-
结肠癌是好发于乙状结肠与直肠交界处的一种常见的消化道恶性肿瘤。据统计,每年全世界结肠癌新增病例为100万例左右,我国新增病例约占其中的1/5;近年来,在我国,随着人们生活习惯的改变,结肠癌的发病率逐年递增[1-2]。目前,放疗是治疗结肠癌的主要手段之一,但部分患者肿瘤组织的放射敏感性低,肿瘤周围组织的放射耐受性差,这些导致结肠癌的放疗疗效较差[3]。因此,寻找可提高结肠癌细胞放射敏感性基因治疗的靶点,探索其分子机制具有重要的临床意义。长链非编码RNA(long non-coding RNA,LncRNA)在生物体中具有重要的生物学功能,其中LncRNA微小RNA-503宿主基因(microRNA503 host gene,MIR503HG)在多种肿瘤中扮演重要的抑癌基因角色[4-6]。然而,LncRNA MIR503HG对结肠癌细胞放射敏感性的影响仍然未知。微小RNA(miRNA)是由约22个核苷酸组成的非编码单链RNA,已有大量的研究结果证实,miRNA在正常组织和肿瘤中的表达存在显著差异[7]。近期的一项研究结果显示,miRNA-224-5p硫酸软骨素合酶1(microRNA-224-5p chondroitin sulfate synthas 1,miR-224-5pCHSY1)在胃癌中过表达,通过抑制胃癌组织中miR-224-5pCHSY1的表达可降低癌细胞的侵袭、迁移及增殖,促进肿瘤细胞凋亡[8]。然而LncRNA MIR503HG、miR-224-5pCHSY1在结肠癌中的表达情况及其与放射敏感性的关系尚未明确。Dai等[9]使用StarBase软件预测LncRNA MIR503HG的靶基因时发现,LncRNA MIR503HG与miR-224-5pCHSY1具有互补结合的核苷酸位点,故推测LncRNA MIR503HG可通过靶向miR-224-5pCHSY1调控结肠癌细胞的放射敏感性。诱导肿瘤细胞凋亡是放疗的主要机理之一,而细胞凋亡又受其相关基因调控,其中Bcl-2和Bax是细胞凋亡相关基因Bcl-2家族中两个功能相反的成员。本研究通过检测LncRNA MIR503HG的表达及其对结肠癌细胞的放射敏感性的调控作用,探讨了LncRNA MIR503HG与miR-224-5pCHSY1的关系,并分析了二者对结肠癌细胞凋亡的影响,以期为结肠癌的诊疗提供理论依据。
-
qRT-PCR检测结果显示,与放射敏感组患者相比,放射抵抗组患者结肠癌组织中LncRNA MIR503HG的相对表达量明显降低(1.40±0.36对0.72±0.17)(图1A),miR-224-5pCHSY1的相对表达量明显升高(1.06±0.25对1.54±0.27)(图1B),且差异均有统计学意义(t=8.247、6.529,均P<0.05),相关性分析结果显示,LncRNA MIR503HG和miR-224-5pCHSY1的相对表达量呈负相关(r=−0.241,P<0.001)(图1C)。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) ( 图1D) ,miR-224-5pCHSY1的表达均明显增加(t=1.238、1.930、2.037、1.742,均P<0.05)(图1E),且HCT116细胞中LncRNA MIR503HG的相对表达量最低,miR-224-5pCHSY1的相对表达量最高,因此选择HCT116细胞株进行后续实验。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2、4、6、8 Gy时,HCT116R细胞的SF均明显高于HCT116细胞[(78.28±7.46)%对(53.92±6.54)%、(58.23±5.54)%对(8.72±2.48)%、(9.42±2.23)%对(2.40±0.12)%、(5.67±1.22)%对(0.52±0.13)%],且差异有统计学意义(t=2.134、2.566、2.761、4.335,均P<0.05)(图1F)。HCT116R细胞的单击多靶模型参数值见表1,HCT116R细胞的SER为0.837,表明HCT116R细胞的放射敏感性明显低于HCT116细胞。
图 1 LncRNA MIR503HG与miR-224-5pCHSY1在放射抵抗结肠癌组织及细胞株中的表达
Figure 1. Expression of LncRNA MIR503HG and miR-224-5pCHSY1 in radio-resistant colon cancer tissues and cell lines
细胞株 D0(Gy) Dq(Gy) N SF2 SER HCT116 3.842 1.406 1.428 0.729 − HCT116R 4.791 3.512 2.116 0.903 0.837 注:HCT116为结肠癌细胞株;HCT116R为放射抵抗的结肠癌细胞株;D0为平均致死剂量;Dq为准阈剂量;N为外推值;SF2为平均2 Gy剂量照射后的细胞存活分数;SER为放射增敏比;−表示无此项数据 表 1 HCT116R和HCT116细胞株的单击多靶模型参数值
Table 1. Parameter values of the single-hit multi-target model for HCT116R and HCT116 cell lines
-
MIR503HG组HCT116R细胞中LncRNA MIR503HG的相对表达量明显高于mimic-NC组(1.44±0.11对0.77±0.06),且差异有统计学意义(t=4.306,P<0.05),提示转染成功(图2A)。细胞克隆实验检测过表达LncRNA MIR503HG后HCT116R细胞的放射敏感性,结果显示,在照射剂量分别为4、6、8 Gy时,MIR503HG组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)(图2B)。在照射剂量为4 Gy时,与mimic-NC组相比,MIR503HG组HCT116R细胞SF开始出现明显差异,因此后续以4 Gy照射细胞。过表达LncRNA MIR503HG后HCT116R细胞单击多靶模型的参数值见表2,MIR503HG组HCT116R细胞的SER为1.399,这提示过表达LncRNA MIR503HG后可明显增加HCT116R细胞的放射敏感性。
图 2 过表达LncRNA MIR503HG对HCT116R细胞放射敏感性的影响
Figure 2. The effect of overexpression of LncRNA MIR503HG on the radiosensitivity of HCT116R cells
组别 D0(Gy) Dq(Gy) N SF2 SER mimic-NC组 4.882 3.454 2.126 0.995 − MIR503HG组 3.423 1.125 1.460 0.760 1.399 注:LncRNA MIR503HG为长链非编码RNA微小RNA-503宿主基因;HCT116R为放射抵抗的结肠癌细胞株;mimic-NC为过表达LncRNA MIR503HG阴性对照组;MIR503HG为过表达LncRNA MIR503HG组;D0为平均致死剂量;Dq为准阈剂量;N为外推值;SF2为平均2 Gy剂量照射后的细胞存活分数;SER为放射增敏比;−表示无此项数据 表 2 过表达LncRNA MIR503HG后HCT116R细胞的单击多靶模型参数值
Table 2. Parameter values of the single-hit multi-target model of HCT116R cells after LncRNA MIR503HG overexpression
-
AnnexinV-FITC/PI双染法(图3A)及Western blot结果(图3B)显示,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)%,Bcl-2的相对表达量分别为0.89±0.12、0.69±0.10、0.68±0.10、0.34±0.07,Bax的相对表达量分别为0.31±0.08、0.54±0.06、0.56±0.08、0.78±0.06,与mimic-NC组相比,MIR503HG组、mimic-NC+4 Gy组HCT116R细胞的凋亡率明显升高(t=2.003、1.475,均P<0.05),Bcl-2的相对表达量明显下降(t=1.934、1.711,均P<0.05),Bax的相对表达量明显上升(t=2.061、2.579,均P<0.05);与mimic-NC+4 Gy组相比,MIR503HG+4 Gy组HCT116R细胞的凋亡率明显升高(t=5.061,P<0.05),Bcl-2的相对表达量明显下降(t=2.916,P<0.05),Bax的相对表达量明显上升(t=4.115,P<0.05)。这提示过表达LncRNA MIR503HG可促进放射诱导的HCT116R细胞凋亡。
-
StarBase在线数据库预测miR-224-5pCHSY1是LncRNA MIR503HG的潜在下游靶标,由图4可见,miR-224-5pCHSY1与LncRNA MIR503HG存在互补结合核苷酸位点。双荧光素酶报告基因实验结果显示,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的荧光素酶活性无明显变化(0.97±0.25对1.00±0.22),且2组间差异无统计学意义(t=0.291,P>0.05)(图4B)。qRT-PCR检测结果显示,与mimic-NC组相比,MIR503HG组miR-224-5pCHSY1相对表达量明显降低(1.97±0.13对1.12±0.12),且差异有统计学意义(t=3.915,P<0.05)(图4C)。这提示过表达LncRNA MIR503HG可靶向调控miR-224-5pCHSY1并抑制其表达。
-
抑制miR-224-5pCHSY1载体及其相应阴性对照分别转染至HCT116R细胞,结果发现,与anti-miR-NC组相比,anti-miR-224-5p组中miR-224-5pCHSY1的相对表达量明显降低(1.99±0.19对0.92±0.18),差异有统计学意义(t=2.664,P<0.05)(图5A)。在照射剂量分别为4、6、8 Gy时,anti-miR-224-5p组HCT116R细胞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)%],且差异有统计学意义(t=1.281、2.034、2.911,均P<0.05)(图5B)。抑制miR-224-5pCHSY1表达后HCT116R细胞单击多靶模型的参数值见表3,anti-miR-224-5p组HCT116R细胞的SER为1.403。这提示抑制miR-224-5pCHSY1表达可增强HCT116R细胞的放射敏感性。
图 5 抑制miR-224-5pCHSY1表达对HCT116R细胞放射敏感性的影响
Figure 5. Effect of inhibition of miR-224-5pCHSY1 expression on radiosensitivity of HCT116R cells
组别 D0(Gy) Dq(Gy) N SF2 SER anti-miR-NC 4.811 3.209 2.003 0.931 − anti-miR-224-5p 3.315 1.336 1.521 0.716 1.403 注:miR-224-5pCHSY1为miRNA-224-5p硫酸软骨素合酶1;HCT116R为放射抵抗的结肠癌细胞株;anti-miR-NC为miR-224-5pCHSY1抑制组的阴性对照组;anti-miR-224-5p为miR-224-5pCHSY1抑制组;D0为平均致死剂量;Dq为准阈剂量;N为外推值;SF2为平均2 Gy剂量照射后的细胞存活分数;SER为放射增敏比;−表示无此项数据 表 3 抑制miR-224-5pCHSY1表达后HCT116R细胞的单击多靶模型参数值
Table 3. Single-hit multi-target model parameter values of HCT116R cells after inhibition of miR-224-5pCHSY1 expression
-
AnnexinV-FITC/PI双染法(图6A)及Western blot结果(图6B)显示,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) %,Bcl-2的相对表达量分别为0.78±0.04、0.46±0.08、0.48±0.06、0.22±0.04,Bax的相对表达量分别为0.22±0.08、0.40±0.06、0.42±0.09、0.72±0.04,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(t=2.067、1.934,均P<0.05),Bcl-2的相对表达量明显下调(t=2.773、2.530,均P<0.05),Bax的相对表达量明显上调(t=1.592、2.140,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(t=4.026,P<0.05),Bcl-2的相对表达量明显下调(t=7.300,P<0.05),Bax的相对表达量明显上调(t=5.227,P<0.05)。这提示抑制表达miR-224-5pCHSY1可促进放射诱导的HCT116R细胞凋亡。
-
使用qRT-PCR验证转染效率,结果发现,mimic-NC组、MIR503HG组、MIR503HG+miR-NC组、MIR503HG+miR-224-5p组miR-224-5pCHSY1的相对表达量分别为1.97±1.13、1.12±0.112、1.10±0.13、2.00±0.13,与mimic-NC组相比,MIR503HG组和MIR503HG+miR-NC组HCT116R细胞中miR-224-5pCHSY1的相对表达量均明显降低(t=1.934、2.061,均P<0.05);与MIR503HG+miR-NC组相比,MIR503HG+miR-224-5p组中miR-224-5pCHSY1的相对表达量明显增加(t=2.519,P<0.05),这提示转染成功(图7A)。细胞克隆形成实验结果显示,照射剂量为4 Gy时,mimic-NC组、MIR503HG组、MIR503HG+miR-NC组、MIR503HG+miR-224-5p组的细胞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,差异有统计学意义(t=1.573、1.204、1.937,均P<0.05)(图7B)。过表达miR-224-5pCHSY1后HCT116R细胞单击多靶模型的参数值见表4,MIR503HG+miR-224-5p组的HCT116R细胞的SER为0.825,相比MIR503HG组明显降低。这提示过表达miR-224-5pCHSY1可逆转过表达LncRNA MIR503HG对HCT116R细胞放射敏感性的增强作用。
图 7 过表达miR-224-5pCHSY1对HCT116R细胞放射敏感性的影响
Figure 7. Effect of overexpression of miR-224-5pCHSY1 on radiosensitivity of HCT116R cells
组别 D0(Gy) Dq(Gy) N SF2 SER mimic-NC 4.803 3.261 2.010 0.879 − MIR503HG 3.434 1.116 1.464 0.660 1.953 MIR503HG+miR-NC 3.347 0.948 1.447 0.756 − MIR503HG+miR-224-5p 4.652 2.116 1.666 0.811 0.825 注:LncRNA MIR503HG为长链非编码RNA微小RNA-503宿主基因;miR-224-5pCHSY1为miRNA-224-5p硫酸软骨素合酶1;HCT116R为放射抵抗的结肠癌细胞株;mimic-NC为过表达LncRNA MIR503HG的阴性对照组;MIR503HG为过表达LncRNA MIR503HG组;MIR503HG+miR-NC为过表达LncRNA MIR503HG+阴性对照组;MIR503HG+miR-224-5p为过表达LncRNA MIR503HG+过表达miR-224-5pCHSY1组;D0为平均致死剂量;Dq为准阈剂量;N为外推值;SF2为平均2 Gy剂量照射后的细胞存活分数;SER为放射增敏比;−表示无此项数据 表 4 过表达LncRNA MIR503HG联合过表达miR-224- 5pCHSY1后HCT116R细胞单击多靶模型的参数值
Table 4. Parameter values of HCT116R cell single-hit multi-target model after overexpression of LncRNA MIR503HG combined with overexpression of miR-224-5pCHSY1
-
AnnexinV-FITC/PI双染法(图8A)及Western blot结果(图8B)显示,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) %,Bcl-2的相对表达量分别为0.82±0.02、0.43±0.02、0.45±0.05、0.79±0.03,Bax的相对表达量分别为0.30±0.03、0.87±0.04、0.89±0.05、0.30±0.06,与mimic-NC组比较,MIR503HG组和MIR503HG+miR-NC组HCT116R细胞的凋亡率明显升高(t=2.304、2.159,均P<0.05),Bcl-2相对表达量明显降低(t=5.291、3.544,均P<0.05),Bax的相对表达量明显增加(t=4.301、5.296,均P<0.05);与MIR503HG+miR-NC组比较,MIR503HG+miR-224-5p组HCT116R细胞的凋亡率明显降低(t=2.067,P<0.05),Bcl-2的相对表达量明显增加(t=2.561,P<0.05),Bax相对表达量明显降低(t=4.671,P<0.05)。这提示过表达miR-224-5pCHSY1可逆转过表达LncRNA MIR503HG对放射诱导的HCT116R细胞凋亡的促进作用。
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
-
摘要:
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。 -
关键词:
- 结肠肿瘤 /
- 辐射耐受性 /
- RNA,长链非编码 /
- LncRNA MIR503HG /
- miR-224-5pCHSY1
Abstract: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. -
Key words:
- Colonic neoplasms /
- Radiation tolerance /
- RNA, long noncoding /
- LncRNA MIR503HG /
- miR-224-5pCHSY1
-
表 1 HCT116R和HCT116细胞株的单击多靶模型参数值
Table 1. Parameter values of the single-hit multi-target model for HCT116R and HCT116 cell lines
细胞株 D0(Gy) Dq(Gy) N SF2 SER HCT116 3.842 1.406 1.428 0.729 − HCT116R 4.791 3.512 2.116 0.903 0.837 注:HCT116为结肠癌细胞株;HCT116R为放射抵抗的结肠癌细胞株;D0为平均致死剂量;Dq为准阈剂量;N为外推值;SF2为平均2 Gy剂量照射后的细胞存活分数;SER为放射增敏比;−表示无此项数据 表 2 过表达LncRNA MIR503HG后HCT116R细胞的单击多靶模型参数值
Table 2. Parameter values of the single-hit multi-target model of HCT116R cells after LncRNA MIR503HG overexpression
组别 D0(Gy) Dq(Gy) N SF2 SER mimic-NC组 4.882 3.454 2.126 0.995 − MIR503HG组 3.423 1.125 1.460 0.760 1.399 注:LncRNA MIR503HG为长链非编码RNA微小RNA-503宿主基因;HCT116R为放射抵抗的结肠癌细胞株;mimic-NC为过表达LncRNA MIR503HG阴性对照组;MIR503HG为过表达LncRNA MIR503HG组;D0为平均致死剂量;Dq为准阈剂量;N为外推值;SF2为平均2 Gy剂量照射后的细胞存活分数;SER为放射增敏比;−表示无此项数据 表 3 抑制miR-224-5pCHSY1表达后HCT116R细胞的单击多靶模型参数值
Table 3. Single-hit multi-target model parameter values of HCT116R cells after inhibition of miR-224-5pCHSY1 expression
组别 D0(Gy) Dq(Gy) N SF2 SER anti-miR-NC 4.811 3.209 2.003 0.931 − anti-miR-224-5p 3.315 1.336 1.521 0.716 1.403 注:miR-224-5pCHSY1为miRNA-224-5p硫酸软骨素合酶1;HCT116R为放射抵抗的结肠癌细胞株;anti-miR-NC为miR-224-5pCHSY1抑制组的阴性对照组;anti-miR-224-5p为miR-224-5pCHSY1抑制组;D0为平均致死剂量;Dq为准阈剂量;N为外推值;SF2为平均2 Gy剂量照射后的细胞存活分数;SER为放射增敏比;−表示无此项数据 表 4 过表达LncRNA MIR503HG联合过表达miR-224- 5pCHSY1后HCT116R细胞单击多靶模型的参数值
Table 4. Parameter values of HCT116R cell single-hit multi-target model after overexpression of LncRNA MIR503HG combined with overexpression of miR-224-5pCHSY1
组别 D0(Gy) Dq(Gy) N SF2 SER mimic-NC 4.803 3.261 2.010 0.879 − MIR503HG 3.434 1.116 1.464 0.660 1.953 MIR503HG+miR-NC 3.347 0.948 1.447 0.756 − MIR503HG+miR-224-5p 4.652 2.116 1.666 0.811 0.825 注:LncRNA MIR503HG为长链非编码RNA微小RNA-503宿主基因;miR-224-5pCHSY1为miRNA-224-5p硫酸软骨素合酶1;HCT116R为放射抵抗的结肠癌细胞株;mimic-NC为过表达LncRNA MIR503HG的阴性对照组;MIR503HG为过表达LncRNA MIR503HG组;MIR503HG+miR-NC为过表达LncRNA MIR503HG+阴性对照组;MIR503HG+miR-224-5p为过表达LncRNA MIR503HG+过表达miR-224-5pCHSY1组;D0为平均致死剂量;Dq为准阈剂量;N为外推值;SF2为平均2 Gy剂量照射后的细胞存活分数;SER为放射增敏比;−表示无此项数据 -
[1] Liao SF, Chen HC, Chen TC, et al. Robotic multivisceral en bloc resection with reconstruction and multidisciplinary treatment of T4 sigmoid colon cancer-a video vignette[J]. Colorectal Dis, 2021, 23(11): 3047−3048. DOI: 10.1111/codi.15894. [2] Di Buono G, Agrusa A. Response to the comment on "feasibility and safety of laparoscopic complete mesocolic excision (CME) for right-sided colon cancer: short-term outcomes. A randomized clinical study"[J]. Ann Surg, 2021, 274(6): e894−e895. DOI: 10.1097/SLA.0000000000004791. [3] Song X, Ning W, Niu JW, et al. CBX8 acts as an independent RNA-binding protein to regulate the maturation of miR-378a-3p in colon cancer cells[J]. Hum Cell, 2021, 34(2): 515−529. DOI: 10.1007/s13577-020-00477-w. [4] Zhu D, Huang XS, Liang F, et al. Retracted article: LncRNA miR503HG interacts with miR-31-5p through multiple ways to regulate cancer cell invasion and migration in ovarian cancer[J/OL]. J Ovarian Res, 2020, 13(1): 3[2022-06-22]. https://ovarianresearch.biomedcentral.com/articles/10.1186/s13048-019-0599-9. DOI: 10.1186/s13048-019-0599-9. [5] Tian J, Yang L, Wang Z, et al. MIR503HG impeded ovarian cancer progression by interacting with SPI1 and preventing TMEFF1 transcription[J]. Aging (Albany NY), 2022, 14(13): 5390−5405. DOI: 10.18632/aging.204147. [6] Monteiro JP, Rodor J, Caudrillier A, et al. MIR503HG loss promotes endothelial-to-mesenchymal transition in vascular disease[J]. Circ Res, 2021, 128(8): 1173−1190. DOI: 10.1161/CIRCRESAHA.120.318124. [7] Liu LT, Xu Q, Xiong Y, et al. LncRNA LINC01094 contributes to glioma progression by modulating miR-224-5p/CHSY1 axis[J]. Hum Cell, 2022, 35(1): 214−225. DOI: 10.1007/s13577-021-00637-6. [8] Wang Y, Yin HL, Chen X. Circ-LDLRAD3 enhances cell growth, migration, and invasion and inhibits apoptosis by regulating MiR-224-5p/NRP2 axis in gastric cancer[J]. Dig Dis Sci, 2021, 66(11): 3862−3871. DOI: 10.1007/s10620-020-06733-1. [9] Dai SH, Li N, Zhou M, et al. LncRNA EBLN3P promotes the progression of osteosarcoma through modifying the miR-224-5p/Rab10 signaling axis[J/OL]. Sci Rep, 2021, 11(1): 1992[2022-06-22]. https://www.nature.com/articles/s41598-021-81641-6. DOI: 10.1038/s41598-021-81641-6. [10] Yang YS, Wen D, Zhao XF. Correction to: transmembrane protease TMPRSS4 promotes the formation and development of mismatch repair deficient colon cancer liver metastasis[J]. Bull Exp Biol Med, 2021, 172(1): 112. DOI: 10.1007/s10517-021-05343-9. [11] Lee SJ, Lee DE, Choi SY, et al. OSMI-1 enhances TRAIL-induced apoptosis through ER stress and NF-κB signaling in colon cancer cells[J/OL]. Int J Mol Sci, 2021, 22(20): 11073[2022-06-22]. https://www.mdpi.com/1422-0067/22/20/11073. DOI: 10.3390/ijms222011073. [12] Hu LJ, Sun F, Sun ZQ, et al. Apatinib enhances the radiosensitivity of the esophageal cancer cell line KYSE-150 by inducing apoptosis and cell cycle redistribution[J]. Oncol Lett, 2019, 17(2): 1609−1616. DOI: 10.3892/ol.2018.9803. [13] Hsu KS, Adileh M, Martin ML, et al. Colorectal cancer develops inherent radiosensitivity that can be predicted using patient-derived organoids[J]. Cancer Res, 2022, 82(12): 2298−2312. DOI: 10.1158/0008-5472.CAN-21-4128. [14] He HC, Lin KY, Su Y, et al. Overexpression of β-catenin decreases the radiosensitivity of human nasopharyngeal carcinoma CNE-2 cells[J]. Cell Physiol Biochem, 2018, 50(5): 1929−1944. DOI: 10.1159/000494873. [15] He P, Xu YQ, Wang ZJ, et al. LncRNA LINC00210 regulated radiosensitivity of osteosarcoma cells via miR-342-3p/GFRA1 axis[J]. J Clin Lab Anal, 2020, 34(12): e23540. DOI: 10.1002/jcla.23540. [16] Yu CY, Liang Y, Jin YQ, et al. LncRNA GAS5 enhances radiosensitivity of hepatocellular carcinoma and restricts tumor growth and metastasis by miR-144-5p/ATF2[J/OL]. Am J Transl Res, 2021, 13(9): 10896-10907[2022-06-22]. https://pubmed.ncbi.nlm.nih.gov/34650771/. [17] Sun CF, Shen C, Zhang YP, et al. LncRNA ANRIL negatively regulated chitooligosaccharide-induced radiosensitivity in colon cancer cells by sponging miR-181a-5p[J]. Adv Clin Exp Med, 2021, 30(1): 55−65. DOI: 10.17219/acem/128370. [18] Li CR, Liang X, Liu YG. LncRNA USP30-AS1 sponges miR-765 and modulates the progression of colon cancer[J/OL]. World J Surg Oncol, 2022, 20(1): 73[2022-06-22]. https://wjso.biomedcentral.com/articles/10.1186/s12957-022-02529-x. DOI: 10.1186/s12957-022-02529-x. [19] Zheng Y, Zeng JT, Lin DJ, et al. Extracellular vesicles derived from cancer-associated fibroblast carries miR-224-5p targeting SLC4A4 to promote the proliferation, invasion and migration of colorectal cancer cells[J]. Carcinogenesis, 2021, 42(9): 1143−1153. DOI: 10.1093/carcin/bgab055. [20] Wu MH, Deng XB, Zhong Y, et al. MafF is regulated via the circ-ITCH/miR-224-5p axis and acts as a tumor suppressor in hepatocellular carcinoma[J]. Oncol Res, 2020, 28(3): 299−309. DOI: 10.3727/096504020X15796890809840. [21] Qiu F, Zhang MR, Zhou Z, et al. LncRNA MIR503HG functioned as a tumor suppressor and inhibited cell proliferation, metastasis and epithelial-mesenchymal transition in bladder cancer[J]. J Cell Biochem, 2019, 120(6): 10821−10829. DOI: 10.1002/jcb.28373. [22] Wei J, Wang ZY, Zhong CY, et al. LncRNA MIR503HG promotes hypertrophic scar progression via miR-143-3p-mediated Smad3 expression[J]. Wound Repair Regen, 2021, 29(5): 792−800. DOI: 10.1111/wrr.12913. [23] Fu J, Dong GJ, Shi H, et al. LncRNA MIR503HG inhibits cell migration and invasion via miR-103/OLFM4 axis in triple negative breast cancer[J]. J Cell Mol Med, 2019, 23(7): 4738−4745. DOI: 10.1111/jcmm.14344. [24] Khan MT, Yang LJ, More E, et al. Developing tumor radiosensitivity signatures using LncRNAs[J]. Radiat Res, 2021, 195(4): 324−333. DOI: 10.1667/RADE-20-00157.1. [25] Wang Z. LncRNA CCAT1 downregulation increases the radiosensitivity of non-small cell lung cancer cells[J]. Kaohsiung J Med Sci, 2021, 37(8): 654−663. DOI: 10.1002/kjm2.12387.