PKLR在胰腺癌中的表达水平及临床病理学意义

doi:10.19401/j.cnki.1007-3639.2021.08.003

中国癌症杂志 ›› 2021, Vol. 31 ›› Issue (8): 704-713.doi: 10.19401/j.cnki.1007-3639.2021.08.003

PKLR在胰腺癌中的表达水平及临床病理学意义

杨士鹏 ^1,2，刘颖^1,3，王馨悦 ^1,3 ，杨洋 ^1,3 ，全吉淑 ⁴ ，林贞花^1,3

1. 延边大学肿瘤研究中心，吉林延吉 133002 ；
2. 延边大学附属医院外科，吉林延吉 133002 ；
3. 民族地区高发肿瘤病理生物学国家民委重点实验室（延边大学），吉林延吉 133002
4. 延边大学医学院生物化学与分子生物学教研室，吉林延吉 133002

出版日期:2021-08-30 发布日期:2021-09-03
通信作者: 林贞花 E-mail: zhlin720@ybu.edu.cn

Clinical significance of PKLR overexpression in the pancreatic cancer

YANG Shipeng ^1,2 , LIU Ying ^1,3 , WANG Xinyue ^1,3 , YANG Yang ^1,3 , QUAN Jishu ⁴ , LIN Zhenhua^1,3

1. Department of Cancer Research Center, Yanbian University Medical College, Yanji 133002, Jilin Province, China; 2. Department of Surgery, Affiliated Hospital of Yanbian University, Yanji 133002, Jilin Province, China; 3. Key Laboratory of Pathobiology of High Frequency Oncology in Ethnic Minority Areas, Yanbian University, State Ethnic Affairs Commission, Yanji 133002, Jilin Province, China; 4. Department of Biochemistry and Molecular Biology, Yanbian University, Yanji 133002, Jilin Province, China

Published:2021-08-30 Online:2021-09-03
Contact: LIN Zhenhua E-mail: zhlin720@ybu.edu.cn

摘要/Abstract

摘要： 背景与目的：胰腺癌是一种原发于消化系统的恶性肿瘤，发病率男性高于女性，且患者预后差。重组人丙酮酸激酶（pyruvate kinase isozymes R/L，PKLR）属于丙酮酸激酶家族，哺乳动物丙酮酸激酶同工酶有4种：L、R、M1和M2，其与肿瘤的发生、发展密切相关。已经有研究发现，PKLR会促进乳腺癌的转移。探究PKLR蛋白在胰腺癌中的临床病理学意义，分析PKLR对胰腺癌生物学行为的影响及分子机制。方法：运用免疫荧光染色检测PKLR蛋白在胰腺癌细胞中的定位；采用免疫组织化学染色EnVision法检测PKLR在胰腺癌组织中的阴性表达率、阳性表达率和强阳性表达率，并分析PKLR与胰腺癌患者临床病理学特征之间的关系；运用小干扰RNA转染技术敲减PKLR表达观察其在胰腺癌细胞中的蛋白表达水平；噻唑蓝实验检测敲低PKLR后细胞增殖活性的变化；平板克隆实验检测敲减PKLR后BxPC-3和MIA PaCa-2细胞菌落形成数量的变化；应用细胞划痕实验检测PKLR对胰腺癌细胞横向迁移能力的影响；transwell实验检测PKLR对胰腺癌细胞纵向迁移能力的影响；蛋白质印迹法（Western blot）检测PKLR对上皮-间质转化（epithelial-mesenchymal transformation，EMT）相关标志蛋白表达变化的影响。结果：免疫荧光结果显示，PKLR荧光信号定位于BxPC-3和MIA PaCa-2胰腺癌细胞的细胞质中；免疫组织化学染色结果表明，PKLR在胰腺癌组织中的阳性表达率（75.2%）和强阳性表达率（48.6%）显著高于正常胰腺组织（14.3%和7.1%）（P<0.01）；且其表达与胰腺癌患者的组织学分级及淋巴结转移密切相关（P<0.05）；小干扰RNA转染技术敲减PKLR表达后，PKLR在胰腺癌细胞中的蛋白表达水平明显下调；MTT结果显示，敲减PKLR后，细胞增殖活性明显受到抑制，且呈时间依赖性（P<0.05）；平板克隆实验发现，敲减PKLR可显著减少BxPC-3和MIA PaCa-2细胞的集落形成数量；细胞划痕实验和transwell实验检测发现敲减PKLR后胰腺癌细胞的迁移能力明显受到抑制；检测PKLR对EMT标志物的影响发现，下调PKLR表达可明显抑制胰腺癌细胞的增殖及迁移能力，并上调上皮标志物E-cadherin的表达，下调间质标志物vimentin和snail的表达（P<0.05）。结论：PKLR在胰腺癌的发生、发展过程中扮演着重要的促癌角色，我们将做进一步深入研究。

关键词: 胰腺癌, PKLR, 增殖, 迁移, 上皮-间质转化

Abstract: Background and purpose: Pancreatic cancer is a malignant tumor which occurs primarily in the digestive system. The incidence rate is higher in men than in women, and its prognosis is poor. Recombinant human pyruvate kinase isozymes R/L (PKLR) belong to the pyruvate kinase family. There are four kinds of mammalian pyruvate kinase isozymes: L, R, M1 and M2, which are closely related to the occurrence and development of tumor. Studies have shown that PKLR promotes breast cancer metastasis. This study aimed to investigate the clinicopathological significance of PKLR protein in pancreatic cancer, and to analyze the effect of PKLR on the biological behavior of pancreatic cancer and its molecular mechanism. Methods: Immunofluorescence staining was used to detect the localization of PKLR protein in pancreatic cancer cells. The negative expression rate, positive expression rate and strong positive expression rate of PKLR in pancreatic cancer tissues were detected by immunohistochemical staining EnVision method, and the correlation between PKLR and clinicopathological features of pancreatic cancer was analyzed. Small interfering RNA transfection technology was used to knockdown PKLR expression and observe its protein expression in pancreatic cancer cells. Methylthiazolyldiphenyl-tetrazolium bromide assay was used to detect the changes of cell proliferation activity after knockdown of PKLR. Plate cloning assay was used to detect the changes of colony formation of BXPC-3 and Mia PaCa-2 cells after PKLR knockdown. The scratch healing test was used to detect the effect of PKLR on the lateral migration of pancreatic cancer cells. Transwell assay was used to detect the effect of PKLR on the vertical migration of pancreatic cancer cells. The effect of PKLR on the expression of epithelial-mesenchymal transformation (EMT) related marker proteins was detected by Western blot. Results: Immunofluorescence results showed that PKLR fluorescence was localized in the cytoplasm of BxPC-3 and MIA PaCa-2 pancreatic cancer cells. Immunohistochemical staining showed that the positive expression rate of PKLR in pancreatic cancer tissues (75.2%) and strong positive expression rate (48.6%) were significantly higher than those in normal pancreatic tissues (14.3% and 7.1%) (P<0.01). The expression was closely related to histological grading and lymph node metastasis of pancreatic cancer (P<0.05). After knockdown of PKLR expression by small interfering RNA transfection, the protein expression level of PKLR in pancreatic cancer cells was significantly downregulated. MTT results showed that cell proliferation was significantly inhibited in a time-dependent manner (P<0.05) after knockdown of PKLR. Plate cloning experiment showed that knockdown of PKLR could significantly reduce the number of colony formation of BXPC-3 and Mia PaCa-2 cells; Scratch healing test and transwell test showed that the migration ability of pancreatic cancer cells was significantly inhibited after knockdown of PKLR. The effect of PKLR on EMT markers was detected. Downregulation of PKLR expression could significantly inhibit the proliferation and migration of pancreatic cancer cells, upregulate the expression of E-cadherin, and downregulate the expression of vimentin and Snail (P<0.05). Conclusion: PKLR plays an important role in the development and progression of pancreatic cancer. We will do further research.

Key words: Pancreatic cancer, PKLR, Proliferation, Migration, Epithelial-mesenchymal transformation

杨士鹏, 刘颖, 王馨悦, 杨洋, 全吉淑, 林贞花 . PKLR在胰腺癌中的表达水平及临床病理学意义[J]. 中国癌症杂志, 2021, 31(8): 704-713.

YANG Shipeng , LIU Ying , WANG Xinyue , YANG Yang , QUAN Jishu , LIN Zhenhua . Clinical significance of PKLR overexpression in the pancreatic cancer[J]. China Oncology, 2021, 31(8): 704-713.

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PKLR在胰腺癌中的表达水平及临床病理学意义

Clinical significance of PKLR overexpression in the pancreatic cancer

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