Regulation of microRNA-7-5p and LRP6 by Epstein-Barr Virus-Encoded RNAs in Burkitt's Lymphoma Cell Line Akata

Epstein-Barr virus (EBV)-encoded small non-coding RNAs (EBERs) are abundantly expressed in various EBV-associated malignancies, and play critical roles in cell proliferation, tumorigenesis, and apoptosis resistance. However, the mechanism how EBERs regulate cell function awaits further clarification. In this study, we investigated the effect of EBERs on the expression of cellular microRNA (miRNA) and mRNA expression. To test the effect of EBERs while unaffected by other EBV genes, we used EBERs-deleted recombinant EBV infected Burkitt's lymphoma cell line (Akata(+)EBERs(-)) as well as EBV-infected (Akata(+)) and EBV uninfected (Akata(-)) cell lines. They all have the same genetic backgrounds. First, 15 different cellular miRNAs which have reverse complementary sequences to EBERs and have reported targets were selected by bioinformatics analysis. When RT-PCR was carried out for the 16 miRNAs using RNAs from Akata(+), Akata(-), and Akata(+)EBERs(-) cells, hsa-miR-7-5p was the only one showing down-regulated expression in Akata(+) than in Akata(-) and Akata(+)EBERs(-) cells. Bioinformatics and mRNA microarray analyses for Akata(+), Akata(-), and Akata(+)EBERs(-) cell lines were then carried out to predict putative targets of hsa-miR-7-5p. Among the 6 predicted targets of hsa-miR-7-5p, only low density lipoprotein receptor-related protein 6 (LRP6) was up-regulated in EBERs-expressing cells when tested by RT-PCR and Western blot. However, luciferase reporter assay showed that the 3'-UTR of LRP6 was not directly targeted by hsa-miR-7-5p. Our data suggest that both hsa-miR-7-5p and LRP6 are regulated by EBERs in Akata cells, and these genes may partly mediate the tumorigenic function of EBERs in Burkitt's lymphoma.

Expression of Epstein-Barr Virus Gene Products, Bcl-2 and p53 Proteins in Nasopharyngeal Carcinomas

The authors studied EBV genome expression in 40 conventionally processed samples of nasopharyngeal carcinomas (NPC), using in situ hybridization for EBERs and immunohistochemistry for LMP, Bcl-2 and p53 proteins. The NPCs consisted of 6 keratinizing squamous cell carcinomas (KSCs), 13 nonkeratinizing carcinomas (NKCs) and 21 undifferentiated carcinomas (UCs). The results were summarized as follows: 1) EBERs were expressed in 80.0% of all the NPCs (32/40). As for the subtypes, they were detected in 92.3% of NKCs (12/13), in 90.5% of the UCs (19/21), and in 16.7% of the KSCs (1/6). In positive cases, the nuclei of tumor cells displayed uniformly strong staining. 2) LMP was expressed in 10.0% of all the NPCs (4/40), all of which were UC. The LMP expression in the UCs was not correlated to the expression of EBERs, Bcl-2 and p53 proteins. 3) Bcl-2 protein was detected in 85.0% of all the NPCs (34/40). As for the subtypes, they were detected in 92.3% of the NKCs (12/13), in 90.5% of the UCs (19/21), and in 50.0% of the KSCs (3/6). 4) p53 protein was detected in 75.0% of all the NPCs (30/40). As for the subtypes, they were detected in 81.0% of the UCs (17/21), in 69.2% of the NKCs (9/13), and in 66.7% of the KSCs (4/6). 5) In the NPCs the expression of EBER showed a significantly positive correlation with that of p53 or Bcl-2 protein. The above results indicate that the association of EBV with NPC is chiefly with poorly differentiated and undifferentiated carcinomas. Additionally, carcinomas commonly display widespread, strong immunoreactivity of Bcl-2 and p53 proteins over tumor cells. In conclusion, these observations indicate that the EBV-association in NPC appears to contribute to the overexpression of tumor-related genes during carcinogenesis.

Epstein-Barr Viral RNA(EBERs) Expression in Conventional Malignant Lymphoma and Polymorphic Reticulosis of Upper Aerodigestive Tract

The author examined the immunophenotype and expression of Epstem-Barr virus RNA (EBERs) used in the situ hybridization technique in 20 cases of conventional malignant lymphoma and 28 cases of polymorphic reticulosis and malignant lymphoma with features of polymorphic reticulosis occured in the upper aerodigestive tract including the upper digestive tract(palatine tonsil), and upper respiratory tract(nasal cavity, paranasal sinus, nasopharynx). The results obtained were summarized as followings; 1. The favorable site of malignant lymphoma that occured in the upper aerodigestive tract was in the palatine tonsil(11 out of 20 cases, 55%), those of polymorphic reticulosis and malignant lymphoma with features of polymorphic reticulosis were nasal cavity and nasopharynx(19 out of 28 cases, 78%). 2. The immunophentype of malignant lymphoma that occured in the upper aerodigestive tract was mostly B-cell phenotype (15 out of 20 cases, 75%), and that of polymorphic reticulosis and malignant lymphoma with features of polymorphic reticulosis was predominantly T-cell phenotype(22 out of 28 cases, 79%). 3. The EBERs positivity of malignant lymphoma that occured in the upper aerodigestive tract was 25%(5 out of 20 cases), but that of polymorphic reticulosis and malignant lymphoma with features of polymorphic reticulosis was 57%(16 out of 28 cases). 4. The positive cases for EBERs revealed angiocentricity with necrosis(16 out of 21 cases, 76%), predominantly T-cell phenotype(19 out of 21 cases, 90%), and favorably involved the nasal cavity and nasopharynx(16 out of 21 cases, 76%). Based on the above results, it was concluded that polymorphic reticulosis and malignant lymphoma with features of polymorphic reticulosis that occurred in the upper respiratory tract was an EBV-positive angiocentric T-cell lymphoma favorably involving the nasal cavity and nasopharynx.