Effects of Pin1 on the Proliferation and Apoptosis of Cervical Cancer SiHa Cells by Regulating Key Enzymes of Lipid Metabolism
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摘要:
目的 探讨下调Pin1表达对宫颈癌SiHa细胞的脂质合成及增殖、凋亡的影响。 方法 Western Blot检测Pin1在Hela、SiHa和H8细胞的表达情况;慢病毒转染技术构建Pin1低表达稳转细胞株,根据不同处理分为对照组(shPin1-NON)、敲低组1(shPin1-1)和敲低组2(shPin1-2),GEPIA2分析脂质代谢关键酶(ACC1、FASN)在宫颈癌及非肿瘤组织中的表达;Western Blot和qRT-PCR检测ACC1、FASN 蛋白和mRNA的表达;Western Blot检测Caspase-8、BCL-2、C-myc 蛋白表达;CCK-8检测细胞增殖能力;克隆形成实验检测细胞集落形成能力;流式细胞术检测细胞凋亡情况。 结果 Pin1在宫颈癌细胞中高表达,且在SiHa细胞中表达最高(P < 0.05);Pin1低表达慢病毒有效下调了Pin1表达水平;宫颈癌中ACC1、FASN mRNA表达高于非肿瘤组织;shPin1-1组和shPin1-2组ACC1、FASN 蛋白和mRNA表达均低于shPin1-NON组(P < 0.05);shPin1-1组和shPin1-2组相较于shPin1-NON组Caspase-8蛋白表达被上调,而BCL-2、C-myc的表达均被抑制;shPin1-1组和shPin1-2组细胞增殖能力低于shPin1-NON组(P < 0.05);shPin1-1组和shPin1-2组细胞集落形成能力小于shPin1-NON组(P < 0.05)。shPin1-1组和shPin1-2组细胞总凋亡率相较shPin1-NON组显著增加(P < 0.05)。 结论 Pin1低表达有效下调宫颈癌SiHa细胞脂质代谢关键酶,抑制细胞增殖,诱发细胞凋亡,为靶向治疗宫颈癌提供新思路。 -
关键词:
- 宫颈癌 /
- 肽基脯氨酰同分异构酶 /
- 脂肪酸合成酶
Abstract:Objective To investigate the effect of down-regulation of Pin1 expression on lipid synthesis, proliferation and apoptosis of cervical cancer SiHa cells. Methods Western Blot was used to detect the expression of Pin1 in Hela, SiHa and H8 cells; Lentiviral transfection technology constructed Pin1 low-expression stable transduction cell line, which was divided into the control group (shPin1-NON), knockdown group1 (shPin1-1) and knockdown group2 (shPin1-2) according to different treatments; GEPIA2 was used to analyze the expression of key enzymes of lipid metabolism (ACC1, FASN) in cervical cancer and non-tumor tissues and the expression of ACC1 and FASN in cervical cancer and non-tumor tissues; Western Blot and qRT-PCR were used to detect the ACC1, FASN protein and mRNA expression; Western Blot was used to detect the Caspase-8, BCL-2, C-myc protein expression; CCK-8 was used to detect the cell proliferation; Clone formation assay was used to detect the cell colony formation ability and flow cytometry was used to detect the cell apoptosis. Results Pin1 was highly expressed in cervical cancer cells, and the highest expression was in SiHa cells; Pin1 low expression lentivirus effectively down-regulated the Pin1 expression level; ACC1 and FASN mRNA expressions in cervical cancer were higher than those in non-tumor tissues; The protein and mRNA expressions of ACC1 and FASN in shPin1-1 group and shPin1-2 group were lower than those in the shPin1-NON group; the shPin1-1 and shPin1-2 groups were up-regulated compared with the shPin1-NON group, while the expression of BCL-2 and C-myc was up-regulated. The expression was inhibited; The proliferation ability of shPin1-1 group and shPin1-2 group was lower than that of shPin1-NON group; The colony formation ability of shPin1-1 group and shPin1-2 group was lower than that of shPin1-NON group. Compared with the shPin1-NON group, the total apoptotic rate of cells in the shPin1-1 and shPin1-2 groups was significantly increased. (P < 0.05). Conclusion Low expression of Pin1 can effectively down-regulate fatty acid synthase in cervical cancer SiHa cells, inhibit cell proliferation, and induce cell apoptosis, which provides a new idea for targeted therapy of cervical cancer. -
Key words:
- Cervical cancer /
- Peptidylprolyl isomerase /
- Fatty acid synthase
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系统性红斑狼疮(systemic lupus erythematosus,SLE)是一种慢性自身免疫性疾病,临床表现多样,可累及多个器官系统,包括肾脏、皮肤、关节、血液系统以及神经精神系统等[1-2]。肾脏损伤是系统性红斑狼疮最常见、最严重的表现之一。合并肾脏损伤的SLE患者可高达60%,未及时诊治可进展为严重性肾损伤和急慢性肾功能衰竭,导致SLE患者死亡率升高[3-4]。SLE合并肾损伤的临床表现包括蛋白尿、血尿以及肾功能不全等,其诊断金标准为肾脏活检。SLE肾损伤的发病机制包括先天性和适应性免疫系统的激活、自身抗体直接攻击、抗dsDNA抗体等形成的免疫复合物沉积于肾脏等[5]。本研究对296例SLE患者进行抗dsDNA抗体等自身抗体及其他实验室指标的检测,分析比较其差异,探讨各项指标在SLE肾损伤患者中的临床应用价值。
1. 资料与方法
1.1 一般资料
选取2014年至2019年昆明医科大学第一附属医院收治的SLE患者共296例,男性25例,女性271例,平均(36.51±13.98)岁。分为SLE合并肾损伤组和未合并肾损伤组,SLE合并肾损伤组患者74例,男性6例,女性68例,平均(35.09±14.46)岁;未合并肾损伤组患者222例,男性19例,女性203例,平均(36.98±13.81)岁。两组间年龄、性别比较,差异无统计学意义(P > 0.05)。
1.2 纳入和排除标准
纳入标准:所有患者符合1997年美国风湿病协会(american college of rheumatology,ACR)修订的SLE分类诊断标准[6]。
排除标准:妊娠和哺乳期、合并严重感染、合并其他自身免疫性疾病、合并严重脏器功能不全、恶性肿瘤等患者。
1.3 方法
患者入院后,取空腹静脉血2~3 mL,低温离心取上清,保存于−20 ℃备用。采用间接免疫荧光法以及免疫印迹法测量自身抗体;采用自动生化分析仪检测血常规、肝肾功能指标、免疫球蛋白IgG、IgM、IgA及补体C3、C4等实验室指标。
1.4 统计学处理
采用SPSS软件进行统计分析。呈正态分布或近似正态分布的计量资料以均数±标准差(
$\bar x \pm s $ )表示,两组间比较采用独立样本t检验;非正态分布的计量资料以四分位数表示,两组间比较采用非参数检验;计数资料以率(%)表示,采用χ2检验。P < 0.05为差异有统计学意义。2. 结果
2.1 自身抗体阳性率比较
SLE合并肾损伤组血清抗dsDNA抗体、抗核小体抗体及抗组蛋白抗体的阳性率显著高于未合并肾损伤组,差异有统计学意义(P < 0.05),抗U1-RNP抗体的阳性率显著低于未合并肾损伤组,差异有统计学意义(P < 0.05),两组血清ANA抗体、抗SmD1抗体、抗SSA-RO 60KD抗体、抗SSA-RO 52KD抗体及抗SSB-La抗体比较,差异无统计学意义(P > 0.05),见表1。
表 1 两组自身抗体阳性率比较[n(%)]Table 1. Comparison of the positive rate of autoantibodies between the two groups [n(%)]检测指标 SLE合并肾损伤组阳性例数 未合并肾损伤组阳性例数 χ2 P ANA抗体(n = 296) 73(98.6) 215(96.8) 0.171 0.679 抗dsDNA抗体(n = 296) 42(56.8) 73(32.9) 13.315 0.000 抗核小体抗体(n = 224) 41(69.5) 81(49.1) 7.293 0.007 抗组蛋白抗体(n = 224) 40(67.8) 81(49.1) 6.122 0.013 抗SmD1抗体(n = 296) 42(56.8) 124(55.9) 0.018 0.892 抗U1-RNP抗体(n = 296) 25(33.8) 107(48.2) 4.667 0.031 抗SSA-RO 60KD抗体(n = 296) 51(68.9) 139(62.6) 0.960 0.327 抗SSA-RO 52KD抗体(n = 296) 28(37.8) 101(45.5) 1.324 0.250 抗SSB-La抗体(n = 296) 16(21.6) 54(24.3) 0.225 0.636 2.2 其他各项实验室指标水平的比较
2.2.1 血液学检查指标水平比较
SLE合并肾损伤组血清白细胞、中性粒细胞水平显著高于未合并肾损伤组,差异有统计学意义(P < 0.05),红细胞、血红蛋白水平显著低于未合并肾损伤组,差异有统计学意义(P < 0.05),淋巴细胞、血小板水平比较,差异无统计学意义(P > 0.05),见表2。
表 2 两组血液学检查指标水平比较[M(P25,P75)]Table 2. Comparison of hematological indexes between the two groups [M(P25,P75)]检测指标 SLE合并肾损伤组(n = 74) 未合并肾损伤组(n = 222) Z/t P 白细胞(×109/L) 5.45(3.90,7.42) 4.52(3.18,5.91) −2.536 0.011 中性粒细胞(×109/L) 4.08(2.50,5.61) 2.97(1.85,4.33) −3.443 0.001 淋巴细胞(×109/L) 1.01(0.57,1.48) 1.05(0.67,1.45) −0.438 0.661 红细胞(×1012/L) 3.75(3.16,4.21) 4.02(3.54,4.48) −2.523 0.012 血红蛋白(g/L) 106(89,124.25) 115(97,130) −2.262 0.024 血小板[×109/L,($\bar x\pm s $)] 173.05 ± 83.72 187.19 ± 82.96 −1.267 0.206 2.2.2 其他实验室指标水平比较
SLE合并肾损伤组尿素、肌酐、尿酸、钾、氯、钙离子水平显著高于未合并肾损伤组,差异有统计学意义(P < 0.05),SLE合并肾损伤组总蛋白、白蛋白、球蛋白、ALT、AST、总胆红素、直接胆红素、间接胆红素、钠离子、免疫球蛋白IgG、IgA及补体C3水平显著低于未合并肾损伤组,差异有统计学意义(P < 0.05),IgM、C4,差异无统计学意义(P > 0.05),见表3。
表 3 两组其他实验室指标水平比较[M(P25,P75)]Table 3. Comparison of other laboratory indexes between the two groups [M(P25,P75)]检测指标 SLE合并肾损伤组(n = 74) 未合并肾损伤组(n = 222) Z P 总蛋白(g/L) 60.15(48.38,68.63) 67.30(58.98,73.68) −4.357 0.000 白蛋白(g/L) 27.05(22.33,34.10) 33.95(27.48,37.90) −5.001 0.000 球蛋白(g/L) 30.90(24.15,36.23) 32.05(27.40,38.30) −2.052 0.04 ALT(IU/L) 11.85(7.68,21.28) 15.40(10.59,26.05) −2.545 0.011 AST(IU/L) 18.10(13.28,27.65) 19.95(14.60,28.58) −1.368 0.171 总胆红素(μmol/L) 5.65(3.38,7.53) 6.65(4.70,9.88) −2.865 0.004 直接胆红素(μmol/L) 2.35(1.50,3.28) 3.00(2.18,4.70) −3.274 0.001 间接胆红素(μmol/L) 3.05(1.80,4.43) 3.40(2.20,5.33) −2.127 0.033 尿素(μmol/L) 6.78(4.24,11.88) 4.39(3.11,6.51) −4.686 0.000 肌酐(μmol/L) 79.15(59.43,144.58) 62.45(53.68,80.15) −4.235 0.000 尿酸(μmol/L) 391(301.55,506.33) 303.60(248.80,368.65) −4.909 0.000 钾(mmol/L) 3.99(3.76,4.54) 3.74(3.51,4.00) −5.034 0.000 钠(mmol/L) 139.55(136.55,142.33) 140.15(137.90,142.73) −1.384 0.166 氯(mmol/L) 106.85(104.05,110.08) 105.70(103.60,108.03) −2.059 0.039 钙(mmol/L) 2.05(1.95,2.18) 2.17(2.06,2.26) −3.969 0.000 IgG(g/L) 12(8.10,15.50) 13.80(10.10,18.80) −2.564 0.010 IgM(g/L) 0.96(0.69,1.41) 1.02(0.64,1.52) −0.542 0.588 IgA(g/L) 2.13(1.39,3.22) 2.22(1.58,3.29) −0.878 0.38 C3(g/L) 0.50(0.31,0.70) 0.65(0.43,0.86) −3.412 0.001 C4(g/L) 0.08(0.04,0.14) 0.10(0.05,0.16) −1.789 0.074 3. 讨论
SLE是一种累及多器官系统的自身免疫性疾病,肾脏受累最为常见。肾脏损伤是SLE最严重的表现之一,可出现肾小球、肾小管间质和肾脏血管的永久性损害,最终进展为终末期肾病[7]。早期的诊断和免疫抑制剂的治疗对于SLE肾损伤患者的预后起到关键性作用。SLE合并肾损伤的主要特征包括自身抗体的产生、免疫复合物沉积以及免疫介导的肾脏损伤,导致细胞增殖和凋亡增加,并诱发破坏正常肾单位的炎症和纤维化过程。自身抗体是SLE的重要临床特征,抗dsDNA抗体是SLE的特异性抗体,约70%的SLE患者可表现为阳性,而健康人群及其他自身免疫性疾病患者阳性率小于0.5%[8-9]。抗dsDNA抗体常与SLE肾损伤的发生相关,其水平通常与疾病活动相关[10-11]。有报道表明,在SLE患者中,针对核成分的自身抗体,抗dsDNA、抗核小体和抗组蛋白抗体同时阳性与SLE肾损伤的发病和活动性显著相关,可作为提示肾脏受累的一个重要性指标[12-13],与本研究结果一致。SLE合并肾损伤的患者体内产生抗dsDNA抗体,其通过与肾脏细胞表面蛋白结合,激活下游信号通路,释放炎症和纤维化介质,诱导炎症和纤维化过程[14-15]。有研究基于动物模型和SLE患者的数据,证实只有抗核小体抗体复合物,特别是抗DNA抗体复合物,而不是单一特异性抗体,才能在体内结合肾小球基底膜并诱发蛋白尿[16-17]。MRL/lpr狼疮小鼠肾小球沉积抗体的洗脱IgG中含有抗dsDNA、抗核小体和抗组蛋白抗体,这些抗体的数量与蛋白尿的发生呈正相关[18]。
SLE的发生和进展与机体的细胞免疫及体液免疫失衡相关,当机体免疫调节失衡时,机体的炎症指标、补体水平等实验室指标出现异常。许多研究结果表明,肾功能指标如血尿酸、肌酐与SLE肾损伤的发生呈正相关,其他免疫相关成分如补体C3、C4与其呈负相关[19-20]。本研究结果表明,SLE合并肾损伤组血清尿素、肌酐及尿酸水平显著高于未合并肾损伤组,补体C3水平显著低于未合并肾损伤组,可能对SLE的肾损伤的发生和预后评价起到重要作用。尿素、肌酐及尿酸等作为肾功能的主要检测指标可以直接、有效地反映SLE患者肾脏累及程度,但其对于早期肾脏损伤的提示不佳,易延误诊治[19]。补体C3是血清中含量最高的补体成分,主要是由巨噬细胞、淋巴组织等合成,与SLE的病情活动相关,SLE合并肾损伤组补体C3水平下降,机制可能为机体免疫失衡时,补体被激活,其与自身抗原抗体复合物结合并沉积,导致机体内补体被大量消耗[21]。
综上所述,抗dsDNA、抗核小体、抗组蛋白等自身抗体、补体C3及多项实验室指标水平与SLE患者肾损伤的发生密切相关,可作为评估SLE肾损伤的免疫学指标。
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表 1 qRT-PCR检测基因的引物序列
Table 1. Primer sequences of qRT-PCR detection genes
基因 引物序列(5′-3′) Pin1 正向 CTGCCTTCAGCAGAGGTCAGATC 反向 CAGTGCGGAGGATGATGTGGATG ACC1 正向 TACCTTCTTCTACTGGCGGCTGAG 反向 GCCTTCACTGTTCCTTCCACTTCC FASN 正向 GTACACAGACAAAGCCCATTTT 反向 TTTGGTTTACATCTGCACTTGG β-actin 正向 TAGTTGCGTTACACCCTTTCTTG 反向 TCACCTTCACCGTTCCAGTTT -
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