| 1. Background and ObjectiveGastric cancer is a common human gastrointestinal cancer which is a serious threat to the health of the people of Guangxi. Currently, the primary treatment for gastric cancer is surgery, chemotherapy, radiotherapy and so on. Despite diagnosis and chemotherapy drugs have made significant progress, the therapeutic effects of gastric cancer remains poorly. Gene therapy has become a new tool in the field of treatment and research in the cancer. As one of the most important transcription factors during the cell cycle and apoptosis, E2F-1is a well-characterized member of the E2F gene family. A lot of studies have demonstrated that disorders of E2F-1signaling pathways will lead to the cancer. In our previous studies, we found that E2F-1was a very stable and conservative gene, and it was overexpressed in gastric cancer tissues. This research suggests that E2F-1may play an important role in the occurrence and development of gastric cancer. Moreover, we constructed a silencing eukaryotic vector targeting E2F-1, and transfected into human gastric cancer cell line MGC-803. The result showed that growth rate of gastric cancer cells was slower than control group, and apoptosis rate of cells increased significantly. We also found that more gastric cancer cells were stoped at M phase when E2F-1was suppressed. It can be concluded that E2F-1gene served as an oncogene in vitro. In order to further investigate anti-tumor effects of silencing E2F-1gene in vivo, lentiviral vector of RNA interference (RNAi) of the E2F-1gene will be constructed and used in preliminary animal experiments. At the same time, we will use semi-quantitative reverse transcription PCR (RT-PCR), Western blot and yeast two-hybrid system to reveal its mechanism.2Methods2.1According to our previous research, the double-stranded DNA oligo (E2F-1-shRNA) which can cause most RNA interference (RNAi) effect to E2F-1was made. The shRNA sequences were annealed and linked with linearized into the pLentiLox3.7lentiviral vector (pLL3.7). The recombinants were named as pLL3.7-shRNA-E2F-1, and were identified by double restriction digestion with XbaI/NotI and DNA sequencing. Moreover, the recombinant lentivirus (Lv-shRNA-E2F-1) was harvested from293T cells when the pLL3.7-shRNA-E2F-1co-transfected with lentiviral packing materials into them after48h. The virus particles were collected. Virus titer was determined by hole dilution method. In addition, the negative control sequences which had no significant homology to any human gene sequences were constructed. This complementary hairpin DNA oligo were synthesized, annealed, and ligated into linearized pLL3.7vector, and virus particles were collected by criteria method mentioned above. The negative control recombinants were named as pLL3.7-shRNA-NC. The negative control virus particles were named as Lv-shRNA-NC.2.2Human gastric cancer cells MGC-803in logarithmic growth phase were obtained by centrifugation and stored. Five BALB/C nude mice were inplanted subcutaneously in flanks with200μl (1×106cells per nude) MGC-803cells to produce the tumor. When the maximum diameter of the tumor was longer than1cm, we pulled out the tumor, and cut it into several small pieces (1-2mm3) which were tansplanted into the rest of nude mouse subcutaneously to establish the nude mouse gastric cell carcinoma model. They were randomly divided into experimental group (Lv-shRNA-E2F-1group), negative control group (Lv-shRNA-NC group) and PBS group. The corresponding agents were injected100μl per time (5×108TU/ml) for a consecutive of6times on every two days, and measured the tumor volumes at the same time. The animals were sacrificed at11days, and the subcutaneous tumors were analyzed. Firstly, the tumor growth-curve was drawn, and the weight of tumor was measured. Secondly, the morphological changes of tumor cells were observed by the optical microscope. Thirdly, the expression of E2F-1in tumor was detected by immunohistochemistry, semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) and western blotting respectively. The apoptosis of tumor was measured by in situ end labeling technique (TUNEL).2.3In our previous studies, we found that PTEN expression was increased when the E2F-1was downregulated in MGC-803cells in virto by using the gene chip assay. Therefore, to investigate the mechanisms of E2F-1-shRNA induction of transplant tumor cell apoptosis, we observed the expression of some apoptosis-associated genes by semi-quantitative RT-PCR and western blot, such as PTEN, caspase-3, caspase-9and NF-κB in this research.2.4According to the results of our pilot experiments, we decide to remove the E2F-1activatied domain, and construct a bait vector of truncated E2F-1gene (309-1241bp) for yeast two-hybrid system. Above all, PCR primers were designed, and the gene sequence of truncated E2F-1was amplified from the PCMV-E2F-1-HA which was conserved in our lab by using PCR. Secondly, the truncated fragment was cloned into pGBKT7, and resulting recombinant plasmid was named as pGBKT7-E2F-1. This recombinant plasmid was transfered into AH109, and the autonomic activation and cytotoxicity of recombinants was determined by selective petri dish method. After that, The cDNA library of human gastric mucosa which was also conserved in our lab was screened with pGBKT7-E2F1as a bait plasmid by yeast two-hybrid system, and His+/Leu+/Trp+/Ade+/LacZ+positive yeast clones were obtained. In addtion, the inserted fragments of identified positive clones were confirmed and sequenced by using yeast turning test and BLAST tools.3Results3.1The small hairpin RNA sequences (shRNA) were successfully inserted into pLentiLox3.7vector by double restriction digestion, and the sequences were identified by DNA sequencing. The shRNA of E2F-1gene of the recombinant lentiviral vector was successfully packed into293T cells. The recombinant lentivirus was harvested from293T cells, and the titer of the virus of Lv-shRNA-E2F-1and Lv-shRNA-NC was3×109TU/ml and2×109TU/ml respectively.3.2The model of human gastric cancer in nude mice was successfully established by subcutaneous transplantation. The formation rate of transplanted tumour in Lv-shRNA-E2F-1, Lv-shRNA-NC and PBS group was90%,90%and80%respectively. The tumor growth-curve showed that the tumor growth rate in the Lv-shRNA-E2F-1treatment group was much slower than that in the Lv-shRNA-NC and PBS group (P<0.05).The weight of transplant tumor in Lv-shRNA-E2F-1group (0.637±0.061g) was lighter than Lv-shRNA-NC (2.225±0.079g) and PBS group (2.334±0.087g). The tissues got from the nude mice haven been diagnosed that was malignant tumor under microscope after HE stain. Immunohistochemistry results showed that there were a few of brown granules in Lv-shRNA-NC and PBS group, while they were almost not found in the Lv-shRNA-E2F-1group (P＜0.05). Compared with Lv-shRNA-NC and PBS group, the expression level of E2F-1mRNA and protein were decreased in the Lv-shRNA-E2F-1group (P＜0.05). The apoptotic rate in the Lv-shRNA-E2F-1treatment group was18.7±3.6%, significantly higher than that in the other two groups (6.5±0.7%for Lv-shRNA-NC and5.8±0.3%for PBS, P＜0.05). There was no difference to find between the Lv-shRNA-NC and PBS group in all the experiment mention above (P＞0.05).3.3To investigate the mechanisms of E2F-1-shRNA induction of transplant tumor cell apoptosis, we used semi-quantitative RT-PCR and western blot to examine the mRNA and protein expression of PTEN, Caspase-3, Caspase-9and NF-κB. Densitometry showed that PTEN, Caspase-3and Caspase-9mRNA expression in the Lv-shRNA-E2F-1group was higher while NF-κB was lower than that of the Lv-shRNA-NC and PBS groups (P＜0.05), and no difference was found between Lv-shRNA-NC and PBS groups (P＞0.05). In addition, E2F-1-shRNA induced cleavage of pro-caspase-3(35kDa) and pro-caspase-9(47kDa) into other multiple, cleaved, maturation products, but only the17kDa form of cleaved caspase-3and37kDa form of cleaved caspase-9were observered in tumor tissue. Densitometry showed that PTEN, p17cleaved caspase-3and p37cleaved caspase-9protein expression in the Lv-shRNA-E2F-1group was higher while NF-κB, pro-caspase-3and pro-caspase-9expression was lower than that in the Lv-shRNA-NC and PBS groups (P<0.05), and no difference was found between the Lv-shRNA-NC and PBS groups (P>0.05).3.4The bait vector pGBKT7-E2F-1was constructed successfully. The result of selective petri dish method showed pGBKT7-E2F-1could not autonomously activate the reporter gene, and it did not have cytotoxicity on AH109yeasts. After the cDNA library of human gastric mucosa was screened with pGBKT7-E2F1, we could obtain20His+/Leu+/Trp+/Ade+/LacZ+positive yeast clones.18different candidate gene sequences were confirmed by yeast turning test and BLAST analysis in NCBI, including IRF7, CB, GUK1, ATPIF1, RPSA, SERBP1, ASS1, MESDC2, WISP2, RCN1, CDC42EP1, EXOSC7, EFEMP1, FSTL3, MT2A, LGALS1, SIVA1and one unknown gene.4Conclusions4.1Lentiviral shRNA expression vector targeting E2F-1gene has been successfully constructed, which provides a stable vector for further study of the relationship between gastric cancer and E2F-1gene.4.2Intra-tumor injection of retrovirus Lv-shRNA-E2F-1has significantly inhibitory effect on the growth of human gastric subcutaneous tumor of Nude Mice. This lentiviral expression vetor would be an effective tool for gastric cancer treatment.4.3E2F-1-shRNA could increase PTEN, Caspase-3and Caspase-9expression while NF-κB expression was decreased. This result indicate that blocking E2F-1expression probably upregulates PTEN expression directly or indirectly, decreases NF-κB expression, activates Caspase-3and Caspase-9via PTEN/NF-Kb signaling pathway, and inhibits the growth of MGC-803cells in vivo.4.4The bait vector pGBKT7-E2F-1was constructed successfully. There was not self-activation activity and cytotoxicity.18different proteins interacting with E2F-1have been successfully screened from the cDNA library of human gastric mucosa by yeast two-hybrid system. At the same time, the prophase work related, we could illuminate mechanism How E2F-1silencing inhibited the growth of gastric cancer.Innovation1. So far, the effect of RNA interference targeting E2F-1on gastric cancer is still not clear. In present study, we investigated how E2F-1-shRNA affected the human gastric cancer cells growth in vivo by using lentivirus-mediated RNA interference system. Moreover, this study also for the first time searched the protein interacting with E2F-1by yeast two-hybrid system analysis. Combining with our previous research, the mechanism how E2F-1silencing inhibited the growth of gastric cancer could be illuminated. This study lays the foundation for treatment of gastric cancer through downregulation of E2F-1expression.2. Yeast two-hybrid sysstem is an effective tool to explore the gene function. This study for the first time searched the protein interacting with E2F-1at home and abroad by using yeast two-hybrid system, and helped clarify the role of E2F-1in gastric cancer. |
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