The Functional Mechanism Of Snail2 And G9a During EMT In Liver Cells

Liver cancer is one of the most common malignant tumor in clinical, the fifth of malignant tumor. Surgical resection and liver transplantation is the main method for the treatment of liver cancer, however liver cancer is coated and vascular invasion,resulting in intrahepatic and extrahepatic metastases which makes the patient’s survival is less than 10% over five years. Autopsy found that patients with extrahepatic metastasis of liver cancer rate is 40%-71.6% which is a major cause of death in patients. Therefore understanding the mechanism is crucial for improving liver cancer survival of patients. Tumor metastasis is an extremely complex pathological process, whereas, the invasion of tumor cells is a prerequisite for tumor metastasis and Epithelia-mesenchymal transition(EMT) is important for malignant tumor cell invasion and migration. To reverl the molecular mechanisms regulating the EMT process has become a key in the study of mechanism of tumor metastasis.E-cadherin expression of down- regulation is a sign of EMT process events, at the same time accompanied by N-cadherin up-regulation, vimentin and fibronectin specific high expression of stromal cell protein. Specific transcription factors down-regulate E-cadherin which can induce the expression of EMT process of tumor cells, so as to promote tumor invasion and metastasis. Snail is one of the key factors of the effect induced by EMT process in the process of tumor invasion and metastasis.However, in the process of EMT in cancer of the liver cells, the mechanism underlying Snail induced EMT in liver cancer cells has not been demonstrated yet. In addition, The change of epigenetic information is also an important mechanism to control the EMT process of malignant tumor cells. All kinds of EMT transcriptional inhibitory factors use different apparent genetic mechanism experiment to regular E-cadherin expression. Our previous research has shown that the occurrence of liver cancer development is closely related to the abnormally high expression of G9 a.While in the breast tumor cells, by inhibiting the expression of Snail family proteins interacting with G9 a mediate E-cadherin, to promote the invasion and metastasis oftumor cells. However, the role that Snail and G9 a played in the process of invasion and metastasis of liver cancer has not yet been elucidated. So we speculated that in the process of invasion and metastasis of hepatocellular carcinoma, Snail2, which by raising G9 a inhibiting the transcription of E-cadherin mucin, thus promotes EMT process in liver cancer cells.We first used TGF-βto induce QSG-7701 and HL-7702 in human normal liver cells generating EMT phenomenon. RT-PCR experiments show that in the course of EMT of hepatic cells, the expression of E-cadherin mRNA decreased significantly,however, N-cadherin, fibronectin and vimentin, their mRNA expression level apparently increased. At the same time, the mRNA level of the Snail2 was also highly expressed but the m RNA level of Snail1 gene was not up. In addition, we also found that the Snail2 gene is highly expressed in HCC tissues compared to adjacent tissues in 33 clinical liver cancer tissue samples. Next, we further verify whether Snail2 interacting with G9 a inhibit E-cadherin expression. We first construct the Snail2full-length(Snail2-FL) and N-terminal region 600bp(Snail2-ND) and C terminal region 200bp(Snail2-CD) deletion mutants, and G9 a full-length(G9a-FL) and N-terminal region 2039bp(G9a-ND) and C terminal region 1832bp(G9a-CD)deletion mutants. Co-ip confirmed, Snail2-FL and G9a-FL in vivo can be combined.Snail2-FL and G9a-CD can be combined, but the Snail2-FL and G9a-ND did not interact with each other. And Snail2-ND and G9a-CD are not combined. These all show that the C-terminal region was responsible for their interaction with each other.At the same time, the CHIP experiment also showed that suggest that Snail2 and G9 a are involved in TGF-β–mediated H3K9me2 within the E-cadherin. In summary, this paper initially confirmed that Snail2 interacting with G9 a, will be raised in the E-cadherin gene, leading the E-cadherin gene methylation, resulting in suppression of the expression of E-cadherin and thereby inducing EMT.