葡萄糖调节蛋白78对肝癌细胞放射敏感性的调控作用及其机制

doi:10.13481/j.1671-587X.20210410

吉林大学学报(医学版) ›› 2021, Vol. 47 ›› Issue (4): 888-895.doi: 10.13481/j.1671-587X.20210410

葡萄糖调节蛋白78对肝癌细胞放射敏感性的调控作用及其机制

杨洁¹,贺武斌²,安妮³,孔雯聪⁴,苏荣健³,王学哲¹()

^1.锦州医科大学附属第一医院检验科，辽宁锦州 121000
^2.锦州医科大学附属第一医院外科学实验室，辽宁锦州 121000
^3.锦州医科大学基础医学院细胞生物学教研室，辽宁锦州 121000
^4.锦州医科大学基础医学院病理学教研室，辽宁锦州 121000

收稿日期:2020-11-05 出版日期:2021-07-28 发布日期:2021-07-22
通讯作者: 王学哲 E-mail:wxz8080@163.com
作者简介:杨洁（1995－），女，江苏省淮安市人，在读硕士研究生，主要从事肝癌治疗及其分子机制方面的研究。
基金资助:
国家自然科学基金项目(81172048);辽宁省教育厅自然科学基金项目(2015020326)

Regulatory effect of glucose-regulating protein 78 on radiotherapy sensitivity of liver cancer cells and its mechanism

Jie YANG¹,Wubin HE²,Ni AN³,Wencong KONG⁴,Rongjian SU³,Xuezhe WANG¹()

^1.Department of Clinical Laboratory，First Affiliated Hospital，Jinzhou Medical University，Jinzhou 121001，China
^2.Department of Surgery Laboratory，First Affiliated Hospital，Jinzhou Medical University，Jinzhou 121001，China
^3.Department of Cell Biology，School of Basic Medical Sciences，Jinzhou Medical University，Jinzhou 121000，China
^4.Department of Pathology，School of Basic Medical Sciences，Jinzhou Medical University，Jinzhou 121000，China

Received:2020-11-05 Online:2021-07-28 Published:2021-07-22
Contact: Xuezhe WANG E-mail:wxz8080@163.com

摘要/Abstract

摘要： 目的

探讨葡萄糖调节蛋白78（GRP78）对肝癌细胞放射敏感性的影响，初步阐明其分子机制。

方法

采用实时荧光定量PCR（RT-qPCR）法和Western blotting法检测肝癌SMMC-7721、HepG2、PLC、Huh7和QGY-7703细胞中GRP78 mRNA和蛋白表达水平，筛选GRP78高表达和低表达的肝癌细胞作为研究对象。采用RNA干扰技术在GRP78高表达的人肝癌SMMC-7721细胞中靶向沉默GRP78，命名为siGRP78- SMMC-7721（siGRP78组），以siNC-SMMC-7721为对照组（siNC组）；采用基因重组技术在GRP78低表达的人肝癌HepG2细胞中过表达GRP78，命名为Flag-GRP78-HepG2（Flag-GRP78组），以空载体3×Flag-HepG2为对照组（3×Flag组）。各组细胞分别给予0、2、4、6、8和10 Gy剂量X射线照射后，采用MTT法检测肝癌细胞存活率，5-乙炔基-2'脱氧尿嘧啶核苷（EdU）荧光染色法检测肝癌细胞增殖率，Western blotting法检测肝癌细胞中磷脂酰肌醇3激酶/蛋白激酶B（PI3K/Akt）信号通路中磷酸化PI3K（p-PI3K）和磷酸化Akt（p-Akt）蛋白表达水平。

结果

RT-qPCR法和Western blotting法检测，SMMC-7721细胞中GRP78 mRNA和蛋白表达水平最高，HepG2细胞中GRP78 mRNA和蛋白表达水平最低，选取SMMC-7721和HepG2细胞作为研究对象。MTT法检测，随着放射剂量的增加，肝癌细胞存活率逐渐降低；与siNC组比较，6 Gy以上剂量X射线照射后，siGRP78组细胞存活率明显降低（P<0.01）；与3×Flag组比较，4 Gy以上剂量X射线照射后，Flag-GRP78组细胞存活率明显升高（P<0.05或P<0.01）。EdU荧光染色法检测，与siNC组比较，siGRP78组细胞增殖率明显降低（P<0.05）；与3×Flag组比较，Flag-GRP78组细胞增殖率明显升高（P<0.05）。Western blotting法检测，与siNC组比较，siGRP78组细胞中p-PI3K和p-Akt蛋白表达水平明显降低（P<0.01）；与3×Flag组比较，Flag-GRP78组细胞中p-PI3K和p-Akt蛋白表达水平明显升高（P<0.01）。

结论

高表达水平的GRP78可降低肝癌细胞放射敏感性，其作用机制可能与GRP78激活PI3K/Akt信号通路有关。

关键词: 葡萄糖调节蛋白78, 肝肿瘤, 放射敏感性, 细胞凋亡, 磷脂酰肌醇3激酶, 蛋白激酶B

Abstract: Objective

To investigate the effect of glucose-regulating protein 78 （GRP78） on the radiotherapy sensitivity of liver cancer cells， and to preliminarily explore its molecular mechanism.

Methods

Real-time fluorescence quantitative PCR（RT-qPCR） and Western blotting methods were used to detect the expression levels of GRP78 mRNA and protein in the liver cancer SMMC-7721， HepG2， PLC， Huh7 and QGY-7703 cells， then the high and low GRP78 expression liver cells were selected as the research objects. GRP78 was targeted silenced by RNA interference technique in highly expressed human liver cancer SMMC-7721 cells， which was named siGRP78-SMMC-7721（siGRP78 group）， and siNC-SMMC-7721 was used as control group（siNC group）. GRP78 was over-expressed in the low-expression liver cancer HepG2 cells by gene recombination technique， which was named Flag-GRP78-HepG2（Flag-GRP78 group），and empty vector 3×Flag-HepG2 was used as control group（3×Flag group）.After irradiation with 0， 2， 4， 6， 8 and 10 Gy X-ray， the survival rates of liver cancer cells in various groups were determined by MTT assay. The proliferation rates of liver cancer cells in various groups were determined by EdU fluorescence staining. Western blotting method was used to detect the expression levels of phospatidylinositol-3（PI3K）/protein kinase B（Akt） signaling pathway proteins.

Results

The results of RT-qPCR and Western blotting methods showed that the expression levels of GRP78 mRNA and protein were the highest in SMMC-7721 cells and the lowest in HepG2 cells，so the SMMC-7721 and HepG2 cells were selected as the subjects.The MTT assay results showed that the survival rates of liver cancer cells were decreased with the increase of radiation dose；compared with siNC group， the survival rates of cells in siGRP78 group were significantly reduced after X-ray irradiation above 6 Gy（P<0.01）；compared with 3×Flag group， the survival rates of cells in Flag-GRP78 group were significantly increased after X-ray irradiation above 4 Gy （P<0.05 or P<0.01）.The EdU fluorescence staining results showed that compared with siNC group， the proliferation rate of cells in siGRP78 group was significantly decreased （P<0.05）；compared with 3×Flag group， the cell proliferation rate in Flag-GRP78 group was significantly increased （P<0.05）.The results of Western blotting method showed that compared with siNC group， the expression levels of phosphorylated PI3K（p-PI3K）and phosphorylated Akt（p-Akt） proteins in the cells in siGRP78 group were significantly decreased（P<0.01）. Compared with 3×Flag group， the expression levels of p-PI3k and p-Akt in the cells in Flag-GRP78 group were significantly increased（P<0.01）.

Conclusion

High expression GRP78 can decrease the radiosensitivity of liver cancer cells， and its mechanism may be related to the activation of PI3K/Akt signaling pathway.

Key words: glucose-regulating protein 78, liver neoplasms, radiotherapy sensitivity, apoptosis, phosphatidylinositol 3 kinase, protein kinase B

中图分类号:

R735.7

杨洁,贺武斌,安妮,孔雯聪,苏荣健,王学哲. 葡萄糖调节蛋白78对肝癌细胞放射敏感性的调控作用及其机制[J]. 吉林大学学报(医学版), 2021, 47(4): 888-895.

Jie YANG,Wubin HE,Ni AN,Wencong KONG,Rongjian SU,Xuezhe WANG. Regulatory effect of glucose-regulating protein 78 on radiotherapy sensitivity of liver cancer cells and its mechanism[J]. Journal of Jilin University(Medicine Edition), 2021, 47(4): 888-895.

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Group	Survival rate
Group	(D/Gy) 0	2	4	6	8	10
siNC	100.00±5.16	90.56±4.18	76.27±4.83	60.95±3.95	46.68±4.63	35.45±3.47
siGRP78	100.00±3.48	88.26±3.99	69.97±4.30	40.19±4.03^*	25.05±2.47^*	15.95±1.94^*
3×Flag	100.00±4.11	85.76±4.52	72.18±4.64	47.09±4.42	33.37±4.30	12.15±2.03
Flag-GRP78	100.00±3.82	90.38±4.37	81.11±5.14^△	67.39±3.23^△△	51.71±3.57^△△	37.52±2.44^△△

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葡萄糖调节蛋白78对肝癌细胞放射敏感性的调控作用及其机制

Regulatory effect of glucose-regulating protein 78 on radiotherapy sensitivity of liver cancer cells and its mechanism

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