结直肠癌中DPD与LC3、P62表达的相关性及其临床意义

doi:10.19401/j.cnki.1007-3639.2022.01.003

摘要/Abstract

摘要：

背景与目的：结直肠癌是常见的消化系统恶性肿瘤之一,应用5-氟尿嘧啶（5-fluorouracil,5-FU）治疗结直肠癌常因耐药或不良反应影响其疗效,二氢嘧啶脱氢酶（dihydropyrimidine dehydrogenase,DPD）是5-FU代谢的关键酶,其表达量或降解率可能成为影响5-FU疗效的因素,自噬是细胞内蛋白质降解的重要途径。检测结直肠癌中自噬关键因子微管相关蛋白轻链3（light chain 3,LC3）、P62和DPD的表达,探讨其相关性及临床意义,为逆转5-FU耐药提供新思路。方法：采用免疫组织化学EnVision法检测2013—2014年滨州医学院附属医院病理科存档的157例结直肠癌石蜡包埋组织,采用蛋白质印迹法（Western blot）及实时荧光定量聚合酶链反应（real-time fluorescence quantitative polymerase chain reaction,RTFQ-PCR）检测2020年滨州医学院附属医院病理科接收的44例结直肠癌新鲜组织中LC3、P62和DPD的表达,分析其相关性及其临床意义。结果：结直肠癌组织中LC3、P62和DPD的mRNA及蛋白表达量均高于正常黏膜组织（P<0.05）。P62与DPD的表达呈正相关,LC3与P62、DPD的表达均呈负相关（P<0.05）。P62^–/LC3^–组中DPD蛋白表达量明显高于其他3组,而P62^–/LC3⁺组中DPD蛋白表达量则低于其他3组（P<0.05）。P62⁺/LC3^–组与P62⁺/LC3⁺组中DPD蛋白表达量虽不同,但差异无统计学意义（P>0.05）。结直肠癌中P62蛋白表达与T分期及淋巴结转移呈正相关;LC3蛋白表达与肿瘤直径及T分期呈正相关;DPD蛋白表达则与组织学类型密切相关（P<0.05）。LC3、P62、DPD及淋巴结转移与结直肠癌患者预后密切相关,四者均是结直肠癌患者预后的独立危险因素。结论：LC3和P62可影响DPD的表达,自噬-溶酶体途径可能是DPD降解的重要途径,其有可能通过DPD降解进而影响结直肠癌对5-FU的耐药。

关键词: 结直肠癌, 二氢嘧啶脱氢酶, 自噬, 轻链3, P62

Abstract:

Background and purpose: Colorectal cancer is one of the most common malignancies. The efficacy of 5-fluorouracil (5-FU) in the treatment of colorectal cancer is often affected by drug resistance or toxic side effects. Dihydropyrimidine dehydrogenase (DPD) is the key enzyme in the metabolism of 5-FU, and the expression or degradation rate of 5-FU may be a factor affecting the efficacy of 5-FU. Autophagy is an important pathway of intracellular protein degradation. This study aimed to detect the expression of microtubule-associated protein light chain 3 (LC3), P62 and DPD in colorectal cancer, and to explore their correlation and clinical significance, so as to provide a new way to reverse 5-FU resistance. Methods: Immunohistochemical envision method was used to detect the expressions of LC3, p62 and DPD in 157 colorectal cancer paraffin tissues archived by Department of Pathology, Binzhou Medical University Hospital from 2013 to 2014. Western blot and real-time fluorescence quantitative polymerase chain reaction (RTFQ-PCR) were used to detect the expressions of LC3, p62 and DPD in 44 colorectal cancer fresh tissues received by Department of Pathology, Binzhou Medical University Hospital in 2020. The correlation and clinical significance were analyzed. Results: The expressions of LC3, P62 and DPD were significantly higher in colorectal carcinoma than in normal tissues (P<0.05). The expressions of P62 was positively correlated with the expressions of DPD, and the expressions of LC3 was negatively correlated with the expressions of P62 and DPD (P<0.05). The expression of DPD was significantly higher in P62^-/LC3^- group than in the other three groups, while the expression of DPD was significantly lower in P62^-/LC3⁺ group than in the other three groups (P<0.05). The protein expression of DPD in P62⁺/LC3^- and P62⁺/LC3⁺ group were different, but the difference was not statistically significant (P>0.05). The expression of P62 was positively correlated with T stage and lymph node metastasis. The expression of LC3 was positively correlated with tumor size and T stage. The protein expression of DPD was closely related to histological type (P<0.05). LC3, P62, DPD and lymph node metastasis were related to the prognosis of colorectal cancer patients. All of them were independent risk factors for the prognosis of colorectal cancer. Conclusion: LC3 and p62 can affect the expression of DPD. Autophagy-lysosome pathway may be an important pathway of DPD degradation, which may affect the resistance of colorectal cancer to 5-FU through the degradation rate of DPD.

Key words: Colorectal cancer, Dihydropyrimidine dehydrogenase, Autophagy, Light chain 3, P62

中图分类号:

贾真真, 何双, 李扬扬, 温菲菲, 许晓阳, 郭宁杰, 吴淑华. 结直肠癌中DPD与LC3、P62表达的相关性及其临床意义[J]. 中国癌症杂志, 2022, 32(1): 24-33.

JIA Zhenzhen, HE Shuang, LI Yangyang, WEN Feifei, XU Xiaoyang, GUO Ningjie, WU Shuhua. Correlations between expressions of DPD, LC3 and P62 in colorectal cancer and their clinical significance[J]. China Oncology, 2022, 32(1): 24-33.

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Gene	Sequence
P62	Forward primer: AGCTGCCCTCAGCCCTCTA
	Reverse primer: GGCTTCTCTTCCCTCCATGTT
DPD	Forward primer: CCAAAGTGAAAGAAGCATTG
	Reverse primer: TGTCACGATGTCCTTATCAA
LC3	Forward primer: GGCGCTTACAGCTCAATGCT
	Reverse primer: CTCCTGGGAGGCATAGACCA
GAPDH	Forward primer: GGCTCTCCAGAACATCATCCCTGC
	Reverse primer: GGGTGTCGCTGTTGAAGTCAGAGG

Item	Case n	LC3	P62	DPD
Normal	157	21 (13.4)	20 (12.7)	33 (21.0)
Tumor	157	79 (50.3)^*	108 (68.8)^*	89 (56.7)^*

Item	Case	P62		r value	P value	LC3		r value	P value
Item	Case	-	+	r value	P value	-	+	r value	P value
DPD				0.160	0.045^*			-0.200	0.012^*
-	68	27	41			26	42
+	89	22	67			52	37

Item	Case	P62		r value	P value	DPD		r value	P value
Item	Case	-	+	r value	P value	-	+	r value	P value
LC3				-0.202	0.011^*			-0.200	0.012^*
-	78	17	61			26	52
+	79	32	47			42	37

Item	Case n	P62⁺/LC3⁺ group	P62⁺/LC3^– group	P62^–/LC3⁺ group	P62^–/LC3^– group
DPD
+	89	30 (63.8)	37 (60.7)	7 (24.2)	15 (88.2)
-	68	17 (36.2)^※△	24 (39.3)^※△	25 (75.8)^*#△	2 (11.8)^*#※