Expression Of HPA MRNA In Gastric Carcinoma And Invasion Inhibition Of Gastric Carcinoma In Vitro

Gastric carcinoma is one of the most common malignant tumors in China, which greatly affects human health due to the uncontrolled cloning growth of tumor cells, ultimately, the cause of death for gastric cancer patients is the invasion and metastasis of carcinoma.Spread of malignant tumors is a multi-step process and many stages of tumor invasion require degradation or breakdown of the physical barriers composed of extracellular matrix (ECM) and basement membrane(BM). The major molecular constituents of the barriers are structural proteins and heparan sulfate proglycan (HSPG). matrix metalloproteinases(MMPs) and heparanase(HPA) facilitate invasion and metastasis of tumor cells by disassembling the ECM and BM. Previous studies have indicated that MMP9 and their natural inhibitor-TIMP1 (tissue inhibitor of metalloproteinasel) play very important roles in the metastasis of gastric carcinoma, but HPA which degrading HSPG was not explored deeply by scientists. Since HPA gene was cloned in 1999, HPA has attracted more and more attention because of its key function in traversing BM process of tumor invasion . Recently, some studies have indicated that HPA mRNA is highly expressed in some human aggressive malignant tumors, such as: breast cancer, lung cancer, and liver cancer, gastric carcinoma is also one of the most aggressive malignant tumors. However, there is very lack of associated reports on the relationship between HPA and gastric carcinoma.At present, it have not been studied that HPA, MMP9 target therapy for gastric carcinoma with antisense RNA technology and sense TIMP1 transfection.In order to explore the relationship between HPA mRNA expression and the metastasis of gastric carcinoma, and to discuss the suppressing effects of antiHPA, antiMMP9 and sense TIMP1 transfection on the invasion and metastasis of human gastric carcinoma cells, in this study, RT-PCR and in situ hybridization were performed to detect the expression of HPA mRNA in fresh surgical specimens of gastric cancer tissues and the relationship of its expression to clinicopathological parameters was analyzed. Based on the results, antisense RNA eukaryotic expressing vectors pcDNA3.1-HPA and pcDNA3.1-MMP9 were constructed, simultaneously recombinant TIMP1 enhanced green fluorescent protein eukaryotic expressing vector PEGFP-C3-TIMPI was constructed. After BGC823 gastric carcinoma cell lines were stably transfected with three different recombinant expressing vectors, the cellular localization of TIMP1 was observed by fluorescent reverse microscope; Cell cycle of BGC823 cells was evaluated using flow cytometry; Both mRNA and protein expressions of HPA, MMP9 and TIMP1 in BGC823 cells were examined with RT-PCR and western blot. The invasion ability of BGC823 cells was detected by method of Boyden chamber experiment in vitro. Ultimately the antimetastatic effects of downregulating HPA, MMP9 was discussed from many aspects including: gene level, protein level, cell cycle, cellular localization and biological behavior of tumor cell, it may provide support to anti-carcinoma clinical treatment with antisense RNA technology. Part oneClinicopathological significance of HPA mRNA expression in gastric carcinoma Methods1. RT-PCR was performed to detect and semi-quantitate expression of HPA mRNA in normal mucosa tissues, gastric cancer tissues and surrounding tissues adjacent to tumor.2. Expression and morphological analyze of HPA mRNA in normal mucosa tissues and gastric cancer tissues were observed with in situ hybridization.3. Statistical analysis SPSS12.0 software was used to perform the x2test, Fisher's exact probability test, t test, analysis of variance. P value less than 0.05 was considered statistically significant.Results1. Expression of HPA mRNA in normal gastric mucosa tissues, surrounding tissues adjacent to tumor and gastric cancer tissues showed a tendency to be higher in order.(PO.05)2. The positive rate of HPA mRNA in the groups with serosal invasion or lymph node metastasis were significantly higher than those in the groups without serosal invasion or lymph node metastasis. (PO.05)3. The expression of HPA mRNA was positively correlated with poorer differentiation.(P<0.05)4. The expression of HPA mRNA had no relationship with gender, age, Borrmann gross type and tumor location. (P >0.05)5. The positive expression of HPA mRNA was located in the cytoplasm of cancer cells. Strong staining was seen in cancer cells invading muscle and serosal layers.Part twoConstruction and identification of eukaryotic expressing vectors for antisenseHPA, MMP9 and sense TIMP1Methods1. antisense HPA, antisense MMP9 and sense TIMP1 cDNA fragments were obtained with RT-PCR from human gastric cancer tissues.2. The PCR products of antisense HPA, antisense MMP9 and sense TIMP1 were purified using DNA purification technology.3. Eukaryotic expressing vector for antisense human HPA was constructed with DNA recombinant technology.4. Eukaryotic expressing vector for antisense human MMP9 was constructed with DNA recombinant technology.5. Eukaryotic expressing vector for sense human TIMP1 was constructed with DNA recombinant technology.6. Three recombinant eukaryotic expressing vectors were identificated with PCR,double restrictive endonuclease digestion and DNA sequence analysis. Results1. AntiHPA fragment with 108bp, AntiMMP9 fragment with 204bp and senseTIMPl fragment with 624bp were amplied with RT-PCR as expected.2. Recombinant pcDNA3.1-AntiHPA> pcDNA3.1-AntiMMP9 and pEGFP-C3-TIMPl were identificated, the results showed: the length of amplied fragments with PCR and the length of digested fragments by BamHI/Hindlll were correct as designed.3. DNA sequence analysis of the inserted fragments revealed the same sequence as that of partial HPA, MMP9 and TIMP1 cDNA published in Genbank, and three target fragments were independently inserted into expressing vectors in orientation.Part threeDown regulation of invasiveness in BGC823 cells by transfections of antisense-HPA, antisenseMMP9 and sense TIMP1Methods1. The cellular localization of TIMP1 protein was observed by fluorescent reverse microscope.2. Cell cycle of transfected BGC823 cells was evaluated using flow cytometry3. Both mRNA and protein expressions of HPA, MMP9 and TIMP1 in BGC823 cells were examined with RT-PCR and western blot.4. The invasion ability of BGC823 cells was detected by method of Boyden chamber experiment in vitro.5. Statistical analysis SPSS12.0 software was used to perform t test and analysis of variance . p value less than 0.05 was considered statistically significant.Results1. The expression of pEGFP-C3-TIMPl fusion protein was located in the cytoplasm of around the karyon, high green fluorescent indensity was observed.2. The G2/M percent of cells transfected antiHPA was increased obviously and S-period frequency was decreased corresspondingly, compared with normalcontrol, apoptosis ratio was also increased, the orther transfected groups had no remarkable variance.3. RT-PCR and Western blot showed: After pcDNA3.1-AntiHPA was transfected to BGC823 by lipofectin reagent, the expressions of HPA mRNA and protein in BGC823 were inhibited remarkably, but the expressions of MMP9 mRNA and protein were not affected by pcDNA3.1-AntiMMP9 transfection. In pEGFP-C3-TIMPl group, TIMP1 mRNA level was significantly higher than normal control group, western blot indicated: immunostaining could be seen in 28 kD and 55kD level ,but only a 28 kD positive staining was seen both in normal control and in pEGFP-C3 group.4. Boyden chamber assay showed: Compared with normal control, the number of cells traversed Matrigel was decreased obviously in both pcDNA3.1-AntiHPA group and pEGFP-C3-TIMPl group (PO.05), but in the following groups: pcDNA3.1, pEGFP-C3 and pcDNA3.1-AntiMMP9, the number of cells traversed matrigel was not notably influenced. (P >0.05)Conclusions1. It is the first time to investigate the expressions of HPA mRNA in gastric carcinoma tissues, surrounding tissues adjacent to tumor and corresponding normal gastric mucosa tissues with RT-PCR and in situ hybridization , the overexpression of HPA mRNA in gastric carcinoma was found.2. The positive expression of HPA mRNA correlates with serosal invasion and lymph node metastasis , hence might be a novel biomarker for invasion and metastasis of gastric carcinoma.3. The frequency of HPA mRNA positive expression is inversely correlated with the grade of carcinoma cell differentiation, it was concluded that HPA might play animportant role in the development of gastric carcinoma.4. It is the first time to construct antisense HPA eukaryotic expressing vector, which should be used to study biological function and target therapy of HPA.5. Antisense MMP9 eukaryotic expressing vector was successfully cloned, which facilitate the exploration of MMP9 gene therapy.6. It is the first time to construct sense TIMP1 eukaryotic expressing vector, which establish the foundation for further studying the effects of TIMP1 in metastasis of gastric carcinoma and antiMMP9 gene therapy of carcinoma.7. Transfection of antisense HPA could suppress invasive potential in vitro of BGC823 cells by downregulating the levels of HPA mRNA and protein in tumor cells.8. It was investigated for the first time that the action of HPA in development of carcinoma with flow cytometry , the result indicated: the SPF of BGC823 cells was decreased by antisense HPA, it implicated that decreased SPF might hold back cell invasion.9. Increased TIMP1 in BGC823 cells could suppress cellular invasive potential in vitro probably due to inhibition of MMP9.