Regulated Mechanisms Of The Dissociative Cytoplasmic Region Of LIFRα Subunit In HL-60

Background and objectiveLeukemia-inhibitory factor (LIF) is a pleiotropic cytokine expressed by multiple tissue types. IT have different biology effects to regulate the process of proliferation and differentiation in multiple cell types, such as inhibit the differentiation of ES cell in vitro stimulate the production of hematoblast ,promote the proliferation of bone cell, the formation of nerve cell, restore the body muscle, participate in the hepatic acute reaction. LIF exerts its action through a heterodimer of the LIF receptor (LIFR) and gp130. LIFRα (gp190) is the low affinity LIF receptor, belonging to the receptor of hematopoietic cytokine super family, which is absent of endogenesis kinase activity. The cytoplasmic domain of gp190 contains three conserved motifs ,termed BOX 1 ,BOX 2 and BOX 3,which contains an YXXQ consensus sequence, especially located in the membrane-distal. YXXQ sequence can specifically associate with the SH2 domain of STAT3. They have different functions in tissue and cells. LIFRα exists in both a membrane-bound and a soluble form, the latter lacking the transmembrane and cytoplasmic domains. Is it exist dissociation receptor in cytoplasm? If we transferred the distinct motif of cytoplasmic receptor to cells, how did the dissociation receptor exert the biology function. It has been indicated that the single motif of one receptor subunit could active the cell proliferation and differentiation. LIF signaling pathway concern the Janus kinase signal transducer and activator of transcription (JAK-STAT) pathway and the Ras-mitogen-activated protein kinase (MAPK) pathway. We have design two recombined eukaryotic expression vector pcDNA3.0-190CT2+3(contains gp190 BOX2+3),pcDNA3.0-190CT3(contains gp190 B0X3), found the different effect of the two molecules after transferred intocells. To validate the previous work and explore the mechanism, our experiment was to constitute recombinant human HL-60 cells expressing BOX2+3 and BOX3. Furthermore, we devised the mutation of three YXXQ sequence located in BOX3 to analyze the related signal pathway. We detected, HL-60 cells which expressed the objective gene stably were inhibited to proliferate and trended to differentiate. In the processes, JAK-STAT3 pathway was activated. In our studies, we conformed the dissociative motif of gpl90 (BOX2+3, BOX3) had different biologic function to HL-60 cells and activated the related signaling pathway. It is beneficial to explore the application value about the single motif of cytoplasmic receptor, induce stem cells to directional differentiation consistently. It is hopeful to acquire a number of specific stem cells.Part I Recombination of human dissociated LIFR (gpl90) cytoplasmic receptor in HL-60 cells and identification its expressionMethods: (1) Enzyme BamHl and Xbal cleaving the recombined plasmid pcDNA3.0-190CT2+3 ^ pcDNA3.0-190CT3,and checking the sequences. (2) Transfected the eukaryotic expression plasmid pcDNA3.0-190CT2+3 ■, pcDNA3.0-190CT3 into HL-60 cells with liposome FuGENE-6. Two control group were setup which were vector pcDNA3.0 tansfected and wild-type HL-60 cells. In the culture processes, we selected the clones with different G-418 concentration. The transfected cells were survival and expanding in presence of neo-gene. We detected the protein expression about trancfected 190CT2+3 and 190CT3 with western blotting and identified the HL-60 cell lines with stable protein expression. (3 Observation of the cell proliferation. Describing the growth curve. The PCNA levels were assayed by western blotting. The levels of CD 15 were assayed by flow cytometer. The levels of STAT3 were assayed by immunohistochemistry. Results: (l)After BamH I and Xba I cleaving, we observed electrophoretic irradiance strip under the UV light. Sequence detection was consistent with the LIFR sequence in Genbank. (2) After subsequent selection by G418,G418-resistent colonies were isolated, namely ,thepcDNA3.0-190CT2+3> pcDNA3.0-190CT3 and pcDNA3.0 HL-60 cell lines. What western blotting analyzed demonstrated that the 190CT had expressed in HL-60 stably. By immunohistochemistry analyzing the LIFR, the positive signal in two test groups (recombined pcDNA3.0-190CT2+^ pcDNA3.0-190CT3 HL-60 cell lines) is enhanced than the signal in two control groups. Cells in two test groups were bigger than wide-type HL-60 cells And the form of nucleus appeared more leafs than the control groups. Statistics assayed that the proportion of leafs-nucleus in 190CT3 HL-60 cell lines than 190CT2+3 HL-60 cell lines. (3) The growth curve showed that the growth speed in the test cells is lower than in the control cells. Flow cytometer assay showed CD 15 expression increased in the test groups. Western blotting assay testified the expression of PCNA decreased in the test groups. Immunohistochemistry analyzed that the expression of STAT3 were increased. Conclusion: we acquired the recombined 190CT2+3> 190CT3 HL-60 cell lines. 190CT3 and 190CT2+3 could promote the differentiation and inhibit the proliferation of HL-60 cells.Part II: Signaling pathway investigation for human dissociated LIFR (gpl90) cytoplasmic receptorMethod: (l)mutant of 190CT3: mutagenic oligonucleotide primers were designed whose sequence is complementary to the gene sequence in the region to be mutated, but with three differences located in Y of YXXQ. The mutagenic oligonucleotide is then allowed to prime new DNA synthesis to create a complementary full-length sequence containing the desired mutation. PCR can be used to couple desired sequences or chemical groups to a target sequence and to produce specific pre-determined mutations in DNA sequences, then it was subcloned into the eukaryotic expression vector pcDNA3.0,namely,pcDNA3.0-MUT. Transfection with pcDNA3-MUT was performed with Fungene-6. The transfected cells were survival and expanding in presence of neo-gene. Then we detected the expression of MUT. Total RNA were abstracted from cells by guanidinium thiocyanate methods and Reserved Transcription PCR was performed to measure the production of MUT. (2)Western blotting examination. We analyzed the levels of related signal molecularas STA^ STAT3(T705)-P> P44/42 and JAK1.(3) immunofluorescence assay. We detected the expression of STAT3 and STAT3(T705)-P by immunofluorescence .Resultes: After PCR reaction, there is one 300-400bp DNA strip. Blast the MUT sequence and the original LIFR sequence, we found that the original TAT were exactly changed to TTC. The MUT4 colony was expressed the higher MUT by RT-PCR. (2) The levels of STAT3 and STAT3 (Y705)-P were obvious increased in two groups(recombined pcDNA3.0-190CT2+3 and 190CT3). The P44/42 levels were oppositely The MUT group had lower expression and none JAK1. (3) STAT3 had strong red- fluorescence signal in the cytoplast in two groups(190CT2+3 and 190CT3). STAT3 (Y705)-P had red- fluorescence signal in the nucleolus. Conclusion: we acquired recombinant pcDNA3.0-MUT HL-60 cell lines. 190CT2+3 and 190CT3, as well as BOX2+3 and B0X3, activated the JAK-STAT3 and inhibited the MAPK pathway.Conclusions1. We testified the dissociated cytoplasmic LIFR, as BOX2+3 and B0X3, could promoted the HL-60 to differentiation. And B0X3 had more intense effect.2. By detection of related signal molecule, we demonstrated the JAK-STAT3 pathway was activated . On the contrary, the MAPK pathway was inhibited.