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前列腺癌长期以来一直是美国男性最常见的肿瘤之一,目前是仅次于肺癌的第二大癌症相关死亡类的肿瘤[1]。在评估的世界范围内前列腺癌的死亡数与中国的前列腺癌死亡数分别为位列第五位和第十位[2],亚洲人群中前列腺癌的发病率低于西方人群。但是,近年来中国的发病率和死亡率迅速增长,发病率的急剧上升导致每年25 000人死亡,5年生存率约54%[3-4]。因此,探究新的肿瘤形成的分子机制对前列腺癌的诊断、治疗及预后是十分迫切的。微小RNA(microRNA,miR)是一种非编码基因,它是通过靶向信使RNA进行转录后的互补修饰进而影响基因的表达水平[5]。miR的表达在人类癌症中广泛失调,表明了其在癌症的发生、进展和治疗耐药中发挥着潜在的作用[6]。已经有研究表明miR-107能够促进结肠癌和胃癌细胞增殖并抑制细胞凋亡而发挥着原癌基因的作用[7-8],然而miR-107在宫颈癌、胰腺癌和乳腺癌的形成中却发挥着抑癌基因的角色[9-11]。本研究的目的是探讨miR-107在前列腺癌中的功能和明确其调控的靶基因,为前列腺癌的发生和潜在机制提供新的见解。
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本研究瞬时转染实验结果显示,通过荧光标记的siRNA(FAM-siRNA)检测后显示miR-107的转染效率高于90%(见图 1,绿色荧光细胞占比>90%)。划痕与侵袭实验检测miR-107组与miR-NC组对前列腺癌细胞(LNCaP和PC3)的伤口愈合和侵袭能力的影响显示,miR-107组的伤口愈合率和侵袭细胞数目小于miR-NC组(P < 0.01)(见图 2、3及表 1)。
分组 n 伤口愈合率/% 侵袭细胞数 LNCaP PC3 LNCaP PC3 miR-NC组 3 65.33±4.50 60.33±5.03 363.00±44.71 397.33±20.25 miR-107组 3 45.66±3.78 40.66±5.13 125.00±27.83 182.66±30.35 t — 5.79 4.74 7.83 10.19 P — < 0.01 < 0.01 < 0.01 < 0.01 表 1 miR-107组与miR-NC组对前列腺癌细胞的伤口愈合与侵袭能力比较(x±s)
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细胞克隆实验显示,miR-107组前列腺癌细胞的增殖能力小于miR-NC组(P < 0.01)(见表 2、图 4)。
分组 n 细胞克隆数 LNCaP PC3 miR-NC组 3 191.66±16.80 201.00±5.03 miR-107组 3 109.33±12.22 107.00±13.22 t — 6.86 7.93 P — < 0.01 < 0.01 表 2 miR-107组与miR-NC组对前列腺癌细胞的增殖能力变化(x±s)
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利用生物信息学分析软件TargetScan显示miR-107与TPD52的结合位点(见图 5),在TPD52野生型(WT)组中miR-107组的荧光素酶活性明显低于miR-NC组(P < 0.01),而在TPD52突变型(MUT)组中2组之间的差异无统计学意义(P>0.05);蛋白免疫印迹实验和免疫荧光实验检测结果表明,miR-107组的TPD52表达明显小于miR-NC组(P < 0.01)(见表 3及图 6、7)。
分组 n 荧光素酶活性 TPD52蛋白相对表达量 平均光密度比(TPD52/Hoechst) WT MUT LNCaP PC3 LNCaP PC3 miR-NC组 5 0.896±0.085 0.884±0.042 0.482±0.039 0.592±0.031 0.882±0.059 1.543±0.079 miR-107组 5 0.274±0.050 0.866±0.032 0.302±0.034 0.280±0.031 0.393±0.028 0.462±0.051 t — 14.00 0.75 7.62 15.52 16.50 25.41 P — < 0.01 > 0.05 < 0.01 < 0.01 < 0.01 < 0.01 表 3 miR-107组与miR-NC组的荧光素酶活性及TPD52表达量变化(x±s)
miR-107靶向肿瘤蛋白D52抑制前列腺癌细胞的增殖和侵袭
miR-107 suppresses cell proliferation and invasion by targeting tumor protein D52 in prostate cancer
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摘要:
目的研究microRNA-107(miR-107)对前列腺癌细胞生物学功能的影响和潜在的作用机制。 方法采用瞬时转染、细胞克隆、细胞划痕、侵袭实验检测miR-107组与对照组(miR-NC组)对前列腺癌细胞生物学功能的影响。此外,通过靶基因预测软件TargetscanHuman7.2筛选出肿瘤蛋白D52(TPD52)作为miR-107的潜在下游靶基因并采用双荧光素酶报告基因实验进行了特异性结合的验证。同时,进一步通过蛋白免疫印迹和免疫荧光实验进行靶基因的表达检测。 结果miR-107组的细胞划痕愈合率、侵袭率、增殖率均低于miR-NC组(P < 0.01)。miR-107组对靶基因TPD52表达量小于miR-NC组(P < 0.01)。 结论miR-107通过靶向TPD52抑制前列腺癌的增殖和侵袭。 Abstract:ObjectiveTo investigate the effects and potential mechanism of miR-107 in prostate cancer. MethodsTransient transfection, cell cloning, cell scratching and invasion experiments were used to detect the effects of miR-107 group and control group(miR-NC) on the biological functions of prostate cancer cells.In addition, we selected tumor protein D52(TPD52) as a potential downstream target gene of miR-107 by TargetscanHuman7.2, and used dual-luciferase reporter assay for verifing the specific binding.Meanwhile, Western blotting and immunofluorescence assays were performed to detected the expression of target gene. ResultsThe healing rate, invasion rate and proliferation rate of cell scratch in miR-107 group were lower than those in miR-NC group(P < 0.01).The expression of target gene TPD52 in miR-107 group was less than that in miR-NC group(P < 0.01). ConclusionsmiR-107 inhibits the proliferation and invasion of prostate cancer by targeting TPD52. -
Key words:
- prostate neoplasms /
- miR-107 /
- tumor protein D52 /
- proliferation /
- invasion
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表 1 miR-107组与miR-NC组对前列腺癌细胞的伤口愈合与侵袭能力比较(x±s)
分组 n 伤口愈合率/% 侵袭细胞数 LNCaP PC3 LNCaP PC3 miR-NC组 3 65.33±4.50 60.33±5.03 363.00±44.71 397.33±20.25 miR-107组 3 45.66±3.78 40.66±5.13 125.00±27.83 182.66±30.35 t — 5.79 4.74 7.83 10.19 P — < 0.01 < 0.01 < 0.01 < 0.01 表 2 miR-107组与miR-NC组对前列腺癌细胞的增殖能力变化(x±s)
分组 n 细胞克隆数 LNCaP PC3 miR-NC组 3 191.66±16.80 201.00±5.03 miR-107组 3 109.33±12.22 107.00±13.22 t — 6.86 7.93 P — < 0.01 < 0.01 表 3 miR-107组与miR-NC组的荧光素酶活性及TPD52表达量变化(x±s)
分组 n 荧光素酶活性 TPD52蛋白相对表达量 平均光密度比(TPD52/Hoechst) WT MUT LNCaP PC3 LNCaP PC3 miR-NC组 5 0.896±0.085 0.884±0.042 0.482±0.039 0.592±0.031 0.882±0.059 1.543±0.079 miR-107组 5 0.274±0.050 0.866±0.032 0.302±0.034 0.280±0.031 0.393±0.028 0.462±0.051 t — 14.00 0.75 7.62 15.52 16.50 25.41 P — < 0.01 > 0.05 < 0.01 < 0.01 < 0.01 < 0.01 -
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