NID1在肾透明细胞癌血管生成中的作用研究

doi:10.11958/20240301

摘要/Abstract

摘要：

目的探讨巢蛋白-1（NID1）在肾透明细胞癌（ccRCC）中的表达变化及对血管生成的影响。方法利用肿瘤基因图谱数据库、细胞系和临床样本分析NID1在ccRCC中的表达，分析NID1与血管生成标志物CD31的关系。通过转染慢病毒构建稳定敲低NID1的786-O细胞系，获取培养上清液作为条件培养基处理人脐静脉内皮细胞（HUVEC）。HUVEC分为DMEM组、空白组、NID1空载组、NID1敲低组，采用CCK-8法检测细胞增殖能力，成管实验检测细胞血管形成能力，划痕实验检测细胞迁移能力。构建右旋糖酐水凝胶空载/敲低NID1肿瘤细胞复合物培养体系小鼠模型，分为NID1空载组和NID1敲低组，采用免疫组化染色检测CD31表达。结果生物信息学分析显示ccRCC中NID1表达增高，与NID1升高呈正相关的500个差异表达基因主要富集在血管生成，且癌组织中NID1和CD31表达呈正相关。经过条件培养基处理后，与NID1空载组相比，NID1敲低组HUVEC迁移和成管能力减弱（P<0.05），增殖能力无变化（P>0.05）。在裸鼠模型中，与NID1空载组相比，NID1敲低组CD31表达明显减少（P<0.05）。结论 NID1在ccRCC中表达增高，同时对血管生成有促进作用。

关键词: 癌, 肾细胞, 细胞外基质, 细胞增殖, 血管新生

Abstract:

Objective To explore the expression of nestin-1 (NID1) in clear cell renal cell carcinoma (ccRCC) and its effect on angiogenesis. Methods The expression of NID1 in ccRCC was analyzed using data from the TCGA database, cell lines and clinical samples. Additionally, the relationship between NID1 and the angiogenesis marker CD31 was investigated. A stable knockdown 786-O cell line with reduced NID1 expression was generated through lentiviral transfection. The conditioned medium obtained from these cells was used to treat human umbilical vein endothelial cells (HUVECs). HUVEC cells were divided into four groups: the DMEM group, the blank group, the NID1 empty vector group and the NID1 knockdown group. Cell proliferation ability was assessed using CCK-8 method, while tube formation ability and migration ability were evaluated through tube formation test and scratch test respectively. A mouse model of dextran hydrogel unloaded/NID1 knockdown tumor cell complex culture system was established and divided into the unloaded NID1 group and the NID1 knockdown group. Immunohistochemical staining was performed to detect the expression of CD31. Results Bioinformatics analysis revealed an increased expression of NID1 in ccRCC tissue. Furthermore, 500 differentially expressed genes positively correlated with elevated levels of NID1 were primarily enriched in angiogenesis-related processes. The results showed a positive correlation between the expression of NID1 and CD31 in cancer tissue. After treatment with conditioned medium derived from the knockdown group, HUVEC cells exhibited weakened migration and tube formation abilities in the empty vector control group (P<0.05), while their proliferation ability remained unchanges compared to the empty vector control group (P>0.05). In vivo experiments using a nude mouse model, the expression of CD31 demonstrated significantly lower levels in the NID1 knockdown group (P<0.05). Conclusion The expression of NID1 is increased in ccRCC, and it can promote angiogenesis.

Key words: carcinoma, renal cell, extracellular matrix, cell proliferation, angiogenesis

中图分类号:

R692.9

图/表 7

图1 NID1在ccRCC细胞中的表达情况 1：HK-2细胞；2：ACHN细胞；3：769-P细胞系；4：786-O细胞；5：CAKI-1细胞；6：OSRC-2细胞。A、B：NID1在HK-2和不同肾癌细胞系中的表达，a与HK-2细胞比较，P<0.05。

Fig.1 Expression of NID1 in ccRCC cells

图2 NID1与血管新生的关系 A：NID1相关基因生物学功能富集；B、C：10例ccRCC癌组织中NID1、CD31的表达及相关性。

Fig.2 Relationship between NID1 and angiogenesis

图3 各组786-O细胞和培养上清液中NID1表达水平检测 A：ncNID1 786-O、shNID1 786-O细胞中NID1表达水平检测；B：正常培养、ncNID1 786-O、shNID1 786-O细胞培养上清液中NID1表达水平检测。

Fig.3 NID1 expression levels detected in 786-O cells and culture supernatant of each group

表1 各组细胞增殖能力、成管数、迁移愈合率比较（n=3，$\bar{x}±s$）

Tab.1 Comparison of proliferation ability, tube number and migration healing rate between four groups of cells

组别	细胞增殖能力/ （OD₄₅₀）	成管数/ （个/视野）	迁移愈合率/%
DMEM组	0.83±0.15	1.67±0.58	7.86±0.49
空白组	0.72±0.03	24.67±6.11^a	18.11±3.35^a
NID1空载组	0.84±0.13	22.67±4.04^a	17.05±0.34^a
NID1敲低组	0.90±0.15	5.67±0.58^b	10.87±0.61^b
F	1.107	30.180^＊＊	24.320^＊＊

图4 NID1表达对HUVEC成管的影响（×10）

Fig.4 Effect of NID1 expression on HUVEC tube formation (×10)

图5 NID1表达对HUVEC迁移的影响

Fig.5 Effect of NID1 expression on HUVEC migration

图6 NID1表达对体内血管生成的影响（CD31染色，×40）

Fig.6 Effect of NID1 expression on angiogenesis in vivo (CD31 staining, ×40)

参考文献 20

[1]	RIZZO M, CALIÒ A, BRUNELLI M, et al. Clinico-pathological implications of the 2022 WHO renal cell carcinoma classification[J]. Cancer Treat Rev, 2023, 116:102558. doi:10.1016/j.ctrv.2023.102558.
[2]	CHEN Y W, WANG L, PANIAN J, et al. Treatment landscape of renal cell carcinoma[J]. Curr Treat Options Oncol, 2023, 24(12):1889-1916. doi:10.1007/s11864-023-01161-5.
[3]	CHOUEIRI T K, KAELIN W G Jr. Targeting the HIF2-VEGF axis in renal cell carcinoma[J]. Nat Med, 2020, 26(10):1519-1530. doi:10.1038/s41591-020-1093-z.
[4]	KUAN M I, CARUSO L B, ZAVALA A G, et al. Human cytomegalovirus utilizes multiple viral proteins to regulate the basement membrane protein nidogen 1[J]. J Virol, 2022, 96(20):e0133622. doi:10.1128/jvi.01336-22.
[5]	MAO X, TEY S K, YEUNG C, et al. Nidogen 1-enriched extracellular vesicles facilitate extrahepatic metastasis of liver cancer by activating pulmonary fibroblasts to secrete tumor necrosis factor receptor 1[J]. Adv Sci(Weinh), 2020, 7(21):2002157. doi:10.1002/advs.202002157.
[6]	ALEČKOVIĆ M, WEI Y, LEROY G, et al. Identification of nidogen 1 as a lung metastasis protein through secretome analysis[J]. Genes Dev, 2017, 31(14):1439-1455. doi:10.1101/gad.301937.117.
[7]	ROKAVEC M, JAECKEL S, HERMEKING H. Nidogen-1/NID1 function and regulation during progression and metastasis of colorectal cancer[J]. Cancers(Basel), 2023, 15(22):5316. doi:10.3390/cancers15225316.
[8]	JONASCH E, DONSKOV F, ILIOPOULOS O, et al. Belzutifan for renal cell carcinoma in von hippel-lindau disease[J]. N Engl J Med, 2021, 385(22):2036-2046. doi:10.1056/NEJMoa2103425.
[9]	BERGQVIST C, EZZEDINE K. Vitiligo:a review[J]. Dermatology, 2020, 236(6):571-592. doi:10.1159/000506103.
[10]	KIM H, SHIM B Y, LEE S J, et al. Loss of Von hippel-lindau(VHL) tumor suppressor gene function: VHL-HIF pathway and advances in treatments for metastatic renal cell carcinoma(RCC)[J]. Int J Mol Sci, 2021, 22(18):9795. doi:10.3390/ijms22189795.
[11]	KAELIN W G Jr. Von Hippel-Lindau disease: insights into oxygen sensing, protein degradation, and cancer[J]. J Clin Invest, 2022, 132(18):e162480. doi:10.1172/JCI162480.
[12]	TELEANU R I, CHIRCOV C, GRUMEZESCU A M, et al. Tumor angiogenesis and anti-angiogenic strategies for cancer treatment[J]. J Clin Med, 2019, 9(1):84. doi:10.3390/jcm9010084.
[13]	JIANG X, WANG J, DENG X, et al. The role of microenvironment in tumor angiogenesis[J]. J Exp Clin Cancer Res, 2020, 39(1):204. doi:10.1186/s13046-020-01709-5.
[14]	YANG J, WANG K, YANG Z. Treatment strategies for clear cell renal cell carcinoma: Past, present and future[J]. Front Oncol, 2023, 13:1133832. doi:10.3389/fonc.2023.1133832.
[15]	ANGULO J C, SHAPIRO O. The changing therapeutic landscape of metastatic renal cancer[J]. Cancers(Basel), 2019, 11(9):1227. doi:10.3390/cancers11091227.
[16]	ASTORE S, BACIARELLO G, CERBONE L, et al. Primary and acquired resistance to first-line therapy for clear cell renal cell carcinoma[J]. Cancer Drug Resist, 2023, 6(3):517-546. doi:10.20517/cdr.2023.33.
[17]	ZHOU S, CHEN S, PEI Y A, et al. Nidogen: a matrix protein with potential roles in musculoskeletal tissue regeneration[J]. Genes Dis, 2022, 9(3):598-609. doi:10.1016/j.gendis.2021.03.004.
[18]	CHENG P, CAO T, ZHAO X, et al. Nidogen1-enriched extracellular vesicles accelerate angiogenesis and bone regeneration by targeting myosin-10 to regulate endothelial cell adhesion[J]. Bioact Mater, 2022, 12:185-197. doi:10.1016/j.bioactmat.2021.10.021.
[19]	HAN N, LI X, WANG Y, et al. HIF-1α induced NID1 expression promotes pulmonary metastases via the PI3K-AKT pathway in salivary gland adenoid cystic carcinoma[J]. Oral Oncol, 2022, 131:105940. doi:10.1016/j.oraloncology.2022.105940.
[20]	LEE H W, XU Y, HE L, et al. Role of venous endothelial cells in developmental and pathologic angiogenesis[J]. Circulation, 2021, 144(16):1308-1322. doi:10.1161/CIRCULATIONAHA.121.054071.