The Mechanism Study On The Regulation Of Egfl8 In The Invasion And Metastasis Of Hepatocellular Carcinoma

Background Hepatocellular carcinoma(HCC) is one of the common malignancies worldwide. It ranks the world's sixth most common cancer and the second most common cause of death from cancer. And in our country,it has become the third most common cancer, with such a high level of incidence and mortality worldwide. This makes it such a serious threat to life and health of our people. Athough the total resection rate of HCC has improved obviously,the overall level of treatment of HCC still has no significance improvement by the measure of 5-year survival rate. The reason lies in the high recurrence and metastasis rate of HCC after the surgery. Obviously, what an important and critical issues currently we are facing with is how to inhibit the invaion and metastasis of HCC in order to reduce the high recurrence rate after surgery.After decades of research, it has been found that a number of factors such as nm23,KAI-1,OPN play an important role in the invasion and metastasis of HCC.Our previous studies have also found Egfl7 promotes invasion and metastasis of HCC by regulating Egfl7 / EGFR / FAK signaling pathway.Epidermal growth factor-like domain 8(Egfl8) is currently the only known Egfl7 homologous genes, both of which constitute a small gene family.EGFL8 is the only known paralog of EGFL7 and the proteins they encode share the same overall domain structure, including an EGF-like domain, a Ca2+ binding EGF-like domain and a N-terminal signal peptide.Therefore,we believe that their consistency on protein structure may strongly suggest that Egfl8 plays a similar role in the development of human malignancies as Egfl7 does.In recent years,we have confirmed Egfl8 expression in human colorectal cancer and gastric cancer tissues was significantly reduced, and the low expression has a close correlation with the metastasis, TNM stage and poor prognosis.But the level of Egfl8 expression in HCC and its function have not been reported yet.Objective To study Egfl8 expression level in HCC tissue and hepatocellular carcinoma cell lines, and also its correlation with clinicopathological features of HCC, the regulation of Egfl8 on apoptosis,invation,athletic ability and other possible aspects.Methods Collection of 34 cases of surgically resected specimens of fresh HCC tissues and corresponding adjacent normal liver tissue, and using q RT-PCR to test Egfl8 expression in both of them.After that,we used q RT-PCR again to test Egfl8 expression in normal hepatic cell line and hepatocellular carcinoma cell lines.At the same time,we analyzed the relation of clinicopathologic features with Egfl8 expression in HCC patients. Furthermore,we intend to use lentivirus-mediated gene transduction technique to build Egfl8 over-expression HCCLM3,and use Western blot to test the level of Egfl8 expression. Finally,we used MTT?Annexin V-APC?Transwell wound scratch assay to observe the change of the proliferation,apoptosis, invasion and athletic ability.Results Real-time PCR showed that Egfl8 showed low expression in 67.6%(23/34) of HCC,and Egfl8 expression in HCC tissues was higher than the adjacent normal tissues(0.008 ± 0.02 versus 0.194 ± 0.05, P = 0.04). Meanwhile, Egfl8 low expression has a close correlation with the liver venous invasion(P = 0.01) and TNM stage(P = 0.00).In addition, Egfl8 expression levels in HCC cell lines was also significantly lower than the hepatic cell lines(P <0.05), and the expression levels in high-invasive potential HCC cell lines were much lower than that in the low ones(P <0.05). Egfl8 over-expression HCCLM3 was successfully constructed by Lentivirusmediated gene transduction technique, and the expression levels in Egfl8 over-expression group were significantly increased compared with the control group(0.853 versus 0.254, P <0.001). The apoptosis rate of Egfl8 over-expression group was significantly increased(10.05 ± 0.26% versus 7.28 ± 0.31%, P <0.001),while the cell invasion ability(Transwell chamber transfer rate: 1.815 ± 0.019 3.732 ± versus 0.061, P <0.001) and migration ability were significantly decreased(24h mobility: 0.03 ± 0.00 versus 0.17 ± 0.04, P = 0.021; 72 h mobility: 0.23 ± 0.03 versus of 0.36 ± 0.05, P = 0.028), which indicated that over-expression of Egfl8 would inhibit the invasion and metastasis of HCC.The curve which was drawn by MTT assay showed that the HCCLM3 proliferation in Egfl8 over-expression group did not change significantly, suggesting that Egfl8 had no significant effect on the proliferation of HCC cells.Conclusion Egfl8 expression is down-regulated in HCC and HCC cell lines,and the level of its expression is closely related to the malignancy of HCC. Egfl8 over-expression promotes the process of apoptosis,and inhibits the process of metastasis and migration of HCC,but has no significant effect on proliferation,suggesting that Egfl8 may be a new valuable molecular marker which can predict the malignancy and invasion of HCC.