【作者】 白冰；

【导师】 孙世龙；

【作者基本信息】 吉林大学 ， 放射医学， 2022， 硕士

【摘要】 肝细胞肝癌（Hepatocellular carcinoma,HCC）作为原发性肝癌中最常见的类型,也是我国最常见的恶性肿瘤之一。放疗作为HCC的主要治疗手段之一,在临床上已得到广泛的应用。但在实际治疗中,常因肿瘤体积较大以及乏氧组织的存在而导致辐射敏感性受到影响。微小RNA（micro RNA,miRNA）通过与靶基因3’UTR结合而调节靶基因的表达,进而参与多种生物过程,包括细胞增殖、凋亡、转移、侵袭,放化疗敏感性等。研究报道称miR-138-5p在肝癌,宫颈癌,骨肉瘤,非小细胞肺癌等不同的恶性肿瘤中呈现低表达状态,并通过调控不同靶基因表达而发挥抑癌作用。然而,miR-138-5p对HCC的辐射敏感性的影响研究尚未见报道。Zeste同源基因增强子2（Enhancer of zeste homolog 2,EZH2）作为一种组蛋白甲基酶,通过催化H3K27me3抑制基因表达。生物信息学分析显示EZH2为miR-138-5p靶基因之一。作为两种不同的表观遗传学调控途径,miR-138-5p及EZH2对于肝癌细胞辐射敏感性的影响仍值得进一步探讨。因此,本研究拟探讨miR-138-5p/EZH2轴在肝癌细胞辐射敏感性中的调控作用,将对提高HCC的放疗疗效提供实验依据和思路。目的:观察miR-138-5p及EZH2对肝癌细胞辐射后细胞克隆形成能力,增殖能力,凋亡,细胞周期阻滞以及细胞迁移侵袭能力的影响;验证miR-138-5p与EZH2之间的靶向调控关系;探讨miR-138-5p/EZH2调控轴对肝癌细胞辐射敏感性影响及作用机制。方法:1.利用生物信息学方法分析miR-138-5p和EZH2在不同分期肝癌组织中表达情况,以及二者对于肝癌患者生存期影响。2.通过转染miR-138-5p mimic构建miR-138-5p过表达细胞模型,利用慢病毒质粒转染方法构建Hep G2-EZH2^LOW细胞模型,利用RT-q PCR以及Western blot法验证模型构建是否成功。利用克隆形成实验检测细胞辐射敏感性,利用MTT实验检测细胞增殖能力,利用流式细胞术检测细胞周期阻滞及凋亡情况,利用细胞划痕实验及Transwell小室检测细胞迁移能力和侵袭能力,Western blot检测相关蛋白表达变化。3.通过双荧光素酶报告实验与染色质免疫共沉淀分析实验证明miR-138-5p及EZH2二者之间的双向调控关系。结果:1.miR-138-5p与肝癌患者疾病分期及生存期关系通过在线分析网站进行生物信息学分析结果显示,miR-138-5p的前体miR-138-1在肝癌组织中处于低表达状态,并且在不同分期的肝癌患者体内的表达水平均较低。miR-138-5p基础表达水平较高的患者生存期明显优于基础表达较低者（P<0.01）。随后细胞水平验证结果显示肝癌细胞中miR-138-5p的表达水平明显低于正常肝细胞L-02（P<0.01）。2.miR-138-5p对Hep G2和Hep3B细胞辐射后反应的影响（1）miR-138-5p对Hep G2和Hep3B细胞辐射敏感性的影响细胞克隆形成实验结果显示,两种肝癌细胞的存活分数（surviving fraction,SF）均随着照射剂量的增加而降低。与对照组相比,同种剂量下,过表达miR-138-5p组细胞的SF值明显降低（P<0.05）。与对照组相比,miR-138-5p过表达组的平均致死剂量D₀值与SF₂值均降低,说明增加miR-138-5p表达能够增强Hep G2和Hep3B细胞辐射敏感性。（2）miR-138-5p对Hep G2和Hep3B细胞辐射后增殖的影响MTT结果显示:在Hep G2和Hep3B两种肝癌细胞中,与对照组相比,未经照射或经8Gy照射48h及72h后,miR-138-5p过表达组细胞增殖明显被抑制（P<0.05）。提示miR-138-5p能够引起辐射后两种肝癌细胞的增殖能力的降低。（3）miR-138-5p对Hep G2和Hep3B细胞辐射后周期进程的影响流式细胞术对细胞周期进程检测结果显示,与对照组细胞相比,miR-138-5p过表达组细胞经0Gy处理后24h,G₀/G₁期百分比明显增加,S期百分比降低（P<0.01）;在两种肝癌细胞经过8Gy辐射之后24h,与对照组相比,miR-138-5p过表达组细胞G₀/G₁期百分比明显增加（P<0.001）,提示发生G₀/G₁期阻滞。（4）miR-138-5p对Hep G2和Hep3B细胞辐射后凋亡的影响利用流式细胞术检测两种肝癌细胞经辐射及miR-138-5p过表达处理后细胞凋亡情况。结果显示,在两种肝癌细胞中,与对照组细胞相比,miR-138-5p过表达组细胞在经过0Gy或8Gy处理后24h凋亡率明显增加（P<0.05）。提示miR-138-5p能够促进辐射后肝癌细胞的凋亡。（5）miR-138-5p对Hep G2和Hep3B细胞辐射后迁移,侵袭能力影响利用细胞划痕实验检测两种肝癌细胞辐射前后迁移能力的变化。结果显示,0Gy处理后24h及48h,与对照组相比,miR-138-5p过表达组细胞的迁移能力明显被抑制（P<0.05）;8Gy处理后的24h及48h,与对照组相比,过表达miR-138-5p组细胞迁移能力也同样明显被抑制（P<0.05）。提示过表达miR-138-5p后Hep G2及Hep3B细胞辐射前后迁移能力减弱。Transwell小室结果显示,经过0Gy或8Gy处理后,与对照组相比,过表达miR-138-5p组侵袭细胞数显著减少（P<0.05）,提示miR-138-5p过表达可抑制辐射前后肝癌细胞的侵袭能力。（6）miR-138-5p对Hep G2和Hep3B细胞辐射后EMT标志物的影响利用Western Blot法检测上皮间质转化（epithelial to mesenchymal transition,EMT）相关标志物E钙粘蛋白（E-cadherin）、N钙粘蛋白（N-cadherin）、波形蛋白（Vimentin）以及EMT标志性的转录因子之一SNAIL（snail family transcriptional repressor 1）的蛋白表达。结果显示,在两种肝癌细胞中,经过0Gy或8Gy处理后,与对照组相比,miR-138-5p过表达组细胞中N-cadherin、Vimentin、SNAIL的表达降低。在Hep3B细胞经过0Gy或8Gy处理后,miR-138-5p过表达组E-cadherin蛋白水平升高,在Hep G2细胞中,0Gy处理后miR-138-5p过表达组E-cadherin蛋白水平同样升高,但经8Gy处理后则无明显变化。结果提示过表达miR-138-5p可以抑制辐射前后两种肝癌细胞的EMT进程。3.miR-138-5p对EZH2靶向调控作用的探究利用在线网站对miR-138-5p靶基因进行预测,筛选出miR-138-5p可能与EZH2具有靶向关系。双荧光素酶报告实验结果显示,与对照组相比,miR-138-5p mimic与psi CHECK2-EZH2-3′UTR共转染后可以显著抑制荧光素酶活性（P<0.05）。与此同时,miR-138-5p mimic或相应的阴性对照与突变型质粒psi CHECK2-EZH2-3′UTR mut共转染后荧光素酶的活性没有发生明显变化。进一步在肝癌细胞中验证结果显示,过表达miR-138-5p后两种肝癌细胞中EZH2的m RNA水平明显降低（P<0.01）,蛋白水平同样被抑制。提示miR-138-5p与EZH2具有靶向调控关系。4.EZH2与肝癌患者疾病分期及生存期关系通过在线分析网站利用生物信息学分析结果显示,EZH2在肝癌组织中处于高表达水平（P<0.001）,并且随着肝癌患者分期的增长,EZH2表达水平亦逐渐升高,并且EZH2基础表达水平较高的患者生存期明显比基础表达较低者差（P<0.001）。随后在细胞水平验证结果显示五种肝癌细胞中EZH2的m RNA水平明显高于L-02（P<0.01）,蛋白表达水平同样高于L-02。提示EZH2在肝癌组织中明显高表达,与miR-138-5p表达呈相反趋势。5.沉默EZH2对Hep G2细胞辐射后反应的影响（1）沉默EZH2对Hep G2细胞的辐射敏感性的影响细胞克隆形成实验结果显示,沉默EZH2后的Hep G2细胞的SF随着照射剂量的增加而逐渐降低。与转染载体质粒组相比,同种剂量下,沉默EZH2组细胞的SF值明显降低（P<0.05）。与转染载体质粒组相比,沉默EZH2组的平均致死剂量D₀值与SF₂值均降低,说明沉默EZH2表达能够增强Hep G2细胞的辐射敏感性,与增加miR-138-5p表达达到的效果趋势相同。（2）沉默EZH2对Hep G2细胞的辐射后增殖的影响根据MTT实验的结果显示:在Hep G2细胞中,与转染载体质粒组相比,经0Gy处理48h后,沉默EZH2组细胞增殖明显被抑制（P<0.05）。经8Gy照射后72h,沉默EZH2组增殖抑制效果显著（P<0.05）。提示沉默EZH2能够引起辐射前后Hep G2的增殖能力的降低。（3）沉默EZH2对Hep G2细胞的辐射后周期进程及凋亡的影响对细胞周期进程检测结果显示,与转染载体质粒组相比,沉默EZH2组细胞经0Gy处理后24h,G₀/G₁期百分比降低,G₂/M期百分比增加（P<0.05）;在经过8Gy辐射之后24h,与转染载体质粒组相比,沉默EZH2组细胞G₀/G₁期百分比明显增加（P<0.05）,提示发生G₀/G₁期阻滞。检测凋亡结果显示,与0Gy组相比,经过8Gy辐射后,各组细胞凋亡率均增加。与转染载体质粒组相比,沉默EZH2组细胞在经过0Gy或8Gy处理后24h凋亡率明显增加（P<0.05）。提示沉默EZH2组能够促进辐射引起的肝癌细胞的凋亡,这与过表达miR-138-5p达到的作用趋势一致。（4）沉默EZH2对Hep G2细胞辐射后迁移,侵袭能力及EMT标志物的影响细胞划痕实验结果显示,0Gy或8Gy处理后24h及48h,与转染载体质粒组相比,沉默EZH2组细胞的迁移能力明显被抑制（P<0.05）。Transwell实验结果显示,经过0Gy或8Gy处理后,与转染载体质粒组相比,沉默EZH2组细胞侵袭细胞数显著减少（P<0.001）,提示沉默EZH2与过表达miR-138-5p后对肝癌细胞辐射前后迁移能力的影响作用一致。随后对EMT相关标志物蛋白表达水平检测结果显示,在经过0Gy或8Gy处理后,与转染载体质粒组相比,沉默EZH2组细胞中N-cadherin、Vimentin、SNAIL的表达降低。以上结果提示在肝癌细胞中,miR-138-5p可通过靶向EZH2增强肝癌细胞辐射敏感性及抑制迁移侵袭等能力。6.EZH2通过H3K27me3对miR-138-5p表达的影响我们的结果显示,在Hep G2细胞中沉默EZH2表达后,miR-138-5p的表达水平明显升高（P<0.01）,而在Hep3B细胞中过表达EZH2之后,miR-138-5p的表达水平明显被抑制（P<0.01）。染色质免疫共沉淀分析（Chromatin Immunoprecipitation,Ch IP）结果显示,与转染载体质粒组相比,Hep G2沉默EZH2后细胞中miR-138-5p启动子部分区域的H3K27me3富集水平明显降低。提示在肝癌细胞中EZH2可通过介导miR-138-5p启动子区域H3K27me3修饰调控miR-138-5p的表达,提示miR-138-5p与EZH2之间存在相互抑制的反馈调控机制。结论:1.miR-138-5p可增强肝癌细胞辐射敏感性,抑制辐射后细胞增殖,促进凋亡,通过影响EMT进程而影响肝癌细胞辐射后迁移侵袭能力。2.抑制EZH2可使肝癌细胞辐射敏感性升高,肝癌细胞辐射后凋亡增多,增殖能力降低,同时,使辐射后肝癌细胞迁移侵袭能力受到抑制。3.miR-138-5p与EZH2之间通过相互抑制的调控机制形成反馈轴,共同调控肝癌细胞辐射敏感性及迁移侵袭等能力。更多还原

【Abstract】 Hepatocellular carcinoma（HCC）is the most common type of primary liver cancer and one of the most common malignant tumors in our country.Radiotherapy,as one of the main treatment methods for HCC,has been widely used in clinical application.However,in actual treatment,the radiation sensitivity is often affected by the large tumor size and the presence of hypoxic tissue.Micro RNA（miRNA,miRNA）regulates the expression of target genes by binding to the 3’UTR of target genes,and then participates in a variety of biological processes,including cell proliferation,apoptosis,metastasis,invasion,radiotherapy or chemotherapy resistance,etc.Studies have reported that miR-138-5p is underexpressed in different malignant tumors such as hepatoma,cervical cancer,osteosarcoma,and non-small cell lung cancer,and plays a tumor suppressor role by regulating the expression of different target genes.However,the effect of miR-138-5p on the radiation sensitivity of HCC has not been reported yet.Enhancer of zeste homolog 2（EZH2）,as a histone methylase,inhibits target gene expression by catalyzing H3K27me3.Bioinformatics analysis showed that EZH2 was one of the target genes of miR-138-5p.As two different epigenetic regulation pathways,the effects of miR-138-5p and EZH2 on the radiosensitivity of hepatocellular carcinoma cells are still worthy of further investigation.Therefore,this study intends to explore the regulatory role of miR-138-5p/EZH2 axis in the radiosensitivity of hepatoma cells,which will provide experimental basis and ideas for improving the radiotherapy efficacy of HCC.Objective:To observe the effects of miR-138-5p and EZH2 on the ability of colony formation,proliferation,apoptosis,cell cycle arrest,migration and invasion of hepatoma cells after radiation;to verify the targeting regulation relationship between miR-138-5p and EZH2;to explore the effect and mechanism of miR-138-5p/EZH2regulatory axis on the radiosensitivity of hepatocellular carcinoma cells.Methods:1.Using bioinformatics methods to analyze the expression of miR-138-5p and EZH2 in different stages of liver cancer tissues,and their effects on the survival of HCC patients.2.The miR-138-5p overexpression cell model was constructed by transfecting miR-138-5p mimic,and the Hep G2-EZH2^LOW cell model was constructed by lentiviral plasmid transfection.RT-q PCR and Western blot were used to verify whether the model was successfully constructed.Radiosensitivity was detected by colony formation assay,cell proliferation ability was detected by MTT assay,cell cycle arrest and apoptosis were detected by flow cytometry,cell migration and invasion ability was detected by wound healing experiment and Transwell method.Western blot method was used to detect related protein expression changes.3.The bidirectional regulatory relationship between miR-138-5p and EZH2 was demonstrated by dual luciferase reporter assay and chromatin immunoprecipitation assay.Results:1.Relationship between miR-138-5p and disease stage and survival in HCC patientsThe results of bioinformatics analysis on the online analysis website showed that miR-138-1,the precursor of miR-138-5p,was lowly expressed in liver cancer tissues,and the expression levels were lower in hepatoma patients with different stages.Patients with higher basal expression levels of miR-138-5p had significantly better survival than those with lower basal expression levels（P<0.01）.Subsequent cell-level verification results showed that the level of miR-138-5p in HCC cells was significantly lower than that in normal hepatocytes L-02（P<0.01）.2.The effect of miR-138-5p on the post-radiation response of Hep G2 and Hep3B cells（1）The effect of miR-138-5p on the radiosensitivity of Hep G2 and Hep3B cellsThe results of cell clone formation experiments showed that the survival fraction（SF）of both hepatoma cells decreased with the increase of radiation dose.Compared with the control group,at the same dose,the SF value of the cells in the overexpression miR-138-5p group was significantly decreased（P<0.05）.Compared with the negative control group,the mean lethal dose D₀ value and SF₂ value of the miR-138-5p overexpression group were both lower,indicating that miR-138-5p could enhance the radiosensitivity of Hep G2 and Hep3B cells.（2）The effect of miR-138-5p on the proliferation of Hep G2 and Hep3B cells after radiationThe results of MTT showed that in Hep G2 and Hep3B cells,compared with the control group,the ability of cell proliferation in the miR-138-5p overexpression group was significantly inhibited with 0Gy or 8Gy radiation for 48h and 72h（P<0.05）.It is suggested that miR-138-5p can reduce the proliferation ability of the two types of hepatoma cells after radiation.（3）The effect of miR-138-5p on the cycle progression of Hep G2 and Hep3B cells after radiationThe detection results of cell cycle progression by flow cytometry showed that,compared with cells in the control group,the percentage of G₀/G₁ phase was significantly increased and the percentage of S phase was decreased in the miR-138-5p overexpression group 24h after 0Gy treatment（P<0.01）;24h after 8 Gy radiation of the two types of hepatoma cells,compared with the control group,the percentage of cells in the G₀/G₁ phase in the miR-138-5p overexpression group was significantly increased（P<0.001）,suggesting that G₀/G₁ phase arrest occurred.（4）The effect of miR-138-5p on apoptosis of Hep G2 and Hep3B cells after radiationFlow cytometry was used to detect the apoptosis of two kinds of hepatoma cells after radiation and miR-138-5p overexpression treatment.The apoptosis rate of cells in the miR-138-5p overexpression group increased significantly at 24h after 0Gy or8Gy treatment（P<0.05）.It is suggested that miR-138-5p can promote the apoptosis of hepatoma cells induced by radiation.（5）The effect of miR-138-5p on the migration and invasion ability of Hep G2 and Hep3B cells after radiationThe wound healing experiment was used to detect the changes in the migration ability of the two types of hepatoma cells after radiation.The results showed that the migration ability of the miR-138-5p overexpression group was significantly inhibited compared with the control group at 24h or 48h after 0Gy treatment（P<0.05）;24h and48h after 8Gy treatment,compared with the control group,the migration ability of the overexpression miR-138-5p group was also significantly inhibited（P<0.05）.It suggested that the migration ability of Hep G2 and Hep3B cells with 0Gy or 8Gy radiation was weakened after overexpression of miR-138-5p.The changes of cell invasion ability were observed by Transwell method.The results showed that after0Gy or 8Gy treatment,compared with the control group,the number of invasive cells in the overexpression miR-138-5p group was significantly reduced（P<0.05）,suggesting that miR-138-5p overexpression can inhibit the invasive ability of hepatoma cells after radiation.（6）The effect of miR-138-5p on EMT markers after radiation of Hep G2 and Hep3B cellsEpithelial to mesenchymal transition（EMT）related markers E-cadherin,N-cadherin,Vimentin and SNAIL（snail family transcriptional repressor 1）protein expression were detected by Western Blot.The results showed that in hepatoma cells,after 0 Gy or 8 Gy treatment,the expressions of N-cadherin,Vimentin,and SNAIL in the miR-138-5p overexpression group were decreased compared with the control group.In Hep3B cells treated with 0Gy or 8Gy,the protein level of E-cadherin in the miR-138-5p overexpression group increased,and in Hep G2 cells,the protein level of E-cadherin in the miR-138-5p overexpression group also increased after 0Gy treatment,but no obvious change after 8Gy treatment.The results suggest that overexpression of miR-138-5p can inhibit the EMT process of the two hepatoma cells after radiation.3.Exploration of miR-138-5p on EZH2 targeting regulationThe target gene of miR-138-5p was predicted by the online website,and it was screened that miR-138-5p may have a targeting relationship with EZH2,and a dual-luciferase reporter gene assay was carried out.The results showed that compared with the control group,co-transfection of miR-138-5p mimic with psi CHECK2-EZH2-3′UTR could significantly inhibit the luciferase activity（P<0.05）.At the same time,the luciferase activity did not change significantly after co-transfection of miR-138-5p mimic or the corresponding negative control with the mutant plasmid psi CHECK2-EZH2-3’UTR mut.Further verification in hepatoma cells showed that the m RNA level of EZH2 in the two hepatoma cells was significantly decreased（P<0.01）after overexpression of miR-138-5p,and the protein level was also inhibited.It is suggested that miR-138-5p has a targeted regulatory relationship with EZH2.4.Relationship between EZH2 and disease stage and survival in HCC patientsThe results of bioinformatics analysis on the online analysis website showed that EZH2 was highly expressed in hepatocellular carcinoma tissue（P<0.001）,and with the increase in the stage of liver cancer patients,the expression level of EZH2gradually increased,and the basal expression level of EZH2 was higher than that of HCC.The survival time of patients with high expression of EZH2 was significantly worse than that of patients with low basal expression（P<0.001）.Subsequent verification results at the cellular level showed that the m RNA level of EZH2 in five types of hepatoma cells was significantly higher than that of L-02（P<0.01）,and the protein expression level was also higher than that of L-02.It is suggested that EZH2 is significantly highly expressed in hepatoma tissues,which is opposite to that of miR-138-5p.5.The effect of silencing EZH2 on the post-radiation response of Hep G2 cells（1）The effect of silencing EZH2 on the radiosensitivity of Hep G2 cellsThe results of cell clone formation experiments showed that the SF of Hep G2cells after silencing EZH2 decreased gradually with the increase of radiation dose.Compared with the transfection vector plasmid group,under the same dose,the SF value of the silenced EZH2 group was significantly decreased（P<0.05）.Compared with the transfection vector plasmid group,the average lethal dose D₀ value and SF₂value of the silencing EZH2 group were both lower,indicating that silencing EZH2expression can enhance the radiation sensitivity of Hep G2 cells,and the trend of the effect achieved by increasing the expression of miR-138-5p is the same.（2）The effect of silencing EZH2 on the proliferation of Hep G2 cells after radiationAccording to the results of MTT experiment,in Hep G2 cells,compared with the transfection vector plasmid group,the cell proliferation in the silenced EZH2 group was inhibited after 48h treatment with 0Gy（P<0.05）.Seventy-two hours after 8Gy radiation,the EZH2 silenced cells had a significant inhibitory effect on proliferation（P<0.05）.It suggested that silencing EZH2 could reduce the proliferation ability of Hep G2 after radiation.（3）The effect of silencing EZH2 on the post-radiation cycle progression and apoptosis of Hep G2 cellsThe detection results of cell cycle progression showed that compared with the transfection vector plasmid group,the percentage of G₀/G₁ phase decreased and the percentage of G₂/M phase increased.Twenty-four hours after 0Gy treatment of cells in the silenced EZH2 group（P<0.05）;After 24h,compared with the transfection vector plasmid group,the percentage of cells in the G₀/G₁ phase in the silenced EZH2group was significantly increased（P<0.05）,suggesting that G₀/G₁ phase arrest occurred.The results of detecting apoptosis showed that compared with the 0Gy group,after 8Gy radiation,the apoptosis rate of each group increased.Compared with the transfection vector plasmid group,the apoptosis rate of the silenced EZH2 group was significantly increased at 24h after 0Gy or 8Gy treatment（P<0.05）.It is suggested that silencing EZH2 group can promote the apoptosis of hepatoma cells induced by radiation,which is consistent with the effect of overexpression of miR-138-5p.（4）The effect of silencing EZH2 on the migration,invasion ability and EMT markers of Hep G2 cells after radiationThe results of wound healing experiment showed that the migration ability of cells in the silenced EZH2 group was significantly inhibited（P<0.05）compared with the transfection vector plasmid group at 24h and 48h after 0Gy or 8Gy treatment.The results of Transwell experiment showed that after 0Gy or 8Gy treatment,compared with the transfection vector plasmid group,the number of invasive cells in the silenced EZH2 group was significantly reduced（P<0.001）,suggesting that silencing EZH2 and overexpression of miR-138-5p have a significant effect on hepatocellular carcinoma.The effect of cell migration ability with or without radiation was consistent.Subsequent detection of EMT-related marker protein expression levels showed that after 0Gy or 8Gy treatment,the expression of N-cadherin,Vimentin,and SNAIL in the silenced EZH2 group was reduced compared with the transfection vector plasmid group.The above results suggest that in hepatoma cells,miR-138-5p can enhance the radiosensitivity and inhibit migration and invasion of HCC cells by targeting EZH2.6.The effect of EZH2 on the expression of miR-138-5p via H3K27me3Our results showed that after silencing EZH2 expression in Hep G2 cells,the expression level of miR-138-5p was significantly increased（P<0.01）,while the expression level of miR-138-5p was significantly increased after EZH2 was overexpressed in Hep3B cells（P<0.01）.The results of chromatin immunoprecipitation（Ch IP）analysis showed that compared with the transfected vector plasmid group,the expression level of H3K27me3 in the partial region of the miR-138-5p promoter in cells after Hep G2 silencing of EZH2 was significantly reduced.It is suggested that EZH2 can regulate the expression of miR-138-5p by mediating H3K27me3modification in the promoter region of miR-138-5p in hepatoma cells,suggesting that there is a feedback regulation mechanism of mutual inhibition between miR-138-5p and EZH2.Conclusion:1.miR-138-5p can enhance the radiation sensitivity of hepatoma cells,inhibit the proliferation after radiation,promote apoptosis,and affect the migration and invasion ability of hepatoma cells after radiation by affecting the EMT process.2.Inhibition of EZH2 can increase the radiation sensitivity of hepatoma cells,increase the apoptosis of hepatoma cells after radiation,reduce the proliferation ability,and at the same time,inhibit the migration and invasion ability of hepatoma cells after radiation.3.A feedback axis is formed between miR-138-5p and EZH2 through a mutual inhibitory regulatory mechanism,which jointly regulates the radiosensitivity,migration and invasion of hepatoma cells.更多还原