长链非编码RNA ARAP1-AS1在肾透明细胞癌中的表达及作用研究

doi:10.19401/j.cnki.1007-3639.2022.01.004

摘要/Abstract

摘要：

背景与目的：长链非编码RNA（long non-coding RNA,lncRNA）ARAP1-AS1在多种肿瘤中异常表达,但其在肾透明细胞癌（clear cell renal cell carcinoma,ccRCC）中的作用尚不清楚。探讨ARAP1-AS1在ccRCC中的生物学作用。方法：通过GEPIA数据库分析ARAP1-AS1在ccRCC组织中的表达及其与临床病理学特征及患者生存率的关系。采用实时荧光定量聚合酶链反应（real-time fluorescence quantitative polymerase chain reaction,RTFQ-PCR）检测ccRCC组织及邻近的非肿瘤组织中ARAP1-AS1的表达水平。将患者分为ARAP1-AS1高表达组和低表达组,分析ARAP1-AS1的表达水平与患者临床病理学特征之间的关系,并进行生存分析。通过细胞计数试剂盒-8（cell counting kit-8,CCK-8）实验、transwell迁移实验及侵袭实验检测ARAP1-AS1对ccRCC细胞体外增殖、迁移及侵袭能力的影响。采用蛋白质印迹法（Western blot）检测Wnt/β-catenin信号通路相关蛋白表达变化。采用BALB/c裸小鼠移植瘤模型分析ARAP1-AS1对ccRCC细胞体内成瘤能力的影响。结果：GEPIA数据库分析结果显示,ARAP1-AS1在ccRCC中高表达,且与患者肿瘤高分期及较差的生存率相关（P均<0.05）。RTFQ-PCR显示,ARAP1-AS1在ccRCC组织及细胞系中高表达,ARAP1-AS1的高表达与肿瘤大小和分期相关（P均<0.05）。ARAP1-AS1高表达患者的总生存率较差（P<0.05）。沉默ARAP1-AS1的表达可以抑制ccRCC细胞增殖、迁移和侵袭（P均<0.05）。沉默ARAP1-AS1可以降低Wnt/β-catenin信号通路相关蛋白的表达水平（P均<0.05）。沉默ARAP1-AS1可使ccRCC细胞体内成瘤能力减弱,并使Ki-67增殖指数降低。结论：ARAP1-AS1可通过激活Wnt/β-catenin信号通路促进ccRCC的进展。

关键词: 肾透明细胞癌, ARAP1-AS1, Wnt/β-catenin

Abstract:

Background and purpose: Long non-coding RNA (lncRNA) ARAP1-AS1 is abnormally expressed in a variety of tumors, but its role in clear cell renal cell carcinoma (ccRCC) is unknown. This study aimed to explore the biological role of ARAP1-AS1 in ccRCC. Methods: The expression level of ARAP1-AS1 in ccRCC tissues and its relationship to patient’s clinicopathological characteristics and survival rate were analyzed using the GEPIA database. The expression level of ARAP1-AS1 was measured in ccRCC and adjacent non-tumor tissues by real-time fluorescence quantitative polymerase chain reaction (RTFQ-PCR). Patients were divided into ARAP1-AS1 high and low expression groups, the relationship between ARAP1-AS1 expression level and patient’s clinicopathological characteristics was analyzed, and survival analysis was performed. The effect of ARAP1-AS1 in vitro proliferation, migration and invasion ability of ccRCC cells was determined by cell counting kit-8 (CCK-8) assay, transwell migration and invasion assay. Changes in the expressions of Wnt/β-catenin signaling pathway-related proteins were detected by Western blot assay. The effect of ARAP1-AS1 on tumorigenic capacity in ccRCC cells in vivo was verified by tumor xenografts in nude mice. Results: The analysis of the GEPIA database showed that ARAP1-AS1 was highly expressed in ccRCC and associated with advanced tumor stage as well as poor survival in patients (all P<0.05). The results of RTFQ-PCR showed that ARAP1-AS1 was highly expressed in ccRCC tissues and cell lines, and high expression correlated with tumor size and stage (all P<0.05). The overall survival rate was poor in patients with high ARAP1-AS1 expression (P<0.05). Knockdown of ARAP1-AS1 expression inhibited the proliferation, migration and invasion of ccRCC cells (all P<0.05). Silencing of ARAP1-AS1 reduced the expression levels of the proteins involved in the Wnt/β-catenin signaling pathway (all P<0.05). Silencing of ARAP1-AS1 attenuated tumorigenic capacity in ccRCC cells and reduced Ki-67 proliferation index (P<0.05). Conclusion: ARAP1-AS1 promotes ccRCC progression through activation of the Wnt/β-catenin signaling pathway.

Key words: Clear cell renal cell carcinoma, ARAP1-AS1, Wnt/β-catenin

中图分类号:

R737.11

雷坤阳, 谢文杰, 孙庭, 刘贻富, 王旭. 长链非编码RNA ARAP1-AS1在肾透明细胞癌中的表达及作用研究[J]. 中国癌症杂志, 2022, 32(1): 34-40.

LEI Kunyang, XIE Wenjie, SUN Ting, LIU Yifu, WANG Xu. Expression and effect of long non-coding RNA ARAP1-AS1 in clear cell renal cell carcinoma[J]. China Oncology, 2022, 32(1): 34-40.

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参考文献 17

[1]	HSIEH J J, PURDUE M P, SIGNORETTI S, et al. Renal cell carcinoma[J]. Nat Rev Dis Primers, 2017, 3: 17009. doi: 10.1038/nrdp.2017.9
[2]	STATELLO L, GUO C J, CHEN L L, et al. Gene regulation by long non-coding RNAs and its biological functions[J]. Nat Rev Mol Cell Biol, 2021, 22(2): 96-118. doi: 10.1038/s41580-020-00315-9
[3]	LUO H C, ZHU G Q, XU J F, et al. HOTTIP lncRNA promotes hematopoietic stem cell self-renewal leading to AML-like disease in mice[J]. Cancer Cell, 2019, 36(6): 645-659. e8. doi: 10.1016/j.ccell.2019.10.011
[4]	KIM J, PIAO H L, KIM B J, et al. Long non-coding RNA MALAT1 suppresses breast cancer metastasis[J]. Nat Genet, 2018, 50(12): 1705-1715. doi: 10.1038/s41588-018-0252-3
[5]	LI M Z, YIN B D, CHEN M L, et al. Downregulation of the lncRNA ASB16-AS1 decreases LARP1 expression and promotes clear cell renal cell carcinoma progression via miR-185-5p/miR-214-3p[J]. Front Oncol, 2020, 10: 617105. doi: 10.3389/fonc.2020.617105
[6]	ZHU K, MIAO C K, TIAN Y, et al. lncRNA MIR4435-2HG promoted clear cell renal cell carcinoma malignant progression via miR-513a-5p/KLF6 axis[J]. J Cell Mol Med, 2020, 24(17): 10013-10026. doi: 10.1111/jcmm.v24.17
[7]	TENG J F, AI X, JIA Z M, et al. Long non-coding RNA ARAP1-AS1 promotes the progression of bladder cancer by regulating miR-4735-3p/NOTCH2 axis[J]. Cancer Biol Ther, 2019, 20(4): 552-561. doi: 10.1080/15384047.2018.1538613
[8]	LU C, WANG X H, ZHAO X W, et al. Long non-coding RNA ARAP1-AS1 accelerates cell proliferation and migration in breast cancer through miR-2110/HDAC2/PLIN1 axis[J]. Biosci Rep, 2020, 40(4): BSR20191764.
[9]	YE Y Q, GU B B, WANG Y, et al. YY1-induced upregulation of long non-coding RNA ARAP1-AS1 promotes cell migration and invasion in colorectal cancer through the wnt/β-catenin signaling pathway[J]. Cancer Biother Radiopharm, 2019, 34(8): 519-528. doi: 10.1089/cbr.2018.2745
[10]	CHEN Y Y, LI Z J, CHEN X G, et al. Long non-coding RNAs: from disease code to drug role[J]. Acta Pharm Sin B, 2021, 11(2): 340-354. doi: 10.1016/j.apsb.2020.10.001
[11]	MING H, LI B W, ZHOU L, et al. Long non-coding RNAs and cancer metastasis: molecular basis and therapeutic implications[J]. Biochim Biophys Acta Rev Cancer, 2021, 1875(2): 188519. doi: 10.1016/j.bbcan.2021.188519
[12]	ZHANG Y, WU D, WANG D. Long non-coding RNA ARAP1-AS1 promotes tumorigenesis and metastasis through facilitating proto-oncogene c-Myc translation via dissociating PSF/PTB dimer in cervical cancer[J]. Cancer Med, 2020, 9(5): 1855-1866. doi: 10.1002/cam4.v9.5
[13]	JIANG Y H, ZHU X Y, GUO Z Y, et al. Increased long non-coding RNA ARAP1-AS1 expression and its prognostic significance in human gastric cancer: a preliminary study[J]. Eur Rev Med Pharmacol Sci, 2020, 24(4): 1815-1820.
[14]	TAO X L, ZHANG Y, LI J P, et al. Low expression of long non-coding RNA ARAP1-AS1 can inhibit lung cancer proliferation by inducing G₀/G₁ cell cycle organization[J]. J Thorac Dis, 2020, 12(12): 7326-7336. doi: 10.21037/jtd
[15]	TREJO-SOLIS C, ESCAMILLA-RAMIREZ A, JIMENEZ-FARFAN D, et al. Crosstalk of the wnt/β-catenin signaling pathway in the induction of apoptosis on cancer cells[J]. Pharmaceuticals (Basel), 2021, 14(9): 871. doi: 10.3390/ph14090871
[16]	XU X L, ZHANG N W, GAO R X, et al. Upregulation of SDHA inhibited proliferation, migration, and invasion of clear cell renal cell carcinoma cells via inactivation of the Wnt/β-catenin pathway[J]. J Recept Signal Transduct Res, 2021: 1-13.
[17]	ZHU L, LIU X L, FU Z Q, et al. LINC00675 suppresses proliferative, migration and invasion of clear cell renal cell carcinoma via the Wnt/β-catenin pathway[J]. Eur Rev Med Pharmacol Sci, 2020, 24(5): 2313-2320.

Clinicopathological feature	ARAP1-AS1 expression		P value
Clinicopathological feature	High (n=32)	Low (n=33)	P value
Age/year			0.378
≤60	13	17
>60	19	16
Gender			0.174
Male	15	21
Female	17	12
Tumor size D/cm			0.032
<4	10	19
≥4	22	14
Tumor stage			0.035
Ⅰ and Ⅱ	12	21
Ⅲ and Ⅳ	20	12
Lymph node metastasis			0.389
Positive	15	12
Negative	17	21
Distant metastasis			0.265
Positive	14	19
Negative	18	14