目的:探讨lncRNA PCAT14对甲状腺癌细胞增殖和侵袭的影响及其分子机制。方法:生物信息学方法预测lncRNA PCAT14表达与肾癌患者临床分期的相关性。选择TPC-1甲状腺癌细胞系转染,分别将PCAT14质粒(实验组)或空载对照质粒(对照组)转至甲状腺癌细胞中。使用qPCR检测PCAT14质粒转染效率。细胞计数实验及克隆形成实验检测转染后细胞增殖活性,Transwell侵袭实验检测转染后甲状腺细胞的侵袭能力。Western blot检测EMT相关标记物的蛋白表达水平。生物信息学方法预测lncRNA PCAT14可能结合的蛋白。结果:TCGA数据库显示,甲状腺癌组织lncRNA PCAT14相对表达水平显著低于癌旁正常组织(P < 0.05)。lncRNA PCAT14在甲状腺癌晚期患者肿瘤组织中相对表达量更低(P < 0.01)。与对照组比较,实验组lncRNA PCAT14相对表达水平显著升高(P < 0.05),细胞增殖活性(P < 0.05)和细胞的侵袭能力显著降低(P < 0.01)。同时,实验组lncRNA PCAT14过表达抑制了EMT相关蛋白的表达。lncRNA PCAT14可能与DBX2结合发挥生物学功能。结论:lncRNA PCAT14在甲状腺癌组织中低表达,与肾癌患者临床分期相关。过表达lncRNA PCAT14可明显抑制肾癌细胞增殖和侵袭,其分子机制可能为lncRNA PCAT14与DBX2结合发挥生物学功能。 Objective: To investigate the effect of lncRNA PCAT14 on the invasion and metastasis of thyroid cancer cells and its molecular mechanism. Methods: Bioinformatics methods predict the correlation between the expression of lncRNA PCAT14 and the clinical stage of renal cancer patients. Select TPC-1 thyroid cancer cell line for transfection, and transfer PCAT14 plasmid (experimental group) or empty control plasmid (control group) to thyroid cancer cells respectively. QPCR was used to detect the transfection efficiency of PCAT14 plasmid. Cell counting experiment and clone formation experiment were used to detect cell proliferation activity after transfection, and Transwell invasion experiment was used to detect the invasion ability of thyroid cells after transfection. Western blot was used to detect the protein expression level of EMT-related markers. Bioinformatics methods predict the protein that lncRNA PCAT14 may bind. Results: The TCGA database showed that the relative expression level of lncRNA PCAT14 in thyroid cancer tissues was significantly lower than that in normal tissues adjacent to cancer (P < 0.05). The relative expression of lncRNA PCAT14 is lower in the cancer tissues of patients with advanced thyroid cancer (P < 0.01). Compared with the control group, the relative expression level of lncRNA PCAT14 of the experimental group was significantly increased (P < 0.05), and the cell proliferation activity (P < 0.05) and cell invasion ability were significantly reduced (P < 0.01). At the same time, the lncRNA PCAT14 overexpression in the experimental group inhibited the expression of EMT-related proteins. lncRNA PCAT14 may combine with DBX2 to exert biological functions. Conclusion: lncRNA PCAT14 is lowly expressed in thyroid cancer tissues and is related to the clinical stage of renal cancer patients. Overexpression of lncRNA PCAT14 can significantly inhibit the proliferation and invasion of renal cancer cells. The molecular mechanism may be that lncRNA PCAT14 combine with DBX2 to exert biological functions.
目的:探讨lncRNA PCAT14对甲状腺癌细胞增殖和侵袭的影响及其分子机制。方法:生物信息学方法预测lncRNA PCAT14表达与肾癌患者临床分期的相关性。选择TPC-1甲状腺癌细胞系转染,分别将PCAT14质粒(实验组)或空载对照质粒(对照组)转至甲状腺癌细胞中。使用qPCR检测PCAT14质粒转染效率。细胞计数实验及克隆形成实验检测转染后细胞增殖活性,Transwell侵袭实验检测转染后甲状腺细胞的侵袭能力。Western blot检测EMT相关标记物的蛋白表达水平。生物信息学方法预测lncRNA PCAT14可能结合的蛋白。结果:TCGA数据库显示,甲状腺癌组织lncRNA PCAT14相对表达水平显著低于癌旁正常组织(P < 0.05)。lncRNA PCAT14在甲状腺癌晚期患者肿瘤组织中相对表达量更低(P < 0.01)。与对照组比较,实验组lncRNA PCAT14相对表达水平显著升高(P < 0.05),细胞增殖活性(P < 0.05)和细胞的侵袭能力显著降低(P < 0.01)。同时,实验组lncRNA PCAT14过表达抑制了EMT相关蛋白的表达。lncRNA PCAT14可能与DBX2结合发挥生物学功能。结论:lncRNA PCAT14在甲状腺癌组织中低表达,与肾癌患者临床分期相关。过表达lncRNA PCAT14可明显抑制肾癌细胞增殖和侵袭,其分子机制可能为lncRNA PCAT14与DBX2结合发挥生物学功能。
甲状腺癌,lncRNA PCAT14,侵袭,转移
Yemei Song, Hui Hua, Xiaoqin Li
Shandong Provincial Key Laboratory, Shandong Cancer Hospital and Institute, Shandong First Medical University (Shandong Academy of Medical Sciences), Jinan Shandong
Received: May 24th, 2021; accepted: Jun. 13th, 2021; published: Jun. 28th, 2021
Objective: To investigate the effect of lncRNA PCAT14 on the invasion and metastasis of thyroid cancer cells and its molecular mechanism. Methods: Bioinformatics methods predict the correlation between the expression of lncRNA PCAT14 and the clinical stage of renal cancer patients. Select TPC-1 thyroid cancer cell line for transfection, and transfer PCAT14 plasmid (experimental group) or empty control plasmid (control group) to thyroid cancer cells respectively. QPCR was used to detect the transfection efficiency of PCAT14 plasmid. Cell counting experiment and clone formation experiment were used to detect cell proliferation activity after transfection, and Transwell invasion experiment was used to detect the invasion ability of thyroid cells after transfection. Western blot was used to detect the protein expression level of EMT-related markers. Bioinformatics methods predict the protein that lncRNA PCAT14 may bind. Results: The TCGA database showed that the relative expression level of lncRNA PCAT14 in thyroid cancer tissues was significantly lower than that in normal tissues adjacent to cancer (P < 0.05). The relative expression of lncRNA PCAT14 is lower in the cancer tissues of patients with advanced thyroid cancer (P < 0.01). Compared with the control group, the relative expression level of lncRNA PCAT14 of the experimental group was significantly increased (P < 0.05), and the cell proliferation activity (P < 0.05) and cell invasion ability were significantly reduced (P < 0.01). At the same time, the lncRNA PCAT14 overexpression in the experimental group inhibited the expression of EMT-related proteins. lncRNA PCAT14 may combine with DBX2 to exert biological functions. Conclusion: lncRNA PCAT14 is lowly expressed in thyroid cancer tissues and is related to the clinical stage of renal cancer patients. Overexpression of lncRNA PCAT14 can significantly inhibit the proliferation and invasion of renal cancer cells. The molecular mechanism may be that lncRNA PCAT14 combine with DBX2 to exert biological functions.
Keywords:Thyroid Cancer, lncRNA PCAT14, Invasion, Metastasis
Copyright © 2021 by author(s) and Hans Publishers Inc.
This work is licensed under the Creative Commons Attribution International License (CC BY 4.0).
http://creativecommons.org/licenses/by/4.0/
甲状腺癌(Thyroid Cancer, THCA)是世界上最常见的内分泌恶性肿瘤,发病率占所有癌症类型的1%~2% [
长链非编码RNA是一种长度在200~100,000核苷酸之间的单链非编码RNA分子 [
甲状腺癌组织样本的大规模lncRNA表达谱数据来源于TCGA,基于RNA测序结果获得512个甲状腺癌组织样本和337个正常对照组织样本的转录组数据。
甲状腺癌细胞(BCPAP, TPC-1)购自中国科学院典型培养物保藏委员会细胞库;1640培养基和DMEM高糖培养基购自美国Corning公司;胎牛血清和胰酶购自Gibco公司;PCAT14质粒和阴性对照质粒购自金唯智;Transwell小室购自美国Corning公司;引物购自上海吉玛公司;jetPRIME转染试剂购自法国Polyplus公司;逆转录试剂盒购自TAKARA公司;结晶紫购自中国国药集团公司;甲醇购自国药集团化学试剂有限公司。TRIzol购自美国Invitorgen公司;RIPA裂解液、5×蛋白上样缓冲液购自北京索莱宝公司;Tris、SDS、甘氨酸购自上海生工生物工程有限公司;抗体购自武汉三鹰生物技术有限公司。
采用TCGA数据库对甲状腺癌和癌旁正常组织中lncRNA PCAT14的差异表达进行分析。采用RNA-蛋白互作预测网站(RNA-Protein Interaction Prediction (RPISeq) (iastate.edu))预测与lncRNA PCAT14结合的蛋白。
用含10%FBS的1640培养基培养BCPAP和TPC-1甲状腺癌细胞,培养条件为恒温37℃,饱和湿度,5% CO2浓度。细胞每隔1~2天进行换液或传代。取对数生长期细胞接种于无菌12孔板内,待细胞融合达40%~50%左右时使用jetPRIME转染试剂进行质粒转染。实验组为PCAT14质粒,阴性对照组为空载质粒,转染6小时后换液。
使用Trizol法提取细胞总RNA,将RNA逆转录成cDNA。以β-actin引物为内参,使用lncRNA PCAT14引物进行qPCR扩增,检测PCAT14在细胞中表达量。实验步骤严格参照实时定量PCR试剂说明书操作。实验数据采用2-ΔΔCt法计算。lncRNA PCAT14引物F:5’-CAAGAATGGGCCATAGTGACG-3’,R:5’-GCACAGCCCTAGATCCCTTA-3’。β-actinF:5’-GGCGGCACCACCATGTACCCT-3’,R:5’-AGGGGCC GGACTCGTCATACT-3’。
取进行转染处理的细胞,分别在转染24 h后第0、24、48、72 h计数。用10 μL枪头吸取已消化悬浮的细胞,从计数板边缘缓缓滴入,使之充满计数板和盖玻片之间的空隙中。显微镜下计算计数板四大格细胞总数,压线细胞只计左侧和上方的。计算公式:细胞数/mL = (4大格细胞总数/4) × 10,000,重复6次,取平均值。
取转染处理24 h后的细胞按照每孔1000个细胞均匀铺于6孔板中,37℃恒温培养箱中孵育8~10天后取出,0.5%结晶紫甲醇溶液固定染色后计算克隆形成率。
下室中加入600 μL无菌PBS放置于培养箱中激活小室,30 min后弃之。Transwell下室中加入600 ul含1% FBS的1640培养基,取实验组及对照组转染24 h后的细胞,用无血清培养基重悬后接种到上室,每个小室4 × 104个细胞。36 h后取出小室,用0.5%结晶紫甲醇溶液固定染色20 min,用清水冲洗小室上层,湿棉棒小心擦去上室膜表面上的细胞,自然晾干。在显微镜下随机选取5~7个视野拍照,image J统计实验结果。
取转染后48 h细胞提取总蛋白。定量后各取5 ug蛋白上样电泳及转膜,室温封闭2 h后剪膜,分别与一抗4℃孵育过夜,三次洗膜后分别与二抗室温孵育1 h,洗膜后用显影液孵育2 min,采用凝胶成像分析仪曝光显影。
所有数据均采用SPSS19.0软件进行分析。符合正态分布的数据资料使用 x ¯ ± s 表示,两独立样本比较采用独立样本t检验,组间比较使用单因素方差分析。所有实验均重复3次,以P < 0.05认为差异有统计学意义。
分析TCGA数据库中512个甲状腺癌组织样本与337个癌旁组织样本lncRNA PCAT14的表达量,结果显示lncRNA PCAT14在甲状腺癌组织中表达量低于癌旁正常组织。差异有统计学意义(P < 0.05,图1)。同时,基于TCGA数据库分析显示lncRNA PCAT14与肿瘤临床分期相关,在Ⅲ期和Ⅳ期甲状腺癌组织中lncRNA PCAT14表达量更低(P < 0.01,图2)。
图1. TCGA数据库中lncRNA PCAT14在甲状腺癌和癌旁组织中的表达
图2. TCGA数据库中不同临床分期肿瘤组织中lncRNA PCAT14的表达量
细胞计数结果显示,转染PCAT14质粒实验组的细胞增殖能力第三天时明显低于空载转染组,分别为26.125 ± 0.215、43 ± 0.364,差异有统计学意义(P < 0.05,图3)。克隆形成实验结果显示,转染PCAT14质粒实验组的细胞克隆形成率低于空载转染组,分别为0.44 ± 0.030、1.03 ± 0.021,差异有统计学意义(P < 0.01,图4)。提示过表达lncRNA PCAT14后可以抑制TPC-1细胞增殖能力。
图3. 细胞计数检测lncRNA PCAT14对TPC-1细胞增殖能力的影响
图4. 克隆形成实验检测lncRNA PCAT14对TPC-1细胞增殖能力的影响
Transwell侵袭实验结果显示,PCAT14转染组每个高倍视野下穿膜细胞数低于空载转染组,分别为0.315 ± 0.012、1.015 ± 0.031,差异有统计学意义(P < 0.01,图5)。提示过表达lncRNA PCAT14后可以抑制TPC-1细胞侵袭能力。
图5. Transwell侵袭实验检测lncRNA PCAT14对TPC-1细胞侵袭能力的影响
Western blot实验结果表明,与空载转染组相比,PCAT14转染组N-cadherin、vimentin和TAZ的蛋白表达水平下降(图6)。提示过表达lncRNA PCAT14抑制了TPC-1细胞发生上皮-间质转化。
RNA-蛋白互作预测网站(RNA-Protein Interaction Prediction (RPISeq) (iastate.edu))显示与lncRNA PCAT14互作的蛋白为DBX2,结合评分中RF = 0.7,SVM- = 0.92。
图6. Western blot检测TPC-1细胞N-cadherin、Vimentin和TAZ蛋白表达
在过去的几十年里,甲状腺乳头状癌的发病率一直在快速上升。虽然甲状腺乳头状癌患者预后一般较好,但肿瘤的侵袭性和转移性是导致预后不良的主要危险因素 [
综上所述,lncRNA PCAT14在甲状腺癌和癌旁组织中有显著差异性表达且在癌组织中低表达,lncRNA PCAT14的相对低表达与甲状腺癌患者临床分期具有相关性。上调甲状腺癌细胞中lncRNA PCAT14的表达显著抑制肿瘤细胞的侵袭转移能力,其分子机制可能是lncRNA PCAT14通过与DBX2结合发挥生物学功能。
宋烨梅,华 慧,李晓琴. lncRNA PCAT14对甲状腺癌细胞侵袭转移的影响Effects of lncRNA PCAT14 on Invasion and Metastasis of Thyroid Cancer Cells[J]. 临床医学进展, 2021, 11(06): 2857-2865. https://doi.org/10.12677/ACM.2021.116415