慢病毒转染调控FoxM1表达对人肝内胆管细胞癌增殖、侵袭及MMPs表达的影响

doi:10.11958/20220114

天津医药 ›› 2022, Vol. 50 ›› Issue (9): 902-906.doi: 10.11958/20220114

慢病毒转染调控FoxM1表达对人肝内胆管细胞癌增殖、侵袭及MMPs表达的影响

刘凌云¹(), 毛涵¹, 朱袭嘉²

1 桂林医学院附属医院肝胆胰外科,肝胆胰外科实验室,广西肝脏损伤与修复分子医学重点实验室（邮编541001）
2 桂林医学院第二附属医院胃肠外科

收稿日期:2022-01-18 修回日期:2022-05-18 出版日期:2022-09-15 发布日期:2022-09-05
作者简介:刘凌云（1982）,男,副主任医师,主要从事肝癌基础及临床方面研究。E-mail： 516001233@qq.com
基金资助:
广西科技基地和人才专项(桂科AD19110005);桂林市科学研究与技术开发计划项目(20190206-1);桂林市科学研究与技术开发计划项目(20210102-1);“广西特聘专家”专项经费资助项目(2019B12);广西科技厅中央引导地方科技发展资金项目(ZY21195024)

Effects of FoxM1 expression regulated by lentiviral transfection on the proliferation and invasion of human intrahepatic cholangiocarcinoma and the expression of MMPs

LIU Lingyun¹(), MAO Han¹, ZHU Xijia²

1 Department of Hepatobiliary and Pancreatic Surgery, Laboratory of Hepatobiliary and Pancreatic Surgery, Guangxi Key Laboratory of Molecular Medicine in Liver Injury and Repair, the Affiliated Hospital of Guilin Medical University, Guilin 541001, China
2 Department of Gastrointestinal Surgery, the Second Affiliated Hospital of Guilin Medical University

Received:2022-01-18 Revised:2022-05-18 Published:2022-09-15 Online:2022-09-05

刘凌云, 毛涵, 朱袭嘉. 慢病毒转染调控FoxM1表达对人肝内胆管细胞癌增殖、侵袭及MMPs表达的影响[J]. 天津医药, 2022, 50(9): 902-906.

LIU Lingyun, MAO Han, ZHU Xijia. Effects of FoxM1 expression regulated by lentiviral transfection on the proliferation and invasion of human intrahepatic cholangiocarcinoma and the expression of MMPs[J]. Tianjin Medical Journal, 2022, 50(9): 902-906.

摘要/Abstract

摘要：

目的通过实验探讨慢病毒转染调控FoxM1表达对人肝内胆管细胞癌（ICC）增殖、侵袭能力及基质金属蛋白酶（MMP）-9和MMP-2表达的影响。方法 Western blot检测ICC细胞株HCCC-9810、RBE及SSP-25的FoxM1蛋白表达水平,选取表达量较低者作为上调FoxM1细胞株,较高者作为下调细胞株;将分别携带FoxM1质粒和shRNA的慢病毒载体转染目标上调和下调ICC细胞株,建立稳定上下调FoxM1的细胞株（Western blot验证）;MTT法检测转染后细胞增殖力,Transwell侵袭实验检测细胞侵袭能力;qPCR检测各组稳定转染细胞株MMP-9及MMP-2 mRNA表达水平。结果 SSP-25的FoxM1蛋白表达最高,HCCC-9810最低,由此选取SSP-25作为下调FoxM1表达目标细胞株,HCCC-9810作为上调FoxM1表达目标细胞株。慢病毒转染成功构建稳定上调（HCCC-9810-FoxM1组）及下调FoxM1细胞株（SSP-25-shFoxM1组）;HCCC-9810-FoxM1组增殖及侵袭能力明显高于HCCC-9810-Control组（均P<0.05）,而SSP-25-shFoxM1组增殖及侵袭能力较SSP-25-Control组明显下降（均P<0.05）;HCCC-9810-FoxM1组中MMP-9及MMP-2 mRNA表达较HCCC-9810-Control组明显升高,而SSP-25-shFoxM1组中MMP-9及MMP-2 mRNA表达较SSP-25-Control组明显降低（均P<0.01）。结论 FoxM1促进ICC细胞增殖及侵袭,可能调控MMP-9及MMP-2表达,也有可能作为ICC潜在的生物标志物。

关键词: 叉头框蛋白M1, 胆管上皮癌, 细胞增殖, 肿瘤侵润, 基质金属蛋白酶9, 基质金属蛋白酶2

Abstract:

Objective To investigate the effects of FoxM1 regulated by lentiviral transfection on the proliferation and invasion of human intrahepatic cholangiocarcinoma (ICC) and the expression levels of MMP-9 and MMP-2 in vitro. Methods Western blot assay was used to detect the FoxM1 protein expression levels of ICC cell lines HCCC-9810, RBE and SSP-25. The higher expression level of cell line was selected as the down-regulated FoxM1 cell line, and the lower one was selected as the up-regulated FoxM1 cell line. FoxM1 plasmid and shRNA lentiviral vector transfection targeting up-regulating and down-regulating ICC cell lines were established a cell line that stably up-regulating or down-regulating FoxM1 cells (verified by Western blot assay). MTT method was used to detect cell proliferation after transfection, and Transwell invasion test was used to detect cell invasion. qPCR was used to detect the expression levels of MMP-9 and MMP-2 mRNA in stably transfected cell lines of each group. Results The protein of FoxM1 expression of SSP-25 was the highest, and HCCC-9810 was the lowest. Therefore, SSP-25 was selected as the target cell line for down-regulating FoxM1 expression, and HCCC-9810 was selected as the target cell line for up-regulating FoxM1 expression. The lentiviral transfection successfully constructed stable overexpression (the HCCC-9810-FoxM1 group) and down-regulated FoxM1 cell line (the SSP-25-shFoxM1 group). The proliferation and invasion ability were significantly higher in the HCCC-9810-FoxM1 group than those of the HCCC-9810-Control group (P<0.05). The proliferation and invasion ability were significantly lower in the SSP-25-shFoxM1 group than those of the SSP-25-Control group (P<0.05). In the HCCC-9810-FoxM1 group, the mRNA expression levels of MMP-9 and MMP-2 were significantly higher than those of the HCCC-9810-Control group, while the mRNA expression levels of MMP-9 and MMP-2 were significantly lower in the SSP-25-shFoxM1 group than those of the SSP-25-Control group (P<0.05). Conclusion FoxM1 promotes the proliferation and invasion of ICC cells. FoxM1 may affect the expression levels of MMP-9 and MMP-2. FoxM1 may serve as a potential biomarker of ICC.

Key words: forkhead box protein M1, cholangiocarcinoma, cell proliferation, neoplasm invasiveness, matrix metalloproteinase 9, matrix metalloproteinase 2

中图分类号:

R735.7

图/表 7

表1 qPCR检测引物序列

Tab.1 qPCR sequence of primers

基因名称	引物序列（5′→3′）	产物大小（bp）
FoxM1	上游：AAGCCAGGCTGGAAGAACTC	150
FoxM1	下游：CTGCTGCTTAAACACCTGGTC	150
MMP-9	上游：TCGTGGTTCCAACTCGGTTT	189
MMP-9	下游：GGGTGTAGAGTCTCTCGCTG	189
MMP-2	上游：GGACTTAGACCGCTTGGCTT	177
MMP-2	下游：GTGTTCAGGTATTGCATGTGCT	177
GAPDH	上游：CGGGAAGGAAATGAATGGGC	179
GAPDH	下游：GCCCAATACGACCAAATCAGAGAA	179

图1 ICC细胞株中FoxM1蛋白表达情况

Fig.1 Expression levels of FoxM1 protein in ICC cell lines

图2 慢病毒转染后ICC细胞株绿色荧光蛋白表达图

Fig.2 Representative graph of green fluorescent protein expression in ICC cell lines after lentivirus transfection

图3 ICC稳定转染细胞株FoxM1表达情况 1：HCCC-9810-Control组;2：HCCC-9810-FoxM1组;3：SSP-25-Control组;4：SSP-25-shFoxM1组。

Fig.3 Expression of FoxM1 in ICC stably transfected cell lines

表2 各组ICC细胞增殖能力对比（n=3,OD490,$\bar{x}±s$）

Tab.2 Comparison of the proliferation ability between the four groups of ICC cells

组别	24 h	48 h	72 h
HCCC-9810-Control组	0.36±0.01	0.76±0.07	1.21±0.08
HCCC-9810-FoxM1组	0.34±0.02	1.07±0.10	2.08±0.08
t	1.107	4.544^＊	11.322^＊＊
SSP-25-Control组	0.35±0.01	0.81±0.06	1.65±0.09
SSP-25-shFoxM1组	0.36±0.01	0.59±0.06	1.02±0.09
t	0.725	3.908^＊	7.288^＊＊

图4 Transwell小室侵袭实验（×100）

Fig.4 Transwell cell invasion test (×100)

表3 FoxM1表达变化对ICC细胞MMP-9及MMP-2 mRNA相对表达量的影响

Tab.3 Effects of FoxM1 expression changes on the relative expression levels of MMP-9 and MMP-2 mRNA in ICC cells （n=3,$\bar{x}±s$）

组别	MMP-9	MMP-2	FoxM1
HCCC-9810-Control组	1.08±0.12	1.00±0.09	1.05±0.11
HCCC-9810-FoxM1组	2.29±0.25	2.40±0.31	2.71±0.28
t	10.464^＊＊	15.640^＊＊	15.044^＊＊
SSP-25-Control组	1.05±0.05	1.04±0.03	1.03±0.04
SSP-25-shFoxM1组	0.47±0.01	0.53±0.08	0.27±0.11
t	10.521^＊＊	15.992^＊＊	18.603^＊＊

参考文献 16

[1]	MEJIA J C, PASKO J. Primary liver cancers: intrahepatic cholangiocarcinoma and hepatocellular carcinoma[J]. Surg Clin North Am, 2020, 100(3):535-549. doi: 10.1016/j.suc.2020.02.013.
[2]	SIRICA A E, GORES G J, GROOPMAN J D, et al. Intrahepatic cholangiocarcinoma:continuing challenges and translational advances[J]. Hepatology, 2019, 69(4):1803-1815. doi: 10.1002/hep.30289.
[3]	SPOLVERATO G, KIM Y, ALEXANDRESCU S, et al. Management and outcomes of patients with recurrent intrahepatic cholangiocarcinoma following previous curative-intent surgical resection[J]. Ann Surg Oncol, 2016, 23(1):235-243. doi: 10.1245/s10434-015-4642-9.
[4]	HALASI M, GARTEL A L. FOX(M1) news--it is cancer[J]. Mol Cancer Ther, 2013, 12(3):245-254. doi: 10.1158/1535-7163.MCT-12-0712.
[5]	BORHANI S, GARTEL A L. FOXM1:a potential therapeutic target in human solid cancers[J]. Expert Opin Ther Targets, 2020, 24(3):205-217. doi: 10.1080/14728222.2020.1727888.
[6]	SANDERS D A, ROSS-INNES C S, BERALDI D, et al. Genome-wide mapping of FOXM1 binding reveals co-binding with estrogen receptor alpha in breast cancer cells[J]. Genome Biol, 2013, 14(1):R6. doi: 10.1186/gb-2013-14-1-r6.
[7]	XIA L, HUANG W, TIAN D, et al. Upregulated FoxM1 expression induced by hepatitis B virus X protein promotes tumor metastasis and indicates poor prognosis in hepatitis B virus-related hepatocellular carcinoma[J]. J Hepatol, 2012, 57(3):600-612. doi: 10.1016/j.jhep.2012.04.020.
[8]	AI C, ZHANG J, LIAN S, et al. FOXM1 functions collaboratively with PLAU to promote gastric cancer progression[J]. J Cancer, 2020, 11(4):788-794. doi: 10.7150/jca.37323.
[9]	CAVDAR Z, CANDA A E, TERZI C, et al. Role of gelatinases (matrix metalloproteinases 2 and 9), vascular endothelial growth factor and endostatin on clinicopathological behaviour of rectal cancer[J]. Colorectal Dis, 2011, 13(2):154-160. doi: 10.1111/j.1463-1318.2009.02105.x.
[10]	QIU Y, CAO X, LIU L, et al. Modulation of MnSOD and FoxM1 is involved in invasion and EMT suppression by isovitexin in hepatocellular carcinoma cells[J]. Cancer Manag Res, 2020, 12:5759-5771. doi: 10.2147/CMAR.S245283.
[11]	GURO H, KIM J W, CHOI Y, et al. Multidisciplinary management of intrahepatic cholangiocarcinoma: current approaches[J]. Surg Oncol, 2017, 26(2):146-152. doi: 10.1016/j.suronc.2017.03.001.
[12]	RUZZENENTE A, CONCI S, VALDEGAMBERI A, et al. Role of surgery in the treatment of intrahepatic cholangiocarcinoma[J]. Eur Rev Med Pharmacol Sci, 2015, 19(15):2892-2900.
[13]	TASSI R A, TODESCHINI P, SIEGEL E R, et al. FOXM1 expression is significantly associated with chemotherapy resistance and adverse prognosis in non-serous epithelial ovarian cancer patients[J]. J Exp Clin Cancer Res, 2017, 36(1):63. doi: 10.1186/s13046-017-0536-y.
[14]	WEILER S, PINNA F, WOLF T, et al. Induction of chromosome instability by activation of Yes-associated protein and forkhead box M1 in liver cancer[J]. Gastroenterology, 2017, 152(8):2037-2051.e22. doi: 10.1053/j.gastro.2017.02.018.
[15]	YU C P, YU S, SHI L, et al. FoxM1 promotes epithelial-mesenchymal transition of hepatocellular carcinoma by targeting Snai1[J]. Mol Med Rep, 2017, 16(4):5181-5188. doi: 10.3892/mmr.2017.7223.
[16]	XUE Y J, XIAO R H, LONG D Z, et al. Overexpression of FoxM1 is associated with tumor progression in patients with clear cell renal cell carcinoma[J]. J Transl Med, 2012, 10:200. doi: 10.1186/1479-5876-10-200.

慢病毒转染调控FoxM1表达对人肝内胆管细胞癌增殖、侵袭及MMPs表达的影响

Effects of FoxM1 expression regulated by lentiviral transfection on the proliferation and invasion of human intrahepatic cholangiocarcinoma and the expression of MMPs

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