放射敏感性不同的鼻咽癌细胞株X线辐射后MiR-7及其上下游调控基因的表达变化
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摘要
研究背景：中国的广东、广西、福建、湖南等地为鼻咽癌多发区。发病年龄大多为中年人,亦有青少年患病者。病因与种族易感性、遗传因素及EB病毒感染等有关,鼻咽癌恶性程度较高,早期即可出现颈部淋巴结转移。鼻咽癌的主要治疗手段为放疗,高分化的鼻咽癌细胞对X线的敏感性较低分化的鼻咽癌细胞差,放射抵抗的癌细胞是导致患者局部控制率不佳及复发的重要因素。
MicroRNA是一类小片段、非编码RNA,通过与靶基因mRNA 3端非编码区域非完全互补配对结合,使靶基因沉默,阻碍蛋白合成,参与细胞增殖、分化、凋亡、肿瘤发生及DNA损伤等生物学过程。高分化的鼻咽癌细胞株CNE-1和低分化的鼻咽癌细胞株CNE-2存在多种microRNA的表达差异,其中miR-7是差异最显著的microRNA之一。MiR-7参与调控细胞发育和EGFR通路的激活,而EGFR通路和放射敏感性密切相关。
EGFR (epidermal growth factor receptor)是一种跨细胞膜糖蛋白,是酪氨酸激酶受体erbB家族成员。EGFR与配体结合后,受体酪氨酸激酶自磷酸化,激活包括RAS通路在内的下游信号通路,调控细胞增殖、分化。同一类型的肿瘤细胞,高分化细胞EGFR表达较低分化细胞高,EGFR表达增高,增强了细胞的放射抵抗性。放射线辐射后,细胞通过促使EGFR核内转移,启动D-NHEJ修复受损DNA,通过EGFR激活下游信号通路,促进细胞增殖,共同达到放射抵抗的效应。临床上,某些肿瘤患者放射治疗后,局部控制率不高,也与EGFR高表达有关。
HOX基因家族在胚胎发育,肢体数量与形态,腰骶部脊髓的发育,运动神经元的分化起着重要的调节作用。在某些器官中,HOX基因家庭具有特异性表达谱,原发于这些器官的肿瘤及其转移至远处的肿瘤,均与其来源的正常组织细胞有相同的HOX基因表达谱。HOX基因家族编码DNA转录调节蛋白,如果HOX基因表达混乱,将会导致血液肿瘤或实体瘤的发生。在组织器官发育完成后,HoxD10仍然起着重要的调控作用,这种作用体现在抑制新生血管生成,抑制肿瘤形成,抑制肿瘤远处转移。虽然可被microRNA抑制,但HoxD10作为编码转录调节蛋白的HOX基因家族成员之一,也可在转录水平调节microRNA的表达。已知HoxD10在乳腺癌细胞中调节miR-7的表达。
因此,抑制EGFR是降低肿瘤细胞放射抵抗性中-个重要的环节,miR-7在调节EGFR转录过程中有重要的作用,而miR-7上游调控因子HoxD10也可能与放射敏感性有关,但活体鼻咽癌细胞中miR-7与HoxD10结合验证未有报道。本研究着重从基因表达水平,初步探讨放射敏感性不同的鼻咽癌细胞X线辐射后miR-7与EGFR基因表达,miR-7与HoxD10基因表达的相关性,并验证HoxD10与miR-7的启动子序列在鼻咽癌细胞中是否结合,从而确定在鼻咽癌细胞中HoxD10是否参与了miR-7的表达调控。
第一部分：鼻咽癌细胞株状态测定
目的：绘制南方医院肿瘤中心提供鼻咽癌细胞株CNE-1、CNE-2的生长曲线,确定其对数生长期,并检测对数生长期细胞的放射敏感性。
方法：用MTT法,经酶标仪检测,得出细胞不同生长天数的0D值,输入Excel软件,绘制CNE-1、CNE-2细胞的生长曲线；使用克隆计数法,计算细胞受X线辐射后细胞存活分数,采用GraphPad Prism 5.0软件,按线性二次模型拟合两种细胞存活曲线,求出相应的数学模型参数α、β、α／β值。
结果：CNE-1的对数生长期为5～7天,CNE-2细胞对数生长期为3～5天；CNE-1细胞放射敏感性较CNE-2细胞低。
第二部分：X线辐射后鼻咽癌细胞株CNE-1、CNE-2的miR7及EGFR表达变化
目的：通过检测放射敏感性不同的鼻咽癌细胞株X线辐射后,miR-7及EGFR的表达变化,探讨二者表达是否有相关性。
方法：两种细胞均分为对照组(未辐射)、2Gy组和8Gy组,X线照射后10小时,用Trizol法提取细胞总RNA。茎环法特异性逆转录miR-7, Oligo dT逆转录EGFR mRNA,设计特异性PCR引物,进行染料法实时定量PCR,以对照组CNE-1细胞为参照样本,△△Ct法得出各样品miR-7和EGFR的相对数。
结果：放射抵抗的细胞株CNE-1低剂量X线照射后miR-7升高明显,高剂量照射后升高不明显。放射敏感的细胞株CNE-2 X线照射后,miR-7表达下降,以低剂量照射下降更为明显。放射抵抗的细胞株CNE-1低剂量X线照射后EGFR表达增加,且随着照射剂量的增加而增加。放射敏感的细胞株CNE-2 X线照射后,EGFR表达也增加,但未随着照射剂量的增加而增加。
第三部分：X线辐射后上游调控因子HoxD10的表达变化
目的：通过检测放射敏感性不同的鼻咽癌细胞株x线辐射后,HoxD10的表达变化,探讨其与miR-7的表达变化的相关性。
方法：两种细胞均分为对照组(未辐射)、2Gy组和8Gy组,x线照射后10小时,用Trizol法提取细胞总RNA。Oligo dT逆转录mRNA,设计HoxD10特异性PCR引物扩增cDNA片段,进行染料法实时定量PCR,以对照组CNE-1细胞为参照样本,△△Ct法得出各样品HoxD10的相对数。
结果：与对照组相比,x线辐射后,各组HoxD10表达均显著下降。两种细胞受X线辐射后,HoxD10表达均明显下降。
第四部分：miR-7上游启动子序列的预测、与HoxD10调控因子结合的验证
目的：查找miR-7上游2Kb范围内启动子所在区域、可能与调控因子的结合位点及其序列。验证活体细胞中HoxD10调控因子与预测的启动子区域是否结合。
方法：通过PubMed查找关键词或题目或摘要为miR-7文献。通过miRbase查找miR-7在人类染色体中的位置,再通过UCSC查找上游2Kb范围内启动子区域的特征。甲醛固定后,超声破碎细胞,将细胞DNA破碎至1000bp左右的片段,加入Protein A及HoxD10抗体,提取HoxD10蛋白结合的DNA片段,设计扩增启动因子所在DNA片段的PCR引物,扩增提取的DNA片段,验证HoxD10蛋白结合的DNA片段是否与启动子所在片段相同。
结果：miR-7的上游启动子所在区域为-958至-968、-1019至-1028两个位点,CHIP实验结果证实HoxD10与上述两个点结合。
Background:Nasopharyngeal carcinoma (NPC) is a common malignancy in Guangdong, Guangxi, Hunan, Fujian province of China. The patients mainly are mid-age, but adolescent patients are not rare. Etiological factors of NPC include race, region and EB virus infection. Metabasis of NPC can be observed in earlier period. The primary therapy of NPC is radiotherapy. Well differentiated NPC cells are more radiosensitive than poor differentiated ones. Low local control rate of some patients is resulting from the different radiosensitivity.
MicroRNAs (miRNAs), about 22 nucleotides in length, are endogenous, noncoding RNAs that can play important regulatory roles in animals by targeting mRNAs for cleavage or translational repression. These microRNAs are diverse in sequence and expression patterns, and are evolutionarily widespread. MicroRNAs have been validated its important role in regulation to cell proliferation, differentiation, apoptosis, tumorigenesis and DNA damage. Some of the miRNAs are overexpressed in cancers, in which they can promote tumor growth, suggesting common mechanisms of miRNA-regulated cell cycle control. Targeted components of the DNA damage response (DDR) pathway, miRNAs are also associated with the response of a tumor to radiation. There is different expression pattern of microRNAs in variously differentiated NPC cell lines. MiR-7 is one of the outstanding. MiR-7 regulates activation of EGFR pathway which concerned with radiosensitivity.
Epidermal growth factor receptor (EGFR) is a kind of transmembrane proteins and member of ERBB family. Connection of EGFR and its ligand will result in autophosphorylation of receptor tyrosine kinase following by downstream pathways activation included RAS pathway to regulate proliferation and differentiation. Level of EGFR expressing in well differentiated cells is higher than poor differentiated cells, and for this reason, the former is more radiosensitive than latter. After radiation, EGFR translocates from the surface membrane to the nucleus, where it may directly priming DNA-PK dependent non-homologous end-joining (D-NHEJ) system to recover DNA damage. Activation of EGFR pathway improves cell proliferation to resist radiation. Clinical data showed low local control of NPC patients is resulted from high EGFR expression.
Homeobox (Hox) genes control rostral-caudal patterning during embryogenesis. It is a primary marker of the lumbosacral region and play an key role in the developing lumbar spinal cord and embryonic limb morphogenesis. Tissues express unique Hox gene expression profiles that persist upon primary and metastatic tumors. Hox genes encode DNA transcription regulatory proteins known for their role in cellular differentiation during embryonic development, aberrant expression of these genes has been associated with hematologic and solid neoplasms. HoxDIO expression was higher in quiescent as compared to tumor-associated angiogenic endothelium, so HoxD10 suppress tumor formation and metastasize. MicroRNAs can suppress transcription of HoxD10, and HoxD10 can also suppress microRNAs transcription. MiR-7 expression is positively regulated by HoxD10 in breast cancer.
Suppression of EGFR is a key point in radiosensitivity increasing and miR-7 and HoxD10 may be included in. The regulator of miR-7 in NPC cells is not reported. The objectives of this study are to approach the relationship between miR-7 and EGFR, miR-7 and HoxD10 in various radiosensitivities NPC cell lines in gene expression level, and to validate whether HoxD10 regulate miR-7 transcription in NPC cells.
Part I Status test of NPC cell lines
OBJECTIVE:To determine logarithmic growth phase and radiosensitivity of CNE-1 and CNE-2 provided by cancer center of Nanfang hospital.
METHODS:ELISA Reader was used to get OD values of NPC cell lines treated by MTT method in different growth days. OD values were typed in Excel to draw cell growth curve. Survival factions (SF) of NPC cell lines after X-ray radiation were counted by clone numeration method. GraphPad Prism 5.0 software treats SF data with linear-quadratic model to get parameters of radiosensitivity of NPC cell lines.
RESULTS:Logarithmic growth phase of CNE-1 and CNE-2 were 5-7 days and 3-5 days, respectively. CNE-1 is less radiosensitive than CNE-2.
Part II Expression patterns of miR-7 and EGFR in CNE-1, CNE-2 after X-ray radiation
OBJECTIVE:To investigate the expression patterns of miR-7 and EGFR in various radiosensitivity nasopharynx cancer cells after X-ray radiation.
METHODS:Nasopharynx cancer cell lines CNE1 and CNE2 irradiated by using different dose X-ray. Total RNAs of cell lines were extracted by Trizol in 10 hours after radiation. The reverse transcription of miR-7 was done by Stem-loop primer and EGFR mRNA was reversely transcript by oligo dT. Non-irradiated CNE1 cell was reference sample and relative quantity of other samples were counted by△△Ct method after real time PCR used SyBR Green.
RESULTS:MiR-7 increase in radioresistant NPC cell lines when cells radiated by X-ray. MiR-7 obviously increases when radioresistant NPC cell radiated by low dose X-ray compared with it radiated by high dose X-ray. In radiosensitive NPC cells, miR-7 decreases when cells were radiated by X-ray, and low dose radation made its decrease significant compared with high dose. Expression level of EGFR in radioresistant NPC cells increased with the X-ray dose increase, and in radiosensitive NPCE cells, it was not.
Part III Expression patterns of HoxD10 in CNE-1, CNE-2 after X-ray radiation
OBJECTIVE:To investigate the expression patterns of HoxD10 in various radiosensitivity nasopharynx cancer cells after X-ray radiation.
METHODS:Nasopharynx cancer cell lines CNE1 and CNE2 irradiated by using different dose X-ray. Total RNAs of cell lines were extracted by Trizol in 10 hours after radiation. The reverse transcription of HoxD10 was done oligo dT. Non-irradiated CNE1 cell was reference sample and relative quantity of other samples were counted by AACt method after real time PCR used SyBR Green.
RESULTS:HoxD10 decreased in both cell lines when they radiated by X-ray. The expression level of HoxD10 in radiosensitive NPC cells was higher than that in radioresistant NPC cells.
Part IV Prediction and ascertain HoxD10 as the regulator of miR-7 in NPC cells
OBJECTIVE:To predict the promoter location in upstream of miR-7 and its regulator, and ascertain their connection.
METHODS:Research articles whose keyword or title or abstract included miR-7 in PubMed. Find the location of miR-7 in human chromatin and the characteristic of promoter in 2kb upstream of miR-7 in UCSC. NPC cell was fixed by formaldehyde and hypersound break the DNA into about 1000bp fractions. Primers designed to amplify the fraction contained promoter of miR-7 were used in PCR to amplify the sample of DNA broken fraction and integrated DNA.
RESULT:Promoters of miR-7 locate in-958 to-968 and-1019 to-1028 upstream of miR-7. HoxD10 bind the two points in NPC cells.
MicroRNA是一类小片段、非编码RNA,通过与靶基因mRNA 3端非编码区域非完全互补配对结合,使靶基因沉默,阻碍蛋白合成,参与细胞增殖、分化、凋亡、肿瘤发生及DNA损伤等生物学过程。高分化的鼻咽癌细胞株CNE-1和低分化的鼻咽癌细胞株CNE-2存在多种microRNA的表达差异,其中miR-7是差异最显著的microRNA之一。MiR-7参与调控细胞发育和EGFR通路的激活,而EGFR通路和放射敏感性密切相关。
EGFR (epidermal growth factor receptor)是一种跨细胞膜糖蛋白,是酪氨酸激酶受体erbB家族成员。EGFR与配体结合后,受体酪氨酸激酶自磷酸化,激活包括RAS通路在内的下游信号通路,调控细胞增殖、分化。同一类型的肿瘤细胞,高分化细胞EGFR表达较低分化细胞高,EGFR表达增高,增强了细胞的放射抵抗性。放射线辐射后,细胞通过促使EGFR核内转移,启动D-NHEJ修复受损DNA,通过EGFR激活下游信号通路,促进细胞增殖,共同达到放射抵抗的效应。临床上,某些肿瘤患者放射治疗后,局部控制率不高,也与EGFR高表达有关。
HOX基因家族在胚胎发育,肢体数量与形态,腰骶部脊髓的发育,运动神经元的分化起着重要的调节作用。在某些器官中,HOX基因家庭具有特异性表达谱,原发于这些器官的肿瘤及其转移至远处的肿瘤,均与其来源的正常组织细胞有相同的HOX基因表达谱。HOX基因家族编码DNA转录调节蛋白,如果HOX基因表达混乱,将会导致血液肿瘤或实体瘤的发生。在组织器官发育完成后,HoxD10仍然起着重要的调控作用,这种作用体现在抑制新生血管生成,抑制肿瘤形成,抑制肿瘤远处转移。虽然可被microRNA抑制,但HoxD10作为编码转录调节蛋白的HOX基因家族成员之一,也可在转录水平调节microRNA的表达。已知HoxD10在乳腺癌细胞中调节miR-7的表达。
因此,抑制EGFR是降低肿瘤细胞放射抵抗性中-个重要的环节,miR-7在调节EGFR转录过程中有重要的作用,而miR-7上游调控因子HoxD10也可能与放射敏感性有关,但活体鼻咽癌细胞中miR-7与HoxD10结合验证未有报道。本研究着重从基因表达水平,初步探讨放射敏感性不同的鼻咽癌细胞X线辐射后miR-7与EGFR基因表达,miR-7与HoxD10基因表达的相关性,并验证HoxD10与miR-7的启动子序列在鼻咽癌细胞中是否结合,从而确定在鼻咽癌细胞中HoxD10是否参与了miR-7的表达调控。
第一部分：鼻咽癌细胞株状态测定
目的：绘制南方医院肿瘤中心提供鼻咽癌细胞株CNE-1、CNE-2的生长曲线,确定其对数生长期,并检测对数生长期细胞的放射敏感性。
方法：用MTT法,经酶标仪检测,得出细胞不同生长天数的0D值,输入Excel软件,绘制CNE-1、CNE-2细胞的生长曲线；使用克隆计数法,计算细胞受X线辐射后细胞存活分数,采用GraphPad Prism 5.0软件,按线性二次模型拟合两种细胞存活曲线,求出相应的数学模型参数α、β、α／β值。
结果：CNE-1的对数生长期为5～7天,CNE-2细胞对数生长期为3～5天；CNE-1细胞放射敏感性较CNE-2细胞低。
第二部分：X线辐射后鼻咽癌细胞株CNE-1、CNE-2的miR7及EGFR表达变化
目的：通过检测放射敏感性不同的鼻咽癌细胞株X线辐射后,miR-7及EGFR的表达变化,探讨二者表达是否有相关性。
方法：两种细胞均分为对照组(未辐射)、2Gy组和8Gy组,X线照射后10小时,用Trizol法提取细胞总RNA。茎环法特异性逆转录miR-7, Oligo dT逆转录EGFR mRNA,设计特异性PCR引物,进行染料法实时定量PCR,以对照组CNE-1细胞为参照样本,△△Ct法得出各样品miR-7和EGFR的相对数。
结果：放射抵抗的细胞株CNE-1低剂量X线照射后miR-7升高明显,高剂量照射后升高不明显。放射敏感的细胞株CNE-2 X线照射后,miR-7表达下降,以低剂量照射下降更为明显。放射抵抗的细胞株CNE-1低剂量X线照射后EGFR表达增加,且随着照射剂量的增加而增加。放射敏感的细胞株CNE-2 X线照射后,EGFR表达也增加,但未随着照射剂量的增加而增加。
第三部分：X线辐射后上游调控因子HoxD10的表达变化
目的：通过检测放射敏感性不同的鼻咽癌细胞株x线辐射后,HoxD10的表达变化,探讨其与miR-7的表达变化的相关性。
方法：两种细胞均分为对照组(未辐射)、2Gy组和8Gy组,x线照射后10小时,用Trizol法提取细胞总RNA。Oligo dT逆转录mRNA,设计HoxD10特异性PCR引物扩增cDNA片段,进行染料法实时定量PCR,以对照组CNE-1细胞为参照样本,△△Ct法得出各样品HoxD10的相对数。
结果：与对照组相比,x线辐射后,各组HoxD10表达均显著下降。两种细胞受X线辐射后,HoxD10表达均明显下降。
第四部分：miR-7上游启动子序列的预测、与HoxD10调控因子结合的验证
目的：查找miR-7上游2Kb范围内启动子所在区域、可能与调控因子的结合位点及其序列。验证活体细胞中HoxD10调控因子与预测的启动子区域是否结合。
方法：通过PubMed查找关键词或题目或摘要为miR-7文献。通过miRbase查找miR-7在人类染色体中的位置,再通过UCSC查找上游2Kb范围内启动子区域的特征。甲醛固定后,超声破碎细胞,将细胞DNA破碎至1000bp左右的片段,加入Protein A及HoxD10抗体,提取HoxD10蛋白结合的DNA片段,设计扩增启动因子所在DNA片段的PCR引物,扩增提取的DNA片段,验证HoxD10蛋白结合的DNA片段是否与启动子所在片段相同。
结果：miR-7的上游启动子所在区域为-958至-968、-1019至-1028两个位点,CHIP实验结果证实HoxD10与上述两个点结合。
Background:Nasopharyngeal carcinoma (NPC) is a common malignancy in Guangdong, Guangxi, Hunan, Fujian province of China. The patients mainly are mid-age, but adolescent patients are not rare. Etiological factors of NPC include race, region and EB virus infection. Metabasis of NPC can be observed in earlier period. The primary therapy of NPC is radiotherapy. Well differentiated NPC cells are more radiosensitive than poor differentiated ones. Low local control rate of some patients is resulting from the different radiosensitivity.
MicroRNAs (miRNAs), about 22 nucleotides in length, are endogenous, noncoding RNAs that can play important regulatory roles in animals by targeting mRNAs for cleavage or translational repression. These microRNAs are diverse in sequence and expression patterns, and are evolutionarily widespread. MicroRNAs have been validated its important role in regulation to cell proliferation, differentiation, apoptosis, tumorigenesis and DNA damage. Some of the miRNAs are overexpressed in cancers, in which they can promote tumor growth, suggesting common mechanisms of miRNA-regulated cell cycle control. Targeted components of the DNA damage response (DDR) pathway, miRNAs are also associated with the response of a tumor to radiation. There is different expression pattern of microRNAs in variously differentiated NPC cell lines. MiR-7 is one of the outstanding. MiR-7 regulates activation of EGFR pathway which concerned with radiosensitivity.
Epidermal growth factor receptor (EGFR) is a kind of transmembrane proteins and member of ERBB family. Connection of EGFR and its ligand will result in autophosphorylation of receptor tyrosine kinase following by downstream pathways activation included RAS pathway to regulate proliferation and differentiation. Level of EGFR expressing in well differentiated cells is higher than poor differentiated cells, and for this reason, the former is more radiosensitive than latter. After radiation, EGFR translocates from the surface membrane to the nucleus, where it may directly priming DNA-PK dependent non-homologous end-joining (D-NHEJ) system to recover DNA damage. Activation of EGFR pathway improves cell proliferation to resist radiation. Clinical data showed low local control of NPC patients is resulted from high EGFR expression.
Homeobox (Hox) genes control rostral-caudal patterning during embryogenesis. It is a primary marker of the lumbosacral region and play an key role in the developing lumbar spinal cord and embryonic limb morphogenesis. Tissues express unique Hox gene expression profiles that persist upon primary and metastatic tumors. Hox genes encode DNA transcription regulatory proteins known for their role in cellular differentiation during embryonic development, aberrant expression of these genes has been associated with hematologic and solid neoplasms. HoxDIO expression was higher in quiescent as compared to tumor-associated angiogenic endothelium, so HoxD10 suppress tumor formation and metastasize. MicroRNAs can suppress transcription of HoxD10, and HoxD10 can also suppress microRNAs transcription. MiR-7 expression is positively regulated by HoxD10 in breast cancer.
Suppression of EGFR is a key point in radiosensitivity increasing and miR-7 and HoxD10 may be included in. The regulator of miR-7 in NPC cells is not reported. The objectives of this study are to approach the relationship between miR-7 and EGFR, miR-7 and HoxD10 in various radiosensitivities NPC cell lines in gene expression level, and to validate whether HoxD10 regulate miR-7 transcription in NPC cells.
Part I Status test of NPC cell lines
OBJECTIVE:To determine logarithmic growth phase and radiosensitivity of CNE-1 and CNE-2 provided by cancer center of Nanfang hospital.
METHODS:ELISA Reader was used to get OD values of NPC cell lines treated by MTT method in different growth days. OD values were typed in Excel to draw cell growth curve. Survival factions (SF) of NPC cell lines after X-ray radiation were counted by clone numeration method. GraphPad Prism 5.0 software treats SF data with linear-quadratic model to get parameters of radiosensitivity of NPC cell lines.
RESULTS:Logarithmic growth phase of CNE-1 and CNE-2 were 5-7 days and 3-5 days, respectively. CNE-1 is less radiosensitive than CNE-2.
Part II Expression patterns of miR-7 and EGFR in CNE-1, CNE-2 after X-ray radiation
OBJECTIVE:To investigate the expression patterns of miR-7 and EGFR in various radiosensitivity nasopharynx cancer cells after X-ray radiation.
METHODS:Nasopharynx cancer cell lines CNE1 and CNE2 irradiated by using different dose X-ray. Total RNAs of cell lines were extracted by Trizol in 10 hours after radiation. The reverse transcription of miR-7 was done by Stem-loop primer and EGFR mRNA was reversely transcript by oligo dT. Non-irradiated CNE1 cell was reference sample and relative quantity of other samples were counted by△△Ct method after real time PCR used SyBR Green.
RESULTS:MiR-7 increase in radioresistant NPC cell lines when cells radiated by X-ray. MiR-7 obviously increases when radioresistant NPC cell radiated by low dose X-ray compared with it radiated by high dose X-ray. In radiosensitive NPC cells, miR-7 decreases when cells were radiated by X-ray, and low dose radation made its decrease significant compared with high dose. Expression level of EGFR in radioresistant NPC cells increased with the X-ray dose increase, and in radiosensitive NPCE cells, it was not.
Part III Expression patterns of HoxD10 in CNE-1, CNE-2 after X-ray radiation
OBJECTIVE:To investigate the expression patterns of HoxD10 in various radiosensitivity nasopharynx cancer cells after X-ray radiation.
METHODS:Nasopharynx cancer cell lines CNE1 and CNE2 irradiated by using different dose X-ray. Total RNAs of cell lines were extracted by Trizol in 10 hours after radiation. The reverse transcription of HoxD10 was done oligo dT. Non-irradiated CNE1 cell was reference sample and relative quantity of other samples were counted by AACt method after real time PCR used SyBR Green.
RESULTS:HoxD10 decreased in both cell lines when they radiated by X-ray. The expression level of HoxD10 in radiosensitive NPC cells was higher than that in radioresistant NPC cells.
Part IV Prediction and ascertain HoxD10 as the regulator of miR-7 in NPC cells
OBJECTIVE:To predict the promoter location in upstream of miR-7 and its regulator, and ascertain their connection.
METHODS:Research articles whose keyword or title or abstract included miR-7 in PubMed. Find the location of miR-7 in human chromatin and the characteristic of promoter in 2kb upstream of miR-7 in UCSC. NPC cell was fixed by formaldehyde and hypersound break the DNA into about 1000bp fractions. Primers designed to amplify the fraction contained promoter of miR-7 were used in PCR to amplify the sample of DNA broken fraction and integrated DNA.
RESULT:Promoters of miR-7 locate in-958 to-968 and-1019 to-1028 upstream of miR-7. HoxD10 bind the two points in NPC cells.
引文
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[6]Kefas B, Godlewski J, Comeau L, et al. microRNA-7 inhibits the epidermal growth factor receptor and the Akt pathway and is down-regulated in glioblastoma.[J]. Cancer Res,2008,68(10):3566-3572.
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[12]Szumiel I. Epidermal growth factor receptor and DNA double strand break repair: the cell's self-defence.[J]. Cell Signal,2006,18(10):1537-1548.
[13]Milas L, Mason K A, Ang K K. Epidermal growth factor receptor and its inhibition in radiotherapy:in vivo findings.[J]. Int J Radiat Biol,2003,79(7):539-545.
[14]Baumann M, Krause M. Targeting the epidermal growth factor receptor in radiotherapy:radiobiological mechanisms, preclinical and clinical results[J]. Radiotherapy and Oncology,2004,72(3):257-266.
[15]Li H F, Kim J S, Waldman T. Radiation-induced Akt activation modulates radioresistance in human glioblastoma cells.[J]. Radiat Oncol,2009,4:43.
[16]Mckenna W G, Muschel R J, Gupta A K, et al. The RAS signal transduction pathway and its role in radiation sensitivity.[J]. Oncogene,2003,22(37):5866-5875.
[17]Kim I A, No M, Lee J M, et al. Epigenetic modulation of radiation response in human cancer cells with activated EGFR or HER-2 signaling:potential role of histone deacetylase 6.[J]. Radiother Oncol,2009,92(1):125-132.
[18]Mukherjee B, Mcellin B, Camacho C V, et al. EGFRvIII and DNA double-strand break repair:a molecular mechanism for radioresistance in glioblastoma.[J]. Cancer Res,2009,69(10):4252-4259.
[19]Tsai L P, Liao H M, Chen Y J, et al. A novel microdeletion at chromosome 2q31.1-31.2 in a three-generation family presenting duplication of great toes with clinodactyly.[J]. Clin Genet,2009,75(5):449-456.
[20]Misra M, Shah V, Carpenter E, et al. Restricted patterns of Hoxd10 and Hoxd11 set segmental differences in motoneuron subtype complement in the lumbosacral spinal cord.[J]. Dev Biol,2009,330(1):54-72.
[21]Redline R W, Hudock P, Macfee M, et al. Expression of AbdB-type homeobox genes in human tumors.[J]. Lab Invest,1994,71(5):663-670.
[22]Hu J, Gray C A, Spencer T E. Gene expression profiling of neonatal mouse uterine development.[J]. Biol Reprod,2004,70(6):1870-1876.
[23]Hedlund E, Karsten S L, Kudo L, et al. Identification of a Hoxd10-regulated transcriptional network and combinatorial interactions with Hoxa10 during spinal cord development.[J]. J Neurosci Res,2004,75(3):307-319.
[24]Myers C, Charboneau A, Cheung I, et al. Sustained expression of homeobox D10 inhibits angiogenesis.[J]. Am J Pathol,2002,161(6):2099-2109.
[25]Osborne J, Hu C, Hawley C, et al. Expression of HOXD10 gene in normal endometrium and endometrial adenocarcinoma.[J]. J Soc Gynecol Investig,1998,5(5):277-280.
[26]Carrio M, Arderiu G, Myers C, et al. Homeobox D10 induces phenotypic reversion of breast tumor cells in a three-dimensional culture model.[J]. Cancer Res,2005,65(16):7177-7185.
[27]Negrini M, Calin G A. Breast cancer metastasis:a microRNA story.[J]. Breast Cancer Res,2008,10(2):203.
[28]Han L, Witmer P D, Casey E, et al. DNA methylation regulates MicroRNA expression.[J]. Cancer Biol Ther,2007,6(8):1284-1288.
[29]Sasayama T, Nishihara M, Kondoh T, et al. MicroRNA-10b is overexpressed in malignant glioma and associated with tumor invasive factors, uPAR and RhoC.[J]. Int J Cancer,2009,125(6):1407-1413.
[30]Baffa R, Fassan M, Volinia S, et al. MicroRNA expression profiling of human metastatic cancers identifies cancer gene targets.[J]. J Pathol,2009,219(2):214-221.
[31]Reddy S D, Ohshiro K, Rayala S K, et al. MicroRNA-7, a homeobox D10 target, inhibits p21-activated kinase 1 and regulates its functions.[J]. Cancer Res,2008,68(20):8195-8200.
[32]Schmittgen T D, Livak K J. Analyzing real-time PCR data by the comparative C(T) method.[J]. Nat Protoc,2008,3(6):1101-1108.
[33]Bartel D P. MicroRNAs:target recognition and regulatory functions.[J]. Cell,2009,136(2):215-233.
[34]Filipowicz W, Bhattacharyya S N, Sonenberg N. Mechanisms of post-transcriptional regulation by microRNAs:are the answers in sight?[J]. Nat Rev Genet,2008,9(2):102-114.
[35]He L, Hannon G J. MicroRNAs:small RNAs with a big role in gene regulation.[J]. Nat Rev Genet,2004,5(7):522-531.
[36]Mendell J T. miRiad roles for the miR-17-92 cluster in development and disease.[J]. Cell,2008,133(2):217-222.
[37]Simone N L, Soule B P, Ly D, et al. Ionizing radiation-induced oxidative stress alters miRNA expression.[J]. PLoS One,2009,4(7):e6377.
[38]Weidhaas J B, Babar I, Nallur S M, et al. MicroRNAs as potential agents to alter resistance to cytotoxic anticancer therapy.[J]. Cancer Res,2007,67(23):l 1111-11116.
[39]Chen G, Zhu W, Shi D, et al. MicroRNA-181a sensitizes human malignant glioma U87MG cells to radiation by targeting Bcl-2.[J]. Oncol Rep,2010,23(4):997-1003.
[40]Xu Y, Shao Y, Zhou J, et al. Ultraviolet irradiation-induces epidermal growth factor receptor (EGFR) nuclear translocation in human keratinocytes.[J]. J Cell Biochem,2009,107(5):873-880.
[41]Takamizawa J, Konishi H, Yanagisawa K, et al. Reduced expression of the let-7 microRNAs in human lung cancers in association with shortened postoperative survival.[J]. Cancer Res,2004,64(11):3753-3756.
[42]Chen D J, Nirodi C S. The epidermal growth factor receptor:a role in repair of radiation-induced DNA damage.[J]. Clin Cancer Res,2007,13(22 Pt 1):6555-6560.
[43]Bussink J, van der Kogel A J, Kaanders J H. Activation of the PI3-K/AKT pathway and implications for radioresistance mechanisms in head and neck cancer.[J]. Lancet Oncol,2008,9(3):288-296.
[44]Szumiel I. Epidermal growth factor receptor and DNA double strand break repair: the cell's self-defence.[J]. Cell Signal,2006,18(10):1537-1548.
[45]Bartel D P. MicroRNAs:target recognition and regulatory functions.[J]. Cell,2009,136(2):215-233.
[3]Esquela-Kerscher A, Slack F J. Oncomirs-microRNAs with a role in cancer.[J]. Nat Rev Cancer,2006,6(4):259-269.
[4]Weidhaas J B, Babar I, Nallur S M, et al. MicroRNAs as potential agents to alter resistance to cytotoxic anticancer therapy.[J]. Cancer Res,2007,67(23):11111-11116.
[5]王旭丹,杨惠玲,郭禹标,et al.不同辐射抗拒鼻咽癌细胞微小RNA差异表达的研究[J].中国病理生理杂志,2007(06).
[6]Kefas B, Godlewski J, Comeau L, et al. microRNA-7 inhibits the epidermal growth factor receptor and the Akt pathway and is down-regulated in glioblastoma.[J]. Cancer Res,2008,68(10):3566-3572.
[7]Li X, Carthew R W. A microRNA mediates EGF receptor signaling and promotes photoreceptor differentiation in the Drosophila eye.[J]. Cell,2005,123(7):1267-1277.
[8]Herbst R S. Review of epidermal growth factor receptor biology.[J]. Int J Radiat Oncol Biol Phys,2004,59(2 Suppl):21-26.
[9]Chakravarti A, Dicker A, Mehta M. The contribution of epidermal growth factor receptor (EGFR) signaling pathway to radioresistance in human gliomas:a review of preclinical and correlative clinical data.[J]. Int J Radiat Oncol Biol Phys,2004,58(3):927-931.
[10]Bussink J, van der Kogel A J, Kaanders J H. Activation of the PI3-K/AKT pathway and implications for radioresistance mechanisms in head and neck cancer.[J]. Lancet Oncol,2008,9(3):288-296.
[11]Chen D J, Nirodi C S. The epidermal growth factor receptor:a role in repair of radiation-induced DNA damage.[J]. Clin Cancer Res,2007,13(22 Pt 1):6555-6560.
[12]Szumiel I. Epidermal growth factor receptor and DNA double strand break repair: the cell's self-defence.[J]. Cell Signal,2006,18(10):1537-1548.
[13]Milas L, Mason K A, Ang K K. Epidermal growth factor receptor and its inhibition in radiotherapy:in vivo findings.[J]. Int J Radiat Biol,2003,79(7):539-545.
[14]Baumann M, Krause M. Targeting the epidermal growth factor receptor in radiotherapy:radiobiological mechanisms, preclinical and clinical results[J]. Radiotherapy and Oncology,2004,72(3):257-266.
[15]Li H F, Kim J S, Waldman T. Radiation-induced Akt activation modulates radioresistance in human glioblastoma cells.[J]. Radiat Oncol,2009,4:43.
[16]Mckenna W G, Muschel R J, Gupta A K, et al. The RAS signal transduction pathway and its role in radiation sensitivity.[J]. Oncogene,2003,22(37):5866-5875.
[17]Kim I A, No M, Lee J M, et al. Epigenetic modulation of radiation response in human cancer cells with activated EGFR or HER-2 signaling:potential role of histone deacetylase 6.[J]. Radiother Oncol,2009,92(1):125-132.
[18]Mukherjee B, Mcellin B, Camacho C V, et al. EGFRvIII and DNA double-strand break repair:a molecular mechanism for radioresistance in glioblastoma.[J]. Cancer Res,2009,69(10):4252-4259.
[19]Tsai L P, Liao H M, Chen Y J, et al. A novel microdeletion at chromosome 2q31.1-31.2 in a three-generation family presenting duplication of great toes with clinodactyly.[J]. Clin Genet,2009,75(5):449-456.
[20]Misra M, Shah V, Carpenter E, et al. Restricted patterns of Hoxd10 and Hoxd11 set segmental differences in motoneuron subtype complement in the lumbosacral spinal cord.[J]. Dev Biol,2009,330(1):54-72.
[21]Redline R W, Hudock P, Macfee M, et al. Expression of AbdB-type homeobox genes in human tumors.[J]. Lab Invest,1994,71(5):663-670.
[22]Hu J, Gray C A, Spencer T E. Gene expression profiling of neonatal mouse uterine development.[J]. Biol Reprod,2004,70(6):1870-1876.
[23]Hedlund E, Karsten S L, Kudo L, et al. Identification of a Hoxd10-regulated transcriptional network and combinatorial interactions with Hoxa10 during spinal cord development.[J]. J Neurosci Res,2004,75(3):307-319.
[24]Myers C, Charboneau A, Cheung I, et al. Sustained expression of homeobox D10 inhibits angiogenesis.[J]. Am J Pathol,2002,161(6):2099-2109.
[25]Osborne J, Hu C, Hawley C, et al. Expression of HOXD10 gene in normal endometrium and endometrial adenocarcinoma.[J]. J Soc Gynecol Investig,1998,5(5):277-280.
[26]Carrio M, Arderiu G, Myers C, et al. Homeobox D10 induces phenotypic reversion of breast tumor cells in a three-dimensional culture model.[J]. Cancer Res,2005,65(16):7177-7185.
[27]Negrini M, Calin G A. Breast cancer metastasis:a microRNA story.[J]. Breast Cancer Res,2008,10(2):203.
[28]Han L, Witmer P D, Casey E, et al. DNA methylation regulates MicroRNA expression.[J]. Cancer Biol Ther,2007,6(8):1284-1288.
[29]Sasayama T, Nishihara M, Kondoh T, et al. MicroRNA-10b is overexpressed in malignant glioma and associated with tumor invasive factors, uPAR and RhoC.[J]. Int J Cancer,2009,125(6):1407-1413.
[30]Baffa R, Fassan M, Volinia S, et al. MicroRNA expression profiling of human metastatic cancers identifies cancer gene targets.[J]. J Pathol,2009,219(2):214-221.
[31]Reddy S D, Ohshiro K, Rayala S K, et al. MicroRNA-7, a homeobox D10 target, inhibits p21-activated kinase 1 and regulates its functions.[J]. Cancer Res,2008,68(20):8195-8200.
[32]Schmittgen T D, Livak K J. Analyzing real-time PCR data by the comparative C(T) method.[J]. Nat Protoc,2008,3(6):1101-1108.
[33]Bartel D P. MicroRNAs:target recognition and regulatory functions.[J]. Cell,2009,136(2):215-233.
[34]Filipowicz W, Bhattacharyya S N, Sonenberg N. Mechanisms of post-transcriptional regulation by microRNAs:are the answers in sight?[J]. Nat Rev Genet,2008,9(2):102-114.
[35]He L, Hannon G J. MicroRNAs:small RNAs with a big role in gene regulation.[J]. Nat Rev Genet,2004,5(7):522-531.
[36]Mendell J T. miRiad roles for the miR-17-92 cluster in development and disease.[J]. Cell,2008,133(2):217-222.
[37]Simone N L, Soule B P, Ly D, et al. Ionizing radiation-induced oxidative stress alters miRNA expression.[J]. PLoS One,2009,4(7):e6377.
[38]Weidhaas J B, Babar I, Nallur S M, et al. MicroRNAs as potential agents to alter resistance to cytotoxic anticancer therapy.[J]. Cancer Res,2007,67(23):l 1111-11116.
[39]Chen G, Zhu W, Shi D, et al. MicroRNA-181a sensitizes human malignant glioma U87MG cells to radiation by targeting Bcl-2.[J]. Oncol Rep,2010,23(4):997-1003.
[40]Xu Y, Shao Y, Zhou J, et al. Ultraviolet irradiation-induces epidermal growth factor receptor (EGFR) nuclear translocation in human keratinocytes.[J]. J Cell Biochem,2009,107(5):873-880.
[41]Takamizawa J, Konishi H, Yanagisawa K, et al. Reduced expression of the let-7 microRNAs in human lung cancers in association with shortened postoperative survival.[J]. Cancer Res,2004,64(11):3753-3756.
[42]Chen D J, Nirodi C S. The epidermal growth factor receptor:a role in repair of radiation-induced DNA damage.[J]. Clin Cancer Res,2007,13(22 Pt 1):6555-6560.
[43]Bussink J, van der Kogel A J, Kaanders J H. Activation of the PI3-K/AKT pathway and implications for radioresistance mechanisms in head and neck cancer.[J]. Lancet Oncol,2008,9(3):288-296.
[44]Szumiel I. Epidermal growth factor receptor and DNA double strand break repair: the cell's self-defence.[J]. Cell Signal,2006,18(10):1537-1548.
[45]Bartel D P. MicroRNAs:target recognition and regulatory functions.[J]. Cell,2009,136(2):215-233.