ARC基因在鼻咽癌中的表达及与鼻咽癌细胞放化疗敏感性的研究
发布时间：2018-08-31 07:54
【摘要】：癌变是一个复杂的过程,是由癌基因激活,抑癌基因和细胞死亡机制失活相互作用而出现的。由于细胞凋亡的减少与肿瘤发生密切相关,这提示凋亡过程中起负调控有关的基因可能有致癌性,而促进细胞凋亡有关的基因可能为肿瘤抑制基因。因此,凋亡相关蛋白一直是肿瘤研究的热点领域。本文将分章讨论一新颖抗凋亡蛋白ARC与鼻咽癌发生、发展的关系及对鼻咽癌放疗与化疗敏感性的影响。 目的蛋白水平探讨鼻咽癌组织及鼻咽部炎症组织中ARC的表达与肿瘤T分期、临床分期和有无淋巴结转移等临床病理学参数的关系,并检测ARC在鼻咽癌细胞系中的表达水平及细胞内定位。 方法采用免疫组织化学的方法检测82例鼻咽癌组织、20例鼻咽部慢性炎症组织的石蜡组织标本,分析其在鼻咽癌组织中的表达与临床病理学参数的关系。采用细胞免疫组化检测ARC在鼻咽癌细胞株中的表达定位,Western blot实验方法检测ARC在鼻咽癌细胞株中的表达。SPSS17.0统计软件对实验数据进行统计分析。 结果 1)通过鼻咽癌组织及细胞的免疫组化检测,结果发现ARC表达于细胞浆及细胞核内,其中大部分表达于细胞浆。 2)ARC蛋白在鼻咽癌组织中的表达强度明显高于鼻咽部炎症组织(P0.001)。其中在鼻咽癌组织中,44例为强阳性(53.7%)23例为中度阳性(28%),15例为弱阳性(18.3%)；鼻咽部慢性炎症组织中,16例为弱阳性(80%),4例为中度阳性(20%),无强阳性。 3)ARC蛋白的表达与鼻咽癌的临床分期、病理分型因素相关(P0.001),临床分期越晚、分化程度越低的组织中ARC的表达越高。而与患者年龄、性别及有无淋巴结转移无关(P0.05)。 4)ARC在鼻咽癌细胞株CNE-1、CNE-2、5-8F、6-10B中均有明显表达,而在鼻咽永生化上皮细胞NP69中表达最弱；其相对表达量分别为CNE-1(4.78±0.47)、CNE-2(5.39±0.58)、5-8F (3.54±0.33)、6-10B (3.12±0.26)、NP69(1.67±0.24)。在鼻咽癌细胞株中表达的表达量由高到低依次为CNE-2CNE-15-8F6-10B。 结论以上结果提示ARC在鼻咽癌的发生、发展中可能起重要作用,临床上检测ARC的表达对鼻咽癌恶性程度的预测可能会有所帮助。 目的使用RNA干扰技术及基因克隆技术分别沉默和过表达ARC在鼻咽癌细胞株中的表达,并筛选出稳定转染的细胞株。 方法使用pcDNA6.2-GW/EmGFP/miRNA载体构建表达miRNA对ARC基因的RNA干扰的质粒；使用pEZ-M03载体构建表达ARC基因的质粒。采用脂质体转染法将构建的ARC-miRNA及对照no-targe-miRNA质粒转染高表达ARC的CNE-2细胞；将pEZ-M03-ARC及对照pReceiver-M03CT质粒稳定转染低表达ARC的6-10B细胞。通过荧光显微镜观察质粒转染效率,CCK-8法检测细胞生存率,荧光定量PCR、Western blot法检测ARC表达效果,通过流式细胞仪检测ARC对细胞周期的影响。 结果 1)三种miRNA干扰质粒ARC-miRNA-1、ARC-miRNA-2、 ARC-miRNA-3及阴性对照质粒转染高表达ARC的CNE-2细胞,稳定转染后,荧光显微镜下绿色荧光表达水平均超过90%,细胞存活率分别为ARC-miRNA-1(98.4±1.3%)、ARC-miRNA-2(97.7±0.9%)、 ARC-miRNA-3(97.1±1.1%)、阴性对照组(98.1±1.6%)。pEZ-M03-ARC质粒及阴性对照质粒转染低表达ARC的6-10B细胞,稳定转染后,荧光显微镜下绿色荧光表达水平均超过90%,细胞存活率分别为pEZ-M03-ARC (98.9±0.8%),阴性对照组(98.3±1.2%)。 2)荧光定量PCR检测各组ARC mRNA表达水平。设定空白对照组ARC mRNA的表达量为1, ARC-miRNA-1、ARC-miRNA-2、 ARC-miRNA-3及阴性对照组CNE-2细胞中ARC mRNA的相对表达量分别为0.833±0.126、0.749±0.117、0.244±0.059、0.975±0.083；pEZ-M03-ARC及对照组6-10B细胞中ARC mRNA的相对表达量分别为2.371士0.330、1.149±0.166。 3)用Western blot检测各组ARC基因蛋白表达水平,定空白对照组ARC蛋白的表达量为1,ARC-miRNA-1、ARC-miRNA-2、 ARC-miRNA-3及阴性对照组CNE-2细胞中ARC蛋白的相对表达量分别为0.875±0.144、0.796±0.122、0.271±0.082、0.989±0.130；pEZ-M03-ARC及对照组6-10B细胞中ARC蛋白的相对表达量分别为2.189±0.274、1.157±0.244。 4)稳定转染后检测显示ARC-miRNA-3质粒为RNA干扰效果最强的质粒。其在CNE-2细胞mRNA水平沉默效率为77.03%；蛋白水平沉默效率为72.6%。稳定转染pEZ-M03-ARC质粒后检测显示：其在6-10B细胞mRNA水平的表达较对照组上调2.06倍；蛋白水平比对照组上调1.89倍。 5) ARC-miRNA-3CNE-2细胞的G0-G1、G2-M、S期的比例分别为56.5±4.7%、8.6±2.1%、34.8±2.5%,对照组CNE-2细胞的G0-G1、 G2-M、S期的比例分别为57.4±4.6%、9.1±2.3%、33.5士2.6%,两组之间无明显统计学差异(P0.05)；pEZ-M03-ARC6-10B细胞的G0-G1、 G2-M、S期的比例分别为61.5±3.3%、9.2±1.7%、29.3±2.3%,对照组6-10B细胞的G2-M、S期的比例分别为61.3±3.5%、10.3±2.1%、28.4±2.2%,两组之间无明显统计学差异(P0.05)。 结论成功构建并筛选出miRNA干扰质粒ARC-miRNA-3,并培养出稳定沉默ARC表达的鼻咽癌细胞株；成功构建并筛选出基因克隆质粒pEZ-M03-ARC,并培养出稳定过表达ARC的鼻咽癌细胞株。ARC的表达对鼻咽癌细胞的生长增殖及细胞周期无明显影响。 目的探讨沉默或过表达ARC后对鼻咽癌细胞放射敏感性的影响 方法沉默或过表达ARC的鼻咽癌细胞及对照组细胞分别接受不同剂量梯度的X线照射后,CCK-8法检测细胞生存率,Annexin V法检测细胞凋亡,Caspase3/8活性检测试剂盒检测Caspase3/8酶活性。 结果 1)沉默ARC表达的ARC-miRNA-3及对照组CNE-2细胞接受OGy、2Gy、4Gy、6Gy、8Gy、10Gy不同剂量照射后CCK-8法检测两组细胞存活率。ARC-miRNA-3细胞的细胞存活率明显低于对照组细胞,实验组细胞存活曲线明显低于对照组,2Gy、4Gy、6Gy、8Gy、10Gy ARC-miRNA-3组细胞生存率分别为0.907±0.014、0.814±0.086、0.694±0.069、0.540±0.066、0.387±0.043；对照组细胞生存率分别为0.985±0.018、0.966±0.095、0.870±0.089、0.735±0.063、0.594±0.053(P0.001)；8Gy X线照射后,Annexin V法检测ARC-miRNA-3细胞凋亡比例为52.2±3.4%明显高于对照组细胞36.1±2.2%(P0.001)。 2)pEZ-M03-ARC细胞的细胞存活率明显高于对照组细胞,实验组细胞存活曲线明显高于对照组,2Gy、4Gy、6Gy、8Gy、10Gy ARC-miRNA-3组细胞生存率分别为0.977±0.010、0.954±0.015、0.914±0.029、0.847±0.027、0.743±0.034；对照组细胞生存率分别为0.974±0.013、0.936±0.035、0.863±0.029、0.765±0.030、0.616±0.052(P0.001)；8Gy X线照射后,Annexin V法检测细胞凋亡比例为21.2±2.4%明显低于对照组细胞33.7±2.5%。 3)8Gy X线照射后,定义照组含量为1,ARC-miRNA-3CNE-2细胞的活性caspase-3含量为2.33±0.26,明显高于对照组；ARC-miRNA-3CNE-2细胞的活性caspase-8含量为1.65±0.17,明显高于对照组(P0.001)。 4)8Gy X线照射后,定义照组含量为1,pEZ-M03-ARC6-10B细胞的活性caspase-3含量为0.62±0.14,明显低于对照组；pEZ-M03-ARC6-10B细胞的活性caspase-8含量为0.73±0.11,明显低于对照组(P0.001)。 结论ARC有增强鼻咽癌细胞放射抵抗性的功能,并可能是通过抑制内源性和外源性的凋亡通路来实现的,ARC有望成为增加鼻咽癌放射敏感性的分子治疗靶点。 目的探讨沉默或过表达ARC后对鼻咽癌化疗药物敏感性的影响 方法沉默或过表达ARC的鼻咽癌细胞及对照组细胞分别接受以不同浓度梯度的紫杉醇或顺铂处理48小时后,使用CCK-8法检测细胞生长增殖情况。使用IC30的药物浓度处理细胞,24小时后,使用Annxin V法检测细胞凋亡情况,用Caspase3/8活性检测试剂盒检测Caspase3/8酶活性。 结果 1) ARC-miRNA-3细胞在顺铂中存活率明显低于对照组细胞,实验组细胞生存曲线明显低于对照组(P0.001)。通过SPSS软件计算,ARC-miRNA-3细胞顺铂作用IC50值为(1.995±0.327)×10-5明显低于对照组IC50值(3.006±0.344)×10-5(P0.001)。Annexin V法检测ARC-miRNA-3细胞凋亡比例为56.8±4.4%明显高于对照组细胞33.02±3.7%(P0.001)。 2) pEZ-M03-ARC细胞在顺铂中细胞存活率明显高于对照组细胞,实验组细胞存活曲线明显高于对照组(P0.001)；pEZ-M03-ARC细胞顺铂作用IC5o值为(1.000±0.244)×10-4明显高于对照组IC50值(6.397±0.276)×10-5(P0.001)。Annexin V法检测实验组细胞凋亡比例为24.8±2.8%明显低于对照组细胞35.1±2.6%(P0.001)。 3)顺铂处理后,ARC-miRNA-3CNE-2细胞的活性caspase-3含量与对照组的比例为3.49±0.31；ARC-miRNA-3CNE-2细胞的活性caspase-8含量与对照组的比例为2.17±0.20,均明显高于对照组(P0.001)。 4)顺铂处理后,pEZ-M03-ARC6-10B细胞的活性caspase-3含量与对照组的比例为0.44±0.07; pEZ-M03-ARC6-1OB细胞的活性caspase-8含量与对照组的比例为0.69±0.12,均明显低于对照组(P0.001)。 5)ARC-miRNA-3细胞及对照组细胞在紫杉醇中的存活率无明显差别(P0.05); pEZ-M03-ARC细胞及对照组细胞在紫杉醇中的存活率无明显差别(P0.05)。 结论ARC有增强鼻咽癌细胞化疗抵抗的功能,并可能是通过抑制内源性和外源性的凋亡通路来实现的,ARC有望成为增加鼻咽癌化疗敏感性的分子治疗靶点。
[Abstract]:Carcinogenesis is a complex process, which is caused by the interaction of oncogene activation, tumor suppressor genes and inactivation of cell death mechanism. The decrease of apoptosis is closely related to tumorigenesis, which suggests that genes involved in negative regulation of apoptosis may be carcinogenic, while genes involved in promoting apoptosis may be tumor suppression. Therefore, apoptosis-related proteins have always been a hot topic in tumor research. This article will discuss the relationship between a novel anti-apoptosis protein ARC and the occurrence and development of nasopharyngeal carcinoma, and its effect on radiotherapy and chemosensitivity of nasopharyngeal carcinoma.
Objective To investigate the relationship between the expression of ARC in nasopharyngeal carcinoma (NPC) and the clinical pathological parameters such as T stage, clinical stage and lymph node metastasis, and to detect the expression level and intracellular localization of ARC in NPC cell lines.
Methods Immunohistochemical method was used to detect paraffin-embedded tissues from 82 cases of nasopharyngeal carcinoma and 20 cases of chronic nasopharyngeal inflammation. The expression of.SPSS17.0 in nasopharyngeal carcinoma cell line was analyzed by statistical software.
Result
1) Immunohistochemical staining of nasopharyngeal carcinoma tissues and cells showed that ARC was expressed in cytoplasm and nucleus, most of which were expressed in cytoplasm.
2) The expression of ARC protein in nasopharyngeal carcinoma tissues was significantly higher than that in nasopharyngeal inflammatory tissues (P 0.001). Among nasopharyngeal carcinoma tissues, 44 were strongly positive (53.7%) 23 were moderately positive (28%) and 15 were weakly positive (18.3%).
3) The expression of ARC protein was correlated with the clinical stage and pathological type of NPC (P 0.001). The later the clinical stage, the higher the expression of ARC protein in the tissues with lower differentiation, but it was not correlated with age, sex and lymph node metastasis (P 0.05).
4) ARC was strongly expressed in nasopharyngeal carcinoma cell lines CNE-1, CNE-2, 5-8F, 6-10B, but weakly expressed in immortalized nasopharyngeal epithelial cells NP69. The relative expression levels of ARC were CNE-1 (4.78.47), CNE-2 (5.39.58), 5-8F (3.54.33), 6-10B (3.12.26), NP69 (1.67.24), respectively. The next time is CNE-2CNE-15-8F6-10B..
Conclusion These results suggest that ARC may play an important role in the occurrence and development of nasopharyngeal carcinoma. Detecting the expression of ARC may be helpful to predict the malignant degree of nasopharyngeal carcinoma.
Objective To screen stable transfected nasopharyngeal carcinoma cell lines by silencing and overexpressing ARC with RNA interference and gene cloning.
Methods RNA interference plasmids expressing microRNAs against ARC gene were constructed by pcDNA6.2-GW/EmGFP/microRNA vector, and plasmids expressing ARC gene were constructed by pEZ-M03 vector. ARC-microNA and control no-targe-microRNA plasmids were transfected into CNE-2 cells with high expression of ARC by liposome transfection, pEZ-M03-ARC and control pReceiver-M03CT plasmids were used. The plasmid transfection efficiency was observed by fluorescence microscope, the cell survival rate was detected by CCK-8, the expression of ARC was detected by fluorescence quantitative PCR and Western blot, and the effect of ARC on cell cycle was detected by flow cytometry.
Result
1) Three microRNAs interfered with the expression of ARC-microNA-1, ARC-microNA-2, ARC-microNA-3 and negative control plasmids in CNE-2 cells with high expression of ARC. After stable transfection, the expression of green fluorescence under fluorescence microscope exceeded 90%, and the cell survival rates were ARC-microNA-1 (98.4 (1.3%), ARC-microNA-2 (97.7 (0.9%), ARC-microNA-3 (97.1 (1) and negative control group (97.1). PEZ-M03-ARC plasmid and negative control plasmid were transfected into 6-10B cells with low expression of ARC. After stable transfection, the expression of green fluorescence under fluorescence microscope exceeded 90%. The survival rate of pEZ-M03-ARC cells was 98.9%(98.9%+0.8%) and that of negative control group was 98.3%+1.2%.
2) The expression of ARC mRNA in CNE-2 cells was detected by fluorescence quantitative PCR. The relative expression levels of ARC mRNA in blank control group were 1, ARC-microNA-1, ARC-microNA-2, ARC-microNA-3 and negative control group were 0.833 [0.126], 0.749 [0.117], 0.244 [0.059], 0.975 [0.083], pEZ-M03-ARC and 6-10B cells respectively. The relative expression amounts were 2.371 0.330,1.149 0.166..
3) Western blot was used to detect the expression level of ARC gene protein in each group. The relative expression levels of ARC protein in blank control group were 1, ARC-microNA-1, ARC-microNA-2, ARC-microNA-3 and negative control group CNE-2 cells were 0.875 (+ 0.144), 0.796 (+ 0.122), 0.271 (+ 0.082), 0.989 (+ 0.130), pEZ-M03-ARC and 6-10B cells respectively. The relative expression of protein was 2.189 + 0.274,1.157 + 0.244.
4) After stable transfection, ARC-microNA-3 plasmid was the most effective plasmid for RNA interference. Its silencing efficiency was 77.03% at mRNA level and 72.6% at protein level in CNE-2 cells. After stable transfection of pEZ-M03-ARC plasmid, the expression of ARC-microNA-3 in 6-10B cells was 2.06 times higher than that in control group. Up to 1.89 times.
5) The proportions of G0-G1, G2-M and S-phase of ARC-microNA-3CNE-2 cells were 56.5 (+ 4.7%), 8.6 (+ 2.1%) and 34.8 (+ 2.5%) respectively. The proportions of G0-G1, G2-M and S-phase of CNE-2 cells in control group were 57.4 (+ 4.6%), 9.1 (+ 2.3%) and 33.5 (+ 2.6%) respectively. There was no significant difference between the two groups (P 0.05). The proportions of G2-M and S-phase of 6-10B cells in the control group were 61.3 (+ 3.5%), 10.3 (+ 2.1%) and 28.4 (+ 2.2%) respectively. There was no significant difference between the two groups (P 0.05).
Conclusion The interfering plasmid ARC-microNA-3 was successfully constructed and screened, and the stable silencing cell line of nasopharyngeal carcinoma was cultured. The cloned plasmid pEZ-M03-ARC was successfully constructed and screened, and the stable overexpressing cell line of nasopharyngeal carcinoma was cultured.
Objective to investigate the effect of silencing or over expression of ARC on radiosensitivity of nasopharyngeal carcinoma cells.
Methods Nasopharyngeal carcinoma cells silenced or overexpressed ARC were irradiated with different dose gradient X-ray respectively. Cell survival rate was measured by CCK-8 method, apoptosis was detected by Annexin V method, and caspase 3/8 activity was detected by Caspase 3/8 activity assay kit.
Result
1) The survival rate of ARC-microNA-3 cells was measured by CCK-8 assay after different doses of OGy, 2Gy, 4Gy, 6Gy, 8Gy, 10Gy irradiation. The cell survival rate of ARC-microNA-3 cells was significantly lower than that of control cells, and the cell survival curve of experimental group was significantly lower than that of control group, 2Gy, 4Gy, 6Gy, 8Gy, 10Gy ARC-microNA-3 cells. The survival rates were 0.907 [0.907 [0.907 [0.017 [0.017 [0.014,0.814 [0.81 [0.086,0.694 [0.694 [0.069,0.699 9 9 [, 0.694 [, 0.540 [0.540 [0.066 [0.066 6 6 0.066 6, 0.066 0.060 [0.066 6 0.066 6, 0.387 [0.387 [0.387 [0.040.0403 [.043] 3] 3] respectively; the cell survival rates in control group were 0.985 5 5 5 In the meantime, it is necessary to study the relationship between the two. + 2.2% (P0.001).
2) The cell survival rate of pEZ-M03-ARC cells was significantly higher than that of the control group. The cell survival curves of the experimental group were significantly higher than that of the control group. The cell survival rates of the experimental group, 2Gy, 4Gy, 6Gy, 8Gy, 10Gy ARC-MiNA-3 groups were 0.977 [0.010], 0.954 [0.015], 0.914 [0.029], 0.847 [0.027], 0.743 [0.034], respectively. After 8 Gy X-ray irradiation, the percentage of apoptosis detected by Annexin V method was 21.2+2.4%, which was significantly lower than that of the control group (33.7+2.5%).
3) After 8 Gy X-ray irradiation, the content of active caspase-3 in ARC-microNA-3CNE-2 cells was 1 and 2.33+0.26 respectively, which was significantly higher than that in control group, and the content of active caspase-8 in ARC-microNA-3CNE-2 cells was 1.65+0.17, which was significantly higher than that in control group (P 0.001).
4) After 8 Gy X-ray irradiation, the content of active caspase-3 in pEZ-M03-ARC6-10B cells was 0.62+0.14, which was significantly lower than that in control group, and the content of active caspase-8 in pEZ-M03-ARC6-10B cells was 0.73+0.11, which was significantly lower than that in control group (P 0.001).
Conclusion ARC can enhance the radioresistance of nasopharyngeal carcinoma cells and may be achieved by inhibiting endogenous and exogenous apoptotic pathways. ARC may be a promising molecular therapeutic target for increasing radiosensitivity of nasopharyngeal carcinoma.
Objective to investigate the effect of silencing or over expression of ARC on chemosensitivity of nasopharyngeal carcinoma.
Methods Nasopharyngeal carcinoma cells silenced or overexpressed ARC were treated with paclitaxel or cisplatin at different concentration gradients for 48 hours. Cell growth and proliferation were detected by CCK-8 assay. Cells treated with IC30 were detected by Annxin V assay after 24 hours. Cell apoptosis was detected by Caspase 3/8 activity assay. The Caspase3/8 activity was detected by kit.
Result
1) The survival rate of ARC-microNA-3 cells in cisplatin was significantly lower than that of the control group, and the survival curve of the experimental group was significantly lower than that of the control group (P 0.001). The proportion of apoptotic cells was 56.8 + 4.4%, which was significantly higher than that of the control group (33.02 + 3.7%) (P0.001).
2) The cell survival rate of pEZ-M03-ARC cells in cisplatin was significantly higher than that of the control group, and the cell survival curve of the experimental group was significantly higher than that of the control group (P 0.001). The IC5o value of pEZ-M03-ARC cells treated by cisplatin was (1.000.244)*10-4, which was significantly higher than that of the control group (6.397.276)*10-5 (P 0.001). The apoptosis rate of the experimental group was 2.00% by Annexin V assay. 4.8 + 2.8% was significantly lower than that of the control group (35.1 + 2.6%) (P0.001).
3) After cisplatin treatment, the ratio of active caspase-3 in ARC-microNA-3CNE-2 cells to the control group was 3.49 [0.31], and the ratio of active caspase-8 in ARC-microNA-3CNE-2 cells to the control group was 2.17 [0.20], which was significantly higher than that in the control group (P 0.001).
4) After cisplatin treatment, the ratio of active caspase-3 in pEZ-M03-ARC6-10B cells to control group was 0.44.07, and the ratio of active caspase-8 in pEZ-M03-ARC6-1OB cells to control group was 0.69.12, which was significantly lower than that in control group (P 0.001).
5) There was no significant difference in the survival rate of ARC-microNA-3 cells and control cells in paclitaxel (P 0.05); there was no significant difference in the survival rate of pEZ-M03-ARC cells and control cells in paclitaxel (P 0.05).
Conclusion ARC can enhance the chemotherapeutic resistance of nasopharyngeal carcinoma cells and may be achieved by inhibiting endogenous and exogenous apoptotic pathways. ARC is expected to be a molecular therapeutic target for increasing the chemotherapeutic sensitivity of nasopharyngeal carcinoma cells.
【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R739.63
本文编号：2214370
[Abstract]:Carcinogenesis is a complex process, which is caused by the interaction of oncogene activation, tumor suppressor genes and inactivation of cell death mechanism. The decrease of apoptosis is closely related to tumorigenesis, which suggests that genes involved in negative regulation of apoptosis may be carcinogenic, while genes involved in promoting apoptosis may be tumor suppression. Therefore, apoptosis-related proteins have always been a hot topic in tumor research. This article will discuss the relationship between a novel anti-apoptosis protein ARC and the occurrence and development of nasopharyngeal carcinoma, and its effect on radiotherapy and chemosensitivity of nasopharyngeal carcinoma.
Objective To investigate the relationship between the expression of ARC in nasopharyngeal carcinoma (NPC) and the clinical pathological parameters such as T stage, clinical stage and lymph node metastasis, and to detect the expression level and intracellular localization of ARC in NPC cell lines.
Methods Immunohistochemical method was used to detect paraffin-embedded tissues from 82 cases of nasopharyngeal carcinoma and 20 cases of chronic nasopharyngeal inflammation. The expression of.SPSS17.0 in nasopharyngeal carcinoma cell line was analyzed by statistical software.
Result
1) Immunohistochemical staining of nasopharyngeal carcinoma tissues and cells showed that ARC was expressed in cytoplasm and nucleus, most of which were expressed in cytoplasm.
2) The expression of ARC protein in nasopharyngeal carcinoma tissues was significantly higher than that in nasopharyngeal inflammatory tissues (P 0.001). Among nasopharyngeal carcinoma tissues, 44 were strongly positive (53.7%) 23 were moderately positive (28%) and 15 were weakly positive (18.3%).
3) The expression of ARC protein was correlated with the clinical stage and pathological type of NPC (P 0.001). The later the clinical stage, the higher the expression of ARC protein in the tissues with lower differentiation, but it was not correlated with age, sex and lymph node metastasis (P 0.05).
4) ARC was strongly expressed in nasopharyngeal carcinoma cell lines CNE-1, CNE-2, 5-8F, 6-10B, but weakly expressed in immortalized nasopharyngeal epithelial cells NP69. The relative expression levels of ARC were CNE-1 (4.78.47), CNE-2 (5.39.58), 5-8F (3.54.33), 6-10B (3.12.26), NP69 (1.67.24), respectively. The next time is CNE-2CNE-15-8F6-10B..
Conclusion These results suggest that ARC may play an important role in the occurrence and development of nasopharyngeal carcinoma. Detecting the expression of ARC may be helpful to predict the malignant degree of nasopharyngeal carcinoma.
Objective To screen stable transfected nasopharyngeal carcinoma cell lines by silencing and overexpressing ARC with RNA interference and gene cloning.
Methods RNA interference plasmids expressing microRNAs against ARC gene were constructed by pcDNA6.2-GW/EmGFP/microRNA vector, and plasmids expressing ARC gene were constructed by pEZ-M03 vector. ARC-microNA and control no-targe-microRNA plasmids were transfected into CNE-2 cells with high expression of ARC by liposome transfection, pEZ-M03-ARC and control pReceiver-M03CT plasmids were used. The plasmid transfection efficiency was observed by fluorescence microscope, the cell survival rate was detected by CCK-8, the expression of ARC was detected by fluorescence quantitative PCR and Western blot, and the effect of ARC on cell cycle was detected by flow cytometry.
Result
1) Three microRNAs interfered with the expression of ARC-microNA-1, ARC-microNA-2, ARC-microNA-3 and negative control plasmids in CNE-2 cells with high expression of ARC. After stable transfection, the expression of green fluorescence under fluorescence microscope exceeded 90%, and the cell survival rates were ARC-microNA-1 (98.4 (1.3%), ARC-microNA-2 (97.7 (0.9%), ARC-microNA-3 (97.1 (1) and negative control group (97.1). PEZ-M03-ARC plasmid and negative control plasmid were transfected into 6-10B cells with low expression of ARC. After stable transfection, the expression of green fluorescence under fluorescence microscope exceeded 90%. The survival rate of pEZ-M03-ARC cells was 98.9%(98.9%+0.8%) and that of negative control group was 98.3%+1.2%.
2) The expression of ARC mRNA in CNE-2 cells was detected by fluorescence quantitative PCR. The relative expression levels of ARC mRNA in blank control group were 1, ARC-microNA-1, ARC-microNA-2, ARC-microNA-3 and negative control group were 0.833 [0.126], 0.749 [0.117], 0.244 [0.059], 0.975 [0.083], pEZ-M03-ARC and 6-10B cells respectively. The relative expression amounts were 2.371 0.330,1.149 0.166..
3) Western blot was used to detect the expression level of ARC gene protein in each group. The relative expression levels of ARC protein in blank control group were 1, ARC-microNA-1, ARC-microNA-2, ARC-microNA-3 and negative control group CNE-2 cells were 0.875 (+ 0.144), 0.796 (+ 0.122), 0.271 (+ 0.082), 0.989 (+ 0.130), pEZ-M03-ARC and 6-10B cells respectively. The relative expression of protein was 2.189 + 0.274,1.157 + 0.244.
4) After stable transfection, ARC-microNA-3 plasmid was the most effective plasmid for RNA interference. Its silencing efficiency was 77.03% at mRNA level and 72.6% at protein level in CNE-2 cells. After stable transfection of pEZ-M03-ARC plasmid, the expression of ARC-microNA-3 in 6-10B cells was 2.06 times higher than that in control group. Up to 1.89 times.
5) The proportions of G0-G1, G2-M and S-phase of ARC-microNA-3CNE-2 cells were 56.5 (+ 4.7%), 8.6 (+ 2.1%) and 34.8 (+ 2.5%) respectively. The proportions of G0-G1, G2-M and S-phase of CNE-2 cells in control group were 57.4 (+ 4.6%), 9.1 (+ 2.3%) and 33.5 (+ 2.6%) respectively. There was no significant difference between the two groups (P 0.05). The proportions of G2-M and S-phase of 6-10B cells in the control group were 61.3 (+ 3.5%), 10.3 (+ 2.1%) and 28.4 (+ 2.2%) respectively. There was no significant difference between the two groups (P 0.05).
Conclusion The interfering plasmid ARC-microNA-3 was successfully constructed and screened, and the stable silencing cell line of nasopharyngeal carcinoma was cultured. The cloned plasmid pEZ-M03-ARC was successfully constructed and screened, and the stable overexpressing cell line of nasopharyngeal carcinoma was cultured.
Objective to investigate the effect of silencing or over expression of ARC on radiosensitivity of nasopharyngeal carcinoma cells.
Methods Nasopharyngeal carcinoma cells silenced or overexpressed ARC were irradiated with different dose gradient X-ray respectively. Cell survival rate was measured by CCK-8 method, apoptosis was detected by Annexin V method, and caspase 3/8 activity was detected by Caspase 3/8 activity assay kit.
Result
1) The survival rate of ARC-microNA-3 cells was measured by CCK-8 assay after different doses of OGy, 2Gy, 4Gy, 6Gy, 8Gy, 10Gy irradiation. The cell survival rate of ARC-microNA-3 cells was significantly lower than that of control cells, and the cell survival curve of experimental group was significantly lower than that of control group, 2Gy, 4Gy, 6Gy, 8Gy, 10Gy ARC-microNA-3 cells. The survival rates were 0.907 [0.907 [0.907 [0.017 [0.017 [0.014,0.814 [0.81 [0.086,0.694 [0.694 [0.069,0.699 9 9 [, 0.694 [, 0.540 [0.540 [0.066 [0.066 6 6 0.066 6, 0.066 0.060 [0.066 6 0.066 6, 0.387 [0.387 [0.387 [0.040.0403 [.043] 3] 3] respectively; the cell survival rates in control group were 0.985 5 5 5 In the meantime, it is necessary to study the relationship between the two. + 2.2% (P0.001).
2) The cell survival rate of pEZ-M03-ARC cells was significantly higher than that of the control group. The cell survival curves of the experimental group were significantly higher than that of the control group. The cell survival rates of the experimental group, 2Gy, 4Gy, 6Gy, 8Gy, 10Gy ARC-MiNA-3 groups were 0.977 [0.010], 0.954 [0.015], 0.914 [0.029], 0.847 [0.027], 0.743 [0.034], respectively. After 8 Gy X-ray irradiation, the percentage of apoptosis detected by Annexin V method was 21.2+2.4%, which was significantly lower than that of the control group (33.7+2.5%).
3) After 8 Gy X-ray irradiation, the content of active caspase-3 in ARC-microNA-3CNE-2 cells was 1 and 2.33+0.26 respectively, which was significantly higher than that in control group, and the content of active caspase-8 in ARC-microNA-3CNE-2 cells was 1.65+0.17, which was significantly higher than that in control group (P 0.001).
4) After 8 Gy X-ray irradiation, the content of active caspase-3 in pEZ-M03-ARC6-10B cells was 0.62+0.14, which was significantly lower than that in control group, and the content of active caspase-8 in pEZ-M03-ARC6-10B cells was 0.73+0.11, which was significantly lower than that in control group (P 0.001).
Conclusion ARC can enhance the radioresistance of nasopharyngeal carcinoma cells and may be achieved by inhibiting endogenous and exogenous apoptotic pathways. ARC may be a promising molecular therapeutic target for increasing radiosensitivity of nasopharyngeal carcinoma.
Objective to investigate the effect of silencing or over expression of ARC on chemosensitivity of nasopharyngeal carcinoma.
Methods Nasopharyngeal carcinoma cells silenced or overexpressed ARC were treated with paclitaxel or cisplatin at different concentration gradients for 48 hours. Cell growth and proliferation were detected by CCK-8 assay. Cells treated with IC30 were detected by Annxin V assay after 24 hours. Cell apoptosis was detected by Caspase 3/8 activity assay. The Caspase3/8 activity was detected by kit.
Result
1) The survival rate of ARC-microNA-3 cells in cisplatin was significantly lower than that of the control group, and the survival curve of the experimental group was significantly lower than that of the control group (P 0.001). The proportion of apoptotic cells was 56.8 + 4.4%, which was significantly higher than that of the control group (33.02 + 3.7%) (P0.001).
2) The cell survival rate of pEZ-M03-ARC cells in cisplatin was significantly higher than that of the control group, and the cell survival curve of the experimental group was significantly higher than that of the control group (P 0.001). The IC5o value of pEZ-M03-ARC cells treated by cisplatin was (1.000.244)*10-4, which was significantly higher than that of the control group (6.397.276)*10-5 (P 0.001). The apoptosis rate of the experimental group was 2.00% by Annexin V assay. 4.8 + 2.8% was significantly lower than that of the control group (35.1 + 2.6%) (P0.001).
3) After cisplatin treatment, the ratio of active caspase-3 in ARC-microNA-3CNE-2 cells to the control group was 3.49 [0.31], and the ratio of active caspase-8 in ARC-microNA-3CNE-2 cells to the control group was 2.17 [0.20], which was significantly higher than that in the control group (P 0.001).
4) After cisplatin treatment, the ratio of active caspase-3 in pEZ-M03-ARC6-10B cells to control group was 0.44.07, and the ratio of active caspase-8 in pEZ-M03-ARC6-1OB cells to control group was 0.69.12, which was significantly lower than that in control group (P 0.001).
5) There was no significant difference in the survival rate of ARC-microNA-3 cells and control cells in paclitaxel (P 0.05); there was no significant difference in the survival rate of pEZ-M03-ARC cells and control cells in paclitaxel (P 0.05).
Conclusion ARC can enhance the chemotherapeutic resistance of nasopharyngeal carcinoma cells and may be achieved by inhibiting endogenous and exogenous apoptotic pathways. ARC is expected to be a molecular therapeutic target for increasing the chemotherapeutic sensitivity of nasopharyngeal carcinoma cells.
【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R739.63
【共引文献】
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