The Research of the Influence of RUNX3on the Epithelial-Mesenchymal Transition (EMT) in Human Gastric Cancer Cells and Its Molecular Mechanisms
|Course||Biochemistry and Molecular Biology|
|Keywords||gastric cancer RUNX3 EMT miR-30a Vimentin|
Gastric cancer is the most common human malignancy, and has the second big mortality rate in tumors. In China, people have the high incidence rate and poor prognosis of gastric cancer.The invasion and metastasis of the gastric cancer is the main reason for the death of the patients, so the study of invasion and metastasis of gastric cancer and its molecular mechanism has important theoretical value and clinical significance.The occurrence and development of the malignant tumor’s invasion and metastasis is a complex biological process, including the isolation of the cancer cells from the primary lesion, the invasion into the surrounding tissues, the penetration of the basement membrane into the blood vessels and lymphatic vessels, the oozing of the blood and lymphatic vessels, and the re-formation of metastatic nodules and then proliferating to new metastases. Epithelial mesenchymal transition (EMT) and mesenchymal epithelial transition (MET) has played a key role in this process. EMT was first found during the embryonic development, and later also found that EMT occurs in the process of many tumors’metastasis. EMT is a bioprocess regulated accurately in the embryonic development process both in time and space, but in the tumors the EMT’s imbalance lead to the lost of their proper phenotype of the epithelial cells and the acquirement of the characteristics of interstitial cells, and it cause the tumor cells’invasion and metastasis, meanwhile in this process the marker molecule of epithelial cells E-cadherin decrease and the expression of the marker molecule of interstitial cells Vimentin rise. The invasion and metastasis easily occur in the clinical process of development of gastric cancer, and the treatment and prognosis is very poor, so the study of the molecular mechanism of the invasion and metastasis of gastric cancer and the EMT is an important foundation of the diagnosis and treatment of gastric carcinoma.RUNX3is one of the RUNX protein family members, and RUNX3mainly involved in the development of the gastrointestinal tract,neurogenesis in the process of embryonic development. In recent years it is found that the abnormal expression of RUNX3is closely related with the development of gastric cancer. Qing-Lin Li et al found that RUNX3can promote gastric epithelial cell apoptosis and cell cycle arrest and the inhibition of gastric epithelial cell proliferation to regulate the growth of gastric epithelial cell. Clinical studies have shown:RUNX3is associated with the invasion and metastasis of gastric cancer, but the molecular mechanism about the influence of RUNX3on the invasion and metastasis of gastric cancer is not entirely clear and whether RUNX3could regulate EMT in the invasion and metastasis of gastric cancer has not been reported. In this study, the influence of the tumor suppressor factor RUNX3on EMT and its molecular mechanisms in human gastric cancer cells were researched.The first part:RUNX3influence the invasion and metastasis of the human gastric cancer cellsObjective:To research the influence of RUNX3on the invasion and metastasis in the human gastric cancer cells BGC823and SGC7901.Methods:The eukaryotic expression plasmid of RUNX3was transfected into human gastric cancer cells SGC7901and of BGC823respectively,and after48hours, the RNA and protein were extracted from a part of cells for QRT-PCR and Western blotting, another part of the cells were digested, counted, and the chamber was covered with the matrigel. fifty thousand cells were put into one upper Transwell chamber, and the lower wes joined into the culture medium of NTH3T3cells, after24hours the upper uninvasive cells were removed, then the invasive cells were fixed by methanol and stained by crystal violet and counted.Results:QRT-PCR and Western boltting showed that RUNX3gene significantly expressed in human gastric cancer cells BGC823and SGC7901. In the Transwell experiments, The number of invasive cells in the group with high expression of RUNX3reduced significantly compared to the control.Conclusions:The expression plasmid of RUNX3highly expressed RUNX3protein successly after transfection. The Transwells showed that high expression of RUNX3in gastric cancer cells could inhibit the ability of invasion and metastasis compared to the control. The second part:The regulation of RUNX3on EMT-related genes’expressionObjective:To research the influence of RUNX3on the expression of E-cadherin, Vimentin, Snail and Twist in EMT.Methods:after the expression plasmid RUNX3transfected into human cancer cells, RNA and protein were extracted with mRNA reverse-transcripted into cDNA, Western blotting,RT-PCR and QRT-PCR were used to detect the expression of E-cadherin, Vimentin, Snail and Twist. Interfere the RUNX3gene with stable and special siRNA, after72hours, the expression of E-cadherin and Vimentin was detected.Result:After the RUNX3expression plasmid transfected into SGC7901, Western blotting showed that the protein level of E-cadherin increased slightly, but the protein level of Vimentin decreased highly.RT-PCR and QRT-PCR showed that the RNA level of E-cadherin and Vimentin did not change.After high expression of RUNX3, the transcription factors of EMT Snail and Twist also had no change in the protein and RNA level.Conclusions:High expression of RUNX3can increase the expression of E-cadherin slightly and decrease the expression of Vimentin significantly only in the protein level, and with RUNX3silenced, the Vimentin protein heighten significantly. But the regulation of RUNX3on EMT has nothing to do with the regulation of Snail and Twist, because it is a kind of post-transcriptional regulation.The third part:The molecular mechanism of the RUNX3of regulating Vimentin’s expression Objective:To research the post-transcriptional regulation’s molecular mechanism of RUNX3in the regulation of the expression of Vimentin.Methods:After42hours of Transfecting the expression plasmid of RUNX3,10μM MG-132was put into gastric cancer cells,then6hours laster, protein was extracted, and the expression of Vimentin was detected by Western blotting. Also the samples transfected with RUNX3plasmid were used to do microRNA gene chip screening, and the results would be analyzed by refering to articles and microRNA database.Results:After adding the proteasome inhibitor MG-132,the expression level of Vimentin didn’t resume.The microRNA chip result showed that RUNX3could significantly upregualate the expression of miR-30a, some articles and microRNA databases found that miR-30a was not completely complementary to the3’-UTR of Vimentin’s mRNA, and innibit the translation of Vimentin protein.Conclusions:The regulation of RUNX3about Vimentin is not achieved through the ubiquitin-proteasome pathway.Microarray results showed that RUNX3could upregulate the expression of miR-30a, also databases and literatures suggested miR-30a could suppress the post-transcriptional expression of Vimentin protein, so all this suggested RUNX3could inhibit the expression of Vimentin in post-transcriptional level.The fourth part:RUNX3regulate the expression of Vimentin to influence EMT through miR-30a.Objective:To validate the molecular mechanism of RUNX3suppressing the post-transcriptional expression of Vimentin by the upregulation of miR-30a. Methods:After48hours of transfection of RUNX3plasmid and72hours of interference of RUNX3by siRNA,RNAs were extracted by Trizol and reverse-transcripted into cDNA, and the expression of miR-30family was detected by QRT-PCR. After transfection of miR-30family mimics into gastric cancer cells,protein was extracted for Western blotting to detect the expression of Vimentin. The wild-type and mutant-type luciferase reporter plasmids were constructed by molecular cloning, pMIR-Vimentin3’UTR, pMIR-Vimentin3’UTR MUT1, and pMIR-Vimentin3’UTR MUT2. after24h of the transfection of miR-30a the reporter plasmids were transfected into cells,and48h later luciferase assay was experimented.Results:The QRT-PCR results confirmed:high expression of RUNX3indeed significantly increased the level of miR-30a, and silence of RUNX3expression could inhibit the expression of miR-30a. Also The change of RUNX3did not influence the expression of miR-30e. After miR-30a mimics transfected into gastric cancer cells, Vimentin was significantly reduced in protein level,which was accordant to the group of transfection of RUNX3expression plasmid. Also miR-30e had no effect on the expression of Vimentin protein. Luciferase assay showed that compared with the transfection of miR-1011, expression of luciferase reduced in the wild-type and Mut1mutation Reporter System after the transfection of miR-30a,but in Mut2mutation reporter system after transfection of miR-30a the expression of luciferase restored to the level of the control group.Conclusions:RUNX3can significantly positively regulate the expression of miR-30a,but it is not obvious to the regulation of miR-30e. miR-30a can directly target to the second binding site in the3’UTR of Vimentin mRNA and inhibit the translation of Vimentin, then reduce the expression of Vimentin protein. The molecular mechanism of the tumor suppressor RUNX3influencing EMT is that RUNX3upregulates the expression of miR-30a and thus indirectly inhibits the expression level of Vimentin.The significance and innovationTumor suppressor RUNX3is closely related to the metastasis of the malignant tumor. This research reveales that RUNX3can affect the epithelial-mesenchymal transition (EMT) in the process of metastasis at the cellular level for the first time in human gastric cancer cells, and proves that RUNX3can suppress the expression of the key mesenchymal marker in EMT Vimentin through upregulation of miR-30a, then miR-30a targets to the3’UTR of Vimentin,and so inhibit the occurrence of EMT and the invasion and metastasis of gastric cancer cells. This study finds an important pathway between the relationship of RUNX3and EMT, and supplies a good molecular basis about molecular markers for clinical diagnosis and treatment of gastric cancer.