【作者】 胡雪莲；

【导师】 余瑜；

【作者基本信息】 重庆医科大学 ， 药理学， 2018， 博士

【摘要】 目的肝癌是最常见的恶性肿瘤之一,具有较高的发病率和死亡率。对于晚期肝癌患者,尚缺乏满意的治疗手段。TNBG-5602是本课题组新合成的TNBG衍生物,对肝癌细胞有明显的增殖抑制作用及脂质聚集作用。两种作用之间是否有关联,TNBG-5602是通过何种机制发挥抗肝癌作用的?这些问题尚待阐明。本研究拟采用人基因组siRNA文库进行筛查,找出TNBG-5602可能的作用靶点,并进行验证,以明确TNBG-5602抗肝癌作用的机制。方法1.分别采用结晶紫染色、CCK-8检测TNBG-5602对三种人肝癌细胞株增殖的影响;通过流式细胞术检测TNBG-5602对肝癌细胞周期和细胞凋亡情况;并采用Western blot检测增殖、凋亡相关蛋白的变化;油红O染色检测TNBG-5602对人肝癌细胞脂代谢的影响;Western blot检测脂代谢相关蛋白的变化。2.利用siRNA文库筛选TNBG-5602抗肝癌作用的关键靶点。（1）首先将siRNA文库通过逆转录病毒系统导入QGY-7701细胞株,待病毒与细胞基因组重组后加入BSD及TNBG-5602进行反复筛选,获得对TNBG-5602不敏感（耐药）的肝癌细胞株,命名为7701-T细胞。（2）通过体内外验证证明7701-T细胞对TNBG-5602的敏感性是否较QGY-7701降低。（3）提取7701-T细胞的基因组DNA（gDNA）,PCR扩增和纯化目标片段,测序并分析高度富集的片段,筛选出可能的关键基因。qPCR验证筛选出的基因在7701-T的表达。3.为验证筛选出的基因PTEN在TNBG-5602抗肝癌的过程中是否发挥了重要作用,利用qPCR及Western blot技术检测TNBG-5602对QGY-7701细胞PTEN水平的影响。采用过表达（AdPTEN）和沉默PTEN（AdsiPTEN）的重组腺病毒考察PTEN在TNBG-5602引起的QGY-7701细胞增殖抑制中的作用及对p-Akt、PCNA、Bcl-2和Bad表达的影响。4.为进一步研究TNBG-5602引起细胞内脂质聚集与抑制细胞增值、促进凋亡之间的关系,利用PPARγ激动剂及抑制剂考察PPARγ对TNBG-5602引起的QGY-7701细胞的增殖抑制的影响;及对TNBG-5602引起的脂质聚集作用的影响;最后考察PPARγ与PTEN的关系。结果1.结晶紫染色、CCK-8等检测结果发现本课题组合成的新化合物TNBG-5602对三种肝癌细胞的增殖均具有显著抑制作用。其中TNBG-5602对人肝癌细胞株QGY-7701的抑制作用相对最强,24h、48h、72h的IC₅₀分别为12.64、9.79、8.91μM。TNBG-5602能诱导QGY-7701细胞发生G1期阻滞;还可通过上调凋亡相关因子Bad的表达,下调抗凋亡因子Bcl-2的表达,从而诱导QGY-7701细胞凋亡。同时还发现TNBG-5602可以诱导QGY-7701人肝癌细胞株产生大量脂质聚集。并可引起QGY-7701细胞中PPARγ等脂质合成相关蛋白表达增加。2.利用siRNA文库筛选出TNBG-5602抗肝癌作用的关键靶点。（1）首先通过将siRNA文库质粒与pCL-Ampho质粒一起共转染293PA细胞制备出携带siRNA文库的逆转录病毒,然后将该逆转录病毒感染QGY-7701细胞株,待病毒与细胞基因组重组后加入BSD及TNBG-5602反复进行筛选,筛选出对TNBG-5602不敏感（耐药）的肝癌细胞株,将其命名为7701-T。（2）体外通过细胞生长状态、CCK8、流式细胞术等检测结果的比较显示,7701-T细胞对TNBG-5602的敏感性大大降低,TNBG-5602对7701-T的24h、48h、72h的IC₅₀分别较QGY-7701的提高了3⁴倍。油红O染色显示,相同浓度的TNBG-5602处理7701-T后,与QGY-7701相比,未出现明显的脂质聚集现象。裸鼠体内实验结果显示,TNBG-5602在裸鼠体内对7701-T所致肿瘤的抑制作用较QGY-7701明显减弱。以上体内外对比实验证实,7701-T对TNBG-5602的敏感性较QGY-7701确实大大降低,说明导入的siRNA已在细胞内发挥作用,经TNBG-5602筛选出的细胞因关键基因被沉默而对TNBG-5602的作用不敏感。（3）提取7701-T细胞的gDNA,PCR扩增和纯化目标片段,并进行测序分析。测序结果采用BLAST分析,结果与PTEN完全匹配的序列富集率高达7.43%。提示PTEN可能是TNBG-5602的作用靶点之一。另外与目标序列匹配的还有LRP6、ACADL、LDLRAP1等与脂代谢相关的基因。提示脂代谢相关基因也是较重要的靶点。采用qPCR进行验证,结果与QGY-7701细胞相比,7701-T的PTEN mRNA水平大大降低,被沉默了约80%,与测序的结果相符。说明PTEN是一个可能的关键靶点。3.为验证PTEN在TNBG-5602抗肝癌的过程中是否发挥了重要作用,进行进一步研究。结果表明TNBG-5602可以增加PTEN而降低p-Akt水平;PI3K抑制剂可以增强TNBG-5602对QGY-7701细胞的增殖抑制作用。提示TNBG-5602抗肝癌作用可能是通过上调PTEN导致PI3K/Akt信号通路被抑制而实现的。进一步分别采用过表达和沉默PTEN的重组腺病毒进行研究的结果发现,AdPTEN可以大大提高TNBG-5602对QGY-7701细胞的抗增殖活性;而AdsiPTEN可以减弱TNBG-5602的此作用。此外,AdPTEN可增强TNBG-5602对p-Akt、PCNA、Bcl-2和Bad的影响;而AdsiPTEN可以减弱TNBG-5602对这些蛋白的作用。以上结果强烈提示TNBG-5602引起的抗增殖和诱导凋亡的作用,可能是由于上调PTEN从而抑制PI3K/AKT信号通路的结果。4.随后的研究发现,TNBG-5602引起的QGY-7701细胞的脂质聚集现象可被PPARγ激动剂增强,而被PPARγ抑制剂部分逆转;而且TNBG-5602对QGY-7701细胞的抗增殖作用也可被PPARγ激动剂增强,而被PPARγ抑制剂部分逆转。表明PPARγ参与了TNBG-5602对QGY-7701细胞的脂质聚集及增殖抑制作用。进一步研究表明,TNBG-5602引起的PTEN上调可被PPARγ激动剂增强,而被PPARγ抑制剂部分逆转。由此可推断,TNBG-5602引起的PTEN上调可能是由PPARγ介导的。结论TNBG-5602对肝癌细胞具有明显的增殖抑制和诱导凋亡作用,可能是一个潜在的抗肝癌药物。TNBG-5602的抗肝癌活性可能是通过激活PPARγ引起PTEN上调,从而抑制PI3K/Akt信号通路实现的。更多还原

【Abstract】 ObjectLiver cancer is one of the most common malignancies,and its unsatisfactory prognosis urgently needs to be ameliorated.TNBG-5602,one of our creatively synthesized derivatives of tetrazanbigen,has been found can effectively inhibit the proliferation of and induce apoptosis of liver cancer cells.However,the mechanism of TNBG-5602 remains to be clarified.The aim of this study was to investigate the possible targets of TNBG-5602 on human cancer cells using the siRNA library,and dissected the possible mechanisms underlying the antitumor effects of TNBG-5602.Methods1.Crystal violet staining and CCK-8 assay was used to assess the effect of TNBG-5602 on cell viability.Cell cycle and apoptosis were detected by flow cytometry.Effect of TNBG-5602 on lipid metabolism in human cancer cells by oil red O staining.Western blot was used to detect the markers of proliferation and apoptosis.2.Investigate the possible targets of TNBG-5602 on human liver cancer cells using the siRNA library.（1）Firstly,retrovirus transfection system was used to introduce siRNA library into the QGY-7701 cells,and TNBG-5602-resistant liver cancer cells were screened by BSD and TNBG-5602 repeatedly,which were named as 7701-T cells.（2）Then,the sensitivity of 7701-T to TNBG-5602 was compared in vitro and in vivo.（3）Finally,the gDNA of 7701-T cells was extracted,and target fragments were amplified and purified.RNA sequences were detected,and highly enriched fragments were analyzed.qPCR verifies the expression of the selected genes in 7701-T.3.To verify whether the screened gene PTEN plays an important role in the anti cancer process of TNBG-5602,the effects of TNBG-5602 on the PTEN level of QGY-7701 cells are detected by qPCR and Western blot.The effects of AdPTEN and AdsiPTEN on the proliferation of QGY-7701 cells were investigated by crystal violet staining and CCK-8.Western blot was used to detect the effect of Ad PTEN and AdsiPTEN on the expression of p-Akt,PCNA,Bcl-2 and Bad.4.To further study the relationship between TNBG-5602 induced lipid aggregation and inhibition of proliferation,the effects of PPARγagonists and inhibitors on the proliferation inhibition of QGY-7701 cells induced by TNBG-5602 were investigated by crystal violet staining and CCK-8,and the effects of PPARγagonists and inhibitors on the lipid aggregation induced by TNBG-5602 were examined by using oil red O staining;Western blot was used to detected the effects of PPARγagonists and inhibitors on PTEN.Results1.It was found that TNBG-5602 could inhibit the proliferation of three liver cancer cell lines.Among them,the inhibitory effect of TNBG-5602 on QGY-7701 cells was the strongest,with the IC₅₀0 of 24h,48h and 72h were 12.64,9.79 and 8.91μM respectively.TNBG-5602 could induce G1 arrest in QGY-7701 cells,induce apoptosis of QGY-7701 cells and down-regulate the expression of apoptosis related factor Bad while down-regulate the expression of anti-apoptotic factor Bcl-2.It is also observed that TNBG-5602 could induce large amounts of lipid accumulation in QGY-7701 cells.Western blot results showed that the expression of PPARγand other lipid synthesis related proteins were increased by TNBG-5602 in QGY-7701 cells.2.Using the siRNA library,we screened the key targets of TNBG-5602 on liver cancer cells.（1）First,the retrovirus carrying the siRNA library was prepared by co-transfection of the siRNA library plasmid and the pCL-Ampho plasmid into 293PA cells.Then the retrovirus was infected to QGY-7701 cells.After the virus recombination to the genome of QGY-7701 cells,BSD and TNBG-5602 were used to screen the TNBG-5602-resistant liver cancer cell,which were named as 7701-T cells.（2）In vitro,the results of cell growth state,CCK-8 and flow cytometry analysis showed that the sensitivity of 7701-T to TNBG-5602 was obviously lower than QGY-7701 cells.In vivo,the results of tumor inhibition rate and tissue H&E staining in nude mice showed that the sensitivity of 7701-T to TNBG-5602 was obviously lower than QGY-7701 cells too.（3）The gDNA of 7701-T cells was extracted,and target fragments were amplified and purified.RNA sequences were detected by high-throughput sequencing,and highly enriched fragments were analyzed using BLAST analysis.It is found that the sequence enrichment rate matched to PTEN was 7.43%.It suggests that PTEN may be one of the targets of TNBG-5602.In addition,lipid metabolism related genes such as LRP6,ACADL,and LDLRAP1 were also found.It indicates that lipid metabolism related genes are also important targets.qPCR results showed that,the PTEN mRNA level of7701-T was significantly reduced（silenced about 80%）compared with QGY-7701,which is consistent with the results of sequencing.3.In order to verify whether PTEN plays an important role in the anti cancer process of TNBG-5602,the effects of TNBG-5602 on the PTEN level of QGY-7701cells are detected by qPCR and Western blot.The results showed that TNBG-5602could increase PTEN and decrease p-Akt level,and PI3K inhibitor could enhance the inhibitory effect of TNBG-5602 on QGY-7701 cells proliferation.It suggests that the effect of TNBG-5602 on QGY-7701 cells may be mediated by up regulation of PTEN and inhibition of PI3K/Akt signaling pathway.In order to further verify this idea,we used AdPTEN and AdsiPTEN to investigate the effects of PTEN on the proliferation of of TNBG-5602 on QGY-7701 cells.It was found that AdPTEN could greatly enhance the anti-proliferative activity of TNBG-5602 on QGY-7701 cells,while AdsiPTEN could attenuate the effect of TNBG-5602.In addition,AdPTEN can enhance the effect of TNBG-5602 on p-Akt,PCNA,Bcl-2 and Bad,while AdsiPTEN can weaken the effect of TNBG-5602 on these proteins.These results strongly suggested that the anti-proliferation and apoptosis promotion induced by TNBG-5602 may be due to the up regulation of PTEN and then inhibition of PI3K/AKT signaling pathway.4.Subsequent studies showed that the lipid aggregation of QGY-7701 cells induced by TNBG-5602 can be enhanced by a PPARγagonist while partially reversed by a PPARγinhibitor,and the anti-proliferation effect of TNBG-5602 on QGY-7701cells can also be enhanced by a PPARγagonist while partially reversed by a PPARγinhibitor.It indicates that PPARγis involved in the inhibition of TNBG-5602 on QGY-7701 cell lipid accumulation and proliferation.Further studies show that the up-regulation of PTEN induced by TNBG-5602 can be enhanced a by PPARγagonist while partly reversed by a PPARγinhibitor.The results suggested that the up-regulation of PTEN induced by TNBG-5602 may be mediated by PPARγ.ConclusionOur findings suggested that TNBG-5602 may be a potential anticancer drug for liver cancer,the effects of which may be mediated by activating PPARγand up-regulating PTEN to block the PI3K/Akt signaling pathway.更多还原