乐伐替尼对裸鼠皮下人肝癌细胞移植瘤生长及生物钟基因表达水平的影响

doi:10.3877/cma.j.issn.1674-0793.2019.02.002

中华普通外科学文献(电子版) ›› 2019, Vol. 13 ›› Issue (02) : 90 -96. doi: 10.3877/cma.j.issn.1674-0793.2019.02.002

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论著

乐伐替尼对裸鼠皮下人肝癌细胞移植瘤生长及生物钟基因表达水平的影响

汪朦朦¹, 熊枝繁¹, 何晓晓¹, 邱梦君¹, 杨盛力²^,^†(

)

^1. 430077　武汉，华中科技大学同济医学院附属梨园医院消化内科
^2. 430022　武汉，华中科技大学同济医学院附属协和医院肿瘤中心

收稿日期:2018-09-13 出版日期:2019-04-01
通信作者: 杨盛力
基金资助:
国家自然科学基金青年基金资助项目(81702885); 中国肝炎防治基金会天晴肝病研究基金资助课题项目(TQGB20170005); 湖北省自然科学基金资助项目(2017CFB178); 华中科技大学自主创新基金项目(0118530328)

Effects of lenvatinib on growth and circadian clock gene expression of hepatocellular carcinoma xenografts in nude mice

Mengmeng Wang¹, Zhifan Xiong¹, Xiaoxiao He¹, Mengjun Qiu¹, Shengli Yang²^,^†()

^1. Department of Gastroenterology, Liyuan Hospital of Tongji Medical College of Huazhong University of Science and Technology, Wuhan 430077, China
^2. Cancer Center of Xiehe Hospital Affiliated to Tongji Medical College of Huazhong University of Science and Technology, Wuhan 430022, China

Received:2018-09-13 Published:2019-04-01
Corresponding author: Shengli Yang
About author:
Corresponding author: Yang Shengli, Email: yangshengli2014@yahoo.com

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引用本文：

汪朦朦, 熊枝繁, 何晓晓, 邱梦君, 杨盛力. 乐伐替尼对裸鼠皮下人肝癌细胞移植瘤生长及生物钟基因表达水平的影响[J]. 中华普通外科学文献(电子版), 2019, 13(02): 90-96.

Mengmeng Wang, Zhifan Xiong, Xiaoxiao He, Mengjun Qiu, Shengli Yang. Effects of lenvatinib on growth and circadian clock gene expression of hepatocellular carcinoma xenografts in nude mice[J]. Chinese Archives of General Surgery(Electronic Edition), 2019, 13(02): 90-96.

目的

研究靶向抗肿瘤药物乐伐替尼对裸鼠皮下肝癌移植瘤生长的抑制作用及其对肝癌细胞中生物钟基因表达水平的调控。

方法

利用HepG2人肝癌细胞株构建肝癌裸鼠皮下移植瘤模型。12只成瘤裸鼠随机分为处理组（30 mg·kg^-1·d^-1，1次/d）和对照组（DMSO，100 μl·20g^-1·d^-1，1次/d），每3天测量1次肿瘤体积并绘制移植瘤生长曲线，末次灌胃给药后24 h处死裸鼠并剥离瘤体，比较两组瘤体体积大小及体质量，并采用Real-time PCR法检测肝癌移植瘤组织中生物钟基因BMAL1、CKIε、PER1、PER2、PER3、CRY1、CRY2、CLOCK、NPAS2、REV-Erbα、TIM和DEC2表达水平。

结果

两组裸鼠成瘤率为100.0%。与对照组相比，处理组裸鼠一般状态良好，移植瘤生长缓慢，瘤体体积较小，且给药第9天后生长减慢趋势明显（P<0.05），终末瘤体质量为（0.235±0.43） g，明显小于对照组的（0.633±0.22）g（P=0.006，95%CI=0.167~0.629）；两组裸鼠体质量均随时间的变化而增加，在灌胃给药后第9天时，处理组体质量较对照组大（F=5.194，P=0.034）；处理组裸鼠皮下移植瘤组织中BMAL1、CKIε、PER3和NPAS2 mRNA表达上调，DEC2、PER1、PER2和CRY2 mRNA表达下调，差异有统计学意义（P<0.05）；两组CLOCK、CRY1、REV-Erbα和TIM基因表达水平差异无统计学意义。

结论

乐伐替尼可抑制裸鼠皮下肝癌移植瘤的生长，并且可明显上调肝癌组织中的BMAL1、NPAS2、PER3、CKIε基因表达，同时下调PERl、PER2、DEC2基因的表达水平。

关键词: 乐伐替尼, 癌,肝细胞, 肿瘤移植, 生物钟基因, 小鼠,裸

Objective

To explore the inhibitory effect of lenvatinib on the growth of hepatocellular carcinoma (HCC) in a nude mouse model bearing HepG2 cell xenograft and its regulation on the circadian clock gene expression in HCC tissues.

Methods

HepG2 cells were inoculated intradermally intothe nude mice to establish xenograft models. After tumorigenesis, all of the mice were randomlydivided into two groups including the observation group and the control group, which were given lenvatinib 30 mg·kg^-1·d^-1, once a day and DMSO 100 μl·20 g^-1·d^-1, once a day for 14 days by intragastric administration, respectively. During the experiment, the tumor volume was measured every 3 days to draw the growth curve of transplanted tumor. The nude mice were killed after 2 weeks and the tumor bodies were stripped. The volume and weight of the tumor were compared between the two groups. The expression levels of circadian clock genes (BMAL1, CKIε, PER1, PER2, PER3, CRY1, CRY2, CLOCK, NPAS2, REV-Erbα, TIM and DEC2) were detected by the Real-time polymerase chain reaction ( Real-time PCR).

Results

Compared with the control group, the nude mice treated with lenvatinib were generally in good condition and the transplanted tumor grew more slowly and the volume was smaller; and after 9 days of administration, the growth slowed down significantly (P<0.05). And the final tumor mass was(0.235±0.43) g, which was significantly lower than that in the control group (0.633±0.22) g (P=0.006, 95%CI=0.167-0.629). The body mass of both groups increased with time. On the 9th day after intragastric administration, the body mass of the treated group was larger than that of the control group (F=5.194, P=0.034). Compared with the control group, the expression levels of BMAL1, CKIε, PER3 and NPAS2 genes increased, DEC2, PER1, PER2 and CRY2 decreased in the observation group, and the differences were statistically significant (all P<0.05). However, there were no significant differences in CLOCK, CRY1, REV-Erbα and TIM.

Conclusion

Lenvatinib inhibits the growth of HCC in nude mice, which can up-regulate the expression of BMAL1, CKIε, PER3 and NPAS2 genes, at the same time, down-regulate the expression of DEC2, PER1, PER2 and CRY2 genes in nude mice.

Key words: Lenvatinib, Carcinoma, hepatocellular, Neoplasm transplantation, Biological clock genes, Mice, nude

表1 各生物钟基因实时荧光定量PCR引物序列

基因	上游引物序列	下游引物序列
BMAL1	5'-TGCCACCAATCCATACACAGAAG-3'	5'-TTCCCTCGGTCACATCCTACG-3'
CKIε	5'-TCAGCGAGAAGAAGATGTC-3'	5'-GAAGAGGTTGCGGAAGAG-3
CRY1	5'-CAACCTCCATTCATCTTTCC-3'	5'-CTCATAGCCGACACCTTC-3
CRY2	5'-TGGGCTTCTGGGACTGAG-3'	5'-GGTAGGTGTGCTGTCTTAGG-3'
DEC2	5'-GGGACCAACTGCTTCACACT-3'	5'-TAATCTGTGGGACGGTAGGC-3'
CLOCK	5'-GCAGCAGCAGCAGCAGAG-3'	5'-CAGCAGAGAGAATGAGTTGAGTTG-3'
TIM	5'-GATAGAGGCCCATTCCTGCAT-3'	5'-GAAGGGCTGGGGAACTTAGAC-3'
NPAS2	5'-AACCTCGGCAGCACTTTAAC-3'	5'-GGTTCTGACATGGCTGTGTG-3'
PER1	5'-AGGCAACGGCAAGGACTC-3'	5'-GGCTGTAGGCAATGGAACTG-3'
PER2	5'-CTACAGCAGCACCATCGTC-3'	5'-CCACTCGCAGCATCTTCC-3'
PER3	5'-TGGTGGTGGTGAATGTAAGAC-3'	5'-GGCTGTGCTCATCGTTCC-3'
REV-Erbα	5'-ACAGAATCGAACTCTGCACTTCT-3'	5'-GGGGAGGGAGGCAGGTATT-3'
β-actin	5'-AGTTGCGTTACACCCTTTCTTGAC-3'	5'-GCTCGCTCCAACCGACTGC-3'

表1 各生物钟基因实时荧光定量PCR引物序列

基因	上游引物序列	下游引物序列
BMAL1	5'-TGCCACCAATCCATACACAGAAG-3'	5'-TTCCCTCGGTCACATCCTACG-3'
CKIε	5'-TCAGCGAGAAGAAGATGTC-3'	5'-GAAGAGGTTGCGGAAGAG-3
CRY1	5'-CAACCTCCATTCATCTTTCC-3'	5'-CTCATAGCCGACACCTTC-3
CRY2	5'-TGGGCTTCTGGGACTGAG-3'	5'-GGTAGGTGTGCTGTCTTAGG-3'
DEC2	5'-GGGACCAACTGCTTCACACT-3'	5'-TAATCTGTGGGACGGTAGGC-3'
CLOCK	5'-GCAGCAGCAGCAGCAGAG-3'	5'-CAGCAGAGAGAATGAGTTGAGTTG-3'
TIM	5'-GATAGAGGCCCATTCCTGCAT-3'	5'-GAAGGGCTGGGGAACTTAGAC-3'
NPAS2	5'-AACCTCGGCAGCACTTTAAC-3'	5'-GGTTCTGACATGGCTGTGTG-3'
PER1	5'-AGGCAACGGCAAGGACTC-3'	5'-GGCTGTAGGCAATGGAACTG-3'
PER2	5'-CTACAGCAGCACCATCGTC-3'	5'-CCACTCGCAGCATCTTCC-3'
PER3	5'-TGGTGGTGGTGAATGTAAGAC-3'	5'-GGCTGTGCTCATCGTTCC-3'
REV-Erbα	5'-ACAGAATCGAACTCTGCACTTCT-3'	5'-GGGGAGGGAGGCAGGTATT-3'
β-actin	5'-AGTTGCGTTACACCCTTTCTTGAC-3'	5'-GCTCGCTCCAACCGACTGC-3'

表2 两组裸鼠灌胃不同时间点移植瘤体积比较(mm³，±s)

组别	n	灌胃前	灌胃第3天	灌胃第6天	灌胃第9天	灌胃第12天	灌胃结束24 h
处理组	6	79.32±3.75	157.34±12.11	196.14±27.85	231.6±26.44	213.33±20.75	228.61±29.99
对照组	6	96.17±6.95	180.90±18.28	264.40±32.99	444.68±75.30	462.67±60.25	555.27±73.47
F值	?	2.873	1.040	0.259	4.869	11.512	5.494
P值	?	0.058	0.308	0.145	0.024^a	0.007^a	0.005^a

表2 两组裸鼠灌胃不同时间点移植瘤体积比较(mm³，±s)

组别	n	灌胃前	灌胃第3天	灌胃第6天	灌胃第9天	灌胃第12天	灌胃结束24 h
处理组	6	79.32±3.75	157.34±12.11	196.14±27.85	231.6±26.44	213.33±20.75	228.61±29.99
对照组	6	96.17±6.95	180.90±18.28	264.40±32.99	444.68±75.30	462.67±60.25	555.27±73.47
F值	?	2.873	1.040	0.259	4.869	11.512	5.494
P值	?	0.058	0.308	0.145	0.024^a	0.007^a	0.005^a

图1 两组肝癌HepG2裸鼠移植瘤的生长趋势与对照组相比，处理组瘤体体积较小，生长速度缓慢，且在灌胃给药第9天两组差异有统计学意义（P<0.05）

图2 两组裸鼠终末移植瘤体质量比较　其中A为两组终末瘤体质量散点分布比较，B为离体瘤肉眼观察；处理组终末瘤体质量和体积明显小于对照组（P<0.05）

表3 两组裸鼠灌胃给药后不同时间点体质量变化(g,±s)

组别	n	灌胃前	灌胃第3天	灌胃第6天	灌胃第9天	灌胃第12天	灌胃结束24 h
处理组	6	18.63±0.72	19.72±0.69	20.25±0.37	20.64±0.41	21.03±0.33	21.23±0.44
对照组	6	17.98±0.44	18.63±0.53	19.16±0.45	19.26±0.15	20.46±0.12	21.09±0.48
F值	?	1.684	0.538	0.095	5.194	5.117	3.072
P值	?	0.485	0.281	0.137	0.034^a	0.176	0.765

表3 两组裸鼠灌胃给药后不同时间点体质量变化(g,±s)

组别	n	灌胃前	灌胃第3天	灌胃第6天	灌胃第9天	灌胃第12天	灌胃结束24 h
处理组	6	18.63±0.72	19.72±0.69	20.25±0.37	20.64±0.41	21.03±0.33	21.23±0.44
对照组	6	17.98±0.44	18.63±0.53	19.16±0.45	19.26±0.15	20.46±0.12	21.09±0.48
F值	?	1.684	0.538	0.095	5.194	5.117	3.072
P值	?	0.485	0.281	0.137	0.034^a	0.176	0.765

图3 两组不同时间点灌胃裸鼠体质量变化　两组裸鼠的体质量均随时间的变化而增加，但在灌胃后第9天时，差异有统计学意义（P=0.034）

表4 两组裸鼠皮下肝癌移植瘤组织中生物钟基因相对表达量比较（n=6，±s）

组别	BMAL1	CKIε	CLOCK	CRY1	DEC2	NPAS2
处理组	4.22±0.39	100.81±0.86	1.87±0.17	11.91±0.38	1.75±0.13	5.23±0.10
对照组	3.64±0.09	87.83±0.72	1.86±0.41	12.49±0.39	2.74±0.16	2.94±0.09
F值	1.680	0.044	1.481	0.007	0.012	0.117
P值	0.004	<0.001	0.831	0.342	0.008	<0.001
组别	PER1	PER2	PER3	REV-Erbα	TIM	CRY2
处理组	8.35±0.88	1.39±0.10	0.98±0.08	9.26±0.26	36.10±0.25	1.89±0.07
对照组	10.97±0.29	2.16±0.10	0.73±0.43	8.67±0.23	36.48±0.41	2.71±0.21
F值	3.208	0.009	1.712	0.135	1.359	5.123
P值	0.048	0.006	0.049	0.170	0.473	0.020

表4 两组裸鼠皮下肝癌移植瘤组织中生物钟基因相对表达量比较（n=6，±s）

组别	BMAL1	CKIε	CLOCK	CRY1	DEC2	NPAS2
处理组	4.22±0.39	100.81±0.86	1.87±0.17	11.91±0.38	1.75±0.13	5.23±0.10
对照组	3.64±0.09	87.83±0.72	1.86±0.41	12.49±0.39	2.74±0.16	2.94±0.09
F值	1.680	0.044	1.481	0.007	0.012	0.117
P值	0.004	<0.001	0.831	0.342	0.008	<0.001
组别	PER1	PER2	PER3	REV-Erbα	TIM	CRY2
处理组	8.35±0.88	1.39±0.10	0.98±0.08	9.26±0.26	36.10±0.25	1.89±0.07
对照组	10.97±0.29	2.16±0.10	0.73±0.43	8.67±0.23	36.48±0.41	2.71±0.21
F值	3.208	0.009	1.712	0.135	1.359	5.123
P值	0.048	0.006	0.049	0.170	0.473	0.020

图4 成瘤裸鼠相关生物钟基因表达比较　处理组裸鼠皮下移植瘤组织中BMAL1、CKIε、PER3和NPAS2基因表达水平增高，DEC2、PER1、PER2和CRY2 mRNA基因表达水平降低（P<0.05）；CLOCK、CRY1、REV-Erbα、TIM基因表达水平差异无统计学意义

[1]	Bruix J,Llovet JM. Prognostic prediction and treatment strategy in hepatocellular carcinoma[J]. Hepatology, 2002, 35(3): 519-524.
[2]	Daskalow K,Rohwer N,Raskopf E, et al. Role of hypoxia-inducible transcription factor 1 alpha for progression and chemosensitivity of murine hepatocellular carcinoma[J]. J Mol Med (Berl), 2010, 88(8): 817-827.
[3]	Llovet JM,Bruix J. Molecular targeted therapies in hepatocellular carcinoma[J]. Hepatology, 2008, 48(4): 1312-1327.
[4]	Llovet JM,Ricci S,Mazzaferro V, et al. Sorafenib in advanced hepatocellular carcinoma[J]. N Engl J Med, 2008, 359(4): 378-390.
[5]	Abdel-Rahman O,Fouad M. Sorafenib-based combination as a first line treatment for advanced hepatocellular carcinoma: a systematic review of the literature[J]. Crit Rev Oncol Hematol, 2014, 91(1): 1-8.
[6]	Matsui J,Funahashi Y,Uenaka T, et al. Multi-kinase inhibitor E7080 suppresses lymph node and lung metastases of human mammary breast tumor MDA-MB-231 via inhibition of vascular endothelial growth factor-receptor (VEGF-R) 2 and VEGF-R3 kinase[J]. Clin Cancer Res, 2008, 14(17): 5459-5465.
[7]	Matsui J,Yamamoto Y,Funahashi Y, et al. E7080, a novel inhibitor that targets multiple kinases, has potent antitumor activities against stem cell factor producing human small cell lung cancer H146, based on angiogenesis inhibition[J]. Int J Cancer, 2008, 122(3): 664-671.
[8]	Kudo M,Finn RS,Qin S, et al. Lenvatinib versus sorafenib in first-line treatment of patients with unresectable hepatocellular carcinoma: a randomised phase 3 non-inferiority trial[J]. Lancet, 2018, 391(10126): 1163-1173.
[9]	Kudo M. Lenvatinib in advanced hepatocellular carcinoma[J]. Liver Cancer, 2017, 6(4): 253-263.
[10]	Grodzinski P. Themed issue on cancer nanotechnology[J]. Integr Biol (Camb), 2013, 5(1): 17-18.
[11]	Barclay JL,Tsang AH,Oster H. Interaction of central and peripheral clocks in physiological regulation[J]. Prog Brain Res, 2012, 199: 163-181.
[12]	Eckel-Mahan K,Sassone-Corsi P. Metabolism and the circadian clock converge[J]. Physiol Rev, 2013, 93(1): 107-135.
[13]	Lin YM,Chang JH,Yeh KT, et al. Disturbance of circadian gene expression in hepatocellular carcinoma[J]. Mol Carcinog, 2008, 47(12): 925-933.
[14]	Barnes JW,Tischkau SA,Barnes JA, et al. Requirement of mammalian timeless for circadian rhythmicity[J]. Science, 2003, 302(5644): 439-442.
[15]	Buhr ED,Takahashi JS. Molecular components of the Mammalian circadian clock[J]. Handb Exp Pharmacol, 2013, 217: 3-27.
[16]	Li H,Lu YF,Chen H, et al. Dysregulation of metallothionein and circadian genes in human hepatocellular carcinoma[J]. Chronobiol Int, 2017, 34(2): 192-202.
[17]	Schlumberger M,Tahara M,Wirth LJ, et al. Lenvatinib versus placebo in radioiodine-refractory thyroid cancer[J]. N Engl J Med, 2015, 372(7): 621-630.
[18]	Motzer RJ,Hutson TE,Glen H, et al. Lenvatinib, everolimus, and the combination in patients with metastatic renal cell carcinoma: a randomised, phase 2, open-label, multicentre trial[J]. Lancet Oncol, 2015, 16(15): 1473-1482.
[19]	Fu L,Pelicano H,Liu J, et al. The circadian gene Period2 plays an important role in tumor suppression and DNA damage response in vivo[J]. Cell, 2002, 111(1): 41-50.
[20]	Hoffman AE,Zheng T,Stevens RG, et al. Clock-cancer connection in non-Hodgkin's lymphoma: a genetic association study and pathway analysis of the circadian gene cryptochrome 2[J]. Cancer Res, 2009, 69(8): 3605-3613.
[21]	Gorbacheva VY,Kondratov RV,Zhang R, et al. Circadian sensitivity to the chemotherapeutic agent cyclophosphamide depends on the functional status of the CLOCK/BMAL1 transactivation complex[J]. Proc Natl Acad Sci U S A, 2005, 102(9): 3407-3412.
[22]	Kwon I,Choe HK,Son GH, et al. Mammalian molecular clocks[J]. Exp Neurobiol, 2011, 20(1): 18-28.
[23]	Ao Y,Zhao Q,Yang K, et al. A role for the clock period circadian regulator 2 gene in regulating the clock gene network in human oral squamous cell carcinoma cells[J]. Oncol Lett, 2018, 15(4): 4185-4192.
[24]	Zeng ZL,Wu MW,Sun J, et al. Effects of the biological clock gene Bmal1 on tumour growth and anti-cancer drug activity[J]. J Biochem, 2010, 148(3): 319-326.
[25]	Wang Q,Ao Y,Yang K, et al. Circadian clock gene Per2 plays an important role in cell proliferation, apoptosis and cell cycle progression in human oral squamous cell carcinoma[J]. Oncol Rep, 2016, 35(6): 3387-3394.
[26]	Hoffman AE,Zheng T,Ba Y, et al. The circadian gene NPAS2, a putative tumor suppressor, is involved in DNA damage response[J]. Mol Cancer Res, 2008, 6(9): 1461-1468.
[27]	Partch CL,Green CB,Takahashi JS. Molecular architecture of the mammalian circadian clock[J]. Trends Cell Biol, 2014, 24(2): 90-99.
[28]	Wang X,Yan D,Teng M, et al. Reduced expression of PER3 is associated with incidence and development of colon cancer[J]. Ann Surg Oncol, 2012, 19(9): 3081-3088.
[29]	Yunokawa M,Tanimoto K,Nakamura H, et al. Differential regulation of DEC2 among hypoxia-inducible genes in endometrial carcinomas[J]. Oncol Rep, 2007, 17(4): 871-878.
[30]	Liu Y,Sato F,Kawamoto T, et al. Anti-apoptotic effect of the basic helix-loop-helix (bHLH) transcription factor DEC2 in human breast cancer cells[J]. Genes Cells, 2010, 15(4): 315-325.

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Effects of lenvatinib on growth and circadian clock gene expression of hepatocellular carcinoma xenografts in nude mice

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Effects of lenvatinib on growth and circadian clock gene expression of hepatocellular carcinoma xenografts in nude mice