The Research Of ABCE1 Gene On The Abilities Of Proliferation, Migration And Invasion Of Small Cell Lung Cancer

ObjectiveSmall cell lung cancer is a special kind of lung cancer,which always has lymph or blood metastasis in its early stage.Although radiotherapy and chemotherapy have produced modest benefits for some patients,it is prone to drug resistance and relapse, which affect the health and life of humankind.So it is important to seek correct small cell lung cancer related gene and find out the mechanism of small cell lung cancer.ABCE1 gene is a member of the ATP-binding cassette(ABC)multigene family, whose full-length cDNA sequence encodes 68KD protein.It encodes a protein originally identified for its inhibition of ribonuclease L.ABCE1 locates in cytoplasm and mitochondria.The gene is universally expressed in the human tissues and organs, but it is different in form and level.ABCE1 can inhibit apoptosis and may play a part in promoting protein synthesis,cell proliferation and differentiation.Therefore,we constructed pEGFP and SiRNA expression vector of ABCE1,then transfected vector into small cell lung cancer cell line NCI-H446 to investigate whether ABCE1 is responsible for the proliferation,invasion and metastasis in small cell lung cancer and the change of apoptosis.At the same time,we study the expression differences between adjacent lung tissue,small cell lung cancer cell line,small cell lung cancer tissue and metastasis lymphoid node.MethodsAccording to the sequence of ABCE1,we designed a pair of primer.Total RNA was extracted from small cell lung cancer cell(NCI-H446).CDNA fragment was amplified by RT-PCR and inserted into the vector pEGFP-C₁,which carried enhanced green fluorescent protein.Then the recombinant plasmid pEGFP-ABCE1 was identified by restriction endonuclease and DNA sequencing.According to the sequence of ABCE1 in Gene bank,three target sequences were designed and synthesized for construction of SiRNA vector.After annealing,oligonucleotides were inserted into pSIREN-DNR vector,which carried red fluorescent protein.The recombinant plasmid ABCE1-SiRNA-1,ABCE1-SiRNA-2 and ABCE1-SiRNA-N were identified by restriction endonuclease and DNA sequencing too.Small cell lung cancer cell line(NCI-H446) was cultured with 1640 culture solution containing 10%fetus bovine serous,at 37℃and 5%CO₂.Using FUGENE-HD pEGFP-ABCE1,ABCE1-SiRNA-1,ABCE1-SiRNA-2,ABCE1-SiRNA-N and pEGFP -C₁ were transfected into human small cell lung cancer cell line.48 hours after transfection,we use Western blot and immunohistochemistry to detect the expression of the ABCE1 protein in the transfected cells.The proliferation,invasion,metastasis ability of the cells was assayed by MTT,Transwell cyberoom and mark experiment. Flow cytometry was used to detect whether the interference vector can induce apoptosis of the small lung cancer cell.Meanwhile the expression of ABCE1 was detected by the immunohistochemistry and Western blot in small cell lung cancer tissue, metastasis lymphoid node,adjacent lung tissue and small cell lung cancer cell line.On the basis of previous experiments,we built ABCE1 gene SiRNA expression vector ABCE1-pRNAT-U6.1/Neo-SiRNA-1,ABCE1-pRNAT-U6.1/Neo-SiRNA-2, ABCE1-pRNAT-U6.1/Neo-SiRNA-N.The new vector carry green fluorescent gene and can be used for screening positive clones.Then we transfected ABCE1-pRNAT-U6.1/Neo-SiRNA-1, ABCE1-pRNAT-U6.1/Neo-SiRNA-2,ABCE1-pRNAT-U6.1/Neo-SiRNA-N into NCI-H446 cells,using G418(400ug/ml) for positive screening,then screen ABCE1 gene silencing stable transfection cell lines.ResultsBy restriction endonuclease and Sequencing,eukaryotic expression plasmid of pEGFP-ABCE1,ABCE1-SiRNA and ABCE1-pRNAT-U6.1 /Neo-SiRNA were proved to be successfully constructed.ABCE1 protein was overexpressed in small cell lung cancer tissue,metastasis lymphoid node,and there were significant differences between small cell lung cancer tissue,metastasis lymphoid node and adjacent lung tissue. ABCE1 protein was overexpressed in small cell lung cancer cell line too.After transfected with ABCE1-SiRNA,red fluorescence can be seen in small cell lung cancer cells.The expression of ABCE1 was high in the cell line which was transfected with ABCE1-SiRNA-N,while the expression was low in the cell line which was transfected ABCE1-SiRNA-1,ABCE1-SiRNA-2.The results showed ABCE1-SiRNA-1,ABCE1-SiRNA-2 and ABCE1-SiRNA-N have been transfected into NCI-H446 successfully and have silenced ABCE1 to a certain degree.MTT tests showed that proliferation ability of the cells which transfected with ABCE1-SiRNA-1, ABCE1-SiRNA-2 was much lower than the control group and parental vector ABCE1-SiRNA-N.TransweU cyberoom tests revealed that the cells penetrating the artificial basement membrane in ABCE1-SiRNA-1,ABCE1-SiRNA-2 were fewer than those in ABCE1-SiRNA-N and control group.Mark experiments showed the transfer speed of the cells which were transfected with ABCE1-SiRNA-1,ABCE1-SiRNA-2 was significantly slower than that of the untransfected and transfected with ABCE1-SiRNA-N cells.Flow cytometry tests showed that when transfected with ABCE1-SiRNA-1,ABCE1-SiRNA-2 the apoptosis cells markedly increased.After transfected ABCE1-pRNAT-U6.1/Neo-SiRNA into small cell hmg cancer cells successfully,green fluorescence can be seen in those cells.Through G418 screening,we got ABCE1 gene silencing stable transfection cell lines of small cell lung cancer,the ABCE1 level of the cell lines was less than that of the untransfected cell lines.Conclusion1 The pEGFP-ABCE1,ABCE1-SiRNA and ABCE1-pRNAT-U6.1/ Neo-SiRNA expression vector have been successfully constructed.ABCE1 proteins were overexpressed in small cell lung cancer tissue,metastasis lymphoid node.It may contribute to the occurrence and progression of small cell lung cancer.ABCE1 protein was overexpressed in small cell lung cancer cell line too.2 ABCE1-SiRNA which has been transfected into the NCI-H446 can not only inhibited the expression of ABCE1,but also inhibited cell growth,proliferation ability. At the same time it can reduce the migration and invasion abilities of cells and induce apoptosis.3 We got ABCE1 gene silencing stable transfection cell lines of small cell lung cancer successfully.Our results may provide some ideas for the gene therapy of ABCE1 gene in small cell lung cancer in future.