| Renal cell carcinoma(RCC)is one of the most common renal malignancies.It originated from renal tubular epithelial cells.The incidence rate is second in the urinary system.It is only next to bladder cancer.Renal cell carcinoma accounts for about 2%-3%of adult malignant tumors.Most patients lost the opportunity of surgical treatment.Patients who undergo radical resection will have recurrence or metastasis gradually,which is not sensitive to radiotherapy and chemotherapy.Therefore,it is urgent to find new treatment.In recent years,our research group has used Conditionally replicative adenovirus to regulate small interfering RNA or other therapeutic genes,such as IL-18,IL-24,to treat urinary system tumors,and achieved preliminary results,but it is far from complete elimination of tumors,because the target is not ideal.As a newly discovered actin binding protein,LASP-1(LIM and SH3 protein 1)is not only involved in the reorganization,regulation and migration of cytoskeleton,but also related to the progression of many kinds of malignant tumors,involving in the proliferation,invasion and metastasis of tumors.But its effect on the proliferation and migration of renal cell carcinoma have not been reported.We compared the expression of LASP-1 in renal cell carcinoma tissues and adjacent healthy tissues by immunohistochemistry and Western blot to explore its role in invasion and migration of renal cell carcinoma.At the same time,the LASP-1 gene was used as a target for the treatment of renal cell carcinoma,and the Conditionally replicative adenovirus loaded with siRNA of LASP-1 gene was constructed to observe its targeted therapeutic effect on renal cell carcinoma in vivo and in vitro,so as to find a new target for the treatment of renal cell carcinoma.Part Ⅰ Expression and significance of LASP-1 in renal cell carcinomaObjective To investigate the expression of LASP-1 in human renal cell carcinoma(RCC)and its role in the progression of RCC.Methods Western blot(WB)and immunohistochemistry were used to detect the difference of LASP-1 expression in tumor tissues and adjacent tissues of 82 resected renal carcinoma samples,to understand the relationship between LASP-1 and pathological characteristics of renal tumors.Results LASP-1 expression in RCC tissues was examined by immunohistochemistry.The results showed that the positive rate of LASP-1 staining was 90.24%(74/82)in 82 cases of renal carcinoma and 26.83%(22/82)in 82 cases of tumor-adjacent normal renal tissue.LASP-1 was positively stained in the nucleus of RCC cells.The expression of LASP-1 was correlated with lymph node metastasis,TNM tumor stage and tissue type(P<0.05),but not with tumor size or Furhman grade(P>0.05).We extracted the tissue protein of fresh RCC after resection and detected it by western blot in order to further investigate the correlation between LASP-1 expression and lymph node metastasis in RCC.Western blot analysis showed that the expression level of LASP-1 in adjacent tissues(0.818±0.064)and in the group without lymph node metastasis(1.443±0.154)was significantly lower than that in the group with lymph node metastasis(2.030±0.183),with statistical significance(P<0.05).Conclusion Detection of LASP-1 expression in renal carcinoma tissue can be used to understand renal tumor progression and evaluate prognosis.It also provides a new target for the targeted therapy of renal cancer.Part Ⅱ Construction and purification of conditionally replicative adenovirus expressing siRNA targeting LASP-1 geneObjective The plasmid pBHGE3 was transfected into 293 cells with pZD55-LASP-1 or pCA13-LASP-1 for constructing a new conditionally replicative adenovirus,ZD55-LASP-1 and a new replication-deficient adenovirus,AD-LASP-1.Methods Cell culture plates were covered with HEK293 cells.The plasmid pZD55-LASP-1 and pBHGE3 were infected with HEK293 cells using Effectene DNA transfection reagent.The plasmid pCA13-LASP-1 and pBHGE3 were infected with HEK293 cells using Effectene DNA transfection reagent.The viral plaques of the recombined replication-deficient adenovirus and conditionally replicative adenovirus appeared 1-2 weeks after infection.The DNA extracted from the recombined replication-deficient adenovirus and conditionally replicative adenovirus was verified by PCR.The correct virus was identified for amplification,purified by CSCL gradient centrifugation,and the titer of the virus was measured by 50%tissue culture infection volume(TCID50).Results Enzyme digestion and polymerase chain reaction(PCR)confirmed that ZD55-LASP-1 was not mixed with wild-type adenovirus and contained the target sequence LASP-1 siRNA.The E1 region of AD-LASP-1 is missing and contains the target sequence LASP-1 siRNA.These results indicate that the CRAds expressing LASP-1 siRNA(ZD55-LASP-1)had been constructed successfully.The titers of ZD55-LASP-1 and AD-LASP-1 were 4×1010 PFU/ml and 2×1010 PFU/ml respectively.The identified virus was amplified in the HEK 293 cell,and purified,and storaged at-80℃.Conclusion The successful recombination of ZD55-LASP-1,a virus targeting LASP-1 siRNA sequence,provides a basis for further gene therapy of renal cancer.Part III Conditionally replicative adenovirus armed with siRNA against LASP-1 gene for renal cancer therapy in vitro and in vivoObjective To study the effect of conditionally proliferating adenovirus ZD55-LASP-1 expressing LASP-1 siRNA on renal cell carcinoma in vivo and in vitro.It provides the experimental foundation in renal cancer Gene-Viro therapy.Methods In vitro:Western Blot was used to detect the expression of E1A protein in tumor cells and normal cells infected with ZD55-LASP-1,and the effect on the expression of LASP-1 protein in renal cancer cells.The cytotoxicity of ZD55-LASP-1 was detected by CCK8 assay.Cell invasion was detected by Matrigel invasion assay.The migration of 786-0 cells was evaluated by Migration assay.The apoptosis effect was determined by AnnexinV-FITC/PI staining.In vivo:The model of human renal carcinoma transplanted with 786-0 cells in nude mice was established.When tumors reached 100-150mm3,mice were divided randomly into four groups(8 mice for each group)and were treated by intratumoral injection of PBS,Ad-LASP-1,ZD55-EGFP or ZD55-LASP-1(100ul,contain virus 5×108 pfu,every other day for three times).The tumor volume was measured every seven days and time-volume growth curve was drawed.All animals were killed after 7 weeks and tested as follows:LASP-1 protein and E1A protein expression of tumor xenografts were detected by immunohistochemistry or immunofluorescence.The apoptosis of tumor xenografts was measured by Terminal dUTP nick-end labeling(TUNEL).We took the livers and lungs of nude mice to observe the change of pathobilolgy.Results:1.Recombinant adenovirus ZD55-LASP-1 replicated selectively in RCC cells.Western Blot results showed that ZD55-LASP-1 and ZD55-EGFP E1A genes were positively expressed in 786-0 cells,suggesting that the two proliferating viruses could proliferate in 786-0 cells.However,neither of the two viruses expressed E1A protein in non-tumor cells(proximal convolute tubular epithelial cells of renal cortex,HK-2 cells),suggesting that the novel proliferating virus ZD55-LASP-1 has the characteristic of targeted proliferation in tumor cells.2.ZD55-LASP-1 inhibited LASP-1 expression in RCC cells.To evaluate the efficiency of silencing LASP-1 expression by ZD55-LASP-1,we infected 786-0 cells with ZD55-LASP-1,ZD55-EGFP or PBS at MOI of 10.Western blot analysis showed that the infection of cancer cells with ZD55-LASP-1 at a MOI of 10 led to significant decrease in LASP-1 protein level.Relative LASP-1 protein level was(0.417±0.052)of the control for 786-0 cells infected with ZD55-LASP-1 at MOI of 10,while the infection of ZD55-EGFP(0.753±0.026)and blank group(0.817±0.018)had little effect on LASP-1 protein level.3.ZD55-LASP-1 inhibited the viability of RCC.To investigate the cytotoxicity of ZD55-LASP-1,CCK-8 assay was performed to examine cell viability.ZD55-LASP-1 exhibited higher cytotoxicity in 786-0 cells than ZD55-EGFP.After 4 days,cell viability of PBS group(2.448±0.128)and ZD55-EGFP group(2.300±0.209)were significantly higher than that of ZD55-LASP-1 group(1.388±0.095).These results indicated that ZD55-LASP-1 inhibited the growth of RCC cells more effectively than ZD55-EGFP.4.ZD55-LASP-1 inhibited the migration and invasion of RCC cells.The number of 786-0 cells that migrated the membrane in ZD55-LASP-1 group was(28.833±5.209),significantly less than that of ZD55-EGFP group(79.333±4.459),and PBS group(80.167±5.113).The difference was statistically significant(P<0.01).The number of 786-0 cells that invaded the membrane in ZD55-LASP-1 group was(22.433±7.299),significantly less than that of ZD55-EGFP group(69.587±4.249),and PBS group(70.610±6.519).The difference was statistically significant(P<0.01).These results suggest that the new recombinant adenovirus ZD55-LASP-1 inhibits the invasion and migration of 786-0 cells by silencing LASP-1 gene.5.ZD55-LASP-1 increased the apoptosis rate of RCC cells.To confirm that ZD55-LASP-1 induced apoptosis of RCC cells,we detected AnnexinV-FITC/PI staining.The results showed that the apoptosis rate of ZD55-EGFP treated group(8.932±0.663)%and PBS group(8.724±0.745)%were significantly less than that in ZD55-LASP-1 treated group(19.134±0.871)%(P<0.05).6.ZD55-LASP-1 may influences invasion and metastasis via the EMT pathway.In order to explore the mechanism of LASP-1 in the invasion and metastasis of renal carcinoma,we studied related proteins in the EMT pathway.The ZD55-LASP-1 could down-regulate the expression of LASP-1 protein in 786-0 cells remarkably.The relative expression of E-cadherin protein in ZD55-LASP-1 group(0.848±0.020)was significantly higher than that in ZD55-EGFP group(0.671±0.018)and PBS group(0.691±0.037),and the difference was statistically significant(P<0.05).The relative expression of N-cadherin,Vimentin protein was significantly lower(0.449±0.047),(0.477±0.029)than that of ZD55-EGFP group(0.613±0.018),(0.598±0.069)and control group(0.633±0.045),(0.635±0.015),the difference was statistically significant(P<0.05).Therefore,it is speculated that LASP-1 may promote the progression of renal cancer through EMT.7.ZD55-LASP-1 can effectively inhibit the growth of xenograft tumor in nude mice.Based on the in vitro data above,we investigated the effects of ZD55-LASP-1 treatment on tumor growth in vivo.Our research demonstrated that the potent antitumor activity of ZD55-LASP-1 is more evident than that of ZD55-EGFP and Ad-LASP-1 in renal cancer xenograft tumors.The tumor volume of the ZD55-LASP-1 group was(355.4±47.4 mm3),which is much smaller than that of the ZD55-EGFP group(541.2±56.1 mm3,P<0.05),the Ad-LASP-1 group(720.1±74.5 mm3,P<0.05)and PBS group(1051.1±122.7mm3,P<0.05).8.Immunofluorescence technique for E1A protein indicated that ZD55-LASP-1 and ZD-55-EGFP group can express E1A protein,but Ad-LASP-1 and PBS can not.It demonstrated ZD55-LASP-1 and ZD55-EGFP can duplicate in renal carcinoma.9.ZD55-LASP-1 markedly reduce LASP-1 expression levels in vivo.Immunohistochemical analysis showed that ZD55-LASP-1 treatment group could significantly inhibit the protein expression of target gene LASP-1 compared with ZD55-EGFP,Ad-LASP-1 and PBS groups.The difference was statistically significant(P<0.05).10.ZD55-LASP-1 promotes tumor cell apoptosis in vivo.TUNEL staining showed markedly more positive cells in the group treated ZD55-LASP-1(45.9±4.6%)that was significant than in the control groups(P<0.05),ZD55-EGFP treated group(34.9±3.2%),Ad-LASP-1 treated group(26.3±3.1%)and PBS treated group(13.8±1.6%).11.Histopathological examination showed that the chromatin in the nucleus of PBS group was loose,nucleoli were seen,and cell proliferation was active.However,in the ZD55-LASP-1,ZD55-EGFP,Ad-LASP-1 treatment group,deep staining of nuclei was seen in varying degrees,and nucleoli were rare,which showed tumor tissue growth inhibition.Some necrosis areas were found in ZD55-LASP-1 and ZD55-EGFP.12.HE staining results of lung tissue showed that no metastasis was found in the lung tissue of ZD55-LASP-1 and AD-LASP-1 treatment groups,but 3 and 1 nude mice in the PBS treatment group and ZD55-EGFP treatment group had lung metastasis respectively,which indicated ZD55-LASP-1 can effectively inhibit distant metastasis of tumor by silencing LASP-1 gene..13.We did not find any change in nude mice liver.Conclusion The conditionally replicative adenovirus ZD55-LASP-1 can replicate in renal carcinoma,inhibit proliferation and induce apoptosis of 786-0 cells and its tumor xenografts by inhibiting LASP-1 gene expression.It provides experimental basis for gene targeting therapy of renal cell carcinoma. |
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