The Influence Of IASPP On The Biological Characteristics Of Gastric Cancer Cells And Its Regulatory Mechanism

Background and ObjectiveThe development of gastric cancer is related to lots of factors, such as the activation of oncogene、tumor suppressor gene mutation or function inhibition, etc. There is close relationship between P53 and human tumor. The wild-type P53 can maintain the normal function of cells, while the mutant-type P53 can lead to cancer. But about two-thirds of gastric cancer had wild-type P53, and it is unclear that how the cancer cells escaped the monitor of wild-type P53.iASPP is a member of ASPP family, it can inhibit the function of wile-type P53. it was considered to be a oncogene because it has exceptionally high expression in a wide variety of tumor. There is less research about iASPP in gastric cancer. It have been reported that its expression is associated with HP infection. But there is not has literature to report the expression、function and own regulatory mechanism of iASPP in gastric cancer.KLF4 is a tumor suppressor gene in gastric cancer. The development of gastric cancer is closely related with the low expression of KLF4. There is function relationship between KLF4、iASPP and P53 because KLF4 can interact with P53 while iASPP is the inhibitor of P53. Gene software analysis showed that there is KLF4 combination sequence in iASPP promoter region. So we speculated that there is regulation relationship between KLF4 and iASPP.The objective of this study is to explore the iASPP expression in gastric cancer; to explore the role of iASPP in the proliferation、apoptosis、 invasion and metastasis of gastric cancer cells; to explore whether there is regulation relationship between iASPP and KLF4.Methods1.The mRNA levels of iASPP in the gastric cancer tissues with metastasis (n=20), gastric cancer tissues without metastasis (n=20), adjacent normal mucosa tissues (n=20), gastric cancer cell lines (MKN45, SGC7901, AGS), and normal gastric mucosa cell line (GES1) were detected by quantitative PCR. The protein levels of iASPP in the gastric cancer tissues with metastasis (n=20), gastric cancer tissues without metastasis (n=20), adjacent normal mucosa tissues (n=20), gastric cancer cell lines (MKN45, SGC7901, AGS), and normal gastric mucosa cell line (GES1) were detected using Western Blot.2.We selected the gastric cancer cell line with high expression of iASPP (MKN45, SGC7901), transfected the interfering lentivirus into the gastric cancer cell lines, then filtrated the stable cell lines, the expression of iASPP was detected which was transfected with interference virus and control lentivirus by Western Blot.3.Cellular proliferation of MKN45 and SGC7901 cell lines with iASPP expression inhibited was detected by MTT and colony formation assays; the cell apoptosis of cells was detected by flow cytometry; Then detected the gastric cancer cells’ invasion and migration ability by the transwell invasion and migration experiment.4.In order to constructed transplantation tumor model in nude mice, we selected the MKN45 cells which were transfected with iASPP interference lentivirus or only empty virus, then injectedt the cells into nude mice subcutaneously, then observed the growth of transplantation tumor of nude mice.5.We detected the protein level of KLF4 and iASPP in gastric cancer cell lines (MKN45、SGC7901) and normal gastric mucosal cells (GES1) by Western Blot, then selected the appropriate cell lines which was negatively correlated with KLF4 and iASPP for the subsequent trials.6.We transfected the over-expression plasmid of KLF4 into the cell lines which we selected, filtrated the stable cell line, then detected the expression of KLF4 after Plasmid transfection by western blot, at the same time we detected the expression of iASPP, observed whether the expression of iASPP was affected by KLF4 expression. Then we transfected iASPP over-expression plasmid into the cells which over-expressed KLF4, detected the iASPP protein level by western blot.7.We detected the cellular proliferation, apoptosis, invasion and metastasis of MKN45 cells after the overexpression of KLF4 By MTT, cell colony formation assay, flow cytometry and transwell experiments, then we transfected the over-expression plasmid of iASPP into the cells with KLF4 over-expression as the remedial experiments, after increased the expression level of iASPP, we observed the changes of cellular proliferation, apoptosis, invasion and metastasis ability.Results1.The results of RT-PCR and Western Blot showed that, the expression level of iASPP in gastric cancer tissues was significantly higher than that in adjacent tissues (P<0.05), furthermore the iASPP expression level in gastric cancer tissues with metastasis was higher than that in gastric cancer tissues without metastasis (P<0.05). The expression of iASPP in gastric cancer cell lines was also higher than in the normal gastric mucosa cells (P <0.05).2. According to the above experiment results, we choosed the gastric cancer cell lines (MKN45, SGC7901) which have a higher level of iASPP expression as the follow-up test object. Fluorescence microscope observation found that there is about more than 95% cells have strong fluorescence expression in iASPP-siRNA-LV transfection group.The results of Western Blot showed that the iASPP expression was significantly down-regulated after we transfected the interference lentiviral of iASPP in MKN45 and SGC7901 cells (P<0.05).3.The results of MTT and cell colony formation assay showed that the inhibition of the iASPP expression could inhibit the growth and proliferation of MKN45 and SGC7901 cells (P<0.05), the results of flow cytometry showed that down-regulated of iASPP expression significantly promotes apoptosis of MKN45 and SGC7901 cells (P<0.05), the experimental results of Transwell showed that the inhibition of the iASPP expression also could reduce the migration and invasion ability of MKN45 and SGC7901 cells (P<0.05).4. The experimental results of transplanted tumor in nude mice showed that, the gastric transplanted tumor size of nude mice in down-regulated iASPP group was significantly smaller than the control group (P<0.05), And the result of Western Blot showed that iASPP expression in transplanted tumor of iASPP interference group was obviously lower than the control group.5.The results of Western Blot showed that KLF4 expression in MKN45 and SGC7901 cells was significantly reduced than that in normal gastric mucosal cells (P<0.05), and we found the expression of KLF4 and iASPP was negatively correlated in the MKN45 of gastric cancer cells, so we selected the MKN45 as the follow-up test object.6.After we transfected he over-expression plasmid of KLF4 into MKN45, the expression levels of KLF4 were significantly up-regulated (P <0.05), meanwhile the expression of iASPP was down-regulated conversely (P<0.05). In addition, after transfecting the iASPP over-expression plasmid, the iASPP expression was obviously up-regulated in cells which over-expressed KLF4 (P<0.05).7.The results of MTT, cell colony formation assay, flow cytometry and Transwell experiments showed that the up-regulated of KLF4 expression can inhibit the proliferation, invasion and migration ability of MKN45 cells and promote the apoptosis of cells (P<0.05). The remedial experiments showed that, the recovered of iASPP expression improved the proliferation, invasion and migration ability of gastric cancer cells and inhibited the apoptosis of cells (P<0.05).Conclusion1.iASPP expression is up-regulated in gastric cancer cells and tissues, it has the characteristics of oncogene. Its expression level is closely related to whether gastric cancers accompany with metastasis.2. We transfected iASPP interference lentivirus successfully. Down-regulating the expression of iASPP could significantly reduce the growth, proliferation, invasion and metastasis of gastric cancer cells, and promote the apoptosis. iASPP down-regulation also can restrain the growth of the nude mouse transplantation tumor in vivo. It suggests that iASPP plays an important role in the process of gastric cancer cell growth, proliferation, apoptosis, invasion and metastasis.3.KLF4 is down-regulated in gastric cancer cells and it is a tumor suppressor gene. After up-regulating the KLF4 expression in gastric cancer cells, resulted to the down-regulation of iASPP expression. It illustrated that iASPP expression level is associated with KLF4 expression.4.The up-regulation of KLF4 expression in gastric cancer cells can inhibit cell proliferation, invasion and metastasis ability, and promote cell apoptosis. Through the remedial experiment, improved the expression of iASPP, we found that cell proliferation, invasion and metastasis abilities were resumed, and inhibited the apoptosis of cells. So we speculated that KLF4 can play its role of the tumor suppressor in gastric cancer by inhibiting the expression of iASPP.5. Previous studies showed that HP infection can influence the expression of iASPP. HP infection can also activate the Notch signal pathway, while the activation of Notch signaling pathway can inhibit the KLF4 expression. Our study confirmed that iASPP express is related with KLF4 expression, and iASPP can interact with P53、P63 and P73. So we speculated that HP infection�'Notch signal pathway activation�'KLF4 expression down-regulation�'iASPP expression up-regulation �'P53, P63, P73 function suppression�'the development of gastric cancer.