调控HER4-PI3K/AKt轴对骨肉瘤恶性生物学行为和干性表达的影响

Effect of Mediating HER4/PI3K/AKt Axis on Proliferation and Stemness Features of Osteosarcoma Cells

目的探讨HER4基因操控对骨肉瘤细胞的恶性生物学特征和干细胞样特点的影响以及可能的机制。方法用Qrt-PCR、western blotting方法分析HER4 mRNA以及蛋白水平。MTT法和集落形成法被用来评价MG-63细胞活性和增殖能力。短发夹RNA(shRNA)干扰预处理、菌落形成、迁移、侵袭和western blotting实验确定HER-4调节骨肉瘤细胞增殖和侵袭/迁移的机制。采用球形成实验和CD133+细胞群检测HER-4诱导的干细胞样特征。结果HER4在骨肉瘤细胞中过表达。Sh-HER4表现出明显的细胞活力、集落形成和侵袭/迁移抑制能力。此外,HER4的敲除显著降低了CD133阳性细胞的球形大小和比例,以及干细胞标志物的表达。从机制上看,HER4通过失活PI3K/AKT通路促进骨肉瘤进展。结论综上所述,这些结果表明HER4是骨肉瘤进展和干细胞调节的一个新靶点,可能对开发治疗骨肉瘤的方法有价值。

Objective To investivage the role of HER4 in malignant proliferation and stemness features of osteosarcoma MG-63 cells.Methods Knockdown of HER4 in osteosarcoma cell lines was performed using sh RNA-HER4(sh-HER4).Reverse transcription-polymerase chain reaction(Qrt-PCR)and Western blotting were employed to defect shRNA interferen efficiency.The clone formation assay and MTT assay were used to investiage the proliferation of osteosarcoma MG-63 cells.The flow cytometry analysis was used to detect the ratio of CD133 positive cells.Mammosphereformation analysis was used to determine self-renewal ability.Apoptosis and stemness-related pmteins were detected by western blotting.Results HER4 is highly expressed in osteosarcoma cell lines(P<0.05).Sh-HER4 blocked clone formation,cell viability,invasive cell numbers and wound healing.Sh-HER4 also downregulated spheroids numbers,CD133 positive cell ratios and downregulated Nanog,Sox-2,Oct3/4 levels.(P<0.05).Conclusion These results suggest a novel role of HER4 in osteosarcoma progression indicating its potential as a therapeutic target for this disease.