Effect Of Gastric Cancer Related Gene GCRG213 On Gastric Cancer In Athymic Mouse And The Correlation With APE/Ref-1

[Background]A differentially expressed new gene, GCRG213, was screened from human intestinal type gastric cancer, paratumor and non-tumor tissues in our laboratory. Through conserved domain database search in GenBank, a putative conserved domain, Human apurinic/apyrimidinic endonuclase/redox-factor 1(APE/Ref-1), was detected in the deduced amino acid sequence of GCRG213—ORF, it shared 61.0% alignment with the C-terminal region of APE/Ref-1 conserved domain. Compared with MKN45 cells transfected with vector, the cells tranfectd with sense fragment of GCRG213 (GCRG213a) had high growth velocity and oncogenicity, transfected with anti-sense fragment of GCRG213(GCRG213b) or GCRG213 RNAi fragment(GCRG213i) had low growth velocity and oncogenicity. Here we futher investigate the effects of these fragments tranfection in vivo, and if GCRG213 was correlated with APE/Ref-1.The adeno-associated virus type 2 (AAV-2) is one of the most promising candidates for gene therapeutic applications. The non-pathogenic nature of the virus, infection of dividing and non-dividing cells, the potential for site-specific integration as well as the low immunogenicity are advantages in favor of AAV-2.[Aims]1. To investigate the results of sense or anti-sense GCRG213 fragment and GCRG213 RNAi fragment tranfection in vivo on gastric cancer.2. To elucidate if GCRG213 was correlated with APE/Ref-1 through comparing their protein expression in tissue and cells.3. To study the expression of APE/Ref-1 in gastric cancer, analyze it's association with clinicopathologic parameters and survival.[Methods]1. The sense and anti-sense fragment of GCRG213 (GCRG213a and GCRG213b), which was introduced the sites of restrictive endonuclease enzyme BamH I , Pst I, and BamH I ,Cla I , was obtained by PCR and inserted into AAV vector respectively. HEK293 (human embryonic kidney cell line 293) cells were co-transfected with 10 ug rAAV vector plasmid, 10 ug phelper plasmid and 10 ug pRC plasmid by calcium phosphate precipitation. HEK293 cells were harvested 72 h after transfection and were subjected to three cycles of freezing and thawing to release virus. rAAV preparations were purified and concentrated using column chromatography. The titration of rAAV particles was assayed using quantitative real-time PCR.2. GCRG-213i with U6 promoter was obtained from IMG-800-GCRG213i-2 plasmid using BamH I and Bgl II, and inserted into pSANV2.0 vector. BHK-21 cells were transfected with new plasmid pSNAV2.0-U6-shRNA by LipofecLamine2000. After G418 selection, the cells were transfected with helper virus HSVl-rc/ A UL2. 48h later, the new rAAV can be purified and concentrated.3. 20 athymic mouses were divided into 5 groups randomly. Every group had 4 mouses. 2X 106MKN45 cells were subcutaneously inoculated in per athymic mouse. After tubercles were formed, 3 groups were respectively injected 1 of 3 rAAVs (IX 1010 virus particles per mouse) into tubercle, as comparison the 2 other groups were respectively injected with NS or rAAV-GFP. 2 weeks after injection athymic mousse were executed. Tumor tubercles were weighed. Expression of GCRG213 mRNA in tumor tissue was detected with RT-PCR.4. To detect GCRG213 and APE/Ref-1 protein in normal gastric mucosa, gastric cancer tissue, normal gastric epithelial cell lines HFE-145 and GES-1, gastric cancer cell lines SGC7901 and MKN45 by IHC (immunohistochemistry). The location and levels of GCRG213 and APE/Ref-1 protein were compared.5. APE/Ref-1 protein was detected and quantitated in MKN45-213a, MKN45-213b and MKN45-213i-2 cells, which was transfected with GCRG213a, GCRG213b and GCRG213i, and in which GCRG213 protein was increased or decreased respectively. To analyze if the changes of GCRG213 protein affectAPE/Ref-1 expression.6. APE/Ref-1 protein was detected in gastric tumor lesion,nonneoplastic mucosa and metastasis lymph node with gastric cancer TMA(tissue microarry) by IHC. Detailed analysis of APE/Ref-1 expression with clinicopathologic parameters and survival was performed.[Results]l.The recombinant adeno-associated virus rAAV-GCRG213-s, rAAV-GCRG213-a and rAAV-U6-shRNA were produced, the titration of rAAV particles was 4 X 10l0v.g./ml, 4 X 1010v.g./ml and 5 X 1012v.g./ml respectively.2. 3 weeks after MKN45 cells were injected, a small tumor tubercles was formed. Compared to groups which were injected rAAV-GFP or NS, tumor growth faster in groups injected rAAV-GCRG213-s, and GCRG213mRNA expression in tumor tissue increased, on the contrary, tumor growth slower in groups injected rAAV-GCRG213-a or rAAV-U6-shRNA, and GCRG213mRNA expression in tumor tissue decreased.3. In normal gastric mucosa and tumor lesion GCRG213 was widespread expression in cytoplasm, cytomembrane and stroma, principally in cytoplasm. But in normal gastric mucosa APE/Ref-1 can be deteced in cytoblast, in tumor lesion in cytoblast and cytoplasm.4. In HFE-145, GES-1, MKN45 and SGC-7901, GCRG213 protein all located in cytoplasm, on the contrary, APE/Ref-1 protein in cytoblast.5. APE/Ref-1 expression at protein level in MKN45-213a, MKN45-213b, MKN45-213i-2 and MKN45 had no significant difference. Gray scale was 121.586 + 8.29(k 124.783 + 11.436, 127.776 + 6.(^ 139.678 +10.987 respectively.6. As displayed by IHC, in nonneoplastic mucosa, tumor lesion and metastatic lymphnode the positive rate of APE/Ref-1 in cytoblast was 96.6%, 97.1% and 98.9%;in cytoplasm was 71.8%, 21.4% and 10.0%;both in cytoblast and cytoplasm was 71.4%, 21.4% and 10.0% respectively. Compared to nonneoplastic mucosa, tumor lesion had lower APE/Ref-1 expression in cytoblast (X2=13.914, P=0.045) andcytoplasm (X2=l3.524, P=0.035);35.9% slightly decreased and 11.2% markedly decreased in cytoblast;42.4% slightly decreased and 21.4% markedly decreased in cytoplasm;In nonneoplastic mucosa and metastatic lymphnode, APE/Ref-1 expression wasn't related with clinicopathologic parameters. In tumor lesion APE/Ref-1 expression level in cytoblast decreased with deeper invasion (P=0.000), lymphnode metastasis (P=0.010) and TNM stage progression (P=0.000), and in femal was lower than in male (P=0.048), in cytoplasm decreased with lymphnode metastasis (P=0.017) and advanced stage (P=0.019). The similar results could be deduced to decreased degree of APE/Ref-1 expression in cytoblast and cytoplasm in tumor lesion. In Kaplan-Meier analysis, the expression level or the expression change status were of no statistical significance to prognosis.[Conclusions]l.No apparente similarity was found between GCRG213 and APE/Ref-1 protein expression.2. The positive rate of APE/Ref-1 expression was high in gastric cancer tissue,nonneoplastic mucosa and metastasis lymph node. In gastric cancer tissue APE/Ref-1 expression is lower, and it decreased with deeper invasion, lymphnode metastasis and TNM stage progression. No significant relation was found between APE/Ref-1 expression and survival.3. Increase or decrease GCRG213 expression could promote or inhibit growth of gastreic cancer. Target gene can be effectively expressed in vivo through AAV vector. After inserting U6 promoter, a AAV vector which could be used as a RNAi vector was constructed.