人内源性反转录病毒-H长末端重复关联蛋白2对骨肉瘤细胞成瘤和转移能力的影响及其机制

Effect of human endogenous retrovirus-H long terminal repeat association protein 2 on tumorigenesis and metastasis of osteosarcoma cells and its mechanism

目的:探讨人内源性反转录病毒-H长末端重复关联蛋白2(HHLA2)对骨肉瘤细胞成瘤和转移能力的影响及其机制。方法:选取2023年6月至2024年6月郑州市骨科医院收治的37例骨肉瘤和癌旁组织作为研究对象,采用荧光定量聚合酶链反应(PCR)和蛋白质免疫分析肿瘤组织和癌旁组织HHLA2蛋白和mRNA水平。采用脂质体转染对照小干扰RNA(siRNA)和HHLA2 siRNA至骨肉瘤细胞U2OS,建立对照组和HHLA2 KD组细胞,采用细胞计数试剂、克隆形成实验和体外移植瘤实验分析两组细胞的柽柳能力;采用划痕实验和Transwell分析两组细胞的迁移和侵袭能力。采用蛋白质免疫印迹分析。组间计量数据比较采用独立样本t检验。结果:癌旁组织HHLA2 mRNA和蛋白质水平(0.95±0.14、0.99±0.21)明显低于骨肉瘤组织HHLA2 mRNA和蛋白质水平(1.94±0.19、1.85±0.26),差异有统计学意义(t=25.550、18.740,P<0.05)。HHLA2对细胞活力的影响:对照组细胞24、36、48 h吸光度值(0.46±0.07、0.92±0.05、1.73±0.07)明显高于HHLA2 KD组细胞(0.37±0.02、0.66±0.05;1.14±0.09),差异有统计学意义(F=6.142,P<0.05)。对照组细胞克隆形成率[(74.17±6.05)%]明显高于HHLA2 KD组细胞[(37.33±4.76)%],差异有统计学意义(t=11.720,P<0.05)。对照组细胞裸鼠成瘤体积和重量[(37.33±4.76)mm 3、(4.78±0.89)g]明显高于HHLA2 KD组细胞[(37.33±4.76)mm 3、(1.99±0.24)g],差异有统计学意义(t=10.160、7.391,P<0.05)。HHLA2对细胞转移的影响:对照组细胞克隆形成率[(83.17±6.33)%、(129.50±17.83)个]明显高于HHLA2 KD组细胞[(52.33±6.77)%、(87.67±6.94)个],差异有统计学意义(t=8.143、5.355,P<0.05)。对照组细胞JAK2、JAK3、STAT2和STAT3蛋白表达水平(1.03±0.11、0.85±0.05、1.13±0.10、1.18±0.10)明显高于HHLA2 KD组细胞(0.56±0.09、0.40±0.05、0.29±0.15、0.38±0.10),差异有统计学意义(t=7.787、14.720、11.770、13.590,P<0.05)。癌旁组织中JAK2、JAK3、STAT2和STAT3蛋白表达水平(0.72±0.11、0.36±0.12、0.67±0.09、0.94±0.11)明显低于骨肉瘤组织(1.03±0.11、1.15±0.23、1.38±0.12、1.52±0.32),差异有统计学意义(t=7.873、11.990、11.770、18.040,P<0.05)。结论:HHLA2在骨肉瘤组织程高表达,促进骨肉瘤细胞的成瘤和转移能力。

Objective:To investigate human endogenous retrovirus subfamily H long terminal repeat associating protein 2(HHLA2),Effect of HHLA2 on tumorigenesis and metastasis of osteosarcoma cells and its mechanism.Methods:37 cases of osteosarcoma and paracancer tissue treated in zhengzhou orthopedic hospital from June 2023 to June 2024 were selected as the study objects.The HHLA2 protein and mRNA levels of tumor tissue and paracancer tissue were analyzed by fluorescence quantitative polymerase chain reaction(PCR)and protein immunoassay.The control small interfering RNA(siRNA)and HHLA2 siRNA were transfected into osteosarcoma cells U2OS by liposome,and the cells in the control group and HHLA2 KD group were established.The tamarix ability of the cells in the two groups was analyzed by cell counting reagent,clonal formation assay and in vitro tumor transplantation assay.The migration and invasion ability of the two groups of cells were analyzed by scratch test and Transwell.Western blotting analysis was performed.Independent-sample t test was used to compare measurement data between groups.Results:The levels of HHLA2 mRNA and protein in paracancer tissues(0.95±0.14),the HHLA2 mRNA and protein levels in osteosarcoma were significantly lower(0.99±0.21)than those in osteosarcoma(1.94±0.19,1.85±0.26,t=25.550,18.740,P<0.05).The effects of HHLA2 on cell viability were as follows:24 h,36 h and 48 h light absorption values of control cells(0.46±0.07,0.92±0.05,1.73±0.07) cells were significantly higher than those in HHLA2 KD group(0.37±0.02,0.66±0.05,1.14±0.09,F=6.142,P<0.05).The cell clonal formation rate[(74.17±6.05)%]in control group was significantly higher than that in HHLA2 KD group[(37.33±4.76)%],and the difference was statistically significant(t=11.720,P<0.05).The tumor volume and weight of nude mice in control group[(37.33±4.76)mm 3,(4.78±0.89)g]were significantly higher than those in HHLA2 KD group[(37.33±4.76)mm 3,(1.99±0.24)g,t=10.160,7.391,P<0.05].Effects of HHLA2 on cell metastasis:The clonal formation rate of the control group[(83.17±6.33)%,(129.50±17.83)cells]was significantly higher than that of the HHLA2 KD group[(52.33±6.77)%,(87.67±6.94)cells,t=8.143,5.355,P<0.05].The expression levels of JAK2,JAK3,STAT2 and STAT3 in control cells(1.03±0.11,0.85±0.05,1.13±0.10,1.18±0.10)were significantly higher than those in HHLA2 KD group(0.56±0.09,0.40±0.05,0.29±0.15,0.38±0.10,t=7.787,14.720,11.770,13.590,P<0.05).The expression levels of JAK2,JAK3,STAT2 and STAT3 proteins in para-cancerous tissues(0.72±0.11,0.36±0.12,0.67±0.09,0.94±0.11)significantly lower than the osteosarcoma tissue(1.03±0.11,1.15±0.23,1.38±0.12,1.52±0.32,t=7.873,11.990,11.770,18.040,P<0.05).Conclusion:HHLA2 is highly expressed in osteosarcoma tissues and promotes tumor formation and metastasis of osteosarcoma cells.