Study On The Anti-tumor Mechanism Of TNBG-5602 Using A Human Genomic Sirna Library

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.