miR-224-5pCHSY1后, anti-miR-224-5p组HCT116R细胞的SER为1.403。anti-miR-NC组、anti-miR-224-5p组、anti-miR-NC+4 Gy组、anti-miR-224-5p+4 Gy组的细胞凋亡率分别为(5.08±0.78)%、(14.48±1.21)%、(13.89±1.36)%、(23.64±1.03)%, 与anti-miR-NC组相比, anti-miR-224-5p组与anti-miR-NC+4 Gy组的细胞凋亡率均明显增加, 且差异有统计学意义(t= 2.067、1.934, 均P<0.05 );与anti-miR-NC+4 Gy组相比, anti-miR-224-5p+4 Gy组的细胞凋亡率明显增加, 差异均有统计学意义(t=4.026 , P<0.05 )。照射剂量为4 Gy时, mimic-NC组、MIR503HG组、MIR503HG+miR-NC组、MIR503HG+miR-224-5p组的SF分别为(0.82±0.17)%、(0.53±0.12)%、(0.54±0.11)%、(0.78±0.15)%, 照射剂量为6 Gy时, 分别为(0.66±0.13)%、(0.38±0.09)%、(0.35±0.08)%、(0.57±0.10)%, 照射剂量为8 Gy时, 分别为(0.49±0.10)%、(0.15±0.06)%、(0.13±0.05)%、(0.43±0.11)%, 与mimic-NC组相比, 照射剂量≥4 Gy时, MIR503HG组HCT116R细胞的SF明显降低(t=1.609、1.533、1.927, 均P<0.05 ), MIR503HG+ miR-NC组HCT116R细胞的SF明显降低(t=1.294、1.490、1.825, 均P<0.05 );与MIR503HG+ miR-NC组相比, 照射剂量≥4 Gy时, MIR503HG+miR-224-5p组HCT116R细胞的SF明显增加, 且差异均有统计学意义(t=1.573、1.204、1.937, 均P<0.05 ), 过表达miR-224-5pCHSY1后, MIR503HG+miR-224-5p组HCT116R细胞的SER为0.825, 相比MIR503HG组明显降低。mimic-NC组、MIR503HG组、MIR503HG+miR-NC组、MIR503HG+miR-224-5p组的细胞凋亡率分别为(11.61±2.10)%、(24.97±0.91)%、(24.81±1.27)%、(16.15±1.10)%, 与mimic-NC组比较, MIR503HG组和MIR503HG+miR-NC组HCT116R的细胞凋亡率明显增高, 且差异有统计学意义(t=2.304、2.159, 均P<0.05 );与MIR503HG+miR-NC组比较, MIR503HG+miR-224-5p组HCT116R的细胞凋亡率明显降低, 且差异有统计学意义(t=2.067, P<0.05)。结论过表达LncRNA MIR503HG可通过抑制miR-224-5pCHSY1表达增加结肠癌细胞的放射敏感性。

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.