解剖学报

解剖学报 ›› 2022, Vol. 53 ›› Issue (4): 488-497.doi: 10.16098/j.issn.0529-1356.2022.04.012

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敲低3-磷酸甘油酸脱氢酶靶向能量代谢逆转骨肉瘤恶性生物学的行为

周红1 康权2 谢圣男1 陈洁1 石雨鹭1 罗庆1*    

  1. 1.重庆医科大学附属儿童医院儿科研究所， 儿童发育疾病研究教育部重点实验室，国家儿童健康与疾病临床医学研究中心，儿童发育重大疾病国家国际科技合作基地， 儿科学重庆市重点实验室，重庆 400014; 2.重庆医科大学附属儿童医院普通外科创伤外科， 重庆 400014
  • 收稿日期:2021-09-27 修回日期:2021-11-17 出版日期:2022-08-06 发布日期:2022-09-11
  • 通讯作者: 罗庆 E-mail:352934430@qq.com
  • 基金资助:
    分化抑制因子(Id)在骨髓间充质干细胞成骨异常分化与骨肉瘤形成中的调控研究;Wnt/β-catenin信号通路调节免疫反应在儿童胆道闭锁发病机制中的研究

Reversing malignant biological behavior of osteosarcoma by knocking down 3-phosphoglycerin dehydrogenase targeting energy metabolism 

ZHOU Hong 1  KANG  Quan2  XIE  Sheng-nan1  CHEN  Jie1  SHI  Yu-lu1  LUO  Qing1*   

  1. 1.Department of Pediatric Research Institute, Ministry of Education Key Laboratory of Child Development and Disorders, National Clinical Research Center for Child Health and Disorders, China International Science and Technology Cooperation Base of Child Development and Critical Disorders, Children’s Hospital of Chongqing Medical University, Chongqing Key Laboratory of Pediatrics, Chongqing 400014, China;  2.Department of General and Trauma  Surgery, Children’s Hospital of Chongqing Medical University, Chongqing 400014, China
  • Received:2021-09-27 Revised:2021-11-17 Online:2022-08-06 Published:2022-09-11
  • Contact: LUO Qing E-mail:352934430@qq.com

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摘要：

目的  探讨敲低3-磷酸甘油酸脱氢酶(PHGDH) 靶向能量代谢对人骨肉瘤143B细胞恶性生物学行为及成骨分化的影响。   方法  Real-time PCR及Western blotting检测PHGDH在成骨细胞hFOB1.19和不同恶性程度骨肉瘤细胞TE85、MG63、143B中的表达。采用脂质体转染法将短发夹RNA（shRNA）-PHGDH重组质粒转染至143B细胞中, Real-time PCR和Western blotting检测PHGDH的表达变化；结晶紫染色法、细胞计数法、CCK-8实验检测细胞增殖；划痕实验检测细胞平行迁移能力，Transwell实验检测细胞垂直迁移及侵袭能力；Annexin V-FITC/PI双染法、DAPI染色法检测细胞凋亡；碱性磷酸酶（ALP）染色和茜素红S染色检测成骨分化作用,Western blotting检测成骨分化指标Runt相关转录因子2(Runx2)、骨钙素(OC)的表达情况；Real-time PCR检测能量代谢相关基因葡萄糖转运蛋白1（(GLUT1）、6-磷酸果糖激酶1（PFK1）、M2型丙酮酸激酶（PKM2）、乳酸脱氢酶A（LDHA)的表达，乳酸检测试剂盒测定乳酸分泌量，三磷酸腺苷（ATP）检测试剂盒检测ATP产生量。   结果  PHGDH在143B细胞中的表达明显高于在hFOB1.19、MG63和TE85细胞中(P<0.01)；转染shRNA-PHGDH重组质粒使143B细胞中的PHGDH表达量降低(P<0.01)、增殖能力降低(P<0.01)、细胞迁移及侵袭能力减低(P<0.01)、凋亡率增高(P<0.01)、ALP染色阳性率增加(P<0.01)、茜素红染色阳性率增加(P＜0.05)、Runx2(P<0.05)和OC的表达增高(P<0.01)、能量代谢相关基因(GLUT1、PFK1、PKM2、LDHA)的表达下调(P<0.01)、乳酸减少(P<0.01)、ATP增多(P<0.05)。   结论  敲低PHGDH可通过能量代谢抑制人骨肉瘤143B细胞增殖、迁移、侵袭, 促进其凋亡, 并促进其成骨分化。

关键词: 3-磷酸甘油酸脱氢酶, 能量代谢, 骨肉瘤, 成骨分化, 实时定量聚合酶链反应, 人

Abstract:

Objective  To investigate the effect of knock-down 3-phosphoglycerin dehydrogenase (PHGDH) targeting energy metabolism on malignant biological behavior and osteogenic differentiation of human osteosarcoma 143B cells.    Methods  Real-time PCR and Western blotting were used to detect the expression of PHGDH in osteoblasts hFOB1.19 and osteosarcoma cells TE85, MG63 and 143B with different malignant degrees. The short hairpin RNA(shRNA)-PHGDH recombinant plasmid was transfected into 143 B cells by liposome transfection method. The expression of PHGDH was detected by Real-time PCR and Western blotting. Crystal violet staining, cell counting and CCK-8 assay were used to detect cell proliferation; wound healing assay was used to detect cell parallel migration ability, and Transwell assay was used to detect cell vertical migration and invasion ability. Annexin V-FITC/PI double staining and DAPI staining were used to detect apoptosis; Alkalinephosphatase(ALP) staining and alizarin red S staining were used to detect osteogenic differentiation. Western blotting was used to detect the expression of Runt related transcription factor 2 (Runx2) and osteocalcin (OC). The expression of genes related to energy metabolism, glucose  transporter-1(GLUT1), 6-phosphofructokinase-1(PFK1), pyruvate kinae subtype M2(PKM2), lactate dehydrogenase A(LDHA) was detected by Real-time PCR. Lactic acid secretion was detected by lactic acid detection kit. Adenosine triphosphate(ATP) production was detected by ATP detection kit.   Results  The expression of PHGDH in 143B cells was significantly higher than that in hFOB1.19, MG63 and TE85 cells (P<0.01). After the transfection of shRNA-PHGDH recombinant plasmid, the expression of PHGDH in 143 B cells decreased (P<0.01), proliferation ability decreased (P<0.01), cell migration and invasion ability decreased (P<0.01), apoptosis rate increased (P<0.01), ALP staining positive rate increased (P<0.01), alizarin red staining positive rate increased (P<0.05), Runx2 (P<0.05) and OC expression increased (P<0.01), expression of genes related to energy metabolism (GLUT1,PFK1,PKM2,LDHA) decreased (P<0.01), lactic acid decreased (P<0.01), ATP increased (P<0.05).   Conclusion  Knocking down of PHGDH can inhibit the proliferation, migration and invasion of human osteosarcoma 143B cells through energy metabolism, promote their apoptosis and promote their osteogenic differentiation.

Key words: 3-Phosphoglycerate dehydrogenase, Energy metabolism, Osteosarcoma, Osteogenic differentiation, Real-time PCR, Human

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