Expression Patterns Of MiR-7, It's Regulator And Target Gene In Nasopharygeal Carcinoma Cell Lines With Different Radiosensitivity After X-ray Radiation

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 linesOBJECTIVE: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 radiationOBJECTIVE: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 radiationOBJECTIVE: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 cellsOBJECTIVE: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.