The Effect And Mechanism Of ABCE1 Up-regulation On Proliferation,migration,and Invasion Via CCL7/receptors Pathway In Non-small Cell Lung Cancer

Objective:Currently lung cancer is one of the malignant tumor with highest morbidity and mortality in the world.According to GLOBOCAN 2012,which issued by international agency for research on cancer,the diagnostic rate of lung cancer is the highest in all of cancers,the mortality rates is also the highest in male all of the world and female in the developed countries.In developing countries the dangerous of lung cancer for women is only behind breast cancer.We need to continuously explore the molecular mechanisms of occurrence,development,invasion and metastasis of lung cancer.ABCE1?ATP-binding cassette E1?belongs to ATP binding cassette superfamily.The expression of ABCE1 is higher in the human lung adenocarcinoma tissue and metastasis lymph node than normal tissues,the level of expression is associated with clinical stage.The migration and invasion ability of small cell lung cancer decreased,when ABCE1 gene was silenced by RNAi technology in vitro.Moreover,some evidence show ABCE1 is a key role to promoting development and metastasis of lung cancer,when the inoculation of lung adenocarcinoma cancer cell with ABCE1overexpressed was executed in nude mice body.These observations supposed ABCE1gene to take a closed relationship with proliferation,invasion and metastasis of lung cancer.Chemokines belongs to a kind of small chemotactic cytokines that can direct cell trafficking and consist of about 70¹00 amino acids with molecular weight between 8¹4 kDa.They conduct their functions via receptors with involvement in cell growth,development,differentiation and apoptosis.It has been proved that they playing an important role in a variety of pathological process,such as inflammation,pathogen infection,trauma repair and tumor formation and transfer.Chemokines triggering downstream signal transduction via receptors prompt epithelial-mesenchymal transition and change the tumor microenvironment,eventually led to the proliferation and metastasis of tumor.Real-time quantitative PCR is recognized to be the most sensitive way of gene expression test.We want to get one or more related-genes of ABCE1 by using PCR Array chip in non-small cell lung cancer cells.Methods:We built a lentivirus packaging vector expressing ABCE1?LV-GV358-ABCE1?and infected H1299 cell lines with LV-GV358-ABCE1.Then we collected cell to extract mRNA and acquired cDNA fragments through reverse transcription reaction.RT² Profiler^TM PCR Array HumanTumor Metastasis chip was applicated to compare the difference between cDNA templates,to screen ABCE1 related-genes which associated with metastasis in lung cancer.Then we affirrmed target one from these genes.Moreover,the test of ABCE1 and target gene protein expression was performed by using Western Blot.We collected and analyzed mRNA and protein expression of target gene and ABCE1 in non-small cell lung cancer,metastasis lymph nodes and carcinoma adjacent tissues,using immunohistochemistry and Real-Time PCR.Then we compared the difference and correlation in target gene and ABCE1.Lentivirus packaging vector expressing target gene should be constructed and infected to H1299 cell lines.The coding regions were confirmed by electrophoresis and DNA sequencing.The proliferation of the cells was assayed by MTT.Flow cytometry was used to detect whether the lentivirus packaging vector expressing target gene can effect apoptosis of the non-small lung cancer cell.The migration and invasion capacity of the cells were assayed by Transwell cell migration and invasion assay.Meanwhile the expression of target gene related-receptor and ABCE1 were detected by Western blot.Results:Chemokine?C-C motif?ligand 7,CCL7 as the target gene was confirmed based on the result of PCR array screening finally.CCL7 overexpressed exist in H1299 with ABCE1 increasing.mRNA and protein of CCL7 and ABCE1 were increased in human NSCLC tissues compared to their corresponding normal tissues,p<0.05.Lentivirus packaging vector expressing CCL7?LV-GV358-CCL7?was proved to be constructed successfully.Compared with empty vector and control group cells,proliferation migration and invasion of CCL7 overexpressed cells was increased significantly,p<0.05,but unvariation of cell apoptosis was occurred in three teams.The receptors of CCL7 including CCR1 CCR2 CCR3 expression increasing were observed in LV-GV358-CCL7 cells,but receptors overexpressed was not happened in other teams.Meanwhile,the level of ABCE1 protein was the same at three teams.Conclusion:We screened ABCE1 tumor metastasis associated genes by PCR Array chip in non-small cell lung cancer cells successfully.CCL7 was chosen to be the target.The expression of ABCE1 and CCL7 was increased in human NSCLC tissues.CCL7 and ABCE1 possess close relationship in NSCLC.CCL7 promotes malignancy of H1299 cells in vitro through CCL7/CCR1-3 axis,including proliferation migration and invasion.The expression of ABCE1 can not be effected by CCL7.