The Role Of Lung Cancer Metastasis Related Protein1(LCMR1) In The Generation Of Apoptosis

Objective: Lung cancer metastasis related protein1(LCMR1) is a novel genecloned from PLA-801, a poorly differentiated human lung large cell carcinomacell line, using a differential display polymerase chain reaction technique in ourlaboratory in2002. The information of this gene has been submitted to NationalCenter for Biotechnology Information (NCBI), and a Genbank accession number(AY148462) has been assigned. In the present study, we employed RNAinterference (RNAi) technique to knock down LCMR1expression andinvestigated the regulatory role of LCMR1in the generation of lung cancer cellapoptosis.Methods:(1) The N-terminal function domain and C-terminal function domainof DEK gene, after sequenced, were cloned into pcDNA3.0-flag plasmid,respectively and the proteins were acquired by using transcription and translationsystem in vitro.(2) The inducing condition of the pGEX-5T/LCMR1prokaryoticexpression vector was optimized to acquire stable expression of GST-LCMR1inE.coli cells.(3) The binding domain of interacting proteins LCMR1and DEK wasanalysed by GST pull-down. The result provided essential clues for theinvestigation of LCMR1function.(4) RNA interference (RNAi) approach wasused to knock down LCMR1in lung cancer cells. Quantitative real-time RT-PCRand Western blot analysis were used to confirm if LCMR1was successfullyknocked down by RNAi at both the mRNA and protein levels.(5) The impacts ofLCMR1knockdown on the apoptosis of lung cancer cells were assessed usingflow cytometry, quantitative real-time RT-PCR and Western blotting.Results:(1) The N-terminal function domain and C-terminal function domainof DEK plasmids were constructed successfully and the proteins weresuccessfully acquired in vitro.(2) The stable expression of GST-LCMR1was obtained by using E.coli system and the fusion protein was successfully purifiedby affinity chromatography using glutathione-agarose resin.(3) The analysis ofGST pull-down showed that LCMR1did have interation with the N-terminalfunction domain of DEK, but no interaction with the C-terminal function domainof DEK, which provided essential clues for the investigation of LCMR1function.(4) The human LCMR1-specific small interfering RNA (siRNA) sequence is5’-CAGUGUCGUCUGAUGCAUAUU-3’, designed using an online siRNA toolprovided by Invitrogen with LCMR1sequence (GenBank code: AY148462) as areference. Quantitative real-time RT-PCR and Western blot analysis revealed thatLCMR1was successfully knocked down by RNAi at both the mRNA and proteinlevels.(5) The inhibition of LCMR1by the siRNA enhanced apoptotic activity inlung cancer cells and LCMR1knockdown increased the expression and activity ofcleaved caspases-3.(6) The apoptosis of LCMR1was associated with cleavedcaspase-8and cleaved caspase-9. LCMR1participated in the regulation ofapoptosis of lung cancer cells and this process required the involvement of p53.LCMR1knockdown resulted in the upregulation of Bax and Bid，butdownregulation of Mcl-1.Conclusions: LCMR1had interation with the N-terminal function domain ofDEK. LCMR1was successfully knocked down by siRNA at both the mRNA andprotein levels. LCMR1suppressed apoptosis of lung cancer cells and this effectwas associated with multiple apoptosis related proteins, including p53, Bax, Bidand Mcl-1. LCMR1participated in both the intrinsic and extrinsic apoptosispathways. LCMR1might serve as a potential molecular target for lung cancertherapies.