【作者】 陈杰；

【导师】 陈钟；

【作者基本信息】 苏州大学 ， 外科学（普外科）（专业学位）， 2023， 博士

【摘要】 背景:肝细胞癌（Hepatocellular carcinoma,HCC）是肝脏恶性肿瘤中最常见的一种,是世界上死亡率第二高的癌症。由于其起病隐匿,进展迅速,HCC在诊断时往往就已经发展为肿瘤过大、血管侵犯和肝外转移。使用铂类药物进行全身化疗是其治疗方式之一。近日,有研究证实,基于奥沙利铂（Oxaliplatin,OXA）的化疗可以有效提高HCC患者的生存率。然而,由于HCC细胞本质上具有很强的增殖、转移和耐药能力,一些患者在Oxaliplatin治疗后仍出现肿瘤快速增长情况。因此,明确HCC进展和化疗耐药作用机制是当务之急。长链非编码RNAs（Long non-coding RNAs,LncRNAs）被定义为含有200多个核苷酸的转录物,它们参与多种生物功能的调节。LncRNAs与癌症治疗耐药性之间的关系,因其复杂的相互作用网络而日益受到重视。随着对lnc RNAs的研究深入,越来越多的研究表明lnc RNAs在HCC耐药过程中发挥了重大作用,因此探索lnc RNAs在HCC中调节肿瘤进展和化疗敏感性的机制极为重要。前列腺癌基因表达标记1（Prostate cancer gene expression marker 1,PCGEM1）被证实参与调节多种癌症的发生和进展。PCGEM1在前列腺癌中充当癌基因,与化疗耐药有关。PCGEM1与肝癌及肝癌耐药的关系鲜有研究。前期研究发现PCGEM1在肝癌组织与正常肝组织表达差异大,探讨其对肝癌及其耐药的影响具有重大意义。第一部分:PCGEM1在奥沙利铂耐药HCC组织中高表达目的:探讨HCC组织中PCGEM1的表达差异。方法:于2020年3月至2022年3月在南通市第一人民医院收集Oxaliplatin-resistant HCC患者（n=6）和Oxaliplatin-sensitive HCC（n=6）组织样本。利用实时荧光定量PCR（Quantitative Real-time PCR,QPCR）检测PCGEM1在各组样品中的表达差异。利用原位杂交技术（In situ hybridization,ISH）对PCGEM1在各组样本中的表达差异进行检测。结果:QPCR和ISH结果显示,和PCGEM1在Oxaliplatin-sensitive HCC组织中相比较,PCGEM1在Oxaliplatin-resistant HCC组织中的表达显著升高。结论:PCGME1在Oxaliplatin-resistant HCC组织的表达显著上调,可能在HCC对Oxaliplatin的耐药过程中起着重要的作用。第二部分:PCGEM1促进肝癌奥沙利铂耐药目的:从整体动物实验水平和离体细胞水平系统地考察PCGEM1对HCC Oxaliplatin耐药的影响。方法:构建Hep3B/Oxaliplatin（Hep3B/OXA）耐药细胞株,QPCR检测PCGEM1在LO-2、Hep3B和Hep3B/OXA这3株细胞中的表达情况。Oxaliplatin干预后CCK-8（Cell Counting Kit 8）实验检测各组细胞的活力及IC₅₀值。构建PCGEM1敲低的Hep3B/OXA稳转细胞株。通过QPCR检测耐药基因（低密度脂蛋白受体相关蛋白1（Recombinant Low Density Lipoprotein Receptor Related Protein 1,LRP1）、多药耐药1（Multidrug resistance,MDR1）和3（MDR3）和多药耐药相关蛋白2（Multidrug resistance protein 2,MRP2））的表达。通过CCK-8、EdU、克隆形成、划痕、Transwell和流式细胞仪等分析sh-PCGEM1对Hep3B/OXA细胞株细胞活力、增殖能力、克隆能力、迁移、侵袭能力、凋亡及对Oxaliplatin耐药性的影响。Hep3B/OXA细胞株中凋亡相关蛋白（Bax、Bcl-2和Cleaved caspase-3）和侵袭相关蛋白（MMP-2和MMP-9）的表达通过western blot（WB）实验评估。然后通过构建裸鼠移植瘤模型,从整体动物层面分析PCGEM1对HCC Oxaliplatin耐药的影响。结果:QPCR实验结果显示PCGEM1在Hep3B和Hep3B/OXA这2株细胞中表达显著升高,尤其是在Hep3B/OXA细胞株中。IC₅₀（Hep3B/OXA）显著高于IC₅₀（Hep3B）和IC₅₀（LO-2）,IC₅₀（LO-2）值最低。Sh-PCGEM1转染Hep3B/OXA后,QPCR结果表明PCGEM1的表达量显著降低,表明转染成功。Sh-PCGEM1显著抑制LRP1、MDR1、MDR3和MRP2的表达。Oxaliplatin干预sh-NC和sh-PCGEM1转染的Hep3B/OXA细胞株后,sh-PCGEM1能够显著降低Oxaliplatin对Hep3B/OXA细胞株的IC₅₀值。细胞功能实验结果证实sh-PCGEM1显著抑制Hep3B/OXA细胞株的活力、增殖能力、克隆形成能力、迁移和侵袭能力。流式细胞术结果表明sh-PCGEM1显著增强Hep3B/OXA细胞株的凋亡能力。WB实验结果显示,PCGEM1的下调明显增强了Bax和Cleaved caspase-3的表达,减弱了Bcl-2、MMP-2和MMP-9的表达。动物实验结果表明,sh-PCGEM1显著抑制Hep3B/OXA移植瘤的生长,sh-PCGEM1+Oxaliplatin进一步抑制Hep3B/OXA移植瘤的生长。QPCR结果表明,PCGEM1在sh-NC组中表达最高,在sh-NC+Oxaliplatin组中表达其次高,在sh-PCGEM1组中表达其次低,在sh-PCGEM1+Oxaliplatin组中表达最低。HE和TUNEL实验结果表明,和sh-NC组相比,sh-NC+Oxaliplatin组肿瘤组织凋亡显著,sh-PCGEM1组肿瘤组织凋亡更加显著,sh-PCGEM1+Oxaliplatin组肿瘤组织凋亡最显著。结论:从整体动物水平,敲低PCGEM1能够显著抑制Hep3B/OXA移植瘤的生长,并且能够增强HCC对Oxaliplatin的敏感性。从离体细胞水平,sh-PCGEM1显著抑制Hep3B/OXA细胞株的活力、增殖能力和转移能力,并且能够促进Hep3B/OXA细胞株凋亡,增强Hep3B/OXA细胞株对Oxaliplatin的敏感性。第三部分:PCGEM1负性调节miR-129-5p促进肝癌奥沙利铂耐药目的:从细胞水平探讨miR-129-5p介导PCGEM1对HCC耐药性的影响。方法:运用生物信息学预测PCGEM1的靶向miRNAs,双荧光素酶报告实验（Dual-luciferase reporter assay）验证PCGEM1的靶向miRNA（miR-129-5p）。QPCR实验检测miR-129-5p在各组细胞株及临床样本中的表达。构建miR-129-5p inhibitor NC和miR-129-5p inhibitor质粒,分别对Hep3B/OXA细胞株进行转染,QPCR检测转染效率。然后在PCGEM1下调的Hep3B/OXA细胞株中分别转染miR-129-5p inhibitor NC和miR-129-5p inhibitor,PCGEM1和miR-129-5p在各组的表达被QPCR检测。QPCR检测miR-129-5p inhibitor介导sh-PCGEM1对Hep3B/OXA细胞中耐药基因LRP1、MDR1、MDR3和MRP2表达的影响。细胞功能实验检测miR-129-5p inhibitor介导sh-PCGEM1对Hep3B/OXA细胞株细胞活力、增殖能力、克隆能力、转移能力、凋亡及对Oxaliplatin耐药性的影响。除此之外,miR-129-5p inhibitor介导sh-PCGEM1对Hep3B/OXA细胞株中凋亡相关蛋白（Bax、Bcl-2和Cleaved caspase-3）和侵袭相关蛋白（MMP-2和MMP-9）表达的影响被WB实验检测。结果:生物信息学预测miR-129-5p是PCGEM1的一个潜在靶向miRNA,双荧光素酶报告实验证实PCGEM1能够靶向miR-129-5p。QPCR结果显示,miR-129-5p在Oxaliplatin-resistant HCC组织及细胞株中的表达水平显著降低。除此之外,miR-129-5p在稳定转染sh-PCGEM1 Hep3B/OXA细胞株中的表达显著高于其在稳定转染sh-NC Hep3B/OXA细胞株中的表达。MiR-129-5p与PCGEM1呈现的负相关被证实。在转染了miR-129-5p inhibitor的Hep3B/OXA细胞株中miR-129-5p表达被抑制,PCGEM1的表达上调。QPCR检测耐药相关基因结果显示和sh-PCGEM1组相比较,miR-129-5p inhibitor能够显著促进LRP1、MDR1、MDR3和MRP2的表达。MiR-129-5p inhibitor能够显著提高Oxaliplatin对Hep3B/OXA细胞株的IC₅₀值。同时,miR-129-5p inhibitor显著提高Hep3B/OXA细胞株的活力、增殖能力、克隆形成能力、迁移和侵袭能力。流式细胞术结果表明,和sh-PCGEM1组相比较,miR-129-5p inhibitor显著抑制Hep3B/OXA细胞株的凋亡能力。在转染sh-PCGEM1的Hep3B/OXA细胞中,miR-129-5p inhibitor显著抑制Bax和Cleaved caspase-3的表达,促进Bcl-2、MMP-2和MMP-9的表达。结论:miR-129-5p在Oxaliplatin-resistant HCC组织和细胞中的表达显著下调。MiR-129-5p inhibitor能够部分逆转sh-PCGEM1对Hep3B/OXA细胞株的活力、增殖能力、转移能力、凋亡情况以及对Oxaliplatin的敏感性。第四部分:ETV1促进PCGEM1/miR-129-5p对肝癌奥沙利铂耐药目的:从细胞水平系统地阐明PCGEM1/miR-129-5p/ETS变异基因1（ETS variantgene 1,ETV1）对HCC Oxaliplatin耐药的影响。方法:Target Scan预测miR-129-5p与ETV1的靶向位点,miR-129-5p与ETV1的靶向关系经双荧光素酶报告实验确认。构建ETV1过表达细胞株,QPCR和WB实验检测转染效率。然后在Hep3B/OXA细胞株（转染sh-PCGEM1+miR-129-5p inhibitor）分别瞬转pc-NC和pc-ETV1,分为5个组别（sh-NC、sh-PCGEM1、sh-PCGEM1+miR-129-5p inhibitor、sh-PCGEM1+miR-129-5p inhibitor+pc-NC和sh-PCGEM1+miR-129-5p inhibitor+pc-ETV1）,QPCR检测各组中PCGEM1、miR-129-5p和ETV1的m RNA水平,WB实验检测各组中ETV1蛋白的表达。QPCR实验检测pc-ETV1介导sh-PCGEM1/miR-129-5p inhibitor对Hep3B/OXA细胞中耐药基因LRP1、MDR1、MDR3和MRP2表达的影响。一系列细胞功能实验检测pc-ETV1介导sh-PCGEM1/miR-129-5p inhibitor对Hep3B/OXA细胞株细胞活力、增殖能力、克隆能力、转移能力、凋亡及对Oxaliplatin耐药性的影响。除此之外,WB试验检测pc-ETV1介导sh-PCGEM1/miR-129-5p inhibitor对Hep3B/OXA细胞株中凋亡相关蛋白（Bax、Bcl-2和Cleaved caspase-3）和侵袭相关蛋白（MMP-2和MMP-9）表达的影响。结果:ETV1被证实是miR-129-5p的靶基因,他们之间存在多个结合位点。QPCR和WB结果显示,ETV1在Oxaliplatin-resistant HCC组织及细胞株中的表达水平显著升高。MiR-129-5p inhibitor逆转了PCGEM1敲低对ETV1表达的影响。表明ETV1与miR-129-5p负性相关,且与PCGEM1正向相关。Hep3B/OXA细胞株中转染pc-ETV1后,ETV1的表达显著升高,证明转染成功。QPCR检测耐药相关基因结果显示,和sh-PCGEM1+miR-129-5p inhibitor组相比较,pc-ETV1能够显著促进LRP1、MDR1、MDR3和MRP2的表达。Pc-ETV1能够显著提高Oxaliplatin对Hep3B/OXA细胞株的IC₅₀值。细胞功能实验结果表明,pc-ETV1显著提高Hep3B/OXA细胞株的活力、增殖能力、克隆形成能力、迁移和侵袭能力,显著抑制Hep3B/OXA细胞株的凋亡能力。和sh-PCGEM1+miR-129-5p inhibitor组相比较,pc-ETV1显著减弱了sh-PCGEM1+miR-129-5p inhibitor对Bax、Cleaved caspase-3、Bcl-2、MMP-2和MMP-9的表达的影响。结论:ETV1在Oxaliplatin-resistant HCC组织和细胞中的表达大幅上调。Pc-EVT1能够部分逆转sh-PCGEM1/miR-129-5p inhibitor对Hep3B/OXA细胞株的活力、增殖能力、转移能力、凋亡情况以及对Oxaliplatin的敏感性。更多还原

【Abstract】 Background:Hepatocellular carcinoma（HCC）is the most common malignant tumor of the liver.Because of its insidious onset and rapid progress,HCC has often developed into tumor oversize,vascular invasion and extrahepatic metastasis at the time of diagnosis.Recently,some studies have confirmed that oxaliplatin-based chemotherapy can effectively improve the survival rate of patients with unresectable HCC.However,due to the strong proliferation,metastasis and drug resistance of HCC cells,some patients still have rapid tumor growth after oxaliplatin treatment.Therefore,it is urgent to clarify the mechanisms of HCC progression and chemotherapy resistance.RNA containing transcripts of more than 200 nucleotides is defined as long non coding RNAs（LncRNAs）.They participate in the regulation of various biological functions.Due to the complex interaction network,lnc RNAs have a wide range of biological activities.With the further study of lnc RNAs,more and more studies show that lnc RNAs play an important role in the drug resistance process of HCC.Therefore,it is very important to explore and study the mechanism of lnc RNA regulating tumor progression and drug sensitivity in HCC.Prostate cancer gene expression marker 1（PCGEM1）has been shown to be involved in regulating the occurrence and progression of various cancers.PCGEM1 acts as an oncogene in prostate cancer and is associated with chemotherapy resistance.There is little research on the relationship between PCGEM1 and liver cancer and liver cancer drug resistance.Previous studies have found significant differences in the expression of PCGEM1 between liver cancer tissue and normal liver tissue,and exploring its impact on liver cancer and its drug resistance is of great significance.Part Ⅰ:High expression of PCGEM1 in oxaliplatin resistant tissues of HCCObjective:To explore whether the differential expression of PCGEM1 appears in HCC tissues.Methods:The tissue samples of Oxaliplatin-resistant HCC patients（n=6）and Oxaliplatin-sensitive HCC（n=6）were collected in The Nantong First People’s Hospital from March 2020 to March 2022.The expression of PCGEM1 in various tissue samples was detected by real-time fluorescent quantitative PCR analysis（QPCR）.Detection of PCGEM1 expression in various tissue samples was carried out through in situ hybridization（ISH）.Results:The results of QPCR and ISH showed that compared with PCGEM1 in Oxaliplatin-sensitive HCC,the expression of PCGEM1 in Oxaliplatin-resistant HCC was significantly increased.Conclusion:PCGME1 expression was significantly increased in Oxaliplatin-resistant HCC,indicating that it might play a key role in the process of oxaliplatin resistance in HCC.Part Ⅱ:PCGEM1 promotes oxaliplatin resistance in HCCObjective:To systematically investigate the effect of PCGEM1 on the Oxaliplatin resistance of HCC at the whole animal experiment level and in vitro cell level.Methods:The expression of PCGEM1 in LO-2,Hep3B and Hep3B/OXA cells was detected by QPCR analysis.After the intervention of oxaliplatin in these three cell lines,cell Counting Kit 8（CCK-8）was used to detect the cell viability and the IC50 value of each group.Hep3B/OXA cells with stable low expression of PCGEM1 were established.The effect of sh-PCGEM1 on the expression of drug resistance genes LRP1,MRP2,MDR1 and MDR3 in Hep3B/OXA cells was evaluated using QPCR.CCK-8,EdU,colony formation assay,wound healing,Transwell test and flow cytometry test were used to detect the effect of sh-PCGEM1 on the cell viability,proliferation,cloning ability,migration,apoptosis and oxaliplatin resistance of each group.In addition,western blotting（WB）experiment was carried out to detect the effect of sh-PCGEM1 on the expressions of apoptotic proteins（Bax,Bcl-2 and cleaved caspase-3）and transfer proteins（MMP-2 and MMP-9）in Hep3B/OXA cell lines.Then,the effect of PCGEM1 on the Oxaliplatin resistance of HCC was analyzed from the whole animal level by constructing nude mouse models of transplanted tumors.Results:The results of QPCR showed that the expression of PCGEM1 was significantly increased in Hep3B and Hep3B/OXA cell lines,especially in Hep3B/OXA cell lines.IC₅₀（Hep3B/OXA）was significantly higher than IC₅₀（Hep3B）and IC₅₀（LO-2）,and IC₅₀（LO-2）was the lowest.After transfected into Hep3B/OXA cells with sh-PCGEM1,the expression of PCGEM1 was significantly reduced detected with QPCR analysis,indicating that the transfection was successful.Sh-PCGEM1 significantly inhibited the expressions of LRP1,MDR1,MDR3 and MRP2.IC₅₀evaluation results showed that sh-PCGEM1 could significantly reduce the IC₅₀value of Oxaliplatin on Hep3B/OXA cell lines.Moreover,sh-PCGEM1 significantly inhibited the proliferation and activity of Hep3B/OXA cell lines,significantly enhanced the apoptosis ability of Hep3B/OXA cell lines,and significantly inhibited the migration and invasion ability of Hep3B/OXA cell lines.WB experiment results showed that the down-regulation of PCGEM1 significantly enhanced the expressions of Bax and caspase-3,and decreased the expressions of Bcl-2,MMP-2 and MMP-9 compared with sh-NC group.The results of animal experiments showed that,sh-PCGEM1 significantly inhibited the growth of Hep3B/OXA transplanted tumors.Sh-PCGEM1+Oxaliplatin further inhibited the growth of Hep3B/OXA transplanted tumors.The results of QPCR showed that the expression of PCGEM1 was the highest in sh-NC group,the second highest in sh-NC+Oxaliplatin group,the second-lowest in sh-NC+Oxaliplatin group,and the lowest in sh-PCGEM1+Oxaliplatin group.Compared with sh-NC group,the apoptosis of tumor tissue in sh-NC+Oxaliplatin group was increased and the degree of tumor deterioration was alleviated.In sh-PCGEM1 group,the apoptosis of tumor tissue was more serious,and the degree of tumor deterioration was further alleviated.Sh-PCGEM1+Oxaliplatin had the most significant apoptosis in tumor tissue,and the degree of tumor deterioration was significantly alleviated.Conclusion:From the overall animal level,sh-PCGEM1 could significantly inhibit the growth of Hep3B/OXA transplanted tumors,and enhanced the sensitivity of HCC to Oxaliplatin.At the cellular level,PCGEM1 knockout alleviated the survival,proliferation and metastasis of Hep3B/OXA cell lines,strengthened the apoptosis of Hep3B/OXA cell lines,and increased the sensitivity of Hep3B/OXA cell lines to Oxaliplatin.Part Ⅲ:PCGEM1 negatively regulates miR-129-5p to promote oxaliplatin resistance in HCCObjective:To explore the effects of miR-129-5p mediated PCGEM1 on the drug resistance of HCC at the cellular level.Methods:Bioinformatics was used to predict the target miRNAs of PCGEM1,and dual-luciferase reporter assay was used to verify the target miRNA（miR-129-5p）of PCGEM1.The expression of miR-129-5p in cell lines and clinical samples of each group was detected by QPCR.The miR-129-5p inhibitor NC and miR-129-5p inhibitor plasmids were constructed and transfected into Hep3B/OXA cell lines respectively.Then miR-129-5p inhibitor NC and miR-129-5p inhibitor were transiently transfected into Hep3B/OXA cell lines stably transfected with sh-PCGEM1.The expression of PCGEM1and miR-129-5p was detected by QPCR.The effects of miR-129-5p inhibitor mediated sh-PCGEM1 on the expressions of drug resistance genes LRP1,MDR1,MDR3 and MRP2in Hep3B/OXA cells was detected by QPCR.Cell function experiments were used to detect the effects of miR-129-5p inhibitor on the cell viability,proliferation,cloning ability,migration,apoptosis and oxaliplatin resistance of Hep3B/OXA cell lines transfected with sh-PCGEM1.In addition,WB experiment was conducted to examine the effects of miR-129-5p inhibitor on the levels of apoptotic proteins（Bax,Bcl-2 and Cleared caspase-3）and transfer proteins（MMP-2 and MMP-9）of Hep3B/OXA cell lines transfected with sh-PCGEM1.Results:There were multiple binding sites between miR-129-5p and PCGEM1,and the dual-luciferase report experiment confirmed that PCGEM1 could target miR-129-5p.The expression level of miR-129-5p in Oxaliplatin-resistant HCC tissues and cell lines was significantly reduced.In addition,miR-129-5p was significantly up-regulated in the stably sh-PCGEM1-transfected Hep3B/OXA cell lines,and miR-129-5p was negatively correlated with PCGEM1.After transient transfer of miR-129-5p inhibitor NC and miR-129-5-p inhibitor in Hep3B/OXA cell lines（transfected with sh-PCGEM1）,QPCR results showed that miR-129-5b inhibitor transfection could significantly reduce the expression of miR-129-5p and promote the expression of PCGEM1.Compared with sh-PCGEM1 group,miR-129-5p inhibitor significantly promoted the expression of LRP1,MDR1,MDR3 and MRP2.The miR-129-5p inhibitor significantly increased the IC₅₀value of Oxaliplatin on Hep3B/OXA cell lines compared with the sh-PCGEM1 group.In addition,miR-129-5p inhibitor significantly increased the activity,proliferation,migration and invasion of Hep3B/OXA cell lines,significantly inhibited the apoptosis of Hep3B/OXA cell lines.WB experiment results showed that miR-129-5p inhibitor inhibited the decrease of Bax and caspase-3 expression and the increase of Bcl-2,MMP-2 and MMP-9 expression in Hep3B/OXA cell lines transfected with sh-PCGEM1.Conclusion:miR-129-5p expression was significantly reduced in Oxaliplatin-resistant HCC tissues and cells.miR-129-5p inhibitor could partially reverse the effects of sh-PCGEM1 on the activity,proliferation,metastasis,apoptosis and Oxaliplatin sensitivity to Hep3B/OXA cell lines.Part Ⅳ:ETV1 promotes effects of PCGEM1/miR-129-5p on HCC resistanceObjective:To systematically elucidate the effects of PCGEM1/miR-129-5p/ETS variant 1（ETV1）on the Oxaliplatin resistance of HCC at the cellular level.Methods:The binding site of miR-129-5p and ETV1 was predicted to use Target Scan,and the binding of miR-129-5-p and target gene ETV1 was verified via the dual-luciferase reporter gene.ETV1 m RNA and protein expressions in cell lines and clinical samples of each group were detected by QPCR and WB tests.ETV1over-expression transient cell lines were constructed.The transfection efficiency was verified by QPCR and WB experiments.Then,pc-NC or pc-ETV1 was transfected into Hep3B/OXA cell lines transfected with sh-PCGEM1+miR-129-5p inhibitor,thus forming sh-NC group,sh-PCGEM1 group,sh-PCGEM1+miR-129-5p inhibitor group,sh-PCGEM1+miR-129-5p inhibitor+pc-NC group and sh-PCGEM1+miR-129-5p inhibitor+pc-ETV1 group.The m RNA expression of PCGEM1,miR-129-5p and ETV1 in each group was detected by QPCR,and the protein expression of ETV1 was detected by WB.The effects of pc-ETV1 mediated sh-PCGEM1/miR-129-5p inhibitor on the expressions of LRP1,MDR1,MDR3 and MRP2 in Hep3B/OXA cells was detected by QPCR.Cell function experiments were used to detect the effects of pc-ETV1 mediated sh-PCGEM1/miR-129-5p inhibitor on the cell viability,proliferation,cloning ability,migration,apoptosis and oxaliplatin resistance of Hep3B/OXA cell lines.In addition,WB experiment examined the effect of pc-ETV1 mediated sh-PCGEM1/miR-129-5p inhibitor on the expressions of apoptosis proteins（Bax,Bcl-2 and Cleared caspase-3）and transfer proteins（MMP-2 and MMP-9）in Hep3B/OXA cell lines.Results:ETV1 was confirmed to be a target gene of miR-129-5p,and there were multiple binding sites between them.Moreover,the results of QPCR and WB showed that the expression level of ETV1 was significantly increased in Oxaliplatin resistant HCC tissues and cell lines.In addition,ETV1 was down-regulated in the stably transfected sh-PCGEM1 Hep3B/OXA cell lines compared with the stably transfected sh-NC Hep3B/OXA cell lines,whereas miR-129-5p inhibitor transfection reversed the expression level of ETV1 m RNA and protein.These results indicated that ETV1 was negatively correlated with miR-129-5p and positively correlated with PCGEM1.After transfected with pc-ETV1 in Hep3B/OXA cell lines,the expression of ETV1 was increased significantly,which proved that the transfection was successful.Compared with sh-PCGEM1+miR-129-5p inhibitor group,pc-ETV1 could significantly promote the expressions of LRP1,MDR1,MDR3 and MRP2 by QPCR.Compared with sh-PCGEM1+miR-129-5p inhibitor group,pc-ETV1 could significantly increase the IC₅₀value of Oxaliplatin.Moreover,pc-ETV1 could significantly increase the activity,proliferation,migration and invasion of Hep3B/OXA cell lines,significantly inhibited the apoptosis of Hep3B/OXA cell lines.Moreover,compared with sh-PCGEM1+miR-129-5p inhibitor group,pc-ETV1 significantly inhibited the expression of Bax and caspase-3,and promoted the expressions of Bcl-2,MMP-2 and MMP-9.Conclusion:ETV1 was over-expressed in Oxaliplatin-resistant HCC tissues and cells compared to controls.Pc-EVT1 could partially reverse the effects of sh-PCGEM1/miR-129-5p inhibitor on the activity,proliferation,metastasis,apoptosis and Oxaliplatin sensitivity to Hep3B/OXA cell lines.更多还原