| Although the incidence of gastric cancer in West countries has obviously declined in recent years, gastric cancer remains one of the most important problems that threats human health throughout the world. However, most of patients with gastric cancer have generally reached an advanced stage, and some of them have organ metastasis and become unresectable. Metastasis remains the major cause of death for gastric cancer patients, and seriously affecting the patient’s quality life and prognosis. The aggressive nature of gastric cancer is a result of a series of intracellular events, including the mutations of oncogenes and tumor suppressor genes, abnormal expression of growth factors and their receptors, and so on. Metastasis regulation involved in multiple genes signal transduction, and tumor angiogenesis is a multi-step complex network collaborative process. Although a large number of previous studies have reported to correlate with the tumor metastasis, its detailed mechanism is still unclear and need us to further study.The novel gene CIAPIN1(Cytokine-induced apoptosis inhibitor1) plays an important role in the development of many types of tumor and is related with tumor MDR, proliferation and prognosis. Previous studies in our laboratory have found that CIAPIN1expression was low expressed in gastric cancer tissues, and involoved in the proliferation and MDR of gastric cancer. CIAPIN1 might be a specific key molecular in the signaling pathway network in gastric cancer development and progression. However, the role of CIAPIN1 in gastric cancer metastasis and angiogenesis has not been studied. Thus, in this research, we want to explore the function and mechanism of CIAPIN1 in gastirc cancer metastasis and angiogenesis, we hope our reserch will provid solid foundation for us to better understand the biological behavior of gastric cancer.Aims: 1. To explore the correlation between CIAPIN1 and gastric cancer metastasis and angiogenesis in clinical samples; 2. To observe the regulation effect of CIAPIN1 on gastric cancer cell invasion, migration and angiogenesis in vitro; 3. To explore the possible molecular mechanism of CIAPIN1 in gastric cancer angiogenesis.Methods: 1. Immunehistochemistry was used to detect the expression level of CIAPIN1 in normal gastric mucosa tissues, gastric cancer tissues and metastasis and non-metastasis gastric cancer tissues and Chi-square test was used to explore the correlation of CIAPIN1 and gastric cancer metastasis; 2. Immunehistochemistry was used to detect the expression level of CIAPIN1, VEGF and CD31-MVD value in gastric cancer tissues and normal gastric mucosa tissues, and Chi-square test was used to investigate the correlation of the expression of CIAPIN1 and VEGF with clinicopathological characteristics of gastric cancer, and Sperman analysis was used to analysis the correlation of the expression of CIAPIN1, VEGF and CD31-MVD value; 3. CIAPIN1 expression vector, CIAPIN1-si RNA vector and control vectors were stably ransfected into SGC-7901 cells, geneticin(G-418) was used to select the resistant colonies, and Western-blot, RT-PCR were used to confirm the transfectin effect; 4. Transwell assay was used to verify the effect of CIAPIN1 on invasion and migrationof gastric cancer cells; 5. We first constructed the Co-HUVECs by co-culture of the cancer cell suspension and human umbilical vein endothelial cell(HUVEC), then used the MTT, cell adhesion and transwell assay to observe the effect of CIAPIN1 on Co-HUVEC’s proliferation, adhesion and migration; used the tube formation assay to observe the effect of CIAPIN1 on gastric cancer angiogenesis; 6. Western-blot was used to detect the level of the molecules associated with angiogenesis, VEGF, CDC42, HIF-1α, IL-6 and IL-8 in CIAPIN1 high and low expressed SGC-7901 cell lines.Results: 1. The expression of CIAPIN1 in gastric cancer tissue was lower than normal gastric mucosa tissues(P=0.0008), and in metastasis gastric cancer tissues was also lower than that in non-metastasis gastric cancer tissues(P<0.0001). Furthermore, the level of CIAPIN1 was closely related to the TNM stage of gastric carcinoma(P=0.0037). 2. VEGF expression was upregulated gastric cancer tissues compared with that in normal gastric mucosa tissues(P<0.0001). The positive rates of VEGF expression was 68%. VEGF expression level was closely related to the tumor differentiation grade of gastric cancer(P=0.0042). Moreover, the CD31-MVD value was significantly increased in gastric cancer tissues(55.82±3.344) and normal gastric mucosa tissues(12.16±1.211)(P<0.0001). Further, Sperman analysis also confirmed that the expression of CIAPIN1 was negative associated with the aberrant expression of VEGF and CD31-MVD value; 3. Western-blot and RT-PCR results showed that pc DNA3.1-CIAPIN1 could up-regulate the expression of CIAPIN1, while psi-CIAPIN1-2 could down-regulate the expression of CIAPIN1 in gastric cancer SGC-7901 cell lines; 4. Transwell assay showed that over expression of CIAPIN1 would obviously reduce the cell number of gastric cancer SGC-7901 cells that migrated through thepolycarbonate membrane(P<0.05), and opposite result was shown in psi-CIAPIN1-2 transfected gastric cancer SGC-7901 cells(P<0.05);5. Used the Co-HUVECs, we found that high expression of CIAPIN1 could inhibit HUVEC growth, cell adhesion and migration, and decrease HUVEC cell tubular number and in tubular length compared with the control group, While down-regulating the expression of CIAPIN1 could receive the similar results(P<0.05); 6. Western-blot results showed that upregulation of CIAPIN1 significantly decreased the expression of CDC42, VEGF, HIF-1α and IL-8 compared with the controls, and downregulation of CIAPIN1 could receive the similar results.Conclusion: 1. The expression of CIAPIN1 in gastric cancer tissues was lower than in normal gastric mucosa tissues, and was significantly lower in metastasis gastric cancer tissues. The expression of CIAPIN1 was negative correlation with the expression level of VEGF and CD31-MVD value in gastric cancer tissue. CIAPIN1 was significantly correlated with gastric cancer metastasis and angiogenesis; 2. In vitro experiments showed that CIAPIN1 could inhibit SGC-7901 invasion, migration and suppress HUVEC proliferation, adhesion, migration and tube formation, the above resluts showed that CIAPIN1 may paly a important role in gastric cancer metastasis and angiogenesis; 3. CIAPIN1 could downregulate CDC42, VEGF, HIF-1α and IL-8 expression, which means that CIAPIN1 inhibit gastric cancer angiogenesis was partly via blocking the expression of HIF-1α and VEGF to achive.As a new apoptosis related gene, CIAPIN1 was reported to plays an important role in cancer development and progression. Our labs pevious studies have found that CIAPIN1 could regulate gastric cancer proliferation and MDR, but its effects on mediating gastric cancer invasion, migration and angiogenesis have not yet been reported. Here, we found that CIAPIN1 is closely associated with gastric cancer metastasis and angiogenesis. The mechanism of inhibiting angiogenesis is partially through inhibiting the expression of HIF-1α and VEGF to achive. Combining with previous results, we guess that CIAPIN1may through two pathways to regulate gastric cancer angiogenesis: one is that CIAPIN1 directly inhibits VEGF expression to regulate gastric cancer angiogenesis, while the other is that CIAPIN1 could via HIF-1α/VEGF pathway indirectly block the angiogenesis of gastric cancer. However, the further study is still need to confirm our hypothesis and to clarify the basic function of the new gene CIAPIN1. |
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