| Backgrounds and objectivesOsteosarcoma is the second of bone malignancies, the mechanismof incidence is unclear. Osteosarcoma always occurs in young people, highdegree of malignancy and the prognosis is poor, About within a fewmonths to lung metastasis and mortality rate is high. So early diagnosis andtreatment are important to cure this tumor.S100protein is a class of small molecules with the EF-hand structureof calmodulin. There are at least25members in the family, with freecalcium or target proteins to produce a variety of physiological effects,such as: to be involved in cell proliferation and apoptosis, and regulateenzyme activity by protein phosphorylation involved in multiple signalingpathways. The genes of21members from the family are located in the lessstable, high incidence of chromosomal rearrangements chromosome1q21.It is reported that S100family is associated with a variety of high incidenceof tumor, such as osteosarcoma, ovarian cancer, breast cancer.This subject intends to study the effect of recombinant proteinhS100A6on proliferation, migration, and invasion of osteosarcoma cells143B with and to explore the molecular mechanisms of this impact whether Wnt/beta-catenin signal pathway and PI3K-AKT signal pathway areinvolved. The study should provide useful data for the in-depth study of themolecular mechanisms of osteosarcoma.Methods1.Preparation and identification of the Recombinant hS100A6:transferred pGST-GST-HRV3C-hS100A6plasmid to the host E. coli BL21by CaCl2method, induced its expression by isopropyl-β-thiogalactoside(IPTG) and then bacteria lysate was prepared by ultrasound on ice;GST-HRV3C-hS100A6was separated and purified by glutathioneSepharose4B beads, then desalinated and,digested overnight byGST-HRV3C protease in4°C; removed the GST tag and GST-HRV3Cprotease by glutathione-Sepharose4B beads. Sodium dodecylsulfate-polyacrylamide gel electrophoresis(SDS-PAGE) and western blotanalysis of recombinant protein, spectrophotometry for proteinquantitation,0.22μm membrane filter,to save it in-80℃.2. Treated cells with different concentrations of hS100A6(1μg/ml,10μg/ml,30μg/ml,60μg/ml,90μg/ml), cell proliferation was detected withMTT method to select the appropriate concentration as a followinterventions concentration. Use MTT to detect recombinant hS100A6tochanges of proliferation; Use transwell to invasion experiments detectchanges of the invasion ability;use transwell to migration experiment detectchanges of mobility;use hoechst33258to detect the changes of apoptosis. 3. Western blot analysis chnangs of β-catenin level and PI3K-AKTsignaling pathway marker protein p-AKT (Ser473) and its downstreamprotein phosphorylation level of GSK3β when hS100A6on humanosteosarcoma cells143B.; Then use of the signaling pathway inhibitors toinhibit the signaling pathway, and then recombinant hS100A6on cellproliferation, migration, invasion and apoptosis.Results1.By restriction enzyme digestion, sequencing analysis confirmed thepGST-HRV3C-hS100A6plasmid was constructed correctly; the relativemolecular mass of about36000protein was expressed in the transformedstrains after added IPTG; After purification, ultrafiltration replacementbuffer and digested with GST-HRV3C, and then removed GST-HRV3C andGST by GS4BB;the results of western blot showed that the protein can berecognized by S100A6antibody specifically; spectrophotometric methodmeasured the protein and the yield was approximately4mg/L bacteriaculture.2. Results of concentration-dependent MTT (1μg/ml to90μg/ml)showed:30μg/ml of the recombinant hS100A6significantly promoted theproliferation of143B cells, while90μg/ml recombinant hS100A6inhibitedtheir proliferation (P0.05). So chose30μg/ml as a follow-up interventionconcentration test.3. Results of MTT assay showed that143B cells treated with recombinant hS100A6from the second day to the fifth day, the A492nmvalue of the experimental group, respectively, increased by56.9%,41.4%,39.3%,72.4%compared with their control group(P0.05), suggesting thatthe recombinant hS100A6promoted the proliferation of143B cells ontime-dependent. Results of transwell invasion experiments showedhS100A6promoted the invasion ability of143B cell: after24hours thenumber of penetrating cells in experimental group increased145.1%thanthe control group (P0.05). Results of transwell migration experimentshowed that after24hours the number of penetrating cells inexperimental group increased of270.3%than the control group (P0.05),suggesting that the recombinant protein hS100A6to promote migration ofthe143B cells. Results of hoechst staining: apoptosis rate of theexperimental group is4.9%and the rate of control group is5.8%.After48h,there is still no significant difference (P>0.05), suggesting that therecombinant protein hS100A6had no effect on apoptosis in143B.4Western blot results showed that:1)143B cells treated with therecombinant hS100A6for24h, β-catenin corrected gray scale values ofthe experimental group and control group was1.69±0.024,0.89±0.051,respectively. Compared with the control group, the β-catenin ofexperimental group increased by89.9%(P<0.01); suggested thatrecombinant hS100A6up-regulated β-catenin levels of the143B cells;2)experimental group, p-GSK3β corrected gray scale values of the experimental group and control group was1.15±0.021and0.25±0.016,the experimental group compared with the control group increased by360.3%(P<0.01); suggested that the recombinant hS100A6up-regulatingGSK3β phosphorylation levels of143B cells;3) p-AKT(Ser473) β-catenincorrected gray scale values of the experimental group and control groupwas0.67±0.021and0.35±0.026, the experimental group compared withthe control group increased by91.4%(P<0.01);5. Western blot showed hS100A6treated with the recombinanthS100A6for24h, p-AKT corrected gray value of1.29±0.022; co-treatedwith LY294002group corrected gray scale value of0.21±0.022, hS100A6treatment group compared with the co-treated group increased by514.3%(P<0.01);10μM concentration of LY294002can effectively inhibit thePI3K-AKT signaling pathway in the143B cells;The use of the PI3K-AKTinhibitor LY294002can blocked the signaling pathway effectively: Resultsof MTT shows that：A492nm value of inhibitor group compared with theexperimental group from the second day of the fourth day, respectivelyget a decrease of37.2%,18.7%,42.9%(P0.05). Results of chamberexperiments showed that the number of penetrating cells of inhibitor groupcompared with the experimental group get a decrease of48.8%after24h(P0.05);in Transwell invasion assay the number of penetrating cells ofthe inhibitor group compared with the experimental group get a decrease of50.2%after24h(P0.05), suggesting that PI3K-AKT involved the mechanism of Recombinant hS100A6promote143B cells proliferation,migration and invasion.Conclusion1. the plasmid pGST-GST-HRV3C-hS100A6, was>>> successfully.in E. coli BL21, pGST-HRV3C-hS100A6was expressed. Afterpurification and digestion, we got the high purity and activie recombinanthS100A6.2.30μg/ml of the recombinant hS100A6can promote theproliferation,invasion and migration process of human osteosarcoma cells143B;90μg/ml of recombinant and S00A6can inhibit their proliferation.3.Exogenous hS100A6can activate intracellular Wnt/β-cateninpathway and PI3K-AKT signaling pathway in human osteosarcoma143Bcells. Both pathways may be involved in the mechanisms of S100A6effects on human osteosarcoma143B cells4The possible mechanism of β-catenin upreglated by S100A6may beinvolved in GSK3β hyperphosphorylation; at the same time, GSK3βhyperphosphorylation may be caused by activation of the PI3K-AKTsignaling pathway... |
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