肿瘤  2014, Vol.34 Issue (1): 39-46   PDF    
基础研究 · Basic Research
miR-26a通过调节TFAP2C的表达而抑制卵巢癌细胞的增殖
王淑芬,董巍蕾,谢 晶,何 璐,周 曦,蔡艳林,刘 杰,谢宛玉     
南华大学附属第一医院妇产科,湖南 衡阳 421001
[摘要]    目的:探讨微小RNA(microRNA-26a,miRNA,miR)-26a通过调节转录因子活化蛋白2C(transcription factor activator protein 2C,TFAP2C)的表达而抑制卵巢癌SKOV3细胞的增殖。方法:将miR-26a模拟物(miR-26a mimic)片段和含野生型TFAP2C基因3’-非翻译区的荧光素酶报告载体(TFAP2C-wt)共转染至SKOV3细胞后,进行荧光素酶活性检测。将miR-26a mimic、靶向TFAP2C基因的短发夹RNA(short hairpin RNA,shRNA)重组载体shRNA-TFAP2C和TFAP2C过表达的重组载体pcDNA3.1-TFAP2C分别转染或不同组合共转染至SKOV3细胞后,蛋白质印迹法检测细胞中TFAP2C的表达水平,MTS法检测细胞的增殖情况。结果:miR-26a mimic和TFAP2C-wt共转染后,SKOV3细胞中的荧光素酶活性强度比对照组[miRNA无关序列阴性对照(miRNA negative control,miR-NC)和含突变TFAP2C基因3’-非翻译区的荧光素酶报告载体(TFAP2C-mut)共转染]下降了约52%(P<0.05)。miR-26a mimic转染组和shRNA-TFAP2C转染组SKOV3细胞中TFAP2C蛋白的表达水平和细胞增殖率均低于相应的对照组(miR-NC和shRNA-NC转染SKOV3细胞),差异有统计学意义(P<0.05);而miR-26a mimic和shRNA-TFAP2C共转染组SKOV3细胞的增殖率低于miR-26a mimic和shRNA-TFAP2C单独转染组,差异有统计学意义(P<0.05)。pcDNA3.1-TFAP2C转染组SKOV3细胞中TFAP2C的表达水平和细胞增殖率高于相应的对照组(pcDNA3.1空载体转染SKOV3细胞),差异有统计学意义(P<0.05);而miR-26a mimic和pcDNA3.1-TFAP2C共转染组SKOV3细胞的增殖率低于pcDNA3.1-TFAP2C转染组,差异有统计学意义(P<0.05)。结论:miR-26a通过调控TFAP2C的表达而抑制卵巢癌SKOV3细胞的增殖。
[关键词]     卵巢肿瘤    微RNAs    细胞增殖    SKOV3细胞
miR-26a inhibits cell proliferation by regulating TFAP2C expression in ovarian cancer cells
WANG Shu-fen, DONG Wei-lei, XIE Jing, HE Lu, ZHOU Xi, CAI Yan-lin, LIU Jie, XIE Wan-yu     
Department of Gynaecology and Obstetrics, First Affiliated Hospital, University of South China, Hengyang 421001, Hunan Province, China
[ABSTRACT]    Objective:To investigate the miR-26a suppressed ovarian cancer SKOV3 cells proliferation by regulating transcription factor activator protein 2C (TFAP2C) expression. Methods:miR-26a mimic and luciferase reporter vector TFAP2C-wt containing wide type of 3’-untranslated region (3’-UTR) were co-transfected into SKOV3 cells and detection of luciferase activity. After SKOV3 cells were transfected or co-transfected in different combination with miR-26a mimic, short hairpin RNA (shRNA) targeting TFAP2C gene recombination vector shRNA-TFAP2C and over-expression of TFAP2C recombination vector pcDNA3.1-TFAP2C, the expression levels of TFAP2C were detected by Western blotting, and the cells proliferation were performed by MTS assay. Results: As compared with the control [miRNA negative control (miR-NC) and luciferase reporter vector TFAP2C-mut containing mutation type of 3’-UTR were co-transfection], luciferase activities of SKOV3 cells after transfection with miR-26a mimic and TFAP2C-wt were decreased by 52% (P < 0.05). The expression levels of TFAP2C and proliferation rates of SKOV3 cells after transfection with miR-26a mimic and shRNA-TFAP2C were lower than those of SKOV3 cells after transfection with miR-NC and shRNA-NC as control (P < 0.05). The proliferation rates of SKOV3 cells after co-transfection with miR-26a mimic and shRNA-TFAP2C were lower than those of SKOV3 cells after transfection with miR-26a mimic and shRNA-TFAP2C alone (P < 0.05). The expression levels of TFAP2C and proliferation rates of SKOV3 cells after transfection with pcDNA3.1-TFAP2C were higher than those of SKOV3 cells after transfection with empty vector pcDNA3.1 as control (P < 0.05). The proliferation rates of SKOV3 cells after co-transfection with miR-26a mimic and pcDNA3.1-TFAP2C were lower than those of SKOV3 cells after transfection with pcDNA3.1-TFAP2C alone (P < 0.05). Conclusion: miR-26a suppresses cell proliferation by targeting TFAP2C expression in ovarian cancer SKOV3 cells.
[KEY WORDS]     Ovarian neoplasms    MicroRNAs    Cell proliferation    SKOV3 cell

微小RNA(microRNA,miRNA,miR)目前已经成为肿瘤研究的热点,其主要作用为在转录后水平对基因表达进行负调控,降解mRNA或阻碍其翻译,从而参与调控个体发育、细胞凋亡、细胞增殖和细胞分化等生命活动[1]。研究表明,miR-26a在肝癌、鼻咽癌、胃癌和乳腺癌组织及细胞中的表达下调,且抑制肿瘤细胞的增殖[2, 3, 4]。最近的研究发现,miR-26a在卵巢癌患者的血清和卵巢癌组织中的表达下调[5]。Anttila等[6]发现,卵巢癌组织中转录因子活化蛋白2C(transcription factor activator protein 2C,TFAP2C)的表达水平越高,卵巢癌患者的预后越差。Salonen等[7]的研究结果也证实了这一观点,并且发现TFAP2C与卵巢癌患者的临床分期呈正相关,进展期卵巢癌患者的TFAP2C的阳性率显著高于早期卵巢癌患者[8]。通过在线软件预测发现,TFAP2C是miR-26a调控的一个靶基因。本研究应用荧光素酶报告活性检测和蛋白质印迹法检测证实miR-26a是否可以调控TFAP2C的表达并抑制卵巢癌细胞的增殖,探讨miR-26a在卵巢癌中的抗肿瘤作用及其可能的分子机制。

1 材料与方法 1.1 主要材料

人卵巢癌SKOV3细胞购自中科院上海生化细胞研究所;RPMI 1640培养液和胎牛血清购自美国Gibco公司;LipofectAMINE 2000转染试剂;miR-26a模拟物(miR-26a mimic)和无关序列阴性对照(microRNA negative control,miR-NC)片段购自美国Ambion公司,miR-26a mimic片段序列为5’-CTTCAAGTAATCCAGGATAGGC-3’,miR-NC片段序列为5’-ATTGGAACGATACAGAGAAGATT-3’;靶向TFAP2C基因的短发夹RNA(short hairpin RNA,shRNA)重组载体shRNA-TFAP2C、阴性对照(negative control,NC)重组载体shRNA-NC、鼠抗人TFAP2C和GAPDH单克隆抗体购自美国Santa Cruz公司;TFAP2C过表达的重组载体pcDNA3.1-TFAP2C、pcDNA3.1空载体对照、含野生型TFAP2C基因3’-非翻译区(3’-untranslated region,3’-UTR)的荧光素酶报告载体TFAP2C-wt和含突变型TFAP2C基因3’UTR的荧光素酶报告载体TFAP2C-mut购自广州复能基因有限公司;双荧光素酶活性检测试剂盒购自美国Promega公司;蛋白质提取试剂盒购自上海BestBio贝博生物公司;BCA蛋白定量试剂和增强型化学发光试剂购自美国Pierce公司;辣根过氧化物酶标记的羊抗鼠IgG(二抗)购自武汉博士德生物工程有限公司;MTS细胞生长增殖/毒性检测试剂盒购自美国Sigma公司。

1.2 细胞培养

人卵巢癌SKOV3细胞用含10%胎牛血清的RPMI 1640培养液,置于37 ℃、CO2体积分数为5%的细胞培养箱中培养。细胞呈单层贴壁生长,细胞融合度为80%~90%时进行常规传代。

1.3 荧光素酶活性检测miR-26a是否与TFAP2C基因3’-UTR结合

收集对数生长期的SKOV3细胞接种于6孔板(细胞密度为5×104个细胞/mL,2 mL/孔细胞悬液),当细胞融合度达60%~70%时,按LipofectAMIN 2000说明书提供的方法分别将miR-26a mimic+TFAP2C-wt、miR-NC+TFAP2C-wt、miR-26a mimic+TFAP2C-mut和miR-NC+TFAP2C-mut共转染至SKOV3细胞。收集转染48 h后的各组细胞,按双荧光素酶活性检测试剂盒说明书提供的方法在单光子检测仪(美国Bio-Rad公司产品)上进行检测,计算相对荧光素酶活性。相对荧光素酶活性=萤火虫荧光素酶活性值/海肾荧光素酶活性值。实验重复3次。

1.4 miR-26a mimic和重组载体shRNA-TFAP2C、pcDNA3.1-TFAP2C转染至SKOV3细胞

收集融合度为80%的SKOV3细胞,用含10%胎牛血清的RPMI 1640培养液调整细胞密度为5×104个细胞/mL,将细胞接种于6孔板(2 mL/孔细胞悬液)或96孔板(100 μL/孔细胞悬液)中,置于细胞培养箱中培养。待细胞融合度为60%~80%时,按LipofectAMIN 2000说明书提供的方法分别将miR-26a mimic、miR-NC、shRNA-TFAP2C、shRNA-NC、pcDNA3.1-TFAP2C、pcDNA3.1、miR-26a mimic+shRNA-TFAP2C和miR-NC+shRNA-NC转染至SKOV3细胞。在6孔板中转染的细胞用于后续的蛋白质印迹法检测,在96孔板中转染的细胞用于后续的MTS法细胞增殖检测。

1.5 蛋白质法检测miR-26a mimic和重组载体shRNA-TFAP2C、pcDNA3.1-TFAP2C转染对SKOV3细胞TFAP2C表达的影响

收集1.4节在6孔板中转染miR-26a mimic、miR-NC、shRNA-TFAP2C、shRNA-NC、pcDNA3.1-TFAP2C和pcDNA3.1空载体对照48 h后的SKOV3细胞,应用蛋白质提取试剂盒提取各组细胞的总蛋白,BCA法测定蛋白质浓度。分别取各组细胞的蛋白质30 μg/孔进行10% SDS-PAGE,将电泳分离后的蛋白转移至聚偏二氟乙烯膜上,用含5%牛血清白蛋白的封闭液4 ℃封闭1 h;加入1∶200稀释的鼠抗人TFAP2C抗体或1∶1 000稀释的鼠抗人GAPDH(内参照)抗体,4 ℃反应过夜;TBST洗膜,加入1∶10 000稀释的辣根过氧化物酶标记的羊抗鼠IgG(二抗)室温反应1 h;TBST洗膜,加入增强型化学发光试剂,X线片曝光、显影和定影。用扫描仪扫描蛋白条带,应用Quantity One软件进行分析,以目的蛋白质条带的灰度值与内参照GADPH蛋白质条带灰度值的比值表示目的蛋白的相对表达水平。实验重复3次。

1.6 MTS法检测miR-26a mimic和重组载体shRNA-TFAP2C、pcDNA3.1-TFAP2C转染对SKOV3细胞增殖的影响

收集1.4节在96孔板中转染miR-26a mimic和miR-NC(对照组)、shRNA-TFAP2C和shRNA-NC(对照组)、pcDNA3.1-TFAP2C和pcDNA3.1(对照组)、miR-26a mimic+shRNA-TFAP2C、miR-NC+shRNA-NC(对照组)、miR-26a mimic+pcDNA3.1-TFAP2C和miR-NC+pcDNA3.1(对照组)48 h后的SKOV3细胞进行细胞增殖活性的检测,在未接种细胞的孔中加入RPMI 1640培养液作为调零孔,加入MTS检测试剂(20 μL/孔),37 ℃反应2 h,加入DMSO(150μL/孔),低速振荡10 min至结晶物充分溶解。用酶联免疫检测仪检测492 nm波长处各孔的吸光度(D)值,计算细胞的增殖活性。细胞的增殖活性=(各转染组-调零孔)/(对照组-调零孔)×100%。每组设6个重复孔,实验重复3次。

1.7 统计学方法

应用SPSS 16.0软件对实验的结果数据进行统计学分析。计量资料以x ± s表示。两组间的比较采用t检验,多组间比较采用单因素方差分析,组间两两比较采用SNK法检验。P<0.05为差异有统计学意义。

2 结果 2.1 TFAP2C是miR-26a调控的靶基因

miR-26a mimic+TFAP2C-wt、miR-NC+TFAP2C-wt、miR-26a mimic+TFAP2C-mut和miR-NC+TFAP2C-mut共转染至SKOV3细胞后进行荧光素酶活性检测的结果(图 1A)显示,miR-26a mimic+TFAP2C-wt共转染组细胞的荧光素酶活性强度比miR-NC+TFAP2C-wt共转染组下降了约52%,差异有统计学意义(P<0.05);而miR-26a mimic+TFAP2C-mut共转染组细胞的荧光素酶活性强度与miR-NC和TFAP2C-mut共转染组之间的差异无统计学意义(P>0.05)。这一结果说明,TFAP2C是miR-26a调控的靶基因。

蛋白质印迹法检测结果(图 1B)显示,miR-26a mimic转染48 h后,卵巢癌SKOV3细胞中TFAP2C的表达水平低于miR-NC转染组(P<0.05)。这一结果表明,高表达miR-26a能抑制卵巢癌SKOV3细胞中TFAP2C的表达,进一步证实TFAP2C是miR-26a调控的靶基因。

图 1 荧光素酶报告载体(A)和蛋白质印迹法(B)检测miR-26a调控TFAP2C的表达 Fig. 1 The expression of transcription factor activator protein 2C (TFAP2C)-regulated by miR-26a was detected by luciferase reporter vector (A) and Western blotting (B), respectively. Figure 1A indicated that miR-26a mimic and TFAP2C-wt, miR-NC and TFAP2C-wt, miR-26a mimic and TFAP2C-mut, miR-NC and TFAP2C-mut co-transfection into ovarian cancer SKOV3 cells for 48 h, the luciferase activity in miR-26a mimic and TFAP2C-wt co-transfection group was lower than that in miR-NC and TFAP2C-wt co-transfection group. Figure 1B indicated that after miR-26a mimic and miR-NC were transfected into ovarian cancer SKOV3 cells for 48 h respectively, the expression level of TFAP2C in miR-26a mimic-transfected group was lower than that in miR-NC-transfected group. *P<0.05, vs miR-NC and TFAP2C-wt co-transfection group (n=3); P<0.05, vs miR-NC (n=3).

2.2 miR-26a过表达和下调TFAP2C表达可抑制卵巢癌SKOV3细胞的增殖

为了验证miR-26a对卵巢癌细胞增殖的影响,将miR-26a mimic、miR-NC、shRNA-TFAP2C和shRNA-NC分别转染至SKOV3细胞中,蛋白质印迹法检测结果(图 2A)显示,shRNA-TFAP2C转染组SKOV3细胞中TFAP2C的表达水平低于shRNA-NC转染组(P<0.05)。

MTS法检测结果(图 2B)发现,miR-26a mimic转染组SKOV3细胞的增殖率明显低于miR-NC转染组,差异有统计学意义(P<0.05);shRNA-TFAP2C转染组SKOV3细胞的增殖率明显低于shRNA-NC转染组,差异有统计学意义(P<0.05);miR-26a mimic+shRNA-TFAP2C共转染组SKOV3细胞的增殖率低于miR-26a mimic或shRNA-TFAP2C单独转染组,差异均有统计学意义(P<0.05)。这些结果表明,过表达miR-26a或下调TFAP2C的表达均能抑制SKOV3细胞的增殖,且过表达miR-26a和下调TFAP2C表达抑制SKOV3细胞增殖的作用更强。

图 2 蛋白质印迹法(A)和MTS法(B)检测miR-26a mimic和shRNA-TFAP2C转染后卵巢癌SKOV3细胞中TFAP2C蛋白的表达及细胞增殖率 Fig. 2 The expression level of transcription factor activator protein 2C (TFAP2C) and proliferation rate of ovarian cancer SKOV3 cells after transfection with miR-26a mimic and shRNA-TFAP2C were detected by Western blotting (A) and (MTS) (B) assay, respectively. Figure 2A indicated that the expression level of TFAP2C in SKOV3 cells after transfection with shRNA-TFAP2C was lower than that in shRNA-NC transfection group (*P<0.05, vs shRNA-NC, n=3). Figure 2B indicated that the proliferation rates of SKOV3 cells after transfection with miR-26a mimics, shRNA-TFAP2C and miR-26a mimic + shRNA-TFAP2C were lower than those in miR-NC, shRNA-NC and miR-NC + shRNA-NC transfection groups, respectively (*P<0.05, vs miR-26a mimic; P<0.05, vs shRNA-TFAP2C, n=3)
2.3 过表达TFAP2C逆转miR-26a对卵巢癌SKOV3细胞的增殖抑制

为了明确miR-26a是否通过调控TFAP2C的表达而影响SKOV3细胞的增殖能力,将pcDNA3.1-TFAP2C和pcDNA3.1分别转染至SKOV3细胞中,蛋白质印迹法检测结果(图 3A)显示,pcDNA3.1-TFAP2C转染组SKOV3细胞中TFAP2C的表达水平明显高于pcDNA3.1转染组,差异有统计学意义(P<0.05)。

MTS法检测结果(图 3B)发现,miR-26a mimic转染组SKOV3细胞的增殖率低于miR-NC转染组,差异有统计学意义(P<0.05);pcDNA3.1-TFAP2C转染组SKOV3细胞的增殖率高于pcDNA3.1转染组,差异有统计学意义(P<0.05);miR-26a mimic+pcDNA3.1-TFAP2C共转染组SKOV3细胞的增殖率低于pcDNA3.1-TFAP2C转染组,差异有统计学意义(P<0.05),说明过表达TFAP2C可能部分逆转miR-26a对SKOV3细胞的增殖抑制作用。

图 3 蛋白质印迹法(A)和MTS法(B)检测miR-26a mimic和pcDNA3.1-TFAP2C转染后卵巢癌SKOV3细胞中TFAP2C蛋白的表达及细胞增殖率 Fig. 3 The expression level of transcription factor activator protein 2C (TFAP2C) and proliferation rate of ovarian cancer SKOV3 cells after transfection with miR-26a mimic and pcDNA3.1-TFAP2C were detected by Western blotting (A) and (MTS) (B) assay, respectively. Figure 3A indicated that the expression level of TFAP2C in SKOV3 cells after transfection with pcDNA3.1-TFAP2C was lower than that in pcDNA3.1 transfection group (*P<0.05, vs pcDNA3.1, n=3). Figure 3B indicated that the proliferation rates of SKOV3 cells after transfection with miR-26a mimics, pcDNA3.1-TFAP2C and miR-26a mimic + pcDNA3.1-TFAP2C were lower than those in miR-NC, pcDNA3.1 and miR-NC + pcDNA3.1 transfection groups, respectively (*P<0.05, vs miR-26a mimic; P<0.05, vs pcDNA3.1-TFAP2C; n=3)
3 讨论

卵巢癌的发病率位居女性生殖系统肿瘤的第6位,每年约有230 000例新发病例[3]。早期卵巢癌患者的生存率较高,但是大多数患者就诊时已是晚期,而且大部分患者对目前的化疗方案会产生耐药,所以化疗药物对卵巢癌患者的治疗成功率较低[9]。每年约有140 000例患者死于卵巢癌,严重威胁着患者的生命[10],迫切需要寻找新的治疗药物提高卵巢癌患者的生存率。

TFAP2是一类非常重要的转录因子,其家族成员包括α、β、γ、δ和ε,分别定位于6p22.3-24、6pl2、20ql3.2、6pl2.1和lp34.3。TFAP2家族通过与其靶基因启动子上的作用元件结合而控制许多靶基因的特异性表达,从而调控胚胎发育、细胞增殖、细胞分化、细胞迁移和细胞凋亡等多种生物学过程[11]。其中TFAP-2γ即是TFAP2C基因编码的蛋白,TFAP2C和肿瘤基因的不稳定性与肿瘤细胞的增殖、抗凋亡、侵袭和药物抵抗密切相关,在肿瘤的发生和发展中起着重要作用。miR-26a存在于2个染色体位点上,miR-26a-1位于3号染色体,miR-26a-2位于12号染色体。研究发现,miR-26a在肝癌中的表达水平下调,其表达水平与肝癌患者的生存时间及其对干扰素治疗的敏感性相关[2]。miR-26a在鼻咽癌中的表达水平下调,且能通过靶基因组蛋白甲基化转移酶抑制鼻咽癌细胞的生长和增殖[3]。同时,miR-26a在乳腺癌组织和乳腺癌细胞中明显下调,miR-26a mimic能明显抑制乳腺癌细胞的增殖,过表达miR-26a能诱导乳腺癌细胞的凋亡[12]

本研究应用生物信息学软件查询、荧光素酶报告基因活性检测和蛋白质印迹法等分子生物学技术证实,TFAP2C是miR-26a调控的靶基因。并且发现,转染miR-26a mimic或下调TFAP2C的表达可抑制卵巢癌SKOV3细胞的增殖;而过表达TFAP2C能部分逆转miR-26a对卵巢癌SKOV3细胞增殖的抑制作用。这一结果说明,miR-26a可通过靶向调控TFAP2C的表达而抑制卵巢癌SKOV3细胞的增殖。本研究初步揭示了miR-26a对卵巢癌SKOV3细胞的抑制机制。最近则有文献报道,miR-26a可通过抑制雌激素受体-α的表达而促进卵巢癌的生长和增殖[13]。因此,进一步研究miR-26a在卵巢癌发生和发展中的作用及其可能的机制,为针对以miR-26a为作用靶点开发治疗卵巢癌的药物奠定实验和理论基础,无疑是十分必要的。

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