Lentivirus-mediated LI-cadherin RNAi Combined With RAd-p53 Molecular Targeted Therapies In Hepatocellular Carcinoma

PartⅠEstablishment,characterization and package of miR-30-based shRNA lentivirus targeted at LI-cadherinPurpose:The study designed the miR-30-based shRNA sequence targeted at LI-cadherin gene and packaged the recombinant lentivirus to study the role of LI-cadherin in the growth and metastasis of HCC.Materials and Methods:Based on miR-30-based shRNA library of Cold Spring Harbour carried by the PSM2 vector,the pLUNIG-miLI-cadherin as the recombinant working plasmid and pLUNIG-miLuciferase as non-specifically interfering control plasmid was established.Ca3(PO4) 2-DNA coprecipitates was used to transfect 293T cells with either the pLUNIG-miLI-cadherin or pLUNIG-miLuciferase and the other three helper plasmids PCMV-VSVG, PMDL-G/P-RRE and PRSV-REV.The virus supernatant was collected and condensed.Q-RT-PCR was used to examine the copies of lentivirus. MHCC97H cells were infected by the lentivirus.The stable transfectants were selected with G418 as titled MHCC97H-miLI-cadherin and MHCC97H-miLuciferase cell line.Q-RT-PCR,Immunocytochemistry and Western blotting were used to investigate whether the expression of LI-cadherin in MHCC97H-miLI-cadherin cells had been efficiently knocked down.Results:The sequences of the working plasmids were analyzed with BLAST method. Two working plasmids were acquired as titled pLUNIG-miLI-cadherin and pLUNIG-miLuciferase.The expression of LI-cadherin mRNA was downregulated to a certain degree.Immunocytochemistry showed the expression of LI-cadherin protein was significantly lower in MHCC97H-miLI-cadherin cells than in MHCC97H -miLuciferase cells.Western blotting further confirmed that the expression of LI-cadherin protein was significantly downregulated in MHCC97H-miLI-cadherin cells. The titre of the condensed lentivirus supernatant was above 10~9 as examined by Q-RT-PCR.The lentivirus was shown to have no function to replicate.Conclusion:The modified lentivirus is proved to be a novel generation with double selection system,which carries miR-30-based shRNA recombinant working plasmids targeted at LI-cadherin or Luciferase.Lentivirus has also been proved to be an ideal vector to induce shRNA into HCC cells.The expression of LI-cadherin protein in MHCC97H has been successfully knocked down with miR-30-based shRNA interfering method.A large quantity of package and production of the recombinant lentivirus with high titres has been successfully performed for the use in in vitro and in vivo experiments.PartⅡCharacterization of changes in biological properties of MHCC97H-miLI-cadherin cellsPurpose:To study the changes in the cell proliferation,adhesion,clone formation, migration,invasion,cell cycle,apoptosis,and related drugs sensitivity of MHCC97H-miLI-cadherin cells.To explore the possible underlying mechanisms.Materials and Methods:Cell proliferation and adhesion assays detected by comparing OD values. Cell cycle assays examined by PI/Rnase staining-Flow Cytometry.Cell apoptosis assays checked by TUNEL.Cloning formation assays measured by soft agar.Cell migration and invasion assays calculated by TRANSWELL methods.Drug sensitivity assays monitored by MTT.Signaling pathways exploration using Western blotting.Results:The cell growth and cell adhesion ability of MHCC97H-miLI-cadherin cells were markedly inhibited as compared to controls.Statistic analyses showed significant difference between MHCC97H-miLI-cadherin cells and controls.The percentage of cells in S phase in MHCC97H-miLI-cadherin cells was significantly less than that in MHCC97H-miLuciferase cells and blank control, whereas the percentage of cells in G0-G1 phase in MHCC97H-miLI-cadherin cells was significantly higher than that in MHCC97H-miLuciferase cells and blank control.The percentage of positive apoptotic cells in MHCC97H-miLI-cadherin cells was 8.52±1.43%,which was significantly higher than 1.55±0.33%in MHCC97H-miLuciferase cells.The number of clone formation,migration and invasion in MHCC97H-miLI-cadherin group was significantly less than that in MHCC97H-miLuciferase and blank control groups.The inhibition rate of MHCC97H-miLI-cadherin group was respectively 93.1%,90.2%and 88.3%. Statistic analysis showed significant difference between MHCC97H-miLI-cadherin group and control groups.The drug sensitivity and inhibition rate of Epirubcin,Carboplatin to MHCC97H-miLI-cadherin cells was significantly elevated as compared to MHCC97H-miLuciferase cells and blank control.Statistic analysis showed significant difference between MHCC97H-miLI-cadherin group and control groups.The drug sensitivity and inhibition rate of rAd-p53 to MHCC97H-miLI-cadherin cells was significantly elevated as compared to MHCC97H-miLuciferase cells and blank control.Statistic analysis showed significant difference between MHCC97H-miLI-cadherin group and control groups.The Wnt/β-catenin pathway was possibly involved in the biological changes of MHCC97H-miLI-cadherin cells.In brief,the downregulation of LI-cadherin activated GSK-3β,then led to the degradation ofβ-catenin,inhibited the expression of CyclinD1 and accordingly increased the expression of Rb.Conclusion:MHCC97H-miLI-cadherin cells show great changes in the cell biological properties as compared to MHCC97H-miLuciferase cells and blank control MHCC97H cells including retarded proliferation;decreased ability in cell adhesion,clone formation,cell migration and invasion;the decrease in the percentage of cell number in S phase and increase in the percentage of cell number in G0-G1 phase;the increase in the cell apoptosis and drug sensitivity.All these results demonstrate that the downregulation of LI-cadherin has obviously altered the tumor properties of the HCC cell line,strongly indicating the oncogene property of LI-cadherin.PartⅢInhibition of growth and metastasis of Hepatocellular Carcinoma by lentivirus-induced RNAi therapy combined with rAd-p53 gone therapy and involved signaling pathwaysPurpose:To study the changes in cancergenesis and metastasis of MHCC97H-miLI-cadherin cells after subcutaneous inoculation in nude mice. To study the changes in metastasis of MHCC97H-miLI-cadherin cells after tail vein injection in nude mice.To study the changes in tumor growth of subcutaneous inoculated HCC and the expression of LI-cadherin in tumor cells after combined gene therapies.To explore the signaling pathway related to LI-cadherin down-regulation.Materials and Methods:MHCC97H-miLI-cadherin cells,MHCC97H-miLuciferase cells,and blank control MHCC97H cells were subcutaneously inoculated into adult nude mice respectively to investigate the difference in cancergenesis and metastasis among the three groups.MHCC97H-miLI-cadherin cells, MHCC97H-miLuciferase cells,and blank control MHCC97H cells were injected into adult nude mice via tail vein respectively to investigate the difference in cancergenesis and metastasis among the three groups.The therapeutic effect of intratumor injection of lenti-miLI-Cadherin with and without rAd-p53 on the growth of subcutaneously inoculated HCC in nude mice was investigated.The expression of LI-cadherin and related molecules in subcutaneously inoculated HCC were investigated after intratumor injection of lenti-miLI-Cad with and without rAd-p53 by IHC and westen blotting assays. The biological safety and off-target effects of combined gene therapies were also investigated with IHC and Q-RT-PCR.Results:The subcutaneously inoculated tumor of MHCC97H-miLI-cadherin group grew much more slowly as compared to MHCC97H-miLuciferase and blank control groups.8 weeks after subcutaneous inoculation,all animals in MHCC97H-miLuciferase and blank control groups showed metastasis in lung, however,no metastasis was observed in MHCC97H-miLI-cadherin group. Similar to subcutaneous inoculation,all animals in MHCC97H-miLuciferase group via tail vein injection showed metastasis in lung and no metastasis was observed in MHCC97H-miLI-cadherin group.The growth of HCC tumor in rAd-p53 group,lenti-miLI-cadherin group and lenti-miLI-cadherin plus rAd-p53 group was remarkably inhibited as compared to TBS group and lenti-miLuciferase group.Among the former three groups, the growth of HCC tumor in lenti-miLI-cadherin plus rAd-p53 group showed the strongest inhibition.Western blotting showed that 4 weeks after the last intratumor injection of lenti-miLI-cadherin,the expression of LI-cadherin was significantly downregulated whereas high expression of LI-cadherin was shown in control groups.The expression of total GSK3βwere high and at the similar level in all five groups while the S9 phosphated GSK3βare apparently dawnregulated in lenti-miLI-cadherin group and lenti-miLI-cadherin plus rAd-p53 group.The expression ofβ-catenin in TBS group,lenti-miLuciferase group were high and at the similar level,which was in contrast to significant downregulation ofβ-catenin in lenti-miLI-cadherin group and lenti-miLI-cadherin plus rAd-p53 group.The expression of Cyclin D1 in TBS group,lenti-miLuciferase group was high and at the similar level,which was in contrast to significant downregulation of Cyclin D1 in lenti-miLI-cadherin group and lenti-miLI-cadherin plus rAd-p53 group and the expression of Rb were upregulated correspondingly.The expression of P53 were high in all five groups especially in rAd-p53 group and lenti-miLI-cadherin plus rAd-p53 group but without any obvious relationship with the LI-cadherin expression.The expression of Bax in rAd-p53 group and lenti-miLI-cadherin plus rAd-p53 group was elevated,whereas the expression of Bcl-XL decreased accordingly. The expression of cleaved caspase 3 in rAd-p53 group,lenti-miLI-cadherin group and lenti-miLI-cadherin plus rAd-p53 group was obviously induced.No risk in biological safety and off-target effects of combined gene therapies were observed using IHC and Q-RT-PCR assays.Conclusion:Downregulation of LI-cadherin expression in MHCC97H cells leads to the marked decrease in the tumor growth and metastasis,providing evidence that LI-cadherin can be therapeutic target for HCC treatment.Intratumor injection of lenti-miLI-cadherin can significantly downregulate the expression of LI-cadherin,which may be associated with inhibiting tumor growth.Intratumor injection of lenti-miLI-cadherin together with rAd-p53 shows greater effects on inhibiting tumor growth,providing evidence to apply combined gene therapies for treatment with HCC.No risk in biological safety and off-target effects of combined gene therapies have been observed during the period of drug admistration.The study of involved signaling pathway suggesting that the downregulation of LI-cadherin was closely related to activation of GSK-3βand degradation ofβ-catenin;indicating that the tumor growth inhibition due to lenti-miLI-cadherin and rAd-p53 therapy was associated with the different signaling pathway to cooperate and increase in the cell apoptosis.