| BackgroundGastric cancer is a common tumor in the digestive system, the mortality of it ranks second in the world. In developed countries, the incidence of gastric cancer in recent years has declined, but in China, according to the latest statistics, gastric cancer mortality of malignant tumor mortality is in third place in urban,while in rural areas, its mortality rate even resides in first. Surgery is still the main treatment, but only30-50%of patients get radical resection. Although there are more drugs treatment at present domestic,the effect has been no significant improved. To investigate the mechanism and its markers of early metastasis of gastric cancer is particularly important.In2009stein et al. have reported the possible function of gene MACC1(Metastasis-associated in colon cancer1) in colon cancer.they found that the expression of MACC1level is involved the survival rate. Patients with low expression of5-year survival rate is80%, while high expression of5-year survival is only15%. The expression of MACC1level is significantly higher in patients with metastasis colon cancer(p<0.0001).Logistic and Cox regression analysis show that MACC1can be used as prognostic indicators of metastasis in colorectal cancer. Followed by a large number of studies reported MACC1not only in colorectal cancer, but also in many solid tumors such as hepatocellular carcinoma, lung cancer, gastric cancer, which involved metastasis. Among them, Some scholars have found the expression of MACC1is higher in hepatocellular carcinoma cell with vascular metastasis. Shimokawa H have been collected for146specimens I pulmonary adenocarcinoma after surgery, they found the expression of MACC1is closely related the prognosis. And there has reported the expression of MACC1associated with peritoneal-disseminated gastric carcinoma. These indicate that the MACC1in the early detection of tumor metastasis and recurrence has an important role in the guidance, most likely to be as a tumor prognostic molecular marker. But the role of MACC1in gastric cancer needs further explore.To investigate the function of MACC1requires a lot of experiments in vitro and vivo, so it is necessary to build a appropriate MACC1expression vector for its structure and function. We have not see the related reports in the domestic. In this study,293FT cells as the template, we successfully amplified MACC1length cDNA sequence and MACC1interference fragment to construct the expression vector and interfere expression vectors,it will be the basis to study the the mechanism and foundation of MACC1in a variety of tumor types. There has reported the expression of MACC1associated with peritoneal-disseminated gastric carcinoma, in order to further study the mechanism of action, for the first time, we successfully establish two cell lines which can stable express of MACC1(pBaBb MACC1/BGC-823)and inhibit the expression of MACC1(sh-MACC1/BGC-823), and they are verified by qRT-PCR and Western blot, these will provide a reliable guarantee to study the the mechanism of MACC1.Gene chips is also called DNA microarrays (DNA microarray),the technology is that lots of probes and molecules sample will be fixed and hybrided in supporter, through detection the strength of hybrid signal we obtain samples of the elements and sequence number information. By designing different probe array, using the specific method of analysis allows the technology with a variety of value, such as gene expression profile determination, mutation detection, polymorphism analysis, genomic library mapping and hybridization sequencing.Along with the human genome project development, the structure genome team study has consummated day by day, but lots of unknown in the function genome team, the gene chip technology has provided the new platform for it, we can screen the genes of differently expressed quickly in different organs、organization and cells, and to further study the role in body. The happening、development and metastasis of gastric cancer is the results of interaction of multiple factors and multiple abnormal gene expression, the specific molecular mechanism is still not clear.Gene expression profile chip is now being applied more widely, the technology is relatively mature, it can detect changes in levels of mRNA expression of the entire genome. Traditional research methods is limited to one or a few genes and their simple regulation of the relationship, unable to fully understand and study tumors occur throughout the complex process of gene regulation. The gene chip is precisely to solve the inadequacies of the traditional research methods, its large-scale and high-throughput method to study thousands of gene expression under physiological and pathological conditions, to predict their function and the complex between their mutual regulation relationship. To investigate the change of tumor related genes in the gene level, through gene chip screen and detection the differences expressed genes in tumor formation、development and metastasis, it is not only to find the cause in the pathological, but also can provide the basis for early diagnosis and prognosis, gene chip technology and clinical close integration will be a certain trend in future.Therefore in this research we through the gene reprint, has constructed the MACC1expression and the silence cell line, they are pBaBb-MACC1/BGC-823and sh-MACC1/BGC-823, And then we use gene chips to detect and analysis the two cell lines, found that different expression of MACC1caused extensive changes in the two cell lines, where LHX9, PDK3and PLEKHA5level and fully consistent with the expression of MACC1trends, IL-8、MYO1A and OST-(3are exactly the opposite of the trend, and thus through literature review, hoping to find the relationship between MACC1regulation and early metastasis of gastric cancer. For the next step function research laid a foundation.Objective1. To establish two cell lines which can stable express of MACC1(pBaBb MACC1/BGC-823)and inhibit the expression of MACCl (sh-MACC1/BGC-823) through the gene reconstruction technology.2. To investigate the different expression of genes between pBaBb-MACC1/BGC-823, sh-MACC1/BGC-823and BGC-823using microarray.3. Looking for differentially expressed genes which is consistent and opposited with the expression of MACC1, to approach the possible relationship between them in cell signaling pathways and regulation mechanism.Methods1. The full-length MACC1cDNA was amplified from hunman embryonic kidney293FT cells and cloned into the pBaBb-puro vector and interference MACC1sequence fragments vector. By cleavage identification sequencing and observing293FT cells gene expression to determine whether the reconstruction was successful. Then we transfect the two vectors into BGC-823, after the end of48h by adding the puromycin (0.5μg/ml) and screening of positive clone, cultivation and amplification in the flasks and propagating the derivation. Collect cell RNA, qRT-PCR and Western blot and detect of MACC1expression in these cells to identify stable cell lines.2. The work of Microarray is completed by Shanghai Kang cheng bio-engineering company. To study the genes of differently expressed genes of gastric cancer cell line BGC-823after MACC1gene transfection with NimbleGen microarray, it contains29250genes. First,we extract the total cellular RNA, reversely transcribe into cDNA, mark with NimbleGen, then hybridizes to NimbleGen to hybridization system and wash with NimbleGen buffer kit, the result scan by Axon Genepox4000B scanner, through the NimbleScan software read data, to input Agilent the GeneSpringGX software further to analyze before standardization analyses. The difference gene screening standard for ratio>2, in order to avoid the experimental error, each cell line duplicates3times respectively.3. To find the genes which up regulation in the pBaBb-MACC1/BGC-823cell line-. down regulation sh-MACC1/BGC-823cell line and down regulation in the pBaBb-MACC1/BGC-823cell line、up regulation sh-MACC1/BGC-823cell line, using qRT-PCR to confirm. Through the literature consult, attempt to find the correlation with MACC1, provide the basis for early gastric cancer metastasis research.Results1. The full-length MACC1cDNA was amplified from hunman embryonic kidney293FT cells successfully and it is completely correct by sequencing. Transfected293FT cells can be seen clearly green fluorescent protein. It is successfully constructed and verified by PCR and sequencing.2. The result of cDNA microarray suggested the changes of the expression profile induced by MACC1and interfere fragment transfection were very wide. These differentlly expressed genes function involve various aspects which include cell apoptosis and cycle regulation、neoplastic progression、enzyme active regulation, cell signal transcription and translation regulation. We find six genes which up regulation in the pBaBb-MACC1/BGC-823cell line、down regulation sh-MACC1/BGC-823cell line and down regulation in the pBaBb-MACC1/BGC-823cell line、up regulation sh-MACC1/BGC-823cell line, the results are consistent with qRT-PCR and statistical analysis.3. Access to a large number of documents, the genes structure、function and other aspects of LHX9、PDK3、PLEKHA5、IL8、MYO1A and OST-β research and analysis to identify possible correlation with MACC1and tumor metastasis, which may be provide direction for studying early gastric cancer metastasis.Conclusion1. pBaBb-MACC1/BGC-823and sh-MACC1/BGC-823cell lines have been successfully established to facilitate further functional study of MACC1gene.2. The result of cDNA microarray suggested the changes of the expression profile induced by MACC1and interfere fragment transfection were very wide in pBaBb-MACC1/BGC-823and sh-MACC1/BGC-823cell lines. But there are only three genes which expression trend consistent with MACCl and completely opposite with it.the results are consistent with qRT-PCR and statistical analysis, those also suggest the microarray results which is reliability and the lower false positive rate.3. The changes of the expression profile induced by MACC1and interfere fragment transfection were very wide. These differentlly expressed genes function involve various aspects which include cell apoptosis and cycle regulation neoplastic progression enzyme active regulation, cell signal transcription and translation regulation.4. Analysis of the differently expressed genes found MACC1genes cause some very important signal transduction pathway in gastric cancer gene expression profile of molecules change, of which MACC1expression is trend exactly the same and opposite the gene may occur in gastric cancer development and with MACC1signal transduction correlation. |
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