Mechanisms Of Transcription Factor PBX1 Reprogramming B16 As Melanoma Stem Cell-like Cells

Background:The skin is the largest organ of the human body and has a very important physiological function.The lack of knowledge about skin tumors and the lack of effective treatment methods make the cases of skin tumors increasing year by year.Because of the high invasiveness,high degree of malignancy and poor prognosis of melanoma,people urgently need to explore effective treatment methods.At present,the research progress of tumor stem cells around the world provides new ideas for the treatment of melanoma.This study will adopt the method of slow virus transduction Pbx1 gene into mice B16 melanoma cells and observe the biological behavior changes of genes reprogrammed cells.We will explore PBX1 of mouse melanoma cells in the activation and the role of stem cell characteristics.And we will study the mechanism of signaling pathways and antitumor drug screening and research for the future.Objective:To study the biological behavior changes and related signal pathway mechanism of transcription factor PBX1 after reprogramming B16,and to provide theoretical and experimental basis for the establishment of molecular early warning,early diagnosis methods and the screening and research of anti-tumor drugs,which is conducive to the treatment of malignant melanoma.Method:B16F10 cells were selected as experimental subjects,and the lentivirus transduction method was used to overexpress Pbx1 gene.Groups were divided according to Wild type control group（Wild）,Vector group（Vector）,overexpressed Pbx1 group（PBX1）,negative control group（nc-shrna）and knockdown Pbx1 group（shPBX1）to verify the gene transduction efficiency functionally.MTT flow cytometry was used to detect the effect of PBX1 on cell proliferation and cell cycle.The migration effect of PBX1 on B16F10 cells was detected by cell scratch assay.The ability of PBX1 to clone B16F10 cells was tested by plate cloning assay.The effect of PBX1 on the drug resistance of B16F10 cells was detected by dacarbazine.Western Blot was used to detect the expression levels of PBX1 on B16F10 cell proliferation and cell cycle,DNA double-strand breakage and repair,and tumor stem cell marker related proteins.Cellular immunofluorescence was used to observe DNA double strand breakage and repair.In vivo experiment,C57BL/6 mice were selected for in vivo tumor-bearing experiment to detect the tumorigenic ability of PBX1 on B16F10 cells.Result:1.The cell lines of Wild type group,Vector group,PBX1 overexpressed B16F10 cell line,nc-shpbx1 negative control group and shPBX1-PBX1-B16F10 knockout group were successfully constructed,and the functional verification showed that the transduction efficiency was more than 90%（P<0.05）.2.PBX1 promoted the proliferation of B16F10 cells and up-regulated its related proteins:MTT results showed that the proliferation rate of 0,1,2,3,and 4 d cells increased gradually,and the proliferation rate of PBX1 and NC-shPBX1 cells was significantly higher in 2 d than that of Vector,and the proliferation rate of shPBX1cells was the lowest（P<0.05）.PBX1 can promote the protein expression levels of AKT,ERK1/2 and STAT3 signaling pathways of cell proliferation,and knock down the expression levels of PBX1.（P<0.05）.3.PBX1 accelerated G₁/S phase transformation of B16F10 cells:the percentage of cells in the PBX1 and NC-shPBX1 groups was significantly increased in the S phase,but decreased in the G₀/G₁ phase（P<0.05）,the expression level of Cyclin CDK2 was up-regulated,and the expression levels of Cyclin D1,Cyclin D3,p21 and p27 were down-regulated（P<0.05）.PBX1 promoted cells to enter the S phase and reduced G₁phase arrest.4.PBX1 promoted the migration of B16F10 cells through the occurrence of EMT:the results of cell scratch experiment showed that the cell migration distance after 24 h of cell treatment,the cell migration distance between PBX1 and NC-shPBX1 group was significantly higher than that of Vector group,and knockdown of Pbx1 reduced the cell migration（P<0.05）.Decreased expression of E-Cadherin and increased expression of Vimentin were related markers of PBX1 and NC-shPBX1 cell migration,and the reverse change of knockdown Pbx1 was observed（P<0.05）.5.PBX1 promoted the clonal formation of B16F10 cells:the clonal formation rate of PBX1 and NC-shPBX1 group was significantly higher than that of Vector group,and the clonal formation rate decreased after knockout of Pbx1（P<0.05）.6.PBX1 increased the drug resistance of Dacarbazine,an anti-tumor drug,in B16F10cells:Cells were treated with 0、1.95312、3.906257、7.8125、15.625、31.25、62.5、125.0、250.0、500.0、1000.0、2000.0μg/mL dacarbazine injection for 24 h.The IC₅₀of Vector group was 163.0±2.3μg/mL.The IC₅₀0 of PBX1 group was 351.6±1.8μg/mL.The IC₅₀0 of NC-shPBX1 group was349.9±2.3μg/mL.The IC₅₀0 of shPBX1group was 64.58±1.6μg/mL,and the difference was statistically significant（P<0.05）.7.PBX1 increased the expression of tumor stem cell markers of B16F10 cells:cells in the PBX1 and NC-shPBX1 groups highly expressed NANOG,CD20,CD44 and ALDH1/2 tumor stem cell markers,and the expression level decreased after knockdown of Pbx1（P<0.05）.8.PBX1-B16F10 cells into a tumor in mice:PBX1 and Vector group cell density of1×10⁶/mL、1×10⁷/mL vaccination 0.1 mL,21 d mice can be obviously mass vaccination in bump,executed peel tissue in mice,clearly visible subcutaneous tumor black,tumor weight PBX1 group was obviously higher than that of Vector group,tumor weight Vector group 2.396±0.7281 g,PBX1 group 4.472±0.8424 g,statistically significant difference（P<0.05）.9.PBX1 promoted repair of B16F10 cell DNA double-strand damage:γH2AX fluorescence focus signal was observed under fluorescence microscope,and the proportion of fluorescence focus of PBX1 group and nc-shpbx1 group was significantly higher than that of Vector group and shPBX1 group（P<0.05）.In the PBX1 and NC-shPBX1 groups,the expression levels of PARP1 116 kDa and 89 kDa were up-regulated,and the expression levels of PARP1 116 kDa and 89 kDa were down-regulated after knockout of Pbx1,with statistically significant differences（P<0.05）.Conclusion:1.PBX1 can successfully transfect B16F10 through lentivirus experiment and exert its biological function.2.PBX1 promotes B16F10 cell proliferation and accelerates G₁/S cell cycle transformation.3.PBX1 can induce the migration of B16F10 cells,accelerate the clone formation of single cells and tumor formation in mice,and increase the resistance of anti-tumor drug Dacarbazine.4.PBX1 accelerates DNA double-strand damage and PARP1 repair pathway in B16F10 cells.5.PBX1 promoted the high expression of tumor stem cell markers NANOG,CD20,CD44 and ALDH1/2 in B16F10 cells.6.PBX1 reprogrammable B16F10 is a melanoma stem-like cells.