The Discovery And Preliminary Functional Exploration Of New Alternative Splicing Variants Of PHB2

Prohibitin 2(PHB2)is an evolutionarily highly conserved protein mainly located in the inner mitochondrial membrane in a complex with PHB1.The PHB complex plays an important role in maintaining mitochondrial structure and genomic stability.Beside in mitochondria,PHB2 also resides in other various subcellular compartments in cells,playing multiple important biological functions and involved in many physiological and pathological processes.However,it remains unclear as to the mechanisms mediating subcellular localization and biological functins of PHB2.Alternative splicing is one of the main mechanisms regulating the expression and function of eukaryotic genes,and it plays an important role to diversify eukaryotic functional proteins.According to the NCBI data,there are two alternative splicing variants of human PHB2,namely full-length V1 and truncated V3.At present,the reports on the biological functions of PHB2 are mostly related to the full-length V1 or not defined to any variants.There have been no reports on the biological functions of V3 or whether there are other alternative splicing variants in PHB2.Previous studies in our lab revealed that subcellular localization and biological roles of PHB2 differs in normal cells and tumor cells.Also,we observed the presence of variant V3 of PHB2 in the cells.So,what’s the role of the alternative splicing variant V3 of PHB2,whether there is difference between the full-length V1 and V3 of PHB2 on the subcellular localization and function,and whether different types of PHB2 variants account for the discrepant localization and biological function of PHB2 in normal cells and tumor cells.Trying to answer the above questions,this study aims to study the alternative splicing variants of PHB2 and starts with amplification of PHB2 variant c DNA by RT-PCR.The results are shown as follows.1.The c DNA of PHB2 was amplified in different cell lines by RT-PCR and agarose gel electrophoresis showed that the size of the amplified bands varies.Then c DNA fragments was recovered from gel and ligated to the T vector followed by DNA sequencing.The results showed that in addition to the alternative splicing variants V1 and V3,there were several other alternative splicing variants,respectively named PHB2-803 bp,PHB2-718 bp,PHB2-709 bp,PHB2-684 bp and PHB2-641 bp.This is the first proof that PHB2 gene undergo complicated alternative splicing in the cells and possesses multiple variants;2.The RT-PCR products of the PHB2 variants were seperated efficiently by high resolution DNA non-denatured polyacrylamide gel electrophoresis,We then developed specific marker for PHB2 variants by ourself.This marker was used to analyze the expression of PHB2 variants in different cell lines,showing that in different cell lines,full-length V1 is the main variant of PHB2,and the expression amount of the other variants is relatively less;3.Four PHB2 variants,PHB2-V1,PHB2-V3,PHB2-803 bp and PHB2-718 bp,were selected to constructed into GFP-fused eukaryotic expression vector.The recombinant plasmids were transfected into HEK293 T cells and demonstrated by Western blot that four GFP-fused PHB2 variants were expressed in the cells indicating that recombinant expression vectors were successfully constructed;4.The recombinant GFP-fused vectors of four PHB2 variants were transfected into human live normal cells L02 and their cellular locations were observed with GFP.Results show that PHB2-V1 is located in the mitochondria in L02 cells as reported,while PHB2-V3 and PHB2-718 bp are located in the nucleus in L02 cells;5.The recombinant expression vectors of four PHB2 variants were transfected into human liver normal cells L02 and human liver cancer cells Bel-7402 respectively,in order to explore the biological effects of four PHB2 variants.(1)MTT assay was used to detect cell activity,Results showed that the cell viability significantly decreased in L02 cells with PHB2-V3 overexpression,while PHB2-V3 overexpression could significantly enhance cell viability of Bel-7402 cell.Interestingly,and overexpressing PHB2-V1 and PHB2-718 bp could significantly reduce the viability of Bel-7402 cell,Whereas had no significant effect in L02 cells;(2)Brd U incorporation assay was applied to detect cell proliferation.The results showed that tendency of Brd U incorporation in L02 cells and Bel-7402 cells with overexpression of PHB2 four variants is consistent to MTT assay,however the differences did not reach the statistical significance,indicating that each variant had no significant effect on the proliferation of both cells.In conclusion,this study was the first proof that PHB2 had a variety of alternative splicing variants in cells and enriched the alternative splice forms of PHB2.The eukaryotic expression vectors of four PHB2 variants were successfully constructed.Preliminary data revealed that celluar localization and biological roles of the four variants might be greatly different.There results provide a good basis for future intensive research on PHB2 alternative splicing variants.