Epstein-Barr virus-encoded miR-BART11-3p modulates the DUSP6-MAPK axis to promote gastric cancer cell proliferation and metastasis.

Epstein-Barr virus (EBV)-encoded miRNAs within the BamHI-A rightward transcript (BART) region are abundantly expressed in EBV-associated gastric cancer (EBVaGC), suggesting that they play roles in tumorigenesis. However, how these viral miRNAs contribute to the development of EBVaGC remains largely obscure. In this study, we found that EBV-encoded miR-BART11-3p targets 3' -UTR of dual-specificity phosphatase 6 (DUSP6) mRNA to upregulate ERK phosphorylation and downregulate JNK and p38 phosphorylation. By doing so, miR-BART11-3p promotes gastric cancer (GC) cell proliferation, migration, and invasion in vitro, and facilitates tumor growth in vivo. Restoration of DUSP6 expression reverses the tumor-promoting activity of miR-BART11-3p in AGS GC cells. Consistently, knockdown of DUSP6 ablates the antitumor effects of miR-BART11-3p inhibitors in EBV-positive GC cells. Furthermore, blocking ERK phosphorylation with trametinib inhibited the proliferation, migration, and invasion of miR-BART11-3p-expressing AGS cells. Administration of a miR-BART11-3p antagomir reduced the growth of EBV-positive xenograft tumors. Together, these findings reveal a novel mechanism by which EBV dysregulates MAPK pathways through an EBV-encoded microRNA to promote the development and progression of EBVaGC, which may be harnessed to develop new therapeutics to treat EBVaGC. IMPORTANCE The Epstein-Barr virus (EBV) is the first human tumor virus found to encode miRNAs, which within the BART region have been detected abundantly in EBV-associated gastric cancer (EBVaGC) and play various roles in promoting tumorigenesis. In our study, we observed that EBV-miR-BART11-3p promotes cell proliferation and induces migration and invasion in GC. Interestingly, we showed that miR-BART11-3p upregulates p-ERK and downregulates p-JNK and p-p38 by directly targeting 3'-UTR of dual-specificity phosphatase 6 (DUSP6). Restoration of DUSP6 rescues the effects generated by miR-BART11-3p in GC cells, and blocking ERK phosphorylation with Trametinib augments JNK and p38 phosphorylation and inhibits the effects of miR-BART11-3p-expressing AGS cells, suggesting that miR-BART11-3p promotes cell proliferation, migration, and invasion by modulating DUSP6-MAPK axis in EBVaGC. The findings presented in this study provide new mechanisms into the tumorigenesis in EBVaGC and new avenues for the development of therapeutic strategies to combat EBVaGC targeting miR-BART11-3p or phospho-ERK.

Identification of transcriptionally-active human papillomavirus integrants through nanopore sequencing reveals viable targets for gene therapy against cervical cancer.

Integration of the human papillomavirus (HPV) genome into the cellular genome is a key event that leads to constitutive expression of viral oncoprotein E6/E7 and drives the progression of cervical cancer. However, HPV integration patterns differ on a case-by-case basis among related malignancies. Next-generation sequencing technologies still face challenges for interrogating HPV integration sites. In this study, utilizing Nanopore long-read sequencing, we identified 452 and 108 potential integration sites from the cervical cancer cell lines (CaSki and HeLa) and five tissue samples, respectively. Based on long Nanopore chimeric reads, we were able to analyze the methylation status of the HPV long control region (LCR), which controls oncogene E6/E7 expression, and to identify transcriptionally-active integrants among the numerous integrants. As a proof of concept, we identified an active HPV integrant in between RUNX2 and CLIC5 on chromosome 6 in the CaSki cell line, which was supported by ATAC-seq, H3K27Ac ChIP-seq, and RNA-seq analysis. Knockout of the active HPV integrant, by the CRISPR/Cas9 system, dramatically crippled cell proliferation and induced cell senescence. In conclusion, identifying transcriptionally-active HPV integrants with Nanopore sequencing can provide viable targets for gene therapy against HPV-associated cancers.

Establishment and characterization of two Epstein-Barr virus-positive gastric cancer cell lines with epitheliotropic M81 strain undergoing distinct viral and altered cellular expression profiles.

Epstein-Barr virus (EBV)-associated gastric cancer (EBVaGC) is a distinct subtype of gastric cancer (GC) distinguished by the presence of the EBV genome and limited viral gene expression within malignant epithelial cells. EBV infection is generally thought to be a relatively late event following atrophic gastritis in carcinogenesis, which implies the heterogeneity of EBVaGC. To facilitate the study of the role of EBV in EBVaGC, we established two EBV-positive GC cell lines (AGS-EBV and HGC27-EBV) with an epitheliotropic EBV strain M81 and characterized viral and cellular gene expression profiles in comparison to SNU719, a naturally derived EBV-positive GC cell line. Like SNU719, AGS-EBV and HGC27-EBV stably maintained their EBV genomes and expressed EBV-encoded small RNAs and nuclear antigen EBNA1. Comprehensive analysis of the expression of EBV-encoded miRNAs within the BamHI-A region rightward transcript region, and the transcripts of EBV latent and lytic genes in cell lines, as well as xenografts, reveals that AGS-EBV and HGC27-EBV cells undergo distinct viral expression profiles. A very small fraction of AGS-EBV and SNU719 cells can spontaneously produce infectious progeny virions, while HGC27-EBV does not. AGS-EBV (both M81 and Akata) cells largely mimic SNU719 cells in viral gene expression profiles, and altered cellular functions and pathways perturbed by EBV infection. Phylogenetic analysis of the EBV genome shows both M81 and Akata EBV strains are closely related to clinical EBVaGC isolates. Taken together, these two newly established EBV-positive GC cell lines can serve as models to further investigate the role of EBV in different contexts of gastric carcinogenesis and identify novel therapeutics against EBVaGC.

Uncovering the Relationship Between Genes and Phenotypes Beyond the Gut in Microvillus Inclusion Disease.

Microvillus inclusion disease (MVID) is a rare condition that is present from birth and affects the digestive system. People with MVID experience severe diarrhea that is difficult to control, cannot absorb dietary nutrients, and struggle to grow and thrive. In addition, diverse clinical manifestations, some of which are life-threatening, have been reported in cases of MVID. MVID can be caused by variants in the MYO5B, STX3, STXBP2, or UNC45A gene. These genes produce proteins that have been functionally linked to each other in intestinal epithelial cells. MVID associated with STXBP2 variants presents in a subset of patients diagnosed with familial hemophagocytic lymphohistiocytosis type 5. MVID associated with UNC45A variants presents in most patients diagnosed with osteo-oto-hepato-enteric syndrome. Furthermore, variants in MYO5B or STX3 can also cause other diseases that are characterized by phenotypes that can co-occur in subsets of patients diagnosed with MVID. Recent studies involving clinical data and experiments with cells and animals revealed connections between specific phenotypes occurring outside of the digestive system and the type of gene variants that cause MVID. Here, we have reviewed these patterns and correlations, which are expected to be valuable for healthcare professionals in managing the disease and providing personalized care for patients and their families.

Progesterone-Induced miR-152 Inhibits the Proliferation of Endometrial Epithelial Cells by Downregulating WNT-1.

Progesterone (P4) is an important ovarian hormone that inhibits estrogen-dependent proliferation of endometrial epithelial cells (EECs). miR-152 has been reported to be a cell cycle regulator. In this study, we first demonstrated that P4 induced the expression of miR-152 in ovariectomized mice and Ishikawa cell. miR-152 was detected in the human endometrial cell lines that were stably transfected with P4 receptor. Results showed that P4 induced its expression through its receptor B subtype. Then, using the specific miRNA mimic and inhibitor, we proved that miR-152 impeded G1/S transition in the cell cycle of EECs and inhibited cellular proliferation via downregulating WNT-1 in mice and human endometrial cancer cell lines (Ishikawa, HEC-1-b, and KLE). miR-152 induced by P4 is an important inhibitor for the proliferation of EECs. miR-152 may be an important tumor suppressor microRNA in endometrial cancer.

[Imaging diagnosis and classification of hepatic cystic echinococcosis].

OBJECTIVE: To investigate the imaging characteristics of hepatic cystic echinococcosis in different imaging examinations and to formulate an imaging classification. METHODS: The data of pre-operative imaging examination of 2 039 cases with hepatic cystic echinococcosis, who underwent operation during the period January 1984 to December 2000, by B mode ultrasonograohy (all cases), X-ray computed tomography (909 cases), and magnetic resonance imaging (24 cases) were analyzed, being compared with the pathological findings of the resected specimens. RESULTS: Imaging examinations succeeded to identify not only the location, size, and form of hydatid cyst in liver, but also the typical pathomorphology of complications. The hepatic echinococcosis can be divided into seven imaging-diagnostic patterns: solitary cyst (1 625 cases, 79.70%), multiple cysts (414 cases, 20.30%), primary hydatid cyst with daughter cysts (1 114 cases, 54.63%), calcified cyst (186 cases, 9.12%), consolidated cyst (28 cases, 1.37%), cyst with infection (391 cases, 19.18%), and ruptured cyst (298 cases, 14.62%). CONCLUSION: A new diagnostic system of hepatic echinococcosis has been formulated based on imaging examination and by reference to clinical manifestation. It helps select appropriate operation program.

[Imaging diagnosis of hepatic alveolar echinococcosis].

OBJECTIVE: To study the characteristics of hepatic alveolar echinococcosis in B mode ultrasonography (BU), computerized tomography (CT), and magnetic resonance imaging (MRI) so as to increase the pre-operative diagnostic rate. METHODS: The data of forty-six cases with hepatic alveolar echinococcosis who underwent operation during the period January 1984 to December 2000 were analyzed and compared with the pathological findings of the resected specimens. All of the cases were examined by BU and CT, and 12 of them were examined by MIR. RESULTS: The imaging characteristics of the 46 cases were divided into three types: infiltration (41 cases, 89.1%), calcification (39 cases, 84.8%), and cavity with liqueficaction (37 cases, 80.4%). Nine imaging signs were found to be specific to hepatic alveolar echinococcosis and of diagnostic significance: halo belt sign, calcification sign, annual ring sign, invacuation sign, cavity with liqueficaction sign, grotto sign, peninsula sign, alveolar sign. With these signs, the diagnostic accuracy rate reached 97.7% in this series. CONCLUSION: There are nine imaging diagnostic signs specific to hepatic alveolar echinococcosis. They help detect the location, pathology of this disease and determine operation program. Identification of only one of them is adequate for diagnosis of hepatic alveolar echinococcosis.

[Reexamination of specific antibodies in sera of cystic echinococccosis patients with IgG negative seroresponse].

OBJECTIVE: To explore the factors of false negative antibody response in patients with echinococcosis granulosus (Eg) for improving immunodiagnosis. METHODS: Indirect ELISA and sandwich ELISA were used to detect the specific antibody of IgG subclass (IgG1, IgG2, IgG3, IgG4) and IgA, IgM, IgE, as well as circulating antigen (CAg) and immunocomplex (CIC) in sera of Eg patients with negative response of total IgG. RESULTS: Among 42 sera with IgG negative seroresponse, 32 were positive with IgG subclass, IgA, IgM or IgE antibody, 10 were negative in all 7 kinds of antibody response. The detection rate of specific IgG1, IgG4, IgA, IgM and IgE was 42.9%, 11.9%, 28.6%, 26.2% and 21.4% respectively, being significantly higher than in sera of the control. IgM level in children was higher than that in adults. IgG subclass in patients with liver Eg was higher than that of pulmonary hydatidosis, when testing with IgG1 combined one or two of the other six Ig antibodies. The highest positive rate (64.3%) was seen in IgG1 + IgA + IgM antibody system. The positive rate of CAg and CIC in IgG negative patients was 28.57% and 30.95%, respectively. CONCLUSION: The factors involved with seronegative response of total IgG in Eg patients might be low specific IgG, variant Ig antibody expression and formation of CIC. Combined detection of IgG1 + IgA + IgM could enhance the sensitivity of serological tests in Eg patients.