| Objective To construct the conditionally replicative adenovirus (CRAds)expressing short interference RNA targeting Ki67gene (Ki67-siRNA) with G250promoter, study the therapic effect on renal cell carcinoma in vitro. The results willpromote better understanding of the anti-tumor effect in vivo and clinical application.Methods The plasmids of pG250-Ki67, pG250-EGFP and pBHGE3were verifiedby restriction enzymes analysis and purified. The pG250-Ki67and pG250-EGFPplasmids were transfected to293cells together with plasmid pBHGE3to abtain therecombined CRAds: G250-Ki67and G250-EGFP. The viral plaques appeared9-12daysafter infection. Extracted DNA from the recombined adenoviruses, verified by PCR.The viral plaques were purified and propagated on low passage HEK293cells. Thefunctional PFU titers were determined by plaque assay on low passage HEK293cells.The expression of E1A protein in786-0, ACHN and HK-2cell lines were detected byWestern blot and immunohistochemistry. Cytotoxic effects of G250-Ki67, G250-EGFPand Ad-Ki67on786-0, ACHN and HK-2cell lines were detected by crystal violetstaining. The effect of Ki67-siRNA gene was observed by RT-PCR analysis. Cellproliferation was assayed by MTT method. The apoptosis of tumor cells was measuredby Annexin V-PE/7-AAD.Results1.The plasmids of pG250-Ki67and pG250-EGFP were constructedsuccessfully and proved by restriction analysis. G250-Ki67and G250-EGFP had beenrecombined successfully and not polluted by wild-type adenovirus tested by PCR. Theirfunctional PFU titers were2.3×1011PFU/ml,2.11×1011PFU/ml and1.97×1011PFU/ml.2. G250-Ki67and G250-EGFP expressed E1A protein in786-0cell lines，but notin ACHN cell lines. There were no expression of E1A protein in786-0cell lines andACHN cell lines which infected by Ad-Ki67. All virus could not expressed E1A proteinin HK-2cell lines. 3. The expression of Ki67gene in786-0cell lines was suppressed by G250-Ki67and Ad-Ki67. G250-Ki67exerted more specific and stronger inhibitory effect on theexpression of Ki67in786-0cells than Ad-Ki67dose（P＜0.05）.4. Cytotoxic effects: G250-Ki67, G250-EGFP and Ad-Ki67infected786-0, ACHNand HK-2cell lines respectively with different MOI. All virus had killing effect on786-0cell lines and ACHN cell lines. Both G250-Ki67and ZD55-Ki67had lethal effecton786-0cell lines, G250-Ki67could kill all786-0cell lines when MOI was10, whilelethal effect of G250-EGFP was poor. G250-Ki67could kill all ACHN cell lines partlywhen MOI was100, while G250-EGFP could not. Ad-Ki67had no obviously lethaleffect on786-0and ACHN cell lines. G250-Ki67, G250-EGFP and Ad-Ki67had noobviously lethal effect on HK-2cell lines.5. G250-Ki67, G250-EGFP and Ad-Ki67could inhibit the proliferation of786-0cell line. The inhibition effect depended on the concentration and time. The inhibitioneffects were weak with the MOI=0.1, improved with increasing concentration, and theeffects reach utmost when the MOI=100. G250-Ki67, G250-EGFP and Ad-Ki67infected786-0cell lines with MOI=10respectively, cell survival rates increased as timewent by, and the inhibition effects reached utmost at the forth day after infection.Ad-Ki67also had inhibition effect on the proliferation of ACHN cells, whileG250-Ki67and G250-EGFP had no obviously inhibition effect on the proliferation ofACHN cells. The result among every group had significant difference (P<0.05).6. Cell apoptosis were detected byAnnexinV-PE/7-AAD. G250-Ki67had the mostpotent apoptosis induction effect on786-0cell line, comparing with the G250-EGFPand Ad-Ki67. Cell apoptosis induced by G250promoter controlled CRAds in ACHNcell lines were weak. G250-Ki67, G250-EGFP and Ad-Ki67had no obviously apoptosisinduction effect on HK-2cell lines.. The result among every group had significantdifference (P<0.05).Conclusion CRAds expressing Ki67-siRNA with G250promoter regulation andcontrol could high amplify and express Ki67-siRNA in renal cancer cells, inhibit renalcancer cells proliferation and induce apoptosis, targeted therapy for the renal cellcarcinoma. It may be used for further investigation of Gene-Viro therapy of cancer. Objective To investigate the effects of G250promoter controlled conditionallyreplicative adenovirus (CRAds) expressing short interference RNA targeting Ki67gene(Ki67-siRNA) on the growth of human renal cell carcinoma in nude mice, provideexperimental foundation in possible clinical use of CRAds for renal cell carcinoma．Methods Human clear cell renal cell carcinoma(786-0) cells were cultured in1640medium to exponential phase of growth and then transplanted under the skin ofBALB/c nude mice to develop the tumor model of human renal cell carcinoma. Afterthe model were successfully developed，40mice were randomly divided into fourgroups：G250-Ki67group, G250-EGFP group, Ad-Ki67group and PBS control group.Different virus included G250-Ki67,G250-EGFP and Ad-Ki67were directly injectedinto tumor on alternate days for3times at the dose of7×108PFU（0.2ml）each time.PBS control group used0.2ml PBS instead.4nude mice in each group were randomlykilled at the7th day after the injection of virus and tumors were dislodged.Immunohistochemistry staining was used to detect the protein expression of Ki67andE1A; the expression of E1A protein was detected by Western Blot; HE staining wasused to observe the tumor cells growth, the apoptosis of tumor cells was measured byTerminal dUTP nick-end labeling (TUNEL). The tumor volume of the rest animals weremeasured regularly to observe the tumor growth conditions. All animals were killed andthe livers were dislodged to detect the toxic effects with HE staining at the35th dayafter the treatment.Results The tumor model of human clear cell renal cell carcinoma weresuccessfully developed. Immunohistochemistry staining shows that the expressions ofki67protein of G250-Ki67group was markedly lower than the rest groups, there weresignificant difference, compared with other groups; Western blot andimmunohistochemistry staining show that there were expressions of E1A protein only inG250-Ki67group and G250-EGFP group, which was imperative for virus replication,the differences between the two groups was significant. The results of HE staining of tumors showed that PBS group stained light color and nucleus was split like adouble-leaf or leaves, which prompted the active cell proliferation. As a comparison,different levels of deeply nucleus staining and few mitotic could be seen in Ad-Ki67group, G250-EGFP group and G250-Ki67group, which indicated tumor growthinhibition. The inhibition effect from strong to weak followed by G250-Ki67group,Ad-Ki67group, G250-EGFP group and PBS group. The apoptosis in each treatmentgroup with TUNEL methods was PBS group（3.78±1.64）%，G250-EGFP group（27.25±1.10）%，AD-Ki67group（31.43±2.57）%，G250-Ki67group（65.32±3.24）%, G250-Ki67group with the remaining differences between the three groups wasstatistically significant （P<0.05).Inhibition of tumor growth curve showed thatG250-Ki67group had the strongest inhibition to tumor growth compared with othergroups. We did not find any change in nude mice liver with HE staining.Conclusion Oncolytic adenovirus G250-Ki67could inhibit tumor proliferationand promote apoptosis. As a result, it could inhibit the growth of tumor model of humanclear cell renal cell carcinoma in nude mice. This provided experimental basis forVirus-Gene therapy of kidney cancer. |
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