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Expression Patterns of miR-7, It’s Regulator and Target Gene in Nasopharygeal Carcinoma Cell Lines with Different Radiosensitivity after X-ray Radiation

Author: ChenZhiXian
Tutor: YuanYaWei
School: Southern Medical University,
Course: Radiation Therapy Oncology
Keywords: Nasopharyngeal Radiosensitivity microRNA miR-7 HoxD10 EGFR
CLC: R739.63
Type: Master's thesis
Year: 2010
Downloads: 126
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Abstract

Background: China's Guangdong, Guangxi, Fujian, Hunan and other places for the NPC-prone areas. Age of onset mostly middle-aged, some young ill. Etiology and racial predisposition, genetic factors and EB virus infection and so on, the higher the degree of malignancy of nasopharyngeal carcinoma, cervical lymph node metastasis can occur early. The main treatment of nasopharyngeal carcinoma radiotherapy, well-differentiated nasopharyngeal carcinoma cells less sensitive X-ray poor differentiation nasopharyngeal carcinoma cells, radiation resistance of cancer cells leading to poor local control rate in patients with relapsed and important factor. MicroRNA is a class of small fragments of non-coding RNA, with the target gene mRNA 3 untranslated region completely complementary pairing non-binding, so that the target gene silencing, impede protein synthesis, involved in cell proliferation, differentiation, apoptosis, tumorigenesis and DNA damage and other biological processes. Well-differentiated nasopharyngeal carcinoma cell line CNE-1 and poorly differentiated nasopharyngeal carcinoma cell line CNE-2 there are several differences in microRNA expression, including miR-7 is the most significant difference between one microRNA. MiR-7 involved in regulation of cell development and activation of the EGFR pathway, and EGFR pathway and radiosensitivity are closely related. EGFR (epidermal growth factor receptor) is a cross-glycoprotein, erbB receptor tyrosine kinase family members. EGFR ligand binding, receptor tyrosine kinase autophosphorylation, including RAS pathway, including activation of downstream signaling pathways, regulation of cell proliferation and differentiation. The same type of tumor cells, well-differentiated cells differentiated cells lower high EGFR expression, EGFR expression increased and enhanced cellular radiation resistance. Radiation exposure, the cells were transferred by promoting nuclear EGFR, start D-NHEJ repair damaged DNA, through EGFR activation of downstream signaling pathways, promote cell proliferation, resistance to jointly achieve the effects of radiation. Clinically, some cancer patients after radiation therapy, local control rate is not high, but also with high EGFR expression. HOX gene family in embryonic development, physical quantity and morphology, the development of the lumbosacral spinal cord, motor neuron differentiation plays an important regulatory role. In some organs, HOX gene expression profiles of families with specificity, primary tumors in these organs and metastasis to distant tumors, are the source of normal tissue cells with the same HOX gene expression. HOX gene family encoding DNA transcription regulatory proteins, HOX gene expression if confusion will result in hematologic malignancies or solid tumors. In tissue and organ development is completed, HoxD10 still plays an important regulatory role, which is reflected in the inhibition of angiogenesis, inhibition of tumor formation, inhibition of tumor metastasis. Although microRNA can be inhibited, but HoxD10 as transcriptional regulator encoded family of HOX genes, can be adjusted at the transcriptional level expression of microRNA. HoxD10 in breast cancer cells known to regulate the expression of miR-7. Therefore, inhibition of EGFR is to reduce the resistance of tumor cells to ionizing radiation - an important aspect, miR-7 EGFR in the regulation of the transcription process has an important role, and miR-7 HoxD10 upstream regulatory factor may also be related with radiation sensitivity, but the living body NPC cells miR-7 binding verification with HoxD10 not been reported. This study focuses on the level of gene expression from the preliminary study of nasopharyngeal carcinoma cells with different radiosensitivity X-ray radiation miR-7 and EGFR gene expression, miR-7 and HoxD10 correlation of gene expression and verify HoxD10 with miR-7's promoter sequence in NPC cells are combined in order to determine whether in NPC cells HoxD10 involved regulating the expression of miR-7. Part I: Determination of nasopharyngeal carcinoma cell line status Objective: Draw Nanfang Hospital Cancer Center nasopharyngeal carcinoma cell line CNE-1, CNE-2 growth curve, determine the logarithmic growth phase, logarithmic phase cells and detect radiation sensitivity. Methods: MTT assay, the microplate detection, derived cells growing number of days 0D different values ??of the input Excel software, draw CNE-1, CNE-2 cell growth curve; using cloning counting method, calculate the cell by X-ray radiation After surviving fraction, using GraphPad Prism 5.0 software, according to the linear quadratic model fit both cell survival curve, find the corresponding mathematical model parameters α, β, α / β values. Results: CNE-1 of the logarithmic phase 5 to 7 days, CNE-2 cells in the logarithmic phase of 3 to 5 days; CNE-1 cell radiosensitivity lower than the CNE-2 cells. Part II: X-ray radiation after nasopharyngeal carcinoma cell line CNE-1, CNE-2 and EGFR expression of miR7 Objective: To detect the different radiosensitivity of nasopharyngeal carcinoma cell line X-ray radiation, miR-7 and EGFR expression, and to explore whether there is correlation between the two expressions. Methods: Two cells were divided into control group (not radiation), 2Gy group and 8Gy group, X-ray irradiation after 10 hours, using Trizol extraction of total cellular RNA. Method-specific stem-loop RT miR-7, Oligo dT RT EGFR mRNA, specific PCR primers were designed for real-time quantitative dye France PCR, the control group CNE-1 cells as a reference sample, △ △ Ct method to get the samples miR-7 and EGFR relative number. Results: Radiation resistant cell line CNE-1 low-dose X-ray irradiation after miR-7 increased significantly, increased after high-dose irradiation is not obvious. Radiosensitive cell line CNE-2 X-ray irradiation after, miR-7 expression decreased to low doses of irradiation decreased more significantly. Radiation resistant cell line CNE-1 low-dose X-ray irradiation increased EGFR expression, and with increasing radiation dose. Radiation-sensitive cell line CNE-2 X-ray irradiation after, EGFR expression was also increased, but as the radiation dose increases. Part III: X-ray radiation after the upstream regulatory factor expression changes HoxD10 Objective: To detect the different radiosensitivity of nasopharyngeal carcinoma cell line x-ray radiation, HoxD10 of expression and its relationship with the expression of miR-7 related sex. Methods: Two cells were divided into control group (not radiation), 2Gy group and 8Gy group, x-ray irradiation after 10 hours, using Trizol extraction of total cellular RNA. Oligo dT reverse transcriptase mRNA, specific PCR primers were designed HoxD10 amplification of cDNA fragments dye method for real-time quantitative PCR, the control group CNE-1 cells as a reference sample, △ △ Ct method to get the relative number of each sample HoxD10. Results: Compared with the control group, x-ray radiation, each group HoxD10 expression were significantly decreased. Two kinds of cells by X-ray radiation, HoxD10 expression were significantly decreased. Part IV: miR-7 promoter sequence was predicted, and the control factor binding HoxD10 verification purposes: Find miR-7 within 2Kb promoter upstream area, and may regulate factor binding sites and sequences. Verify living cells HoxD10 regulatory factors and predict whether the combination of the promoter region. METHODS: PubMed search keyword or title or abstract of miR-7 literature. MiR-7 by miRbase find the location in the human chromosome, and then search through UCSC 2Kb upstream promoter region within the feature. Formaldehyde after ultrasonication cell, the cells crushed to 1000bp DNA fragment of about adding HoxD10 Protein A and antibody binding protein extract HoxD10 DNA fragments amplified initiation factor design where the PCR DNA primers extracted DNA fragment HoxD10 verify whether the binding of the DNA fragment where the promoter fragment of the same. Results: miR-7 is located upstream of the promoter region -958 to -968, -1019 to -1028 two loci, CHIP experimental results confirm HoxD10 combination with the above two points.

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