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Effects of UHRF1 on Estrogen Receptor and Proliferation, Invasion and Migration of BCPAP Cells in Thyroid Papillary Carcinoma

  • Objective To investigate the effects of ubiquitin-like PDH and ring finger domain 1 (UHRF1) on the expression ratio of estrogen receptor (ER) α/ERβ, and to explore the experimental mechanism of UHRF1 affecting the proliferation, invasion and migration of BCPAP cells in papillary thyroid carcinoma. Methods The protein and mRNA expressions of UHRF1, ERα and ERβ in normal thyroid Nthy-ori3-1 cells and thyroid papillary carcinoma BCPAP cells were detected by Western blot and qRT-PCR. BCPAP cells were treated with Scrambled siRNA and UHRF1 siRNA, respectively. The expressions of ERα and ERβ mRNAs were detected by qRT-PCR. MTT and Transwell were used to determine the proliferation, invasion and migration in each group of BCPAP cells. Results Compared with Nthy-ori3-1 cells, the expressions of UHRF1 and ERα proteins and mRNAs in BCPAP cells were significantly up-regulated (P<0.05), while the expressions of ERβ protein and mRNA were significantly down-regulated (P<0.05). Compared with the control group and Scrambled siRNA group, the expression of ERα mRNA in BCPAP cells transfected with UHRF1 siRNA was significantly decreased (P<0.05), while the expression of ERβ mRNA was significantly increased (P<0.05). The proliferation, invasion and migration of BCPAP cells transfected with UHRF1 siRNA were significantly decreased (P<0.05). Conclusion UHRF1 upregulates ERα/ERβ expression ratio and promotes proliferation, invasion and migration of BCPAP cells in papillary thyroid carcinoma.
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  • [1] DALAL V, KAUR M, BANSAL A. Papillary carcinoma thyroid with anastomosing channels: an unusual morphology. J Lab Physicians,2017,9(2): 140–142. doi: 10.4103/0974-2727.199631
    [2] SIEGEL R, MA J M, ZOU Z H, et al. Cancer statistics, 2014. CA cancer J Clin,2014,64(1): 9–29. doi: 10.3322/caac.21208
    [3] 何二松. 甲状腺乳头状癌术后的综合治疗研究进展. 临床合理用药杂志,2019,12(14): 180–181.
    [4] QIU Y B, LIAO L Y, JIANG R, et al. PES1 promotes the occurrence and development of papillary thyroid cancer by upregulating the ERα/ERβ protein ratio. Sci Rep,2019,9(1): 1032[2019-12-15]. https://doi.org/10.1038/s41598-018-37648-7.
    [5] XUE B, ZHAO J, FENG P, et al. Epigenetic mechanism and target therapy of UHRF1 protein complex in malignancies. Onco Targets Ther,2019,12: 549–559. doi: 10.2147/OTT.S192234
    [6] GAO S P, SUN H F, LI L D, et al. UHRF1 promotes breast cancer progression by suppressing KLF17 expression by hypermethylating its promoter. Am J Cancer Res,2017,7(7): 1554–1565.
    [7] ZHOU L, SHANG Y, JIN Z, et al. UHRF1 promotes proliferation of gastric cancer via mediating tumor suppressor gene hypermethylation. Cancer Biol Ther,2015,16(8): 1241–1251. doi: 10.1080/15384047.2015.1056411
    [8] GE T T, YANG M, CHEN Z, et al. UHRF1 gene silencing inhibits cell proliferation and promotes cell apoptosis in human cervical squamous cell carcinoma CaSki cells. J Ovarian Res,2016,9(1): 42[2029-12-21].https://doi.org/10.1186/s13048-016-0253-8. doi: 10.1186/s13048-016-0253-8
    [9] CHEN G G, VLANTIS A C, ZENG Q, et al. Regulation of cell growth by estrogen signaling and potential targets in thyroid cancer. Curr Cancer Drug Targets,2008,8(5): 367–377. doi: 10.2174/156800908785133150
    [10] DAI Y J, QIU Y B, JIANG R, et al. Concomitant high expression of ERα36, GRP78 and GRP94 is associated with aggressive papillary thyroid cancer behavior. Cell Oncol,2018,41(3): 269–282. doi: 10.1007/s13402-017-0368-y
    [11] YI J W, KIM S J, KIM J K, et al. Upregulation of the ESR1 gene and ESR ratio (ESR1/ESR2) is associated with a worse prognosis in papillary thyroid carcinoma: the impact of the estrogen receptor α/β Expression on clinical outcomes in papillary thyroid carcinoma patients. Ann Surg Oncol,2017,24(12): 3754–3762. doi: 10.1245/s10434-017-5780-z
    [12] PARK Y R, LEE J, JUNG J H, et al. Absence of estrogen receptor is associated with worse oncologic outcome in patients who were received neoadjuvant chemotherapy for breast cancer. Asian J Surg,2020,43(3): 467–475.
    [13] BOGUSH T A, BASHARIBA A A, BOGUSH E A, et al. Estrogen receptors alpha and beta in ovarian cancer: expression level and prognosis. Dokl Biochem Biophys,2018,482(1): 249–251. doi: 10.1134/S1607672918050058
    [14] ALHOSIN M, OMRAN Z, ZAMZAMI M A, et al. Signalling pathways in UHRF1-dependent regulation of tumor suppressor genes in cancer. J Exp Clin Cancer Res,2016,35(1): 174[2019-12-15]. https://doi.org/10.1186/s13046-016-0453-5.
    [15] HWANG T, MATHIOS D, MCDONALD K L, et al. Integrative analysis of DNA methylation suggests down-regulation of oncogenic pathways and reduced somatic mutation rates in survival outliers of glioblastoma. Acta Neuropathol Commun,2019,7(1): 88[2019-12-16]. https://doi.org/10.1186/s40478-019-0744-0.
    [16] GAO L, QI X, HU K, et al. Estrogen receptor β promoter methylation: a potential indicator of malignant changes in breast cancer. Arch Med Sci,2016,12(1): 129–136.
    [17] KIM S J, KIM T W, LEE S Y, et al. CpG methylation of the ERalpha and ERbeta genes in breast cancer. Int J Mol Med,2004,14(2): 289–293.
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Effects of UHRF1 on Estrogen Receptor and Proliferation, Invasion and Migration of BCPAP Cells in Thyroid Papillary Carcinoma

    Corresponding author: LIU Zhi-min, liuzm9999@aliyun.com
  • Department of Biochemistry and Molecular Biology, Institute of Molecular Medicine and Cancer, College of Basic Medical Sciences, Chongqing Medical University, Chongqing 400016, China

doi: 10.12182/20200560501

Abstract:  Objective To investigate the effects of ubiquitin-like PDH and ring finger domain 1 (UHRF1) on the expression ratio of estrogen receptor (ER) α/ERβ, and to explore the experimental mechanism of UHRF1 affecting the proliferation, invasion and migration of BCPAP cells in papillary thyroid carcinoma. Methods The protein and mRNA expressions of UHRF1, ERα and ERβ in normal thyroid Nthy-ori3-1 cells and thyroid papillary carcinoma BCPAP cells were detected by Western blot and qRT-PCR. BCPAP cells were treated with Scrambled siRNA and UHRF1 siRNA, respectively. The expressions of ERα and ERβ mRNAs were detected by qRT-PCR. MTT and Transwell were used to determine the proliferation, invasion and migration in each group of BCPAP cells. Results Compared with Nthy-ori3-1 cells, the expressions of UHRF1 and ERα proteins and mRNAs in BCPAP cells were significantly up-regulated (P<0.05), while the expressions of ERβ protein and mRNA were significantly down-regulated (P<0.05). Compared with the control group and Scrambled siRNA group, the expression of ERα mRNA in BCPAP cells transfected with UHRF1 siRNA was significantly decreased (P<0.05), while the expression of ERβ mRNA was significantly increased (P<0.05). The proliferation, invasion and migration of BCPAP cells transfected with UHRF1 siRNA were significantly decreased (P<0.05). Conclusion UHRF1 upregulates ERα/ERβ expression ratio and promotes proliferation, invasion and migration of BCPAP cells in papillary thyroid carcinoma.

  • 甲状腺乳头状癌(papillary thyroid carcinoma, PTC)约占甲状腺癌总数80%,是最常见的一种甲状腺癌[1]。近年来,PTC发病率的增长速度位居女性肿瘤首位[2],目前已知PTC的发生发展会受到激素、遗传、环境等因素的影响[3]。有研究报道,雌激素在PTC的发展中发挥关键作用,而雌激素对PTC细胞的作用主要由雌激素受体(ER)α和ERβ介导,其中ERα表达与细胞异常增殖和恶性肿瘤的发展有关,相反,ERβ可抑制细胞增殖、迁移和侵袭[4]

    泛素样含PDH和环指域1(UHRF1)是与细胞增殖和表观遗传调节相关的多结构域蛋白,是一种众所周知的肿瘤标志物[5]。近年来研究发现UHRF1在乳腺癌[6]、胃癌[7]、宫颈癌[8]等肿瘤中不仅自身表达失调,而且抑制抑癌基因,参与多种肿瘤的增殖、侵袭和迁移等过程。目前,UHRF1在PTC发展中的作用机制尚不清楚。鉴于ERβ在PTC发展中发挥抑癌基因的作用,我们猜测:UHRF1能否调控抑癌基因ERβ表达,进而改变ERα/ERβ表达比率并参与PTC发生发展过程?这种调控机制国内外鲜见报道。

    本研究以人PTC的BCPAP细胞为模型,探讨UHRF1对雌激素受体ERα/ERβ表达和PTC发展的影响,为PTC临床治疗和靶向研究提供更多的理论依据。

1.   材料与方法
  • 胎牛血清(FBS)和RPMI1640培养基购自Gibco公司;胰蛋白酶购自Hyclone公司;RIPA裂解液、PMSF、SDS-PAGE凝胶试剂盒和免疫印迹化学发光剂ECL购自碧云天生物技术有限公司;RNA提取试剂盒和定量PCR试剂盒SYBR Premix Ex TaqTM购自TaKaRa公司;兔抗人UHRF1和β-actin多克隆抗体购自Bioworld公司;兔抗人ERα和ERβ多克隆抗体购自Abcam公司;Lipofectamine RNAiMAX购自Invitrogen公司;PCR引物、Scrambled siRNA、UHRF1 siRNA序列由上海生工生物工程有限公司合成;MTT试剂盒购自美国Sigma公司;Transwell小室和Matrigel基质胶购自美国BD公司。

  • 将本实验室保存的人PTC细胞BCPAP和人甲状腺滤泡上皮正常细胞Nthy-ori3-1贴壁培养于含100 mL/L FBS、100 U/mL青霉素和100 μg/mL链霉素的RPMI1640培养基,在37 ℃、体积分数为5%CO2的细胞培养箱环境下培养细胞,根据细胞生长状态换液或传代,随后按照Lipofectamine RNAiMAX使用说明书转染对数生长期的BCPAP细胞。转染前一天将BCPAP细胞按3×105/孔接种于6孔板内培养,待细胞融合度达到60%~80%时转染siRNA。UHRF1 siRNA正义链:5′-GUGGCAAGAAUAGCAAGUATT-3′,反义链:5′-UACUUGCUAUUCUUGCCACTT-3′;Scrambled siRNA正义链:5′-UUCUCCGAACGUGUCACGUTT-3′,反义链:5′- ACGUGACACGUUCGGAGAATT-3′。用无血清无抗生素的RPMI1640培养基稀释 UHRF1 siRNA、Scrambled siRNA和Lipofectamine RNAiMAX,室温静置5 min后,再将两者混匀室温静置20 min,随后将混合溶液加入对应细胞孔并置于37 ℃、体积分数为5%CO2的细胞培养箱环境下培养6 h后,更换含100 mL/L FBS的RPMI1640培养基继续培养48 h后收取细胞。将PTC细胞BCPAP分为空白对照组、Scrambled siRNA组和UHRF1 siRNA组。

  • 用RIPA裂解液和PMSF提取培养48 h后Nthy-ori3-1和BCPAP细胞与转染后的各组细胞总蛋白,测定UHRF1、ERα和ERβ的蛋白浓度。配置分离胶和浓缩胶后,每孔加入30 μg蛋白进行电泳,随后将分离的目的条带电转移到PVDF膜上,50 g/L脱脂奶粉封闭PVDF膜2 h,PBST洗膜后放入一抗稀释液 4 ℃孵育过夜,PBST洗膜后再将膜放入二抗稀释液37 ℃孵育2 h, PBST洗膜后发光显影,分析实验数据,以目的条带与β-actin灰度值的比值为目的蛋白的相对含量。实验重复3次。

  • 首先用RNA提取试剂盒提取培养48 h后Nthy-ori3-1和BCPAP细胞与转染后的各组细胞总RNA,然后逆转录成cDNA,最后按照SYBR Premix Ex TaqTM说明书进行扩增,引物设计如下:UHRF1上游引物5′-CAGGCAGACAAGCTGTTGG-3′,下游引物:5′-AACAGCTCCTGGATCTTCCG-3′;ERα上游引物:5′-AGCCTCCTGTCTACCGACTC-3′,下游引物:5′-GCAGGAGAAAGGAGCATGGA-3′;ERβ上游引物:5′-TTGACATGCTCCTGGCAACT-3′,下游引物: 5′-GGCCCAGCTGTGTGATTACT-3′;β-actin上游引物:5′-GGAGAAGATGACCCAGATCA-3′,下游引物5′-GATCTTCATGAGCTAGTCAG-3′。所有基因均使用β-actin为内参,实验所用引物均由上海生工生物工程有限公司合成,实验重复3次,用公式2ΔΔCt计算基因相对表达量。

  • 将对数生长期的BCPAP细胞,经胰酶消化后制成单细胞悬液,以5×103/孔的细胞数接种于96孔板,培养于37 ℃、体积分数为5%CO2孵育箱中,孵育24 h;细胞贴壁后分组处理细胞,继续培养48 h,每孔加入20 μL MTT (5 mg/mL)继续培养4 h。终止培养弃去培养液,每孔加入150 μL DMSO,充分溶解,震荡10 min,显色,570 nm处测定每孔吸光度值,以空白对照组细胞活力为100%,计算各组细胞增殖率,公式:增殖率=(实验组值-空白孔组值)/(空白对照组值-空白孔组值)×100%。实验重复3次。

  • 侵袭实验:无血清RPMI1640培养基按8∶1稀释Matrigel胶后,在Transwell小室上室中每孔均匀铺入60 μL,小室放于24孔培养板中,37 ℃孵育1 h。转染24 h后收集空白对照组、Scrambled siRNA组和UHRF1 siRNA组的细胞。按1×105/孔细胞密度接种于上室,并加入100 μL RPMI1640无血清培养基,将600 μL含100 mL/L FBS的RPMI1640培养基加入下室。37 ℃孵箱培养24 h后甲醇固定30 min,1 g/L结晶紫染色10 min,计数200倍光镜下随机选取的5个视野的细胞数目,实验重复3次。

    迁移实验:在实验中不铺Matrigel胶,取出在37 ℃、体积分数为5%CO2环境下培养24 h后的小室,其余步骤与侵袭实验一致。

  • 实验数据以$\bar x$ ± s表示。组间比较采用t检验、单因素方差分析,P<0.05为差异有统计学意义。

2.   结果
  • 图1。与Nthy-ori3-1细胞比较,BCPAP细胞中的UHRF1和ERα的蛋白表达水平增高,而ERβ蛋白表达水平降低,差异均有统计学意义(P<0.05)。在Nthy-ori3-1细胞中,ERα蛋白表达水平明显低于ERβ蛋白表达水平,ERα/ERβ蛋白比率为0.44,而在BCPAP细胞中,ERα蛋白表达水平高于ERβ蛋白表达水平,ERα/ERβ蛋白比率为2.37。

    Figure 1.  The protein expressions of UHRF1, ERα and ERβ in Nthy-ori3-1 and BCPAP cells detected by Western blot

  • 图2。BCPAP细胞中UHRF1 mRNA表达水平(16.91±1.59)高于Nthy-ori3-1细胞(6.36±0.73)。与Nthy-ori3-1细胞比较,BCPAP细胞中ERα mRNA表达水平增高,而ERβ mRNA表达水平降低,差异有统计学意义(P<0.05)。在Nthy-ori3-1细胞中,ERα mRNA表达水平明显低于ERβ mRNA表达水平,ERα mRNA/ERβ mRNA比率为0.35,而在BCPAP细胞中,ERα mRNA表达水平明显高于ERβ mRNA表达水平,ERα mRNA/ERβ mRNA比率为2。结果与蛋白表达结果一致。

    Figure 2.  The mRNA expressions of ERα and ERβ in Nthy-ori3-1 and BCPAP cells detected by qRT-PCR

  • 图3。将Scrambled siRNA和UHRF1 siRNA转染入BCPAP细胞后,与空白对照组和Scrambled siRNA组相比,UHRF1 siRNA组细胞ERβ蛋白和mRNA表达水平升高的同时,ERα蛋白和mRNA表达水平降低,导致ERα/ERβ比率降低,差异均有统计学意义(P<0.05)。结果说明:UHRF1抑制ERβ表达的同时增加ERα表达,进而上调ERα/ERβ比率。

    Figure 3.  The protein and mRNA expression levels of ERα and ERβ in 3 groups of BCPAP cells

  • 将Scrambled siRNA和UHRF1 siRNA转染入BCPAP细胞后,MTT检测到各组细胞增殖率变化,与空白对照组(100%)和Scrambled siRNA组〔(99.67±9.02)%〕相比,UHRF1 siRNA组细胞增殖率〔(79.67±6.11)%〕降低,差异有统计学意义(P<0.05),空白对照组和Scrambled siRNA组差异无统计学意义。结果说明UHRF1促进BCPAP细胞增殖。

  • 与空白对照组和Scrambled siRNA组相比,UHRF1 siRNA组细胞侵袭(图4)和迁移(图5)均降低,差异均有统计学意义(P<0.05),空白对照组和Scrambled siRNA组差异无统计学意义。结果说明UHRF1促进BCPAP细胞侵袭和迁移。

    Figure 4.  The invasion of BCPAP cells detected by Transwell. ×200

    Figure 5.  The migration of BCPAP cells detected by Transwell. ×200

3.   讨论
  • PTC是一种与雌激素相关的内分泌系统肿瘤,经PTC动物模型研究证实雌激素可以促进PTC的发展[9];体外研究也表明雌激素对培养的PTC细胞有促增生作用[10];这说明雌激素在PTC的发展中起促进作用。雌激素对PTC细胞的作用主要由雌激素受体ERα和ERβ介导,ERα和ERβ由独立不同的基因编码,虽有相似的结构,但在结构上存在一定的差异,使得ERα和ERβ可能具有不同的功能[11]。我们近期研究成果表明:与雌激素相关的肿瘤如乳腺癌[12]和卵巢癌[13]一样,与正常甲状腺组织和细胞比较,ERα在PTC组织和细胞中表达增高,而ERβ表达下降,ERα/ERβ比率显著增高,ERα促进细胞增生、侵袭和转移,与PTC恶性临床病理特征(大肿瘤、包膜浸润、淋巴结转移和高TNM分期)呈正相关,而ERβ抑制细胞增生、侵袭和转移,与PTC的恶性临床病理特征呈负相关,即与乳腺癌和卵巢癌等雌激素相关肿瘤相似,ERα和ERβ在PTC的发展中也表现出相反的作用[4]。越来越多的研究显示:雌激素信号主要依赖于ERα和ERβ表达水平及其平衡,当促增生的ERα相对于抗增生的ERβ占优势即ERα/ERβ比率上调显著大于1时,就会导致雌激素反应器官肿瘤的发生[11,13]

    UHRF1基因位于染色体19p13.3,是一种重要的表观遗传学调节因子,可招募表观遗传学标记酶如甲基化转移酶结合到TSG的启动子区,形成抑制性复合体抑制这些TSG的表达,从而参与肿瘤的发生发展过程[14]。人类基因组数据分析显示:大多数基因的启动子区都具有CpG富集区即CpG岛。启动子区CpG岛低水平或缺乏甲基化与染色质松弛状态有关,有利于DNA与转录复合物相互作用,激活基因表达。相反,基因启动子区CpG岛的高甲基化与染色质紧密状态有关,导致基因表达降低或沉默[15]。研究表明:在乳腺癌[16-17]中,DNA甲基化转移酶的表达增高与ERβ基因启动子区CpG岛高甲基化以及ERβ mRNA表达降低或沉默密切相关。本研究中,UHRF1抑制ERβ表达水平,结合文献报道结果说明:在PTC中,UHRF1可能招募DNA甲基化转移酶到ERβ基因启动子区域,下调或沉默ERβ mRNA表达。但是UHRF1对ERβ表达水平的具体调控机制尚需进一步研究。

    有研究报道:在乳腺癌细胞中,ERβ对ERα基因表达和启动子活性有抑制作用,ERβ通过与转录因子Sp1相互作用,招募NCoR和SMRT共抑制因子,结合到ERα基因转录起始位点上游启动子GC区而下调ERα的表达[18]。提示:在乳腺癌等雌激素相关肿瘤的发展中,作为肿瘤抑制基因的ERβ表达逐渐降低,减弱了它对ERα基因表达的抑制作用,使ERα基因表达上调,与ERα/ERβ比率增高密切相关。本研究结果显示:在BCPAP细胞中,UHRF1抑制ERβ表达水平的同时促进ERα表达水平,进而上调了ERα/ERβ表达比率,提示此结果的产生可能是由于上述调控机制所导致。

    在本实验中,UHRF1上调了ERα/ERβ表达比率并且也促进了PTC细胞BCPAP增生、侵袭和迁移,分析有以下原因:①UHRF1作为癌基因本身促进BCPAP细胞增殖、侵袭和迁移;②UHRF1抑制了ERβ表达水平,因ERβ能够显著抑制ERα表达水平,减弱了它对ERα基因表达的抑制作用,使得ERα/ERβ比率增高,促进BCPAP细胞增殖、侵袭和迁移,PTC细胞BCPAP增殖、侵袭和迁移可能是上述两个机制的综合结果。

    综上,调节UHRF1表达可以改变ERα/ERβ表达比率,影响BCPAP细胞增殖、侵袭和迁移,说明UHRF1有望成为PTC治疗和预后的一个潜在靶点。但UHRF1通过表观遗传学改变ERα/ERβ表达比率的分子调控机制,需要后续更为深入的研究,这些研究将为PTC临床治疗提供更为广阔的前景。

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