miR-150-5p与NCAPG的靶向关系及其对肝细胞肝癌Huh7细胞的抑制作用

doi:10.13481/j.1671-587X.20230116

摘要/Abstract

摘要：

目的探究miR-150-5p与非SMC凝聚体Ⅰ复合物亚基G（NCAPG）的靶向关系及其对肝细胞肝癌（HCC）细胞生物学功能的影响，为HCC的临床诊断和治疗提供潜在靶点。方法通过双荧光素酶报告基因实验验证miR-150-5p与NCAPG的靶向关系。将HCC Huh7细胞根据转染质粒不同分为模拟物阴性对照（mimic NC）组、miR-150-5p模拟物（miR-150-5p mimic）组、miR-150-5p模拟物+过表达阴性对照（miR-150-5p mimic+ovNC）组和miR-150-5p模拟物+过表达NCAPG（miR-150-5p mimic+ovNCAPG）组。实时荧光定量PCR（RT-qPCR）法检测各组细胞中NCAPG mRNA表达水平，Western blotting法检测各组细胞中NCAPG蛋白表达水平，5-乙炔基-2'-脱氧尿嘧啶核苷（EdU）实验检测各组细胞EdU阳性率，流式细胞术检测各组细胞凋亡率，Transwell实验检测各组细胞中迁移和侵袭细胞数。将裸鼠分为agomiR-NC组、agomiR-150-5p组、agomiR-150-5p+过表达阴性对照（agomiR-150-5p+ovNC）组和agomiR-150-5p+过表达NCAPG（agomiR-150-5p+ovNCAPG）组，Huh7细胞皮下成瘤，检测各组裸鼠肿瘤体积和质量。结果双荧光素酶报告基因实验证实NCAPG是miR-150-5p的靶基因。与mimic NC组比较，miR-150-5p mimic组Huh7细胞中NCAPG mRNA和蛋白表达水平及EdU阳性率明显降低（P<0.05），细胞凋亡率明显升高（P<0.05），迁移细胞数和侵袭细胞数明显减少（P<0.05）；与miR-150-5p mimic+ovNC组比较，miR-150-5p mimic+ovNCAPG组Huh7细胞中NCAPG mRNA和蛋白表达水平及EdU阳性率明显升高（P<0.05），细胞凋亡率明显降低（P<0.05），迁移细胞数和侵袭细胞数明显增加（P<0.05）。与agomiR-NC比较，agomiR-150-5p组裸鼠Huh7细胞移植瘤体积和质量明显减少（P<0.05）；与agomiR-150-5p+ovNC组比较，agomiR-150-5p+ovNCAPG组裸鼠Huh7细胞移植瘤体积和质量明显增加（P<0.05）。结论 miR-150-5p通过靶向结合抑制NCAPG表达进而抑制HCC细胞增殖、迁移、侵袭和肿瘤生长，促进细胞凋亡。

关键词: 非SMC凝聚体Ⅰ复合物亚基G, 肝细胞肝癌, miR-150-5p, 细胞增殖, 细胞迁移, 细胞侵袭

Abstract:

Objective To explore the targeting relationship between miR-150-5p and non-SMC condensate Ⅰ complex subunit G （NCAPG） and its effect on the biological function of hepatocellular carcinoma （HCC） cells， and to provide the potential targets for the clinical diagnosis and treatment of HCC. Methods The targeting relationship between miR-150-5p and NCAPG was verified by dual-luciferase reporter gene experiment. The Huh7 cells were divided into mimic negative control （mimic NC）group， miR-150-5p mimic group， miR-150-5p mimic+overexpression negative control （miR-150-5p mimic+ovNC）group and miR-150-5p mimic+overexpression NCAPG （miR-150-5p mimic+ovNCAPG） group. The expression levels of NCAPG mRNA in the cells in various groups were detected by real-time fluorescence quantitative PCR （RT-qPCR） method， and the expression levels of NCAPG protein in the cells in various groups were detected by Western blotting method； 5-ethynyl-2'-deoxyuridine （EdU） assay was used to detect the EdU positive rates of cells in various groups， flow cytometry was used to detect the apoptotic rates， and the numbers of migration and invasion cells were detected by Transwell assay. The nude mice were divided into agomiR-NC group， agomiR-150-5p group， agomiR-150-5p+overexpression negative control（agomiR-150-5p+ovNC） group and agomiR-150-5p+overexpression NCAPG （agomiR-150-5p+ovNCAPG） group. The Huh7 cells formed subcutaneous tumors， and the tumor volumes and tumor weights of nude mice in various groups were measured. Results The dual luciferase reporter gene assay results confirmed that NCAPG was the target gene of miR-150-5p. Compared with mimic NC group， the expression levels of NCAPG mRNA and protein and the EdU positive rate in the Huh7 cells in miR-150-5p mimic group were significantly decreased （P<0.05），the apoptotic rate was significantly increased （P<0.05），and the numbers of migration and invasion cells were significantly decreased（P<0.05）； compared with miR-150-5p mimic +ovNC group， the expression levels of NCAPG mRNA and protein and the EdU positive rate in the Huh7 cells in miR-150-5p mimic +ovNCAPG group were significantly increased （P<0.05），the apoptotic rate was significantly decreased （P<0.05），and the numbers of migration and invasion cells were significantly increased（P<0.05）. Compared with agomiR-NC group， the transplanted tumor volume and tumor weight of the nude mice in agomiR-150-5p group were significantly reduced （P<0.05）； compared with agomiR-150-5p+ovNC group， the transplanted tumor volume and tumor weight of the nude mice in agomiR-150-5p ovNCAPG group were significantly increased （P<0.05）. Conclusion MiR-150-5p inhibits the HCC cell proliferation， migration， invasion and tumor growth through targeting binding and inhibitiing the NCAPG expression， and promotes the apoptosis.

Key words: Non-SMC condensate Ⅰ complex subunit G, Hepatocellular carcinoma, MiR-150-5p, Cell proliferation, Cell migration, Cell invasion

中图分类号:

R735.7

金俊伊,李木,胡耀元,王熠辉. miR-150-5p与NCAPG的靶向关系及其对肝细胞肝癌Huh7细胞的抑制作用[J]. 吉林大学学报(医学版), 2023, 49(1): 122-130.

Junyi JIN,Mu LI,Yaoyuan HU,Yihui WANG. Targeting relationship between miR-150-5p and NCAPG and its inhibitory effect on hepatocellular carcinoma Huh7 cells[J]. Journal of Jilin University(Medicine Edition), 2023, 49(1): 122-130.

图/表 9

表1

图1

表2

图2

图3

图4

图5

表3

图6

参考文献 25

1	SIEGEL R L， MILLER K D， FUCHS H E， et al. Cancer statistics， 2021［J］. CA Cancer J Clin， 2021， 71（1）： 7-33.
2	LIU W W， LIANG B， LIU H L， et al. Overexpression of non‑SMC condensin Ⅰ complex subunit G serves as a promising prognostic marker and therapeutic target for hepatocellular carcinoma［J］. Int J Mol Med， 2017， 40（3）： 731-738.
3	LIU K K， LI Y M， YU B， et al. Silencing non-SMC chromosome-associated polypeptide G inhibits proliferation and induces apoptosis in hepatocellular carcinoma cells［J］. Can J Physiol Pharmacol， 2018， 96（12）： 1246-1254.
4	GONG C W， AI J Y， FAN Y， et al. NCAPG promotes the proliferation of hepatocellular carcinoma through PI3K/AKT signaling［J］. Onco Targets Ther， 2019， 12： 8537-8552.
5	OURA K， MORISHITA A， MASAKI T. Molecular and functional roles of microRNAs in the progression of hepatocellular carcinoma-A review［J］. Int J Mol Sci， 2020， 21（21）： 8362.
6	CUI X， JIANG X Y， WEI C S， et al. Astragaloside Ⅳ suppresses development of hepatocellular carcinoma by regulating miR-150-5p/β-catenin axis［J］. Environ Toxicol Pharmacol， 2020， 78： 103397.
7	BRAY F， FERLAY J， SOERJOMATARAM I， et al. Global cancer statistics 2018： GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries［J］. CA Cancer J Clin， 2018， 68（6）： 394-424.
8	KULIK L， EL-SERAG H B. Epidemiology and management of hepatocellular carcinoma［J］. Gastroenterology， 2019， 156（2）： 477-491.
9	SCHLABE S， ROCKSTROH J K. Advances in the treatment of HIV/HCV coinfection in adults［J］. Expert Opin Pharmacother， 2018， 19（1）： 49-64.
10	HUO T I， HSU C Y， LIU P H. Magic mirror on the wall： which is the best biomarker for hepatocellular carcinoma？［J］. Hepatology， 2018， 67（6）： 2482-2483.
11	HARTKE J， JOHNSON M， GHABRIL M. The diagnosis and treatment of hepatocellular carcinoma［J］. Semin Diagn Pathol， 2017， 34（2）： 153-159.
12	SUN J Y， YIN T L， ZHANG X Y， et al. Therapeutic advances for patients with intermediate hepatocellular carcinoma［J］. J Cell Physiol， 2019， 234（8）： 12116-12121.
13	YERUKALA SATHIPATI S， HO S Y. Novel miRNA signature for predicting the stage of hepatocellular carcinoma［J］.Sci Rep，2020，10（1）：14452.
14	MOHAMED A A， ALI-ELDIN Z A， ELBEDEWY T A，et al. MicroRNAs and clinical implications in hepatocellular carcinoma［J］. World J Hepatol， 2017， 9（23）： 1001-1007.
15	CHEN F， LI X F， FU D S， et al. Clinical potential of miRNA-221 as a novel prognostic biomarker for hepatocellular carcinoma［J］. Cancer Biomark， 2017， 18（2）： 209-214.
16	HUNG J H， LI C H， YEH C H， et al. microRNA-224 down-regulates glycine N-methyltransferase gene expression in Hepatocellular Carcinoma［J］. Sci Rep， 2018， 8（1）： 12284.
17	JIANG C， HE Z L， HU X H， et al. MiRNA-15a-3p inhibits the metastasis of hepatocellular carcinoma by interacting with HMOX1［J］. Eur Rev Med Pharmacol Sci， 2020， 24（24）： 12694-12700.
18	YU W S， WANG Z G， GUO R P， et al. Hepatocellular carcinoma progression is protected by miRNA-34c-5p by regulating FAM83A［J］. Eur Rev Med Pharmacol Sci， 2020， 24（11）： 6046-6054.
19	CHEN X X， XU X N， PAN B， et al. miR-150-5p suppresses tumor progression by targeting VEGFA in colorectal cancer［J］.Aging （Albany NY），2018，10（11）： 3421-3437.
20	LU W， ZHANG H H， NIU Y Q， et al. Long non-coding RNA linc00673 regulated non-small cell lung cancer proliferation， migration， invasion and epithelial mesenchymal transition by sponging miR-150-5p［J］. Mol Cancer， 2017， 16（1）： 118.
21	JIANG L L， REN L L， CHEN H， et al. NCAPG confers trastuzumab resistance via activating SRC/STAT3 signaling pathway in HER2-positive breast cancer［J］. Cell Death Dis， 2020， 11（7）： 547.
22	WU Y， LIN Y， PAN J F， et al. NCAPG promotes the progression of lung adenocarcinoma via the TGF-β signaling pathway［J］. Cancer Cell Int，2021，21（1）： 443.
23	WANG Y， GAO B， TAN P Y， et al. Genome-wide CRISPR knockout screens identify NCAPG as an essential oncogene for hepatocellular carcinoma tumor growth［J］. FASEB J， 2019， 33（8）： 8759-8770.
24	GUO Z Y， ZHU Z T. NCAPG is a prognostic biomarker associated with vascular invasion in hepatocellular carcinoma［J］. Eur Rev Med Pharmacol Sci， 2021， 25（23）： 7238-7251.
25	AI J Y， GONG C W， WU J J， et al. microRNA‑181c suppresses growth and metastasis of hepatocellular carcinoma by modulating NCAPG［J］. Cancer Manag Res， 2019， 11： 3455-3467.

Gene	Primer sequence
miR-150-5p	Forward: 5'-TCGGCGTCTCCCAACCCTTGTAC-3' Reverse: 5'-GTCGTATCCAGTGCAGGGTCCGAGGT-3'
NCAPG	Forward: 5'-ATTGCTTTGTATTGGTGTGCCCTTTG-3' Reverse: 5'-ACAACTGGAATGCTCTGGATGTAACTC-3'
GAPDH	Forward: 5'-CGGAGTCAACGGATTTGGTCGTAT-3' Reverse: 5'-AGCCTTCTCCATGGTGGTGAAGAC-3'
U6	Forward: 5'-TGCGGGTGCTCGCTTCGGCAGC-3' Reverse: 5'-CCAGTGCAGGGTCCGAGGT-3'

Group	NCAPG
Group	mRNA	Protein
Mimic NC	1.000±0.000	0.957±0.062
MiR-150-5p mimic	0.382±0.068^*	0.425±0.022^*
MiR-150-5p mimic+ovNC	0.351±0.028	0.415±0.037
MiR-150-5p mimic+ovNCAPG	0.987±0.075^△	0.725±0.051^△

Group	Tumor volume
Group	(t/d) 3	6	9	12	15	18	21
AgomiR-NC	4.18±0.87	8.24±1.48	55.35±8.77	98.72±21.35	174.25±42.88	382.54±87.66	689.09±211.94
AgomiR-150-5p	3.44±0.66	5.86±1.08	15.82±3.42^*	27.52±5.77^*	35.47±4.82^*	56.45±9.21^*	123.24±19.96^*
AgomiR-150-5p+ovNC	3.12±0.48	5.77±0.75	14.33±2.95	21.85±4.32	32.35±5.89	51.08±8.77	116.04±19.04
AgomiR-150-5p+ovNCAPG	4.56±0.74	7.52±0.98	28.52±1.77^△	42.74±8.77^△	68.75±18.72^△	108.95±30.27^△	229.86±81.88^△

[1]	陈星竹,于明岳,刘双,李佳凝,谢尊玄,刘金瑶,刘玉艳. 载氟聚碳酸丙烯酯牙贴片再矿化性能及其对小鼠成纤维L929细胞的毒性作用[J]. 吉林大学学报(医学版), 2022, 48(6): 1429-1436.
[2]	张庆宇,徐庭瑞,矫君君,夏德庚,张天翼,张莉,马宁. 富血小板纤维蛋白和羟基磷灰石复合物的制备方法及其性能评价[J]. 吉林大学学报(医学版), 2022, 48(6): 1448-1454.
[3]	谢先顺,王伟,蒋海兵. miR-431-3p对胃癌细胞增殖和凋亡的影响及其靶向调控CTDP1基因表达机制[J]. 吉林大学学报(医学版), 2022, 48(6): 1555-1565.
[4]	孙红霞,刘春旭,安学俊,崔光华,王靖宇,佟双喜,杨晓秋. 北五味子多糖对人膀胱癌T24细胞增殖和凋亡的影响及其机制[J]. 吉林大学学报(医学版), 2022, 48(5): 1216-1222.
[5]	张蒨,李静. 过表达TLR4基因对胃癌细胞自噬的抑制作用及其机制[J]. 吉林大学学报(医学版), 2022, 48(5): 1238-1246.
[6]	黄晓妹,王洪伟,韩贞艳,韦秋园,黄素静. lncRNA GHET1通过Wnt/β-catenin信号通路对子痫前期滋养层细胞生物学行为的影响[J]. 吉林大学学报(医学版), 2022, 48(5): 1324-1332.
[7]	胡连涛,邓文俊,鹿士振,孙跞,李学斌,黎楚豪,王欣然,张春斌,李玥,王伟群. CC趋化因子配体20在肝细胞癌组织中的表达及其在肝细胞肝癌预后评估中作用的生物信息学分析[J]. 吉林大学学报(医学版), 2022, 48(4): 1010-1017.
[8]	王志娟,张明姝,叶丽平. 血小板衍生生长因子D通过ERK信号通路对肺癌H1299细胞增殖、迁移和侵袭的影响及其机制[J]. 吉林大学学报(医学版), 2022, 48(4): 898-904.
[9]	马琰迪,卢香云,何尚峰,俞雪燕,胡云华,高海霞,陈云昭,禹洁,王文洁,李锋,崔晓宾. m6A甲基化修饰结合蛋白YTHDF2在食管癌组织中的表达及其对食管癌细胞增殖和迁移的影响[J]. 吉林大学学报(医学版), 2022, 48(4): 962-970.
[10]	汪国武,姚远,张雨,徐娜,刘芳. miR-152降低低密度脂蛋白受体表达对子宫内膜癌细胞增殖和侵袭的抑制作用[J]. 吉林大学学报(医学版), 2022, 48(3): 591-599.
[11]	李官虎,郎庆旭,刘纯岩,刘沁,耿梦柔,李晓倩,王珍琦. 丙戊酸联合X射线照射对乳腺癌MDA-MB-231细胞增殖的抑制作用及其机制[J]. 吉林大学学报(医学版), 2022, 48(3): 622-629.
[12]	颜培玉,张爱臣,章宏,李杨,张萌萌,洛梦泽,潘颖. 脂肪间充质干细胞对卵巢早衰模型大鼠的治疗作用及其机制[J]. 吉林大学学报(医学版), 2022, 48(3): 648-656.
[13]	刘翠兰,胡凤爱,刘晶,王丹,邱长云,柳敦江,赵娣. 脂联素受体激动剂AdiopRon对胶质瘤细胞生物学行为的影响及其机制[J]. 吉林大学学报(医学版), 2022, 48(3): 702-710.
[14]	吴壮志,贺小宁,陈思祺. miR-124-3p对口腔鳞状细胞癌细胞增殖和侵袭的抑制作用及其机制[J]. 吉林大学学报(医学版), 2022, 48(3): 718-727.
[15]	李小燕,张维,何杰. REG1A通过调控Wnt/β-catenin信号通路对肺腺癌细胞增殖和迁移的促进作用[J]. 吉林大学学报(医学版), 2022, 48(2): 444-453.