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摘要
• 摘要:   目的  探讨miR-503-5p通过靶向调控E2F3对宫颈癌Hela细胞增殖、侵袭、迁移和上皮间质化的影响。  方法  将宫颈癌HeLa细胞分为对照组、mimic-NC组、miR-503-5p mimic组、E2F3组、mimic+E2F3组，并通过Lipofectamine 2000将质粒分别或者联合转染进入各组HeLa细胞，运用基因预测软件预测靶基因，荧光素实验验证靶向关系，RT-PCR检测miR-503-5p和E2F3的表达，MTT法检测细胞增殖，Western blot检测Ki67、增殖细胞核抗原（proliferating cell nuclear antigen，PCNA）、E-cadherin和N-cadherin的表达，Transwell检测细胞侵袭，划痕实验检测细胞迁移。将裸鼠分为对照组和miR-503-5p mimic组，在裸鼠右后肢腹侧皮下分别注射0.2 mL转染mimic-NC或miR-503-5p mimic的宫颈癌HeLa细胞悬液，第30天颈椎脱位法处死裸鼠，测量肿瘤质量，并用免疫组化方法检测肿瘤组织中Ki67和Vimentin表达情况。  结果  宫颈癌HeLa细胞中miR-503-5p的表达量明显下调，miR-503-5p与E2F3在3′UTR区存在结合位点， miR-503-5p直接靶向作用于E2F3，过表达miR-503-5p抑制E2F3表达； miR-503-5p过表达降低细胞生长速度、Ki67和PCNA表达量，减少侵袭细胞数目，增宽划痕、降低愈合率，上调E-cadherin表达、下调N-cadherin表达（P<0.01）；miR-503-5p过表达减小移植瘤体积、减轻移植瘤重量，减少Ki67和Vimentin的阳性所占比率（P<0.01）。  结论  miR-503-5p通过靶向调控E2F3抑制宫颈癌HeLa细胞增殖、侵袭、迁移和上皮间质化。

  关键词:
  • miR-503-5p / 
  • E2F3 / 
  • 宫颈癌HeLa细胞 
  Abstract:   Objective  To investigate the effect of miR-503-5p on the proliferation, invasion, migration and epithelialization of cervical cancer HeLa cells via targeting E2F3.  Methods  Four ccervical cancer HeLa cells groups were set up including control group, mimic-NC group, miR-503-5p mimic group, E2F3 group, miR-503-5p mimic+E2F3 group (mimic+E2F3 group). The plasmids were separately or jointly transinfected into cervical cancer Hela cells of each group by Lipofectamine 2000, After transinfection, the target gene was predicted by gene prediction software, the targeting relationship was verified by fluorescein experiment, the expression of miR-503-5p and E2F3 was detected by RT-PCR, cell proliferation was detected by MTT assay, expression of Ki67, proliferating cell nuclear antigen (PCNA), E-cadherin and N-cadherin were detected by Western blot, cell invasion was detected by Transwell, and cell migration was detected by scratch test. Nude mice were divided into control group and miR-503-5p mimic group, and 0.2 mL of cervical cancer HeLa cell suspension transfected with mimic-NC or miR-503-5p mimic was injected subcutaneously into the ventral side of the right hind limb of nude mice. Thirty days post injection, the nude mice were sacrificed by cervical dislocation. The tumor weight was weighed by an electronic balance, and the expression of KI67 and Vimentin in the tumor tissue was detected by immunohistochemistry.  Results  The expression level of miR-503-5p in cervical cancer HeLa cells was down-regulated, miR-503-5p directly targeted E2F3 by binding with E2F3 at binding sites in the 3'UTR region. Over-expressing of miR-503-5p inhibited the expression of E2F3, significantly decreased cell growth rate and the expression level of Ki67 and PCNA, decreased the number of invasive cells, widened the scratches, reduced the healing rate, up-regulated the expression of E-cadherin and also down-regulated the expression of N-cadherin (P<0.01). Over-expressing of miR-503-5p significantly reduced the volume and weight of transplanted tumors, and decreased the proportion of positive Ki67 and Vimentin (P<0.01).  Conclusion  miR-503-5p inhibits the proliferation, invasion, migration and epithelialization of cervical cancer HeLa cells by targeting E2F3.

  Key words:
  • miR-503-5p / 
  • E2F3 / 
  • Cervical cancer HeLa cells 
HTML全文
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  图  1  RT-PCR检测miR-503-5p表达水平（n=9）

  ** P<0.01 , vs normal cervical cells

  Figure  1.  miR-503-5p mRNA expression level was detected by RT-PCR(n=9)

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  图  2  miR-503-5p靶向作用于E2F3（n=9）

  A: Targeting relationship was detected by luciferase activity; B: The expression of miR-503-5p was detected by RT-PCR; C: The expression of E2F3 was detected by RT-PCR. **P < 0.01, vs. control group; ## P< 0.01, vs. miR-503-5p mimic group

  Figure  2.  miR-503-5p targets to E2F3 (n=9)

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  图  3  各组细胞增殖情况（n=9）

  A: Cell proliferation was detected by MTT; B: Ki67 and PCNA protein expression levels were detected by Western blot. **P<0.01, vs. control group; ## P<0.01, vs. miR-503-5p mimic group

  Figure  3.  Cell proliferation in each group (n=9)

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  图  4  Transwell检测细胞侵袭情况（n=9）

  ** P<0.01, vs. control group; ## P<0.01, vs. miR-503-5p mimic group

  Figure  4.  Cell invasion was detected by Transwell (n=9)

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  图  5  划痕实验检测细胞迁移能力（n=9）。 ×200

  ** P<0.01, vs. control group; ## P<0.01, vs. miR-503-5p mimic group

  Figure  5.  The migration ability of each group was tested by scratch test (n =9). ×200

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  图  6  Western blot检测E-cadherin和N-cadherin的表达（n=9）

  **P<0.01, vs. control group; ## P<0.01, vs. miR-503-5p mimic group

  Figure  6.  The expression levels of E-cadherin and N-cadherin were detected by Western blot (n=9)

  下载: 全尺寸图片 幻灯片
  

  图  7  免疫组化检测Ki67和Vimentin（n=9）。 ×400

  **P<0.01, vs. control group

  Figure  7.  Ki67 and Vimentin were detected by immunohistochemistry (n=9) . ×400

  下载: 全尺寸图片 幻灯片
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