Effects of <i>MCCC2 </i>knockdown on proliferation, migration and apoptosis of DU145 prostate cancer cells

CHEN Xue; HUANG Zehao; ZHENG Chengjian

doi:10.12206/j.issn.1006-0111.202104020

Volume 39 Issue 3

May 2021

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CHEN Xue, HUANG Zehao, ZHENG Chengjian. Effects of MCCC2 knockdown on proliferation, migration and apoptosis of DU145 prostate cancer cells[J]. Journal of Pharmaceutical Practice and Service, 2021, 39(3): 215-220. doi: 10.12206/j.issn.1006-0111.202104020

Citation:

CHEN Xue, HUANG Zehao, ZHENG Chengjian. Effects of MCCC2 knockdown on proliferation, migration and apoptosis of DU145 prostate cancer cells[J]. Journal of Pharmaceutical Practice and Service, 2021, 39(3): 215-220. doi: 10.12206/j.issn.1006-0111.202104020

Effects of MCCC2 knockdown on proliferation, migration and apoptosis of DU145 prostate cancer cells

doi: 10.12206/j.issn.1006-0111.202104020

CHEN Xue^{1, 2
,},
HUANG Zehao^{1
,
,},
ZHENG Chengjian^{2
,
,}

1.
School of Pharmacy, Fujian University of Traditional Chinese Medicine, Fuzhou 350122, China
2.
School of Pharmacy, Naval Medical University, Shanghai 200433, China

Received Date: 2021-04-03
Rev Recd Date: 2021-05-03

Available Online: 2021-05-25

Publish Date: 2021-05-25

Abstract

Objective To investigate the change of biological characteristics after stable knockdown of the coding gene of 3-methylcrotonyl-coenzyme A carboxylase β subunit (MCCC2) expression in DU145 by lentivirus shRNA. Methods Three groups were included in this study. shNC was the control group in which MCCC2 was negatively knocked down in DU145. shMCCC2 was the experimental group in which MCCC2 was knocked down. DU145 was the blank group without any treatment. The expression of MCCC2 was assessed by Western blot and qPCR. The proliferation of DU145 cells was detected by CCK8 assay. The migration ability of DU145 was detected by transwell. The apoptosis of DU145 cells was detected by flow cytometry. Results The expression level of MCCC2 in shMCCC2 group was significantly lower than that in shNC group (0.22 ± 0.02 vs 0.61 ± 0.06, P < 0.001). The proliferation (2.24 ± 0.04 vs 3.13 ± 0.15) and migration (23.96 ± 1.85 vs 49.73 ± 0.63) of DU145 cells in shMCCC2 group was significantly lower than that in shNC group, whereas the apoptosis (12.64 ± 0.30 vs 3.68 ± 0.02) of DU145 cells in shMCCC2 was significantly higher than that in shNC group. Conclusion MCCC2 knockdown significantly inhibited the proliferation and migration, and induced apoptosis of DU145 cells, which indicated that the down-regulation of MCCC2 is correlated with the change of tumor biological characteristics of DU145 cell line and can be a potential target for the treatment of prostate cancer.
- MCCC2,
- prostate cancer,
- proliferation,
- migration,
- apoptosis

References

[1]	HA CHUNG B, HORIE S, CHIONG E. The incidence, mortality, and risk factors of prostate cancer in Asian men[J]. Prostate Int,2019,7(1):1-8. doi: 10.1016/j.prnil.2018.11.001
[2]	唐志柳, 白洁, 顾丽娜, 等. 2000-2010年我国前列腺癌和乳腺癌流行状况的系统性综述[J]. 中国肿瘤, 2013, 22(04):260-265.
[3]	王宁, 刘硕, 杨雷, 等. 2018全球癌症统计报告解读[J]. 肿瘤综合治疗电子杂志, 2019, 5(01):87-97.
[4]	ABULA A, SHAO W, TUSONG H, et al. Protein expression information of prostate infection based on data mining[J]. J Infect Public Health,2020,13(10):1533-1536. doi: 10.1016/j.jiph.2019.07.019
[5]	GRÜNERT S C, STUCKI M, MORSCHER R J, et al. 3-methylcrotonyl-CoA carboxylase deficiency: clinical, biochemical, enzymatic and molecular studies in 88 individuals[J]. Orphanet J Rare Dis,2012,7:31. doi: 10.1186/1750-1172-7-31
[6]	MORSCHER R J, GRÜNERT S C, BÜRER C, et al. A single mutation in MCCC₁ or MCCC₂ as a potential cause of positive screening for 3-methylcrotonyl-CoA carboxylase deficiency[J]. Mol Genet Metab,2012,105(4):602-606. doi: 10.1016/j.ymgme.2011.12.018
[7]	HOLZINGER A, RÖSCHINGER W, LAGLER F, et al. Cloning of the human MCCA and MCCB genes and mutations therein reveal the molecular cause of 3-methylcrotonyl-CoA: carboxylase deficiency[J]. Hum Mol Genet,2001,10(12):1299-1306. doi: 10.1093/hmg/10.12.1299
[8]	GALLARDO M E, DESVIAT L R, RODRÍGUEZ J M, et al. The molecular basis of 3-methylcrotonylglycinuria, a disorder of leucine catabolism[J]. Am J Hum Genet,2001,68(2):334-346. doi: 10.1086/318202
[9]	PANG J, LIU W P, LIU X P, et al. Profiling protein markers associated with lymph node metastasis in prostate cancer by DIGE-based proteomics analysis[J]. J Proteome Res,2010,9(1):216-226. doi: 10.1021/pr900953s
[10]	MARQUES R B, DITS N F, ERKENS-SCHULZE S, et al. Modulation of androgen receptor signaling in hormonal therapy-resistant prostate cancer cell lines[J]. PLoS One,2011,6(8):e23144. doi: 10.1371/journal.pone.0023144
[11]	LIU Y, YUAN Z, SONG C. Methylcrotonoyl-CoA carboxylase 2 overexpression predicts an unfavorable prognosis and promotes cell proliferation in breast cancer[J]. Biomarks Med,2019,13(6):427-436. doi: 10.2217/bmm-2018-0475
[12]	JUNG C W, LEE B H, KIM J H, et al. Uneventful clinical courses of Korean patients with methylcrotonylglycinuria and their common mutations[J]. J Hum Genet,2012,57(1):62-64. doi: 10.1038/jhg.2011.116
[13]	AL-SHAMSI A, HERTECANT J L, SOUID A K, et al. Whole exome sequencing diagnosis of inborn errors of metabolism and other disorders in United Arab Emirates[J]. Orphanet J Rare Dis,2016,11(1):94. doi: 10.1186/s13023-016-0474-3
[14]	RIPS J, ALMASHANU S, MANDEL H, et al. Primary and maternal 3-methylcrotonyl-CoA carboxylase deficiency: insights from the Israel newborn screening program[J]. J Inherit Metab Dis,2016,39(2):211-217. doi: 10.1007/s10545-015-9899-4
[15]	ZANDBERG L, VAN DYK H C, VAN DER WESTHUIZEN F H, et al. A 3-methylcrotonyl-CoA carboxylase deficient human skin fibroblast transcriptome reveals underlying mitochondrial dysfunction and oxidative stress[J]. Int J Biochem Cell Biol,2016,78:116-129. doi: 10.1016/j.biocel.2016.07.010
[16]	FICICIOGLU C, PAYAN I. 3-methylcrotonyl-CoA carboxylase deficiency: metabolic decompensation in a noncompliant child detected through newborn screening[J]. Pediatrics,2006,118(6):2555-2556. doi: 10.1542/peds.2006-1659
[17]	BAUMGARTNER M R, ALMASHANU S, SUORMALA T, et al. The molecular basis of human 3-methylcrotonyl-CoA carboxylase deficiency[J]. J Clin Invest,2001,107(4):495-504. doi: 10.1172/JCI11948
[18]	STADLER S C, POLANETZ R, MAIER E M, et al. Newborn screening for 3-methylcrotonyl-CoA carboxylase deficiency: population heterogeneity of MCCA and MCCB mutations and impact on risk assessment[J]. Hum Mutat,2006,27(8):748-759. doi: 10.1002/humu.20349
[19]	YANG L, YANG J, ZHANG T, et al. Identification of eight novel mutations and transcript analysis of two splicing mutations in Chinese newborns with MCC deficiency[J]. Clin Genet,2015,88(5):484-488. doi: 10.1111/cge.12535
[20]	FONSECA H, AZEVEDO L, SERRANO C, et al. 3-methylcrotonyl-CoA carboxylase deficiency: mutational spectrum derived from comprehensive newborn screening[J]. Gene,2016,594(2):203-210. doi: 10.1016/j.gene.2016.09.003
[21]	COZZOLINO C, VILLANI G R, FRISSO G, et al. Biochemical and molecular characterization of 3-methylcrotonylglycinuria in an Italian asymptomatic girl[J]. Genet Mol Biol,2018,41(2):379-385. doi: 10.1590/1678-4685-gmb-2017-0093
[22]	STENSON P D, MORT M, BALL E V, et al. The Human Gene Mutation Database: building a comprehensive mutation repository for clinical and molecular genetics, diagnostic testing and personalized genomic medicine[J]. Hum Genet,2014,133(1):1-9. doi: 10.1007/s00439-013-1358-4

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前列腺癌（prostate cancer，PCa）是全球范围内第二位最常见的男性癌症，也是全球第二大致死癌症^[1]。据统计，我国前列腺癌的发病率呈逐年上升的趋势，其中，上海地区该病的发病率和病死率较高，但总体而言，亚洲国家前列腺癌的发病率明显低于西方国家^[1-3]。在前列腺癌的治疗中，局限性前列腺癌常规行根治性前列腺切除术或放射治疗，而对于进展期患者则需要接受内分泌治疗即雄激素阻断治疗（androgen deprivation therapy，ADT）。虽然ADT在早期可以明显抑制激素依赖性前列腺癌的进展，但是随着治疗时间的延长，激素依赖性前列腺癌会逐渐失去对ADT的反应而转化成为去势抵抗性前列腺癌（castration-resistant prostate cancer, CRPC），目前对CRPC尚无有效的治疗方法。因此，亟需深入挖掘CRPC病程进展中的关键基因和蛋白，为CRPC提供潜在治疗靶标。

Abula等^[4]进行了PCa蛋白质组学文献的数据挖掘，发现了前列腺肿瘤组织和正常组织中41种差异表达蛋白，并构建了蛋白相互作用网络。其中，3-甲基巴豆酰辅酶A羧化酶β亚基（3-methylcrotonyl-coenzyme A carboxylase β subunit，MCCB）就是其中的重要差异蛋白之一。MCCB是生物素依赖性羧化酶（3-methylcrotonyl-coenzyme A carboxylase，MCC）的两个组成亚基之一，由563个氨基酸组成^[5-8]，MCCC2是该蛋白的编码基因。已有研究报道，MCCC2是一种致癌基因，与包括肝细胞癌、结直肠癌以及乳腺癌在内的多种肿瘤的形成和发展密切相关^[9-11]，有望成为肿瘤治疗干预的潜在靶标。

在本研究中，我们主要探讨了MCCC2与前列腺癌的关系。首先，采用慢病毒与质粒构建了稳定低表达MCCC2的DU145细胞株并通过Western blot和qPCR分析敲减率；然后，系统考察敲减MCCC2对DU145前列腺癌细胞的增殖、迁移以及凋亡的影响，明确下调MCCC2与DU145细胞系肿瘤生物学特性的改变的相关性。

1. 实验材料

1.1. 细胞株

人前列腺癌DU145细胞，来源于海军军医大学附属长海医院泌尿外科，培养于含10%胎牛血清的RPMI 1640培养基中。

1.2. 材料与试剂

CCK-8试剂盒（上海翊圣生物科技有限公司）；兔MCCC2多克隆抗体、GAPDH抗体以及HRP标记山羊抗兔二抗（美国CST公司）；4%多聚甲醛固定液以及结晶紫染色液（碧云天生物技术有限公司）；细胞凋亡试剂盒（南京凯基生物有限公司）。

4. 讨论

围绕CRPC发生发展中的关键基因和蛋白，已有研究者采用蛋白组学和转录组学等多种组学技术深入挖掘PCa中AR等重要信号通路的关键调节蛋白、基因和潜在的疾病标志物，发现MCCB是前列腺肿瘤组织和正常组织中差异表达的关键蛋白之一^[4]，而编码该蛋白的基因MCCC2为AR信号通路调节的关键基因，在人类前列腺癌临床样本中显著过表达，可作为潜在的肿瘤标志物^[10]。

3-甲基巴豆酰基辅酶A羧化酶（MCC）是一种生物素依赖的羧化酶，可将3-甲基巴豆酰基辅酶A催化为3-甲基戊二酰辅酶A^[5-6]，是亮氨酸降解途径的重要一步。MCC包含α（MCCA）和β（MCCB）两个亚基，分别由MCCC1和MCCC2基因编码^[12-13]。MCC在人体内主要表达于肾脏和肝脏中，它的缺陷可引起甲基巴豆酰甘氨酸尿症，严重的可导致婴儿期的死亡^[14-16]。MCC缺陷主要与MCCC1和MCCC2基因的突变有关^[17-21]。人MCCC1位于染色体区域3q25-q27，具有19个外显子，而MCCC2位于染色体区域5q12-q13，由17个外显子组成^[7-8]。迄今为止，人类基因突变数据库中共报道了MCCC1基因的49个突变和MCCC2基因的52个突变^[22]。近年来，越来越多的研究表明，MCCC2参与了包括肝细胞癌、结直肠癌以及乳腺癌在内等多种肿瘤的形成和发展^[9-11]，其在不同肿瘤中发挥的重要作用有待进一步挖掘。

本研究主要探究了MCCC2与前列腺癌的关系。结果表明，敲减MCCC2可显著抑制DU145前列腺癌细胞的增殖和迁移，并诱导细胞凋亡。研究结果提示，MCCC2可能在前列腺癌发生发展过程中发挥重要作用，有望成为治疗前列腺癌的一个潜在新靶标。至今尚无针对其编码蛋白MCCB的选择性抑制剂的报道，是前列腺癌治疗药物发现的潜在新方向。

Reference (22)

[1]	HA CHUNG B, HORIE S, CHIONG E. The incidence, mortality, and risk factors of prostate cancer in Asian men[J]. Prostate Int,2019,7(1):1-8.
[2]	唐志柳, 白洁, 顾丽娜, 等. 2000-2010年我国前列腺癌和乳腺癌流行状况的系统性综述[J]. 中国肿瘤, 2013, 22(04):260-265.
[3]	王宁, 刘硕, 杨雷, 等. 2018全球癌症统计报告解读[J]. 肿瘤综合治疗电子杂志, 2019, 5(01):87-97.
[4]	ABULA A, SHAO W, TUSONG H, et al. Protein expression information of prostate infection based on data mining[J]. J Infect Public Health,2020,13(10):1533-1536.
[5]	GRÜNERT S C, STUCKI M, MORSCHER R J, et al. 3-methylcrotonyl-CoA carboxylase deficiency: clinical, biochemical, enzymatic and molecular studies in 88 individuals[J]. Orphanet J Rare Dis,2012,7:31.
[6]	MORSCHER R J, GRÜNERT S C, BÜRER C, et al. A single mutation in MCCC₁ or MCCC₂ as a potential cause of positive screening for 3-methylcrotonyl-CoA carboxylase deficiency[J]. Mol Genet Metab,2012,105(4):602-606.
[7]	HOLZINGER A, RÖSCHINGER W, LAGLER F, et al. Cloning of the human MCCA and MCCB genes and mutations therein reveal the molecular cause of 3-methylcrotonyl-CoA: carboxylase deficiency[J]. Hum Mol Genet,2001,10(12):1299-1306.
[8]	GALLARDO M E, DESVIAT L R, RODRÍGUEZ J M, et al. The molecular basis of 3-methylcrotonylglycinuria, a disorder of leucine catabolism[J]. Am J Hum Genet,2001,68(2):334-346.
[9]	PANG J, LIU W P, LIU X P, et al. Profiling protein markers associated with lymph node metastasis in prostate cancer by DIGE-based proteomics analysis[J]. J Proteome Res,2010,9(1):216-226.
[10]	MARQUES R B, DITS N F, ERKENS-SCHULZE S, et al. Modulation of androgen receptor signaling in hormonal therapy-resistant prostate cancer cell lines[J]. PLoS One,2011,6(8):e23144.
[11]	LIU Y, YUAN Z, SONG C. Methylcrotonoyl-CoA carboxylase 2 overexpression predicts an unfavorable prognosis and promotes cell proliferation in breast cancer[J]. Biomarks Med,2019,13(6):427-436.
[12]	JUNG C W, LEE B H, KIM J H, et al. Uneventful clinical courses of Korean patients with methylcrotonylglycinuria and their common mutations[J]. J Hum Genet,2012,57(1):62-64.
[13]	AL-SHAMSI A, HERTECANT J L, SOUID A K, et al. Whole exome sequencing diagnosis of inborn errors of metabolism and other disorders in United Arab Emirates[J]. Orphanet J Rare Dis,2016,11(1):94.
[14]	RIPS J, ALMASHANU S, MANDEL H, et al. Primary and maternal 3-methylcrotonyl-CoA carboxylase deficiency: insights from the Israel newborn screening program[J]. J Inherit Metab Dis,2016,39(2):211-217.
[15]	ZANDBERG L, VAN DYK H C, VAN DER WESTHUIZEN F H, et al. A 3-methylcrotonyl-CoA carboxylase deficient human skin fibroblast transcriptome reveals underlying mitochondrial dysfunction and oxidative stress[J]. Int J Biochem Cell Biol,2016,78:116-129.
[16]	FICICIOGLU C, PAYAN I. 3-methylcrotonyl-CoA carboxylase deficiency: metabolic decompensation in a noncompliant child detected through newborn screening[J]. Pediatrics,2006,118(6):2555-2556.
[17]	BAUMGARTNER M R, ALMASHANU S, SUORMALA T, et al. The molecular basis of human 3-methylcrotonyl-CoA carboxylase deficiency[J]. J Clin Invest,2001,107(4):495-504.
[18]	STADLER S C, POLANETZ R, MAIER E M, et al. Newborn screening for 3-methylcrotonyl-CoA carboxylase deficiency: population heterogeneity of MCCA and MCCB mutations and impact on risk assessment[J]. Hum Mutat,2006,27(8):748-759.
[19]	YANG L, YANG J, ZHANG T, et al. Identification of eight novel mutations and transcript analysis of two splicing mutations in Chinese newborns with MCC deficiency[J]. Clin Genet,2015,88(5):484-488.
[20]	FONSECA H, AZEVEDO L, SERRANO C, et al. 3-methylcrotonyl-CoA carboxylase deficiency: mutational spectrum derived from comprehensive newborn screening[J]. Gene,2016,594(2):203-210.
[21]	COZZOLINO C, VILLANI G R, FRISSO G, et al. Biochemical and molecular characterization of 3-methylcrotonylglycinuria in an Italian asymptomatic girl[J]. Genet Mol Biol,2018,41(2):379-385.
[22]	STENSON P D, MORT M, BALL E V, et al. The Human Gene Mutation Database: building a comprehensive mutation repository for clinical and molecular genetics, diagnostic testing and personalized genomic medicine[J]. Hum Genet,2014,133(1):1-9.

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Verification Code

物质	加入量（V/μl）
总RNA	X（X=1000/c，c为上述测定的总RNA的浓度）
Rnase Free dH₂O	16－X
5×Mix	4

物质	加入量（V/μl）
cDNA	1.0
Primer R	0.3
Primer F	0.3
ddH₂O	3.4
SYBR	5.0
总计	10

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