HULC lncRNA实时荧光定量检测方法建立、应用及其在髓系白血病的功能研究
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摘要
目的：
1.建立实时荧光定量PCR检测肝癌高表达长链非编码RNA(highly up-regulated in liver cancer long non-coding RNA, HULC lncRNA)的方法；
2.利用建立的实时荧光定量PCR方法检测HULC lncRNA在实体肿瘤组织、髓系白血病外周血白细胞（WBCs）和肿瘤细胞株的表达量，并比较表达量的差异；
3.利用RNA干扰技术，研究HULC lncRNA对髓系白血病细胞株的相关功能。
方法：
1.根据Genebank中的HULC lncRNA(AY914050.1)全序列，设计HULC lncRNA基因的特异引物，构建HULC lncRNA和β-actin基因的T克隆载体，分别命名为pMD18-T-HULC lncRNA和pMD-β-actin；以pMD18-T-HULC lncRNA和pMD-β-actin为实时荧光定量PCR检测HULC lncRNA的标准品。以SYBR GreenⅡ为染料，建立实时荧光定量PCR检测HULC lncRNA的方法并进行方法学评价；
2.应用此方法检测8种肿瘤细胞株、肝癌组织、其它癌及癌旁组织、粒系白血病病人及健康人外周血WBCs中HULC lncRNA表达水平，并比较其表达量的差异；
3.构建靶向干扰HULC lncRNA的shRNA载体，脂质体转染法将HULC lncRNA-shRNA载体转染白血病细胞株K562，G418筛选获得细胞系HULC lncRNA-shRNA-K562，通过已建立的实时荧光定量方法检测细胞系中HULC lncRNA基因表达水平。通过MTT和流式细胞术观察细胞增殖、细胞周期、凋亡等恶性生物学行为的改变。
结果：
1.建立了HULC lncRNA实时荧光定量PCR检测方法。方法的最低检测限为1.80×10~3拷贝/L；线性范围为1.80×10~3拷贝/L~1.80×10~13拷贝/L；批内变异系数CV<2.0%,批间CV<8.0%。
2. HULC lncRNA在肝癌组织表达量为（2.01±0.32）×10~5拷贝/L，明显高于其它实体肿瘤及其癌旁组织HULC lncRNA表达量（<1.80×10~3拷贝/L）。新发现膀胱癌细胞株T24中表达量高[(1.41±0.54)×10~5拷贝/L]，但在膀胱癌组织中表达低（<1.80×10~3拷贝/L）。也新发现慢性粒细胞白血病细胞株K562有HULC lncRNA表达,与肝癌细胞株HepG2中表达量比较,没有明显差异（Ｐ>0.05）；急、慢性粒性白血病病人外周血WBCs中可检测到HULC lncRNA的明显表达，其中急性粒性白血病病人阳性表达率为80%，慢性粒性白血病病人阳性表达率则为79%；而健康人外周血WBCs均无表达。
3.建立了HULC lncRNA-shRNA-K562(干扰组)和HK-K562(阴性对照组)细胞组。干扰组的HULC lncRNA基因的表达水平和细胞增殖能力较阴性对照组和K562(空白组)明显下降(P<0.05)，而阴性对照组与空白组间无明显差异(P>0.05)；干扰组、阴性对照组和空白组处于G0/G1期的细胞分别为(68.85±0.27)%、(53.05±0.35)%和(53.54±0.33)%；细胞凋亡率分别为(9.6±0.20)%、(1.79±0.23)%和(1.75±0.20)%；干扰组与其他2对照组比较，差异显著(P<0.05)。
结论：
1.成功建立了实时荧光定量PCR检测HULC lncRNA的方法。
2.新发现髓系白血病人外周血WBCs及细胞株K562中存在HULClncRNA表达，HULC lncRNA表达可能在髓系白血病的诊断和治疗上有一定意义。
3.利用RNA干扰技术成功构建了靶向干扰HULC lncRNA的细胞系HULC lncRNA-shRNA-K562，发现HULC lncRNA也参与了髓系白血病细胞株K562的增殖、细胞周期及凋亡的调控，并为HULC lncRNA分子的作用机制研究提供细胞模型。
Objective:
1. To establish a real-time fluorescent quantitative PCR method for thedetection of long non-coding RNA (highly up-regulated in liver cancer,HULC lncRNA).
2. To detect the expressions of HULC lncRNA in solid tumors, WBCsof myeloid leukemia peripheral blood and tumor cell lines by establishedreal-time fluorescent quantitative PCR method and compare the differenceof the expressions in various cancers.
3. To research the function of HULC lncRNA in myelogenousleukemia cell line K562by RNA interference technology.
Methods:
1. Specific primers for HULC lncRNA gene were designed accordingto full HULC lncRNA sequence in Genbank (AY914050.1). The T cloningvectors for the quantitative standard of HULC lncRNA and β-actin genedetection was constructed and named pMD18-T-HULC and pMD-β-actin,respectively. The SYBR Green Ⅱ dye real-time fluorescence quantitativePCR method of HULC lncRNA was established and methodologicallyevaluated.
2. The primary application of this method to detect HULC lncRNA in8types of tumor cell lines, liver cancer, other cancer tissues andparacancerous tissue, WBCs of myeloid leukemia patients was investigatedand compared for the difference of HULC lncRNA expressions.
3. The recombinant interference plasmid HULC-shRNA wasconstructed and transfected into K562cells by lipofectamine mediation,then monoclonal cell line was selected by G418pressure. The expressionsof HULC lncRNA were measured by the real-time fluorescent quantitativePCR. The changes of biological behavior of malignant cells were observedby MTT and flow cytometry.
Results：
1. We had successfully established a real-time fluorescent quantitativePCR for detection of HULC lncRNA. The minimum detection limit ofdeveloped method was1.80×10~3copies/L； The linear range was1.80×10~3copies/L~1.80×10~13copies/L；
2. The expression levels of HULC lncRNA in hepatocellularcarcinoma tissues were8.6×10~5copies/L~1.2×10~7copies/L, significantlyhigher than other cancer and paracancerous tissues (all <1.80×10~3copies/L). It was first found that HULC lncRNA was high expressed inhuman bladder cancer cell line T24[(1.41±0.54)×10~5copies/L], but wasnot detected in bladder carcinoma tissues (<1.80×10~3copies/L).Furthermore, it was also first found that the HULC lncRNA gene wasexpressed in K562cell line, the expressions were no significant differencewith HepG2cell lines (P>0.05). There were significant expressions ofHULC lncRNA gene in peripheral WBCs of acute and chronic myeloidleukemia patients, the positive expression rate was80%in acute leukemiapatients and79%in chronic leukemia patients. However, there was no expression of HULC lncRNA gene in peripheral WBCs of healthy humanperipheral WBCs.
3. The recombinant interference plasmid HULC lncRNA-shRNA wasconstructed and transfected into K562cells. HULC lncRNA-shRNA-K562(interference group) and HK-K562group (negative control group) cell lineswere established. The HULC lncRNA expression levels of interferencegroups and cell proliferation were significantly decreased, and there weresignificant differences compared with those of negative control group andK562(blank group)(all P<0.05)； while those of control group and blankcontrol group were not significantly different (P>0.05). Interference group,negative control group and blank group of cells in G0/G1phase were(68.85±0.27)%(P<0.05, vs HK-K562and K562groups),(53.05±0.35)%and (53.54±0.33)%, respectively； the percentage of apoptotic cells were(9.6±0.20)%(P<0.05, vs HK-K562and K562groups),(1.79±0.23)%and(1.75±0.20)%,separately.
Conclusion:
1. The real-time fluorescence quantitative PCR detection of HULClncRNA is successfully established.
2. The positive HULC lncRNA gene expression is first found inmyeloid leukemia patient’s peripheral blood WBCs and cell line K562, andthe assay of HULC lncRNA gene expression may be with some clinicalsignificance in the diagnosis and treatment of myeloid leukemia.
3. HULC lncRNA-shRNA-K562is constructed by RNA targetinterference technology. HULC lncRNA plays some role in theproliferation, cell cycle and apoptosis of K562cell. It could provide thefoundation for further study of HULC lncRNA gene function in thepathogenesis of myeloid leukemia.
1.建立实时荧光定量PCR检测肝癌高表达长链非编码RNA(highly up-regulated in liver cancer long non-coding RNA, HULC lncRNA)的方法；
2.利用建立的实时荧光定量PCR方法检测HULC lncRNA在实体肿瘤组织、髓系白血病外周血白细胞（WBCs）和肿瘤细胞株的表达量，并比较表达量的差异；
3.利用RNA干扰技术，研究HULC lncRNA对髓系白血病细胞株的相关功能。
方法：
1.根据Genebank中的HULC lncRNA(AY914050.1)全序列，设计HULC lncRNA基因的特异引物，构建HULC lncRNA和β-actin基因的T克隆载体，分别命名为pMD18-T-HULC lncRNA和pMD-β-actin；以pMD18-T-HULC lncRNA和pMD-β-actin为实时荧光定量PCR检测HULC lncRNA的标准品。以SYBR GreenⅡ为染料，建立实时荧光定量PCR检测HULC lncRNA的方法并进行方法学评价；
2.应用此方法检测8种肿瘤细胞株、肝癌组织、其它癌及癌旁组织、粒系白血病病人及健康人外周血WBCs中HULC lncRNA表达水平，并比较其表达量的差异；
3.构建靶向干扰HULC lncRNA的shRNA载体，脂质体转染法将HULC lncRNA-shRNA载体转染白血病细胞株K562，G418筛选获得细胞系HULC lncRNA-shRNA-K562，通过已建立的实时荧光定量方法检测细胞系中HULC lncRNA基因表达水平。通过MTT和流式细胞术观察细胞增殖、细胞周期、凋亡等恶性生物学行为的改变。
结果：
1.建立了HULC lncRNA实时荧光定量PCR检测方法。方法的最低检测限为1.80×10~3拷贝/L；线性范围为1.80×10~3拷贝/L~1.80×10~13拷贝/L；批内变异系数CV<2.0%,批间CV<8.0%。
2. HULC lncRNA在肝癌组织表达量为（2.01±0.32）×10~5拷贝/L，明显高于其它实体肿瘤及其癌旁组织HULC lncRNA表达量（<1.80×10~3拷贝/L）。新发现膀胱癌细胞株T24中表达量高[(1.41±0.54)×10~5拷贝/L]，但在膀胱癌组织中表达低（<1.80×10~3拷贝/L）。也新发现慢性粒细胞白血病细胞株K562有HULC lncRNA表达,与肝癌细胞株HepG2中表达量比较,没有明显差异（Ｐ>0.05）；急、慢性粒性白血病病人外周血WBCs中可检测到HULC lncRNA的明显表达，其中急性粒性白血病病人阳性表达率为80%，慢性粒性白血病病人阳性表达率则为79%；而健康人外周血WBCs均无表达。
3.建立了HULC lncRNA-shRNA-K562(干扰组)和HK-K562(阴性对照组)细胞组。干扰组的HULC lncRNA基因的表达水平和细胞增殖能力较阴性对照组和K562(空白组)明显下降(P<0.05)，而阴性对照组与空白组间无明显差异(P>0.05)；干扰组、阴性对照组和空白组处于G0/G1期的细胞分别为(68.85±0.27)%、(53.05±0.35)%和(53.54±0.33)%；细胞凋亡率分别为(9.6±0.20)%、(1.79±0.23)%和(1.75±0.20)%；干扰组与其他2对照组比较，差异显著(P<0.05)。
结论：
1.成功建立了实时荧光定量PCR检测HULC lncRNA的方法。
2.新发现髓系白血病人外周血WBCs及细胞株K562中存在HULClncRNA表达，HULC lncRNA表达可能在髓系白血病的诊断和治疗上有一定意义。
3.利用RNA干扰技术成功构建了靶向干扰HULC lncRNA的细胞系HULC lncRNA-shRNA-K562，发现HULC lncRNA也参与了髓系白血病细胞株K562的增殖、细胞周期及凋亡的调控，并为HULC lncRNA分子的作用机制研究提供细胞模型。
Objective:
1. To establish a real-time fluorescent quantitative PCR method for thedetection of long non-coding RNA (highly up-regulated in liver cancer,HULC lncRNA).
2. To detect the expressions of HULC lncRNA in solid tumors, WBCsof myeloid leukemia peripheral blood and tumor cell lines by establishedreal-time fluorescent quantitative PCR method and compare the differenceof the expressions in various cancers.
3. To research the function of HULC lncRNA in myelogenousleukemia cell line K562by RNA interference technology.
Methods:
1. Specific primers for HULC lncRNA gene were designed accordingto full HULC lncRNA sequence in Genbank (AY914050.1). The T cloningvectors for the quantitative standard of HULC lncRNA and β-actin genedetection was constructed and named pMD18-T-HULC and pMD-β-actin,respectively. The SYBR Green Ⅱ dye real-time fluorescence quantitativePCR method of HULC lncRNA was established and methodologicallyevaluated.
2. The primary application of this method to detect HULC lncRNA in8types of tumor cell lines, liver cancer, other cancer tissues andparacancerous tissue, WBCs of myeloid leukemia patients was investigatedand compared for the difference of HULC lncRNA expressions.
3. The recombinant interference plasmid HULC-shRNA wasconstructed and transfected into K562cells by lipofectamine mediation,then monoclonal cell line was selected by G418pressure. The expressionsof HULC lncRNA were measured by the real-time fluorescent quantitativePCR. The changes of biological behavior of malignant cells were observedby MTT and flow cytometry.
Results：
1. We had successfully established a real-time fluorescent quantitativePCR for detection of HULC lncRNA. The minimum detection limit ofdeveloped method was1.80×10~3copies/L； The linear range was1.80×10~3copies/L~1.80×10~13copies/L；
2. The expression levels of HULC lncRNA in hepatocellularcarcinoma tissues were8.6×10~5copies/L~1.2×10~7copies/L, significantlyhigher than other cancer and paracancerous tissues (all <1.80×10~3copies/L). It was first found that HULC lncRNA was high expressed inhuman bladder cancer cell line T24[(1.41±0.54)×10~5copies/L], but wasnot detected in bladder carcinoma tissues (<1.80×10~3copies/L).Furthermore, it was also first found that the HULC lncRNA gene wasexpressed in K562cell line, the expressions were no significant differencewith HepG2cell lines (P>0.05). There were significant expressions ofHULC lncRNA gene in peripheral WBCs of acute and chronic myeloidleukemia patients, the positive expression rate was80%in acute leukemiapatients and79%in chronic leukemia patients. However, there was no expression of HULC lncRNA gene in peripheral WBCs of healthy humanperipheral WBCs.
3. The recombinant interference plasmid HULC lncRNA-shRNA wasconstructed and transfected into K562cells. HULC lncRNA-shRNA-K562(interference group) and HK-K562group (negative control group) cell lineswere established. The HULC lncRNA expression levels of interferencegroups and cell proliferation were significantly decreased, and there weresignificant differences compared with those of negative control group andK562(blank group)(all P<0.05)； while those of control group and blankcontrol group were not significantly different (P>0.05). Interference group,negative control group and blank group of cells in G0/G1phase were(68.85±0.27)%(P<0.05, vs HK-K562and K562groups),(53.05±0.35)%and (53.54±0.33)%, respectively； the percentage of apoptotic cells were(9.6±0.20)%(P<0.05, vs HK-K562and K562groups),(1.79±0.23)%and(1.75±0.20)%,separately.
Conclusion:
1. The real-time fluorescence quantitative PCR detection of HULClncRNA is successfully established.
2. The positive HULC lncRNA gene expression is first found inmyeloid leukemia patient’s peripheral blood WBCs and cell line K562, andthe assay of HULC lncRNA gene expression may be with some clinicalsignificance in the diagnosis and treatment of myeloid leukemia.
3. HULC lncRNA-shRNA-K562is constructed by RNA targetinterference technology. HULC lncRNA plays some role in theproliferation, cell cycle and apoptosis of K562cell. It could provide thefoundation for further study of HULC lncRNA gene function in thepathogenesis of myeloid leukemia.
引文
[1]宋皓军,俞秀冲,夏天等.长链非编码RNA与肿瘤的关系及其临床价值[J].细胞生物学杂志,2012,34(7):704~712.
[2] Lin R, Maeda S, Liu C, et al. A large noncoding RNA is a marker for murinehepatocellular carcinomas and a spectrum of human carcinomas [J].Oncogene,2007,26(6):851~858.
[3]李丹,宋咏梅,詹启敏. HULC RNA在肝癌细胞中的表达及其对邻近基因SLC35B3表达的影响[J].中华医学杂志,2010,90(44):3156~3159.
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[8]邓艳华，吴荃，陈华云等.实时荧光PCR法检测HER2基因扩增方法的建立[J].热带医学杂志,2011,11(3):295-297.
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[2]宋皓军,俞秀冲,夏天等.长链非编码RNA与肿瘤的关系及其临床价值[J].中国细胞生物学学报,2012,34(7):704~712.
[3] Lin R, Maeda S, Liu C, et al. A large noncoding RNA is a marker for murinehepatocellular carcinomas and a spectrum of human carcinomas [J].Oncogene,2007,26(6):851~858.
[4] Gabory A, Ripoche MA, Yoshimizu T, et al. The H19gene: regulation andfunction of a non-coding RNA [J]. Cytogenet Genome Res,2006,113(1-4):188~193.
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[8]徐丽华,刘春雷,常玉梅，等.双标准曲线相对定量PCR试验原理与方法[J].生物技术通报,2011,1:70-75.
[9]邓艳华，吴荃，陈华云，等.实时荧光PCR法检测HER2基因扩增方法的建立[J].热带医学杂志,2011,11(3):295-297.
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