miRNA调控TRAIL表达对前列腺癌细胞产生选择性细胞毒作用

发布时间：2018-04-12 00:29

本文选题：前列腺癌 + 腺病毒　；参考：《吉林大学》2014年博士论文

【摘要】：前列腺癌是最常见的男性肿瘤，属于高发性癌症。许多证据表明，肿瘤坏死因子相关的凋亡诱导配体（TRAIL）可诱导癌细胞的凋亡，因此，TRAIL有希望成为前列腺癌治疗的生物制剂。但是，目前调控TRAIL表达的载体缺少肿瘤细胞的特异性，因此对正常细胞，特别是肝细胞也具有细胞毒性作用。本文为解决这个问题，将miRNA反应元件（MRE）miR-143、 miR-145以及miR-122插入腺病毒载体，成功构建Ad-TRAIL-M3腺病毒载体来控制TRAIL的表达。miR-143、miR-145和miR-122在肝细胞中特异性表达，而在前列腺癌中表达水平降低。Ad-TRAIL-M3将TRAIL以MRE调控方式表达，从而在前列腺癌中特异性表达高水平TRAIL，通过诱导凋亡抑制前列腺癌细胞的生长，而在正常细胞中没有表达。随后在PC-3移植瘤小鼠的研究中，更进一步证明了Ad-TRAIL-M3可抑制肿瘤的生长并且不具有细胞毒性，具有较高的生物安全性。这个基于miRNA的基因治疗有希望成为前列腺癌治疗方法。具体研究如下： 1.实验路线 1.1检测miR-143、miR-145和miR-122在原发性前列腺癌和相应的非肿瘤前列腺组织、前列腺癌细胞株、正常前列腺细胞株和肝细胞中的表达量。 1.1.1人正常前列腺上皮细胞株PNT2，人正常肝细胞L-02，激素非依赖性前列腺癌PC-3和激素依赖性前列腺癌LNCaP细胞培养。 1.1.2通过qPCR比较上述两种正常细胞系和人两种前列腺癌细胞系中miR-143、miR-145和miR-122表达水平的差异。 1.1.3收集患者的前列腺癌标本、新鲜癌组织及非癌变前列腺组织。1.1.4通过qPCR检测比较前列腺癌组织和正常前列腺组织中miR-143、miR-145和miR-122表达水平的差异。 1.2检测miR-143、miR-145和miR-122的MRE是否可以用于在前列腺癌细胞中表达目标基因 1.2.1将miR-143、miR-145和miR-122的MRE拷贝到荧光素酶的载体编码区的下游部位，构建psiCheck2-3MRE。1.2.2将psiCheck2和psiCheck2-3MRE转染前列腺癌细胞，48小时后用双荧光素酶报告基因系统确定其荧光素酶活性。 1.3miR-143、miR-145和miR-122的MRE是否限制前列腺癌细胞中腺病毒调控的TRAIL表达 1.3.1将两个miR-143、miR-145和miR-122的MRE拷贝连接进腺病毒空载构建了含MRE的TRAIL表达载体，Ad-TRAIL-M3。 1.3.2将Ad-TRAIL和Ad-TRAIL-M3转染PNT2、L-02、PC-3和LNCaP后，通过免疫印迹法检测TRAIL的表达。 1.3.3将Ad-TRAIL和Ad-TRAIL-M3转染PNT2、L-02、PC-3和LNCaP后，通过ELISA检测TRAIL的表达。 1.4检测Ad-TRAIL-M3调控TRAIL表达是否与miR-143、miR-145和miR-122的表达量相关 1.4.1将合成的miRNA抑制剂和模拟物加入到培养的L-02和PC-3细胞中。通过免疫印迹法检测TRAIL的表达量。 1.5Ad-TRAIL-M3是否能在对正常细胞没显著毒性的情况下杀死前列腺癌细胞 1.5.1将Ad-TRAIL和Ad-TRAIL-M3转染PNT2、L-02，MTT试验检测Ad-TRAIL-M3是否能在对正常细胞没显著毒性的情况下杀死前列腺癌细胞。 1.6Ad-TRAIL-M3是否能选择性的诱导前列腺癌细胞凋亡 1.6.1将Ad-TRAIL和Ad-TRAIL-M3转染L-02和PC-3细胞，用流式细胞仪检测细胞周期，分析Ad-TRAIL-M3是否能选择性的诱导前列腺癌细胞凋亡。 1.6.2免疫印迹法检测活化型caspase3和PARP，确定Ad-TRAIL-M3感染细胞的凋亡激活的信号通路。 1.7Ad-TRAIL-M3在体内对PC-3前列腺癌是否具有抑制作用 1.7.1通过肿瘤部位注射Ad-TRAIL、Ad-TRAIL-M3、Ad-EGFP和PBS，并定期测量肿瘤直径。 1.7.2免疫组化分析肿瘤切片分析TRAIL在PC-3移植瘤中表达情况。 1.8Ad-TRAIL-M3是否能保护肝细胞，免受TRAIL诱导的细胞毒的影响 1.8.1尾静脉给予20只正常小鼠注射Ad-TRAIL、Ad-TRAIL-M3、Ad-EGFP和PBS（n=5），随后收集血样，检测ALT（肝损伤标志物）。 2.结果 2.1miR-143、miR-145和miR-122在前列腺癌细胞中表达下调实验结果显示，与非肿瘤组织相比，在原发性前列腺癌样本中，miR-143、miR-145和miR-122表达下调。同样，与正常前列腺上皮细胞PNT2和胚胎肝细胞L-02相比，在前列腺细胞株PC-3和LNCaP中，miR-143和miR-145表达也降低。miR-122在L-02细胞中高度表达，在前列腺癌细胞细胞株中表达也降低。 2.2miR-143、miR-145和miR-122的MRE限制外源基因在前列腺癌细胞中的表达实验结果显示，与转染psiCheck2组相比，转染psiCheck2-M3的PNT2中荧光强度被强烈抑制。于此相反，在转染psiCheck2-M3和psiCheck2的前列腺癌细胞中，荧光表达无显著差异。 2.3miR-143、miR-145和miR-122的MRE可限制前列腺癌细胞中腺病毒调控的TRAIL表达免疫印迹和ELISA结果显示所有转染Ad-TRAIL的细胞都表达TRAIL蛋白，Ad-TRAIL-M3表达的TRAIL仅在前列腺癌细胞中表达。 2.4Ad-TRAIL-M3调控TRAIL表达取决于miR-143、miR-145和miR-122的丰度免疫印迹结果显示，在转染miR-143、miR-145和miR-122抑制剂的L-02细胞中，TRAIL表达部分恢复。而在PC-3细胞中TRAIL表达降低，miR-143、miR-145和miR-122可被miR-143、miR-145和miR-122模拟物上调。qPCR分析显示，miR-143、miR-145和miR-122的丰度，调控转染Ad-TRAIL-M3的L-02和PC-3细胞中TRAIL的表达水平。 2.5Ad-TRAIL-M3在对正常细胞没显著细胞毒性的情况下降低前列腺癌细胞的存活率 MTT试验显示PNT2和L-02细胞被Ad-TRAIL强烈抑制，但Ad-TRAIL-M3对其没有影响。而Ad-TRAIL和Ad-TRAIL-M3对前列腺癌细胞都有生长抑制作用。 2.6Ad-TRAIL-M3对前列腺癌细胞具有凋亡诱导作用，但对正常细胞没有作用流式细胞仪分析显示，Ad-TRAIL转染后，导致L-02和PC-3细胞G0/G1亚期数升高。而Ad-TRAIL-M3处理仅升高前列腺癌细胞G0/G1亚期数，，对正常肝细胞没有影响。免疫印迹分析活化型caspase3和PARP表明，这两个凋亡相关的标志物在转染Ad-TRAIL-M3或Ad-TRAIL的前列腺癌细胞中特异性被活化。 2.7Ad-TRAIL-M3对小鼠PC-3移植瘤的生长抑制作用结果显示，Ad-TRAIL-M3可抑制PC-3移植瘤生长达80%左右，Ad-TRAIL-M3和Ad-TRAIL对肿瘤生长的抑制作用没有显著的区别。免疫组化分析肿瘤切片显示，经Ad-TRAIL-M3和Ad-TRAIL治疗后，TRAIL在PC-3移植瘤中广泛表达。 2.8Ad-TRAIL-3MRE可保护肝脏不受细胞毒影响尾静脉给予20只正常小鼠注射Ad-TRAIL、Ad-TRAIL-M3、Ad-EGFP和PBS（n=5），随后收集血样，检测ALT（肝损伤标志物）结果显示，注射Ad-TRAIL的小鼠ALT水平明显升高，Ad-TRAIL-M3、Ad-EGFP和PBS组小鼠ALT水平无变化。此外，免疫组化数据显示，Ad-TRAIL-M3治疗组小鼠肝脏中无TRAIL表达。免疫印迹检测活化PARP显示，Ad-TRAIL-M3可诱导肿瘤凋亡，但对正常肝组织没有影响。 3.结论将miRNA反应元件（MRE）miR-143、 miR-145以及miR-122插入腺病毒载体，成功构建Ad-TRAIL-M3腺病毒载体来控制TRAIL的表达，Ad-TRAIL-M3将TRAIL以MRE调控方式表达，从而在前列腺癌中特异性表达高水平TRAIL，通过诱导凋亡抑制前列腺癌细胞的生长，随后在PC-3移植瘤小鼠的研究中，更进一步证明了Ad-TRAIL-M3可抑制肿瘤的生长，而在正常细胞中TRAIL没有表达，不具有细胞毒性，具有较高的生物安全性。
[Abstract]:In order to solve this problem , it has been proved that TRAIL - M3 can inhibit the growth of prostate cancer cells and has no cytotoxicity and high biological safety . The gene therapy based on miRNA is promising to be a treatment method for prostate cancer .

Specific studies are as follows :

1 . Experimental route

1.1 The expression amount of miR - 143 , miR - 145 and miR - 122 in primary prostate cancer and corresponding non - tumor prostate tissue , prostate cancer cell line , normal prostate cell line and liver cell is detected .

1.1 . 1 Human normal prostate epithelial cell line PNT2 , human normal liver cell line L - 02 , hormone - independent prostate cancer PC - 3 , and hormone - dependent prostate cancer LNAs cell culture .

1.1 . 2 Comparison of miR - 143 , miR - 145 , and miR - 122 expression levels in both normal cell lines and human two prostate cancer cell lines was compared by qPCR .

1.1 . 3 The difference in expression levels of miR - 143 , miR - 145 and miR - 122 in prostate cancer tissue and normal prostate tissue was detected by qPCR by collecting prostate cancer specimens from patients , fresh cancerous tissue , and non - cancerous prostate tissue . 1.4 . 4 .

1.2 Detecting whether MREs of miR - 143 , miR - 145 and miR - 122 can be used to express target genes in prostate cancer cells

1.2 . 1 To construct psiCheck2 - 3MREs . 1.2 . 2 transfected prostate cancer cells with psiCheck2 - 3MREs . 1.2 . 2 , and luciferase activity was determined using a dual luciferase reporter gene system after 48 hours of transfection of psiCheck2 - 3MREs . 1.2 . 2 into the downstream site of the vector coding region of luciferase .

1 . Whether the MREs of miR - 143 , miR - 145 and miR - 122 restrict the expression of TRAIL in prostate cancer cells

1.3 . 1 Two miR - 143 , miR - 145 and miR - 122 were ligated into an adenovirus no - load to construct a TRAIL expression vector containing MREs , Ad - TRAIL - M3 .

1.3 . 2 After transfection of Ad - TRAIL and Ad - TRAIL - M3 into PNT2 , L - 02 , PC - 3 , and LNAs , the expression of TRAIL was detected by Western blotting .

1.3 . 3 After transfection of Ad - TRAIL and Ad - TRAIL - M3 into PNT2 , L - 02 , PC - 3 , and LNAs , the expression of TRAIL was detected by ELISA .

1.4 detecting whether the expression of TRAIL expression in Ad - TRAIL - M3 is related to the expression amount of miR - 143 , miR - 145 and miR - 122

1.4 . 1 The synthesized miRNA inhibitor and mimetic were added to the cultured L - 02 and PC - 3 cells . The expression level of TRAIL was detected by Western blotting .

1.5 Ad - TRAIL - M3 can kill prostate cancer cells without significant toxicity to normal cells

1.5 . 1 Ad - TRAIL and Ad - TRAIL - M3 were transfected into PNT2 , L - 02 , and MTT assay was used to detect whether Ad - TRAIL - M3 could kill prostate cancer cells without significant toxicity to normal cells .

1.6Ad - TRAIL - M3 selectively induces apoptosis of prostate cancer cells

1.6 . 1 Ad - TRAIL and Ad - TRAIL - M3 were transfected into L - 02 and PC - 3 cells . Cell cycle was detected by flow cytometry .

1.6 . 2 The activation type caspase3 and parp were detected by Western blotting , and the signal pathway of apoptosis activation of Ad - TRAIL - M3 infected cells was determined .

Inhibitory Effect of 1.7Ad - TRAIL - M3 on PC - 3 Prostate Cancer in vivo

1.7 . 1 Ad - TRAIL , Ad - TRAIL - M3 , Ad - EGFP and PBS were injected through the tumor site and the tumor diameter was measured periodically .

1.7 . 2 The expression of TRAIL in PC - 3 transplanted tumor was analyzed by immunohistochemistry .

1.8 Ad - TRAIL - M3 protects hepatocytes from TRAIL - induced cytotoxicity

1.8 . 1 Ad - TRAIL , Ad - TRAIL - M3 , Ad - EGFP and PBS ( n = 5 ) were injected into the tail vein in 20 normal mice , followed by the collection of blood samples to detect ALT ( liver injury markers ) .

2 . Results

2.1 Expression of miR - 143 , miR - 145 and miR - 122 in Prostate Cancer Cells

Experimental results showed that miR - 143 , miR - 145 , and miR - 122 expression were down - regulated in a sample of primary prostate cancer compared with non - tumor tissues . Similarly , miR - 143 and miR - 145 expression were also reduced in prostate cancer cell lines PC - 3 and LNAs compared with normal prostate epithelial cells PNT2 and L - 02 . Expression of miR - 122 in L - 02 cells was also reduced .

2.2 Expression of miR - 143 , miR - 145 and miR - 122 in Prostate Cancer Cells

The results showed that the fluorescence intensity was strongly inhibited in the PNT2 transfected psiCheck2 - M3 compared with the transfected psiCheck2 group . In contrast , there was no significant difference in fluorescence expression in the transfected psiCheck2 - M3 and psiCheck2 prostate cancer cells .

2.3miR - 143 , miR - 145 and miR - 122 MREs can limit the expression of TRAIL in prostate cancer cells

Western blot and ELISA results show that all cells transfected with Ad - TRAIL express TRAIL protein and TRAIL - M3 expressed TRAIL is only expressed in prostate cancer cells .

2.4Ad - TRAIL - M3 regulates TRAIL expression depending on the abundance of miR - 143 , miR - 145 and miR - 122

Western blot showed that TRAIL expression was partially restored in the L - 02 cells transfected with miR - 143 , miR - 145 and miR - 122 inhibitors , while miR - 143 , miR - 145 , and miR - 122 were up - regulated by miR - 143 , miR - 145 , and miR - 122 . The qPCR analysis showed that miR - 143 , miR - 145 , and miR - 122 were abundant , and that the expression level of TRAIL in L - 02 and PC - 3 cells transfected with Ad - TRAIL - M3 was regulated .

2.5Ad - TRAIL - M3 reduces the survival rate of prostate cancer cells in the absence of significant cytotoxicity to normal cells

MTT assay showed that PNT2 and L - 02 cells were strongly inhibited by Ad - TRAIL , but Ad - TRAIL - M3 had no effect on prostate cancer cells . Ad - TRAIL and Ad - TRAIL - M3 inhibited the growth of prostate cancer cells .

2.6Ad - TRAIL - M3 has an apoptosis - inducing effect on prostate cancer cells , but has no effect on normal cells .

Flow cytometry analysis showed that after Ad - TRAIL transfection , the G0 / G1 substeps of L - 02 and PC - 3 cells were increased . Ad - TRAIL - M3 treatment only increased the G0 / G1 sub - period of prostate cancer cells and had no effect on normal hepatocytes . Western blot analysis of activated caspase3 and parp showed that these two apoptosis - related markers were specifically activated in prostate cancer cells transfected with Ad - TRAIL - M3 or Ad - TRAIL .

Inhibitory effect of 2 . 7Ad - TRAIL - M3 on growth of mouse PC - 3 transplanted tumor

The results showed that Ad - TRAIL - M3 could inhibit the growth of PC - 3 transplanted tumor by about 80 % , Ad - TRAIL - M3 and Ad - TRAIL had no significant difference in tumor growth . After treatment with Ad - TRAIL - M3 and Ad - TRAIL , TRAIL was widely expressed in PC - 3 transplanted tumor .

2.8 Ad - TRAIL - 3MREs protect the liver from cytotoxic effects

Ad - TRAIL , Ad - TRAIL - M3 , Ad - EGFP and PBS ( n = 5 ) were injected into the tail vein in 20 normal mice . Blood samples were collected . The ALT level of Ad - TRAIL - M3 , Ad - EGFP and PBS group was not changed .

3 . Conclusions

The invention further proves that the Ad - TRAIL - M3 can inhibit the growth of the tumor by inducing the apoptosis to inhibit the growth of the prostate cancer cell , and then further proves that the Ad - TRAIL - M3 can inhibit the growth of the tumor in the study of the PC - 3 transplantation tumor mouse , and further proves that the Ad - TRAIL - M3 can inhibit the growth of the tumor , and the TRAIL is not expressed in the normal cell , and has high biological safety .

【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R737.25

【参考文献】