| [background]Gastric cancer is one of the most common malignant tumor in the world, and it has a high incidence in our country. Although the incidence of gastric cancer has decreased in recent years, the mortality rate was still high. The emergence of peritoneal metastasis marked the disease has entered into the advanced course. About 60% patients with advanced gastric cancer died of peritoneal metastasis, which lead to the refractory cancer ascites and cachexia and so on. In the recurrence of gastric cancer after surgery, peritoneal metastasis account in 50%. And it is becoming one of the main cause of deadth. Peritoneal metastasis of gastric cancer is considered to be a multi-molecular involved and complex process. First, the gastric cancer tissue infringe into the serosa of stomach, and into the abdominal cavity, and then the fall off tumor cells will be planted in the peritoneal tissue. This is a whole process of the formation of peritoneal metastasis of gastric cancer. This is widely recognized as the"seed-soil"theory. Once the peritoneal metastasis has emerged in gastric cancer patients, usually the clinical treatment is not very well. So, to find a target molecule which participant in peritoneal metastasis is very important. We can do some target treatment in furture.Fortunately, we have successfully established a high peritoneal metastasis of gastric cancer cell lines GC9811-P in our previous work. Using a phage display library, we isolated a specific peptide that selectively bound to GC9811-P cell, it was named peptide PIII. Further screening of differentially expressed genes, and combinating with affinity chromatography, we have obtained a molecule of peptide PIII receptor—heme oxygenenase-1(HO-1). Initially speculated it was a specific target molecule of peritoneal metastasis of gastric cancer. Heme Oxygenase-1(HO-1 )is a rate-limiting enzyme which catalyzes the oxidation of heme to biologically active products: ferrous iron, carbon monoxide(CO) and biliverdin. And then the biliverdin convert to bilirubin by biliverdin reductase. There are three isoforms of heme oxygenase, HO-1 is the highly inducible enzyme by heme and various other stimuli including oxidative stress. HO-2 is the constitutively expressed isoform. A third isoform HO-3 was only discovered in recent years, preliminary studies have shown that it may be have a similar effect to HO-2. Since 1986 it has been found, a large number of studies have shown that HO-1 participated in various oxidative stress and inflammatory responses. It proved that it has anti-inflammatory, anti-apoptotic and cell protective effect. In recent years, studies have shown that HO-1 was highly expressed in a variety of malignant tumors. And participating in anti-apoptotic in tumors. Also it plays a regulatory role in tumor angiogenesis and metastasis. This subject will test HO-1 expression in the high peritoneal metastasis of gastric cancer cell lines GC9811-P. At the same time, we will construct small interfering RNA eukaryotic expression vector of HO-1. Further to explore the effection in invasion and metastasis of HO-1 siRNA on gastric cancer cell lines GC9811-P in vitro.[Objectives]1,To clear whether HO-1 is high expressed in the high peritoneal metastasis of gastric cancer cell lines GC9811-P.2,To explore whether it would affect the ability of invasion and metastasis in vitro while down-regulate HO-1 expression in GC9811-P.[Methods]1,To detect HO-1 expression in GC9811-P in protein and mRNA levels by Western-blot and RT-PCR respectively.2,To construct a HO-1-specific siRNA eukaryotic expression vector by DNA recombinant technology. 3,To transfect HO-1-siRNA into GC9811-P cells in lipofectamine 2000-mediated, and to detect the variation of HO-1 expression in GC9811-P in protein and mRNA levels by Western-blot and RT-PCR respectively.4,To observe the invasion and metastasis of GC9811-P through wound-healing assay and transwell assay in vitro.[Results]1,The expression of HO-1 in GC9811-P and its parental GC9811. The result of Western-blot show that HO-1 was high expressed in GC9811-P compared with GC9811. The result of RT-PCR also found that HO-1 expression was higher in GC9811-P.2,Construction of HO-1 small interfering RNA expression vector pEGFP-C1/HO-1-siRNA was successfully.3,Getting successful transfect of the HO-1-siRNA of GC9811-P cells, mRNA and protein levels show that HO-1 expression was decreased significantly in the GC9811-P cells, but there was no change in the GC9811-P cells which transfect empty vector.4,Wound-healing assay show that transfect with HO-1-siRNA group GC9811-P cells migration are not obvious, but a large number of negative control group transfect cells and un-transfect control cells moved to the scratch area . Transwell assay show that transfect with HO-1-siRNA group GC9811-P cells in the number of penetrating are (54.2±2.68), while the negative control group transfect cells are (83.2±5.71), un-transfect control group cells are (84.3±4.29). LSD-t test analysis of differences between group of HO-1-siRNA cells are less than the number of negative control group and blank control group(P<0.05).[Conclusion]From this study we can conclude that HO-1 was high expressed in high peritoneal metastasis of gastric cancer cell lines GC9811-P, and if down-regulate its expression by HO-1-siRNA , it would inhibit the invasion and metastasis of GC9811-P in vitro. It indicates that HO-1 plays an important regulatory role in invasion and metastasis of GC9811-P cells in vitro. |
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