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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,随着人们生活习惯的改变,我国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 synthase,miR-224-5pCHSY1)在胃癌中过表达,通过抑制胃癌组织中miR-224-5p的表达可降低癌细胞的侵袭、迁移及增值,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,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。
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RPMI1640培养基、胰蛋白酶、胎牛血清均购自美国Gibco公司,双荧光素酶报告基因载体及其活性检测试剂均购自美国Invitrogen公司,实时定量聚合酶链式反应(real-time quantitative polymerase chain reaction,qRT-PCR)仪购自德国Eppendorf公司,流式细胞仪购自美国BD公司,miR-224-5pCHSY1抑制剂(anti-miR-224-5pCHSY1)、过表达LncRNA MIR503HG、过表达miR-224-5pCHSY1及各自阴性对照(anti-miR-NC、mimic-NC、miR-NC)均购自上海吉玛生物科技有限公司,SYBR Green PCR Master Mix试剂盒、Trizol试剂及反转录试剂盒均购自深圳晶美生物工程有限公司,膜联蛋白V(Annexin V)-异硫氰酸荧光素(fluorescein isothiocyanate,FITC)/碘化丙啶(propidium iodide,PI)双染法细胞凋亡检测试剂盒购自上海碧云天生物技术有限公司。
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结肠癌细胞株(COLO320、SW480、RKO、HCT116)和正常细胞株CCD841均购自美国菌种保藏中心,用含10%胎牛血清的RPMI 1640培养基在温度为37 ℃、含5%CO2浓度的恒温细胞培养箱内培养。
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选取2019年3月至2022年1月于我院收治的48例结肠癌患者作为研究对象。纳入标准:(1)经组织病理及临床检查确诊为结肠癌患者;(2)入组前未接受任何治疗;(3)临床资料完整;(4)接受放化疗及其临床评估。排除标准:(1)合并自身免疫性疾病及其他恶性肿瘤等;(2)心、肺等重要脏器存在功能障碍;(3)患有精神疾病,无法正常沟通。所有受试者均签署了同意书,本研究已经本院伦理委员会批准(批号:KS5219)。放疗后进行结肠癌放射敏感性判定:将肿瘤复发或肿瘤未控患者纳入放射抵抗组(23例),将原发肿瘤或部分肿瘤未出现复发或未控患者纳入放射敏感组(25例)。所有患者均于术中切取结肠癌组织,术后先将结肠癌组织保存于−80 ℃冰箱,然后转入液氮中保存。
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取对数生长期的HCT116细胞株,使用ONCOR直线加速器(西门子股份公司,德国),射线参数:能量为6MV-X射线,剂量率为200 cGy/min,照射源皮距为100 cm,照射射野为25 cm×25 cm。使用RPMI 1640培养基于5% CO2、37 ℃培养箱中继续培养,定期更换培养液,直到对数生长期,再进行传代培养,待稳定传代后进行400 cGy照射细胞,重复以上过程,依次给予600、800、1000、1200 cGy照射细胞,筛选具有稳定放射抵抗性表型的结肠癌细胞株HCT116R,常规培养HCT116R细胞株,待细胞稳定传代5代后进行后续研究。
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复苏CCD841、COLO320、SW480、RKO、HCT116、HCT116R,使用含10%胎牛血清和100 mg/ml链霉素与青霉素的RMPI1640培养基进行培养,所有细胞均置于恒温培养箱中培养,培养条件为37 ℃、5% CO2、饱和湿度,取生长良好的细胞,用0、2、4、6、8 Gy照射剂量的X射线照射细胞。同时取生长良好的HCT116R细胞,胰蛋白酶消化,接种于6孔板中,转染,将LncRNA MIR503HG及其相应阴性对照分别转染至HCT116R细胞,分别记为过表达MIR503HG组(MIR503HG)、过表达对照组(mimic-NC),再使用4 Gy照射剂量照射转染后的HCT116R细胞,分别记为mimic-NC+4 Gy组、MIR503HG+4 Gy组;将anti-miR-224-5pCHSY1及其阴性对照分别转染至HCT116R细胞,分别记为抑制miR-224-5p组(anti-miR-224-5p)、抑制对照组(anti-miR-NC),再使用4 Gy照射剂量照射HCT116R细胞,分别记为anti-miR-NC+4 Gy组、anti-miR-224-5p+4 Gy组;将miR-224-5pCHSY1及其对应阴性对照分别与LncRNA MIR503HG共转染至HCT116R细胞,分别记为过表达LncRNA MIR503HG+过表达miR-224-5pCHSY1组(MIR503HG+miR-224-5p)、过表达LncRNA MIR503HG+过表达对照组(MIR503HG+miR-NC)。转染6 h后将细胞培养液更换为含10%胎牛血清的RPMI1640完全培养基。转染48 h后,使用4 Gy剂量的X射线照射细胞,检测细胞凋亡情况。
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使用Trizol试剂盒提取结肠癌组织及各组细胞总RNA,利用反转录试剂盒合成cDNA,将制备好的cDNA保存于−20 ℃的冰箱中。配置20μl 的PCR反应加样体系:正反向引物各0.8 μl,SYBR染料 12 μl,cDNA 2 μl,用双蒸水补足20μl。置于qRT-PCR仪中检测,设定反应程序:95 ℃ 25 s,95 ℃ 6 min,73 ℃ 28 s,59 ℃ 31 s,共循环40次。使用2−△△Ct方法计算LncRNA MIR503HG、miR-224-5pCHSY1、Bcl-2和Bac的相对表达量。
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收集HCT116与HCT116R细胞,分别接种于6孔板中,分别使用0、2、4、6、8 Gy照射剂量的X射线照射细胞,均设置3次重复,于培养箱中培养,待出现克隆团时,用PBS洗涤、甲醇固定、结晶紫染色,置于显微镜下统计≥50个细胞的有效克隆数,细胞克隆形成率=(克隆数/接种细胞)×100%,细胞存活分数(survival fraction,SF)=受照射细胞克隆形成数/对照细胞克隆形成数×100%。使用GraphPad Prism 7(美国GraphPad软件公司)进行单击多靶模型曲线拟合,计算平均2 Gy照射后的细胞SF(SF2)、平均致死剂量(D0)、准阈计量(Dq)、外推值(N)、放射增敏比(sensitization enhancement ratio,SER)(D0实验组/D0对照组)。
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HCT116R细胞经4 Gy剂量照射48 h后,使用PBS洗涤,悬浮液重悬细胞,调整细胞密度,将一定量的细胞悬液加入检测管中,依次加入5 μlAnnexin V-FITC ,避光孵育,洗涤细胞,4 ℃ 1000 r/min 离心3 min,再次悬浮细胞,加入10 μl PI,采用流式细胞仪检测细胞凋亡情况。
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使用StarBase在线数据库预测得知LncRNA MIR503HG可能吸附miR-224-5pCHSY1。构建含有miR-224-5pCHSY1结合位点的LncRNA MIR503HG野生型荧光素酶报告基因载体及突变型荧光素酶报告基因载体:MIR503HG-Wt、MIR503HG-Mut,将HCT116R细胞置于24孔板中,用anti-miR-NC或anti-miR-224-5p与MIR503HG-Wt、MIR503HG-Mut分别共转染HCT116R细胞,每组设置3个复孔。置于培养箱中培养48 h,静置,同时加入荧光素酶缓冲液,检测荧光素酶活性。
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采用SPSS22.0软件分析数据,作图工具采用Graphpad 5.01。计量资料以(均数±标准差)表示,两组间数据的比较使用t检验,多组间比较使用单因素方差分析,组内多个时间点比较使用重复测量方差分析。使用Pearson相关分析评估LncRNA MIR503HG和miR-224-5pCHSY1表达水平之间的关系。P<0.05为差异有统计学意义。
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qRT-PCR检测结果显示,与放射敏感组相比,放射抵抗组结肠癌组织中LncRNA MIR503HG的表达水平明显降低(1.40±0.36对0.72±0.17)(图1A),miR-224-5pCHSY1的表达水平明显增加(1.06±0.25对1.54±0.27)(图1B),且差异有统计学意义(t=8.247、6.529,均P<0.05),相关性分析结果显示,LncRNA MIR503HG和miR-224-5pCHSY1的表达呈负相关(r=−0.241,P<0.05)(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,miR-224-5pCHSY1的表达明显增加,差异均有统计学意义(t=5.720、6.454,均P<0.05)。在照射剂量分别为4、6、8 Gy时,HCT116R细胞的存活率均明显高于HCT116细胞[(0.78±0.07)对(0.53±0.06)、(0.58±0.05)对(0.35±0.05)、(0.40±0.03)对(0.14±0.02)],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0.830,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:−为无此项数据;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为准阈计量;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 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
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MIR503HG组HCT116R细胞中LncRNA MIR503HG的表达水平明显高于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组的HCT116R细胞SF明显低于mimic-NC组[(0.59±0.10)对(0.79±0.05)、(0.40±0.08)对(0.60±0.10)、(0.18±0.06)对(0.38±0.04)],且差异有统计学意义(t=1.064、1.937、2.650,均P<0.05)(图2B)。照射剂量为4 Gy时,HCT116R细胞SF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可增加HCT116R细胞的放射敏感性
Figure 2. Overexpression of LncRNA MIR503HG increased 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:−为无此项数据;mimic-NC为过表达对照组;MIR503HG为过表达LncRNA MIR503HG组;D0为平均致死剂量;Dq为准阈计量;N为外推值;SF2为平均2 Gy照射后的细胞存活分数;SER为放射增敏比 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 single-click multi-target model of HCT116R cells after LncRNA MIR503HG overexpression
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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的表达水平分别为0.89+0.12、0.69+0.10、0.68+0.10、0.34+0.07,Bax的表达水平分别为0.31+0.08、0.54+0.06、0.56+0.08、0.18+0.06,与mimic-NC组相比,MIR503HG组、mimic-NC+4 Gy组HCT116R细胞的凋亡率明显增加(t=2.003、1.475,均P<0.05),Bcl-2的表达水平明显下调(t=1.934、1.711,均P<0.05),Bax的表达水平明显上调(t=2.061、2.579,均P<0.05);与mimic-NC+4 Gy组相比,MIR503HG+4 Gy组HCT116R细胞的凋亡率明显增加(t=5.061,P<0.05),Bcl-2的表达水平明显下调(t=2.916,P<0.05),Bax的表达水平明显上调(t=4.115,P<0.05),差异均有统计学意义。这提示过表达LncRNA MIR503HG可促进放射诱导的HCT116R细胞凋亡。
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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。通过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的活性无明显变化(0.97±0.25对1.00±0.22),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)(图4C)。这提示过表达LncRNA MIR503HG可靶向调控miR-224-5pCHSY1并抑制其表达。
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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),见图5A。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)],差异有统计学意义(t=1.281、2.034、2.911,均P<0.05),见图5B。抑制表达miR-224-5pCHSY1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1.566。这提示抑制miR-224-5pCHSY1表达可增强结肠癌细胞放射敏感性。
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:−为无此项数据;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为准阈计量;N为外推值;SF2为平均2 Gy照射后的细胞存活分数;SER为放射增敏比 表 3 抑制表达miR-224-5pCHSY1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
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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的表达水平明显下调(t=2.773、2.530,均P<0.05),Bax的表达水平明显上调(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的表达水平明显下调(t=7.300,P<0.05),Bax的表达水平明显上调(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。
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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的表达水平均明显降低(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的表达水平明显增加(t=2.519,P<0.05),这提示转染成功(图5A)。细胞克隆形成实验结果显示,mimic-NC组、MIR503HG组、MIR503HG+miR-NC组、MIR503HG+miR-224-5p组的细胞存活分数,照射剂量为4 Gy时,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SF明显增加,差异有统计学意义(t=1.573、1.204、1.937,均P<0.05),见图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SER为0.824,相比于MIR503HG明显降低。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:−为无此项数据;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且过表达miR-224-5pCHSY1的阴性对照组;MIR503HG+miR-224-5p为过表达LncRNA MIR503HG且过表达miR-224-5pCHSY1组;D0为平均致死剂量;Dq为准阈计量;N为外推值;SF2为平均2 Gy照射后的细胞存活分数;SER为放射增敏比 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 click multi-target model after overexpression of LncRNA MIR503HG combined with overexpression of miR-224-5pCHSY1
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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细胞凋亡率明显增高(t=2.304、2.159,均P<0.05),Bcl-2表达水平明显降低(t=5.291、3.544,均P<0.05),Bax的表达水平明显增高(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的水平明显增高(t=2.561,P<0.05),Bax水平明显降低(t=4.671,P<0.05),差异均有统计学意义。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。
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结肠癌是腹部外科常见的一种起源于大肠上皮组织的恶性肿瘤,随着医学研究的深入,结肠癌早期诊疗手段不断更新,其中放化疗可有效提高患者5年生存率,在临床中已得到广泛应用[10]。对于早期患者,手术切除是临床常用的根治方法;中晚期患者主要依赖放化疗来提高5年生存率[11]。然而,部分中晚期结肠癌患者对放疗的敏感性较差。因此,探讨结肠癌患者放射敏感性的调节机制,寻找潜在治疗靶点具有重要临床意义。对于肿瘤细胞放射敏感性的评价,以往研究多采用单一指标,如SF2或细胞存活率等[12]。本研究使用单击多靶模型3个相关参数(D0、Dq、N值)中的任意2个参数均可在一定程度上反映细胞的放射敏感性[13]。D0表示平均每个细胞引起一次致死事件所需的剂量,D0越小,细胞的放射敏感性越强;Dq为细胞受到损伤所需的准阈剂量,Dq越小,即肩区(低剂量范围内的弯曲部分)缩小,细胞对射线越敏感;外推数N值代表存活曲线肩区宽度,反映细胞内所含的放射敏感区域数[14]。本研究结果显示,HCT116R细胞株的D0、Dq和N值均明显高于HCT116细胞,SER明显小于HCT116细胞株,这提示HCT116R细胞株的放射敏感性较弱。此外,过表达LncRNA MIR503HG后,HCT116R细胞的放射敏感明显增强。抑制表达miR-224-5pCHSY1后,HCT116R细胞的放射敏感性也明显增强。在过表达LncRNA MIR503HG的基础上再过表达miR-224-5pCHSY1后,HCT116R细胞的放射敏感性可有效逆转过表达LncRNA MIR503HG对HCT116R细胞放射敏感性的增强作用。这提示过表达LncRNA MIR503HG、抑制表达miR-224-5pCHSY1均可增强HCT116R细胞株的放射敏感性。
近年来越来越多的研究结果显示,LncRNA会影响癌细胞的放射敏感性,可作为肿瘤诊疗的重要靶点[15]。有研究报道,过表达LncRNA GAS5可抑制肿瘤的生长和转移,增强肝细胞癌的放射敏感性[16]。也有研究结果显示[17],过表达LncRNA ANRIL可通过抑制表达miR-181a-5p降低结肠癌细胞的辐射敏感性。然而,LncRNA MIR503HG对结肠癌放射敏感性的影响尚未见报道。本研究结果显示,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的表达水平明显降低,这提示结肠癌组织的放射敏感性与过表达LncRNA MIR503HG有关。本研究进一步检测了结肠癌细胞株中LncRNA MIR503HG的表达情况,结果显示,与正常细胞株相比,结肠癌细胞株中LncRNA MIR503HG的表达水平均明显较低,且与其他结肠癌细胞株相比,HCT116细胞株中LncRNA MIR503HG的相对表达量最低,因此,本研究后续试验均基于HCT116细胞株进行。本研究利用不同照射剂量照射后发现,随着照射剂量增加,放射抵抗性结肠癌细胞株HCT116R的细胞存活率明显高于HCT116细胞,且HCT116R细胞中LncRNA MIR503HG的相对表达量明显低于HCT116细胞,这进一步证实过表达LncRNA MIR503HG会增强结肠癌细胞的放射敏感性。
前期研究结果表明,miRNA通过不完全碱基互补与靶基因结合,影响肿瘤细胞的增殖、凋亡及放射敏感性等过程,触发周围细胞的坏死和凋亡,且miRNA也是lncRNA作用的重要环节[18]。miR-224-5pCHSY1属于miRNA,与肿瘤进展密切相关[19]。已有研究报道,在肝细胞癌患者血清中,miR-224-5pCHSY1水平明显上调,且与肿瘤TNM分期、淋巴结和远处转移密切相关[20]。越来越多的研究结果证实,LncRNA MIR503HG可作为竞争性内源性RNA吸附miRNA调控靶基因,从而影响肿瘤进展[21]。有学者发现,LncRNA MIR503HG通过调控miR-191参与宫颈癌细胞的增殖、侵袭与凋亡[22]。也有学者发现,LncRNA MIR503HG通过靶向调控miR-103调控三阴性乳腺癌细胞的活力、转移和凋亡[23]。本研究通过Starbase数据库预测,miR-224-5pCHSY1与LncRNA MIR503HG存在结合位点。此外,放射抵抗组结肠癌组织中miR-224-5pCHSY1的表达明显高于放射敏感组患者,HCT116R细胞中miR-224-5pCHSY1的相对表达量明显高于HCT116细胞,LncRNA MIR503HG和miR-224-5pCHSY1的表达呈负相关。这些结论均可推测过表达LncRNA MIR503HG可靶向调控miR-224-5pCHSY1并抑制其表达,从而增强结肠癌细胞的放射敏感性。
细胞凋亡不仅是影响肿瘤形成的一个重要机制,而且是影响肿瘤放疗疗效的重要因素之一。细胞凋亡与肿瘤细胞的放射敏感性密切相关[24]。Bcl-2与Bax是调控细胞凋亡的关键因子,Bax是凋亡促进因子,而Bcl-2属于凋亡抑制因子[25]。本研究将LncRNA MIR503HG过表达载体转染至HCT116R细胞后发现,HCT116R细胞的SF明显低于mimic-NC组,细胞凋亡率明显增加,Bcl-2表达水平明显下调,Bax表达水平明显上调;过表达HCT116R细胞再经4 Gy照射细胞后,细胞SF、细胞凋亡率、凋亡相关蛋白Bcl-2、Bax的变化更加明显。这提示过表达LncRNA MIR503HG可促进放射照射诱导的HCT116R细胞凋亡,从而增加细胞放射敏感性。本研究抑制表达miR-224-5pCHSY1后发现,HCT116R细胞SF明显降低,凋亡率明显增加,凋亡蛋白Bcl-2表达水平明显下调,Bax表达水平明显上调;抑制表达miR-224-5pCHSY1的HCT116R细胞再经4 Gy剂量照射后,细胞SF、细胞凋亡率、凋亡相关蛋白Bcl-2、Bax变化的更加明显。这提示抑制表达miR-224-5pCHSY1可促进辐射诱导的HCT116R细胞凋亡,从而增加细胞放射敏感性。本研究结果显示,在过表达LncRNA MIR503HG的基础上过表达miR-224-5pCHSY1,HCT116R细胞SF明显增加,细胞凋亡率明显降低,凋亡蛋白Bcl-2表达水平明显上调,Bax表达水平明显下调。这提示过表达miR-224-5pCHSY1可逆转过表达LncRNA MIR503HG对辐射诱导的HCT116R细胞凋亡的促进作用,从而再次降低HCT116R细胞的放射敏感性。
综上所述,上调LncRNA MIR503HG或下调miR-224-5pCHSY1均可增强HCT116R细胞的放射敏感性;过表达LncRNA MIR503HG可通过抑制miR-224-5pCHSY1表达增加结肠癌HCT116R细胞的放射敏感性。本研究不足:本研究仅对HCT116R细胞的凋亡进行分析,后续会进一步研究LncRNA MIR503HG异常表达对肿瘤细胞周期分布及侵袭、迁移等其他生物学行为的影响,并对其下游通路进行探索分析,以期更加全面充分地证明LncRNA MIR503HG对结肠癌细胞放射敏感性的影响机制。
利益冲突 所有作者声明无利益冲突
作者贡献声明 张健负责研究命题设计、实验实施、论文撰写、修订;李文军负责方法建立、数据分析、论文审阅
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
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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miR-224-5pCHSY1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例结肠癌患者为研究对象,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MIR503HG组、mimic-NC组、anti-miR-224-5p组、anti-miR-NC组、MIR503HG+miR-224-5p组、MIR503HG+miR-NC组;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率和凋亡率。两组间数据的比较使用t检验,多组间比较使用单因素方差分析,组内多个时间点比较使用重复测量方差分析。 结果 与放射敏感组相比,放射抵抗组结肠癌组织中LncRNA MIR503HG的表达水平明显降低(1.40±0.36对0.72±0.17),miR-224-5pCHSY1的表达水平明显增高(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细胞株LncRNA MIR503HG的相对表达量明显降低,miR-224-5pCHSY1的表达量明显增加,差异均有统计学意义(t=5.720、6.454,均P<0.05)。在照射剂量分别为4、6、8 Gy时,MIR503HG组的HCT116R细胞存活分数(SF)明显低于mimic-NC组[(0.59±0.10)对(0.79±0.05)、(0.40±0.08)对(0.60±0.10)、(0.18±0.06)对(0.38±0.04)],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增加,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),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1.566。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)。mimic-NC组、MIR503HG组、MIR503HG+miR-NC组、MIR503HG+miR-224-5p组的SF,照射剂量为4 Gy时,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0.824,相比于MIR503HG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 radiosensitivity of colon cancer cells by regulating the expression of miR-224-5pCHSY1. Methods 48 patients with colon cancer treated in our hospital from March 2019 to January 2022 were selected as the study subjects. According to the lesions after radiotherapy, they were divided into the radiation resistance group (n=23) and the radiosensitive group (n=25). The expressions of LncRNA MIR503HG and miR-224-5pCHSY1 in colon cancer tissues and cell lines (CCD841, COLO320, SW480, RKO, HCT116) were detected by qRT-PCR. The radiation-resistant colon cancer cell line HCT116R was constructed and divided into MIR503HG group, mimic-NC group, anti-miR-224-5p group, anti-miR-NC group, MIR503HG+miR-224-5p group and MIR503HG+miR-NC group. The targeting effect of LncRNA MIR503HG and miR-224-5pCHSY1 was verified by double luciferase report, and the cell survival rate and apoptosis rate were detected. The comparison of data between two groups was conducted using t-test, the comparison between multiple groups was conducted using one-way analysis of variance, and the comparison between multiple time points within the group was conducted using repeated measurement analysis of variance. Results Compared with radiosensitive group, the expression level of LncRNA MIR503HG in radiation resistance group was significantly lower (1.40±0.36 vs 0.72±0.17), and the expression level of miR-224-5pCHSY1 was significantly higher (1.06±0.25 vs 1.54±0.27) in radioresistant group. The relative expression of LncRNA MIR503HG in CCD841, COLO320, SW480, RKO, HCT116 and HCT116R cell lines was 2.38±0.06, 1.03±0.05, 0.87±0.03, 0.86±0.02, 0.77±0.04, 0.54 ±0.09 respectively. The expression of miR-224-5pCHSY1 was 0.38±0.06, 0.56±0.01, 0.59±0.02, 0.59±0.05, 0.63±0.04, 0.82±0.06 respectively, Compared with the normal cell line CCD841, the relative expression of LncRNA MIR503HG in colon cancer cell lines COLO320, SW480, RKO and HCT116 decreased significantly (t=2.061, 1.665, 4.058, 6.201, all P<0.01), while the expression of miR-224-5pCHSY1 increased significantly (t=1.238, 1.930, 2.037, 1.742, all P< 0.05). Compared with HCT116 cell line, the relative expression of LncRNA MIR503HG in HCT116R was significantly decreased, while the expression of miR-224-5pCHSY1 was significantly increased (t=5.720, 6.454, both P<0.05). When the irradiation doses were 4 Gy, 6 Gy and 8 Gy, the survival fraction (SF) of HCT116R cells in the MIR503HG group was significantly lower than that in the mimic-NC group (0.59±0.10 vs. 0.79±0.05, 0.40±0.08 vs. 0.60±0.10, 0.18±0.06 vs. 0.38±0.04), and the difference was statistically significant (t=1.064,1.937, 2.650, all P<0.05). After overexpression of LncRNA MIR503HG, the HCT116R SER of MIR503HG group was 1.399. The apoptosis rates of HCT116R cells in mimic-NC group, MIR503HG group, mimic-NC+4 Gy group and MIR503HG+4 Gy group were (8.10±0.23)%, (18.44±1.57)%, (17.33±2.35)% and (29.83±1.89)%, respectively. Compared with mimic-NC group, the apoptosis rate of HCT116R cells in MIR503HG group and mimic-NC+4 Gy group was significantly higher than that in MIR503HG group and mimic-NC+4 Gy group(t=2.003, 1.475, both P<0.05). Compared with anti-miR-NC group, the luciferase activity of MIR503HG-Wt in anti-miR-224-5p group increased significantly (1.02±0.20 vs. 1.60±0.25), and the difference was statistically significant. After the mutation of miR-224-5pCHSY1 and LncRNA MIR503HG binding site, there was no significant difference in MIR503HG-Mut activity between anti-miR-224-5p group and anti-miR-NC group (0.97±0.25 vs. 1.00±0.22). Compared with mimic-NC group, the expression level of miR-224-5pCHSY1 in MIR503HG group was significantly lower than that in MIR503HG group (1.97±0.13 vs. 1.12±0.12). The expression level of miR-224-5pCHSY1 in anti-miR-224-5p group was significantly lower than that in anti-miR-NC group (1.99±0.19 vs. 0.92±0.18). When the irradiation doses were 4 Gy, 6 Gy and 8 Gy, the SF of HCT116R cells in anti-miR-224-5p group was significantly lower than that in anti-miR-NC group (0.59±0.08 vs. 0.79±0.12, 0.39±0.06 vs. 0.67±0.07, 0.19±0.04 vs. 0.52±0.04). The difference was 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 anti-miR-224-5p group was 1.566. The apoptosis rates of anti-miR-NC group, anti-miR-224-5p group, anti-miR-NC+4 Gy group and anti-miR-224-5p+4 Gy group were 5.08+0.78, 14.48+1.21, 13.89+1.36 and 23.64+1.03, respectively. Compared with anti-miR-NC group, the apoptosis rates of anti-miR-224-5p group and anti-miR-NC+4 Gy group were significantly higher than those of anti-miR-NC+4 Gy group (t=2.067, 1.934, both P<0.05). Compared with anti-miR-NC+4 Gy group, the apoptosis rate of anti-miR-224-5p+4 Gy group was significantly higher than that of anti-miR-224-5p+4 Gy group (t=4.026, P<0.05). The SF of mimic-NC group, MIR503HG group, MIR503HG+miR-NC group and MIR503HG+miR-224-5p group were 0.82+0.17, 0.53+0.12, 0.54+0.11, 0.78+0.15, when the irradiation dose was 4 Gy, 0.66+0.13, 0.38+0.09, 0.35+0.08, 0.57+0.10 when the radiation dose was 6 Gy, and 0.49+0.10, 0.15+0.06, 0.13+0.05, 0.43+0.11 when the radiation dose was 8 Gy, respectively. Compared with mimic-NC group, the SF of HCT116R cells in MIR503HG group decreased significantly when the irradiation dose was ≥4 Gy (t=1.609, 1.533, 1.927, all P<1.533), and the SF of HCT116R cells in MIR503HG+miR-NC group decreased significantly (t=1.294, 1.490, 1.825, all P<0.05). Compared with MIR503HG+miR-NC group, the SF of HCT116R cells in MIR503HG+miR-224-5p group increased significantly when the irradiation dose was ≥ 4 Gy, and the difference was 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 MIR503HG+miR-224-5p group was 0.824, which was significantly lower than that of MIR503HG. The apoptosis rates of mimic-NC group, MIR503HG group, MIR503HG+miR-NC group and MIR503HG+miR-224-5p group were 11.61±2.10, 24.97±0.91, 24.81±1.27 and 16.15 ±1.10, respectively. Compared with mimic-NC group, the apoptosis rate of HCT116R cells in MIR503HG group and MIR503HG+miR-NC group was significantly higher than that in MIR503HG group and MIR503HG+miR-NC group (t=2.304, 2.159, both P<0.05). Compared with MIR503HG+miR-NC group, the apoptosis rate of HCT116R cells in MIR503HG+miR-224-5p group was significantly lower than that in HCT116R group (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
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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:−为无此项数据;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为准阈计量;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 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 single-click 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:−为无此项数据;mimic-NC为过表达对照组;MIR503HG为过表达LncRNA MIR503HG组;D0为平均致死剂量;Dq为准阈计量;N为外推值;SF2为平均2 Gy照射后的细胞存活分数;SER为放射增敏比 表 3 抑制表达miR-224-5pCHSY1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:−为无此项数据;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为准阈计量;N为外推值;SF2为平均2 Gy照射后的细胞存活分数;SER为放射增敏比 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 click 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:−为无此项数据;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且过表达miR-224-5pCHSY1的阴性对照组;MIR503HG+miR-224-5p为过表达LncRNA MIR503HG且过表达miR-224-5pCHSY1组;D0为平均致死剂量;Dq为准阈计量;N为外推值;SF2为平均2 Gy照射后的细胞存活分数;SER为放射增敏比 -
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