Constructing high-density tissue microarrays with a novel method and a self-made tissue-arraying instrument.

Tissue microarrays (TMAs), also called tissue chips, contain hundreds to thousands of tissue cores obtained from different tissue donor blocks. By using TMA technology, a molecular marker, such as protein, RNA or DNA, can be simultaneously examined in hundreds of different specimens under the same experimental conditions. A growing number of previous studies have introduced different methods for constructing TMAs. Many authors tried to use various methods to implant more tissue cores in a single recipient block, and most of these methods involved reducing the diameter of the tissue cores and/or the spacing between adjacent tissue cores. However, when creating TMAs, it is difficult to reduce the distance between tissue cores to zero except with extremely expensive automatic TMA arrayers. Here, we introduce a novel method to construct a high-density TMA that does not have spacing between the tissue cores. We also introduce a method for preparing a self-made tissue-arraying instrument. With this method and the tissue-arraying instrument, we successfully created a TMA containing 126 tissue cores that were 2 mm in diameter. H&E staining and immunohistochemical staining were performed on the sections cut from the TMA without any tissue spot loss. This method is easy to operate, and the materials for creating the tissue-arraying instrument are inexpensive and can be purchased anywhere. Therefore, this method can be applied in all laboratories.

PLEKHA4 Is a Prognostic Biomarker and Correlated with Immune Infiltrates in Glioma.

Objective: Gliomas are the most common and life-threatening intracranial tumors. Immune infiltration of the tumor microenvironment significantly affects tumor prognosis in glioma. Recently, PLEKHA4 was reported to be upregulated in melanoma and closely associated with tumor genesis and development, but its role in glioma is poorly understood. Our aim was to investigate the expression, functional role, and prognostic value of PLEKHA4 in glioma. Methods: The expression levels of PLEKHA4 in 33 types of cancer in the TCGA (The Cancer Genome Atlas) database were collected via the UCSC Xena browser. The clinical samples of glioma patients were downloaded from the TCGA database. Immunohistochemistry was used to verify PLEKHA4 expression in tumor tissues. We assessed the influence of PLEKHA4 on survival of glioma patients by survival module and GEPIA. Then, we downloaded datasets of glioma from TCGA and investigated the correlations between the clinical characteristics and PLEKHA4 expression using logistic regression. Moreover, we used TIMER to explore the collection of PLEKHA4 expression and immune infiltration level in glioma and to analyze cumulative survival in glioma. Gene Set Enrichment Analysis (GSEA) was performed using the TCGA dataset. Results: PLEKHA4 transcript levels were significantly upregulated in multiple cancer types, including gliomas. Moreover, immunohistochemical analysis verified that PLEKHA4 was overexpressed in gliomas compare to the corresponding normal tissues. Univariable survival and multivariate cox analysis show that increased PLEKHA4 expression significantly correlated with age, tumor grade, IDH mutation status, and 1p/19q codel status, and higher PLEKHA4 had shorter OS, DSS, and PFI. Specifically, PLEKHA4 expression level had significant positive correlations with infiltrating levels of B cell, CD4+ T cells, CD8+ T cells, macrophages, neutrophils, and DCs in glioma, and upregulation of PLEKHA4 expression was significantly related to immune cell biomarkers and immune checkpoint expression in glioma. In addition, several GO and Kyoto Encyclopedia of Genes and Genomes (KEGG) items associated with immune response, JAK STAT signal pathway, and cell cycle were significantly enriched in the high PLEKHA4 expression phenotype pathway. Conclusions: Our findings proposed that PLEKHA4 was an independent prognostic biomarker and correlated with immune infiltrates in glioma, and targeting PLEKHA4 might improve immunotherapy in glioma. Of course, these findings also need basic experiments and further clinical trials to confirm in the future.

YTHDF2 is a novel diagnostic marker of endometrial adenocarcinoma and endometrial atypical hyperplasia/ intraepithelial neoplasia.

Numerous studies show that some biomarkers are aberrantly expressed in endometrial endometrioid adenocarcinoma (EMAC) and endometrial atypical hyperplasia/endometrioid intraepithelial neoplasia (EAH/EIN) compared to endometrial benign lesions. Because of low sensitivity and/or specificity, the utility of these markers to distinguish EMAC and EAH/EIN from benign endometrial lesions is limited. YTH domain family 2 (YTHDF2) is a functional N6-methyladenosine (m6A)-specific reader protein that mainly regulates mRNA stability. Aberrant YTHDF2 expression has been reported in many cancers and plays important functions in tumorigenesis and cancer progression. However, its expression in endometrial benign and malignant lesions has not been investigated. We evaluated YTHDF2 mRNA and protein expression in EMAC and normal endometrium using the UALCAN database and validated the bioinformatic results in EMAC cells using qRT-PCR, Western blot, and immunohistochemical (IHC) staining. We found that YTHDF2 was weakly expressed in normal endometrium, benign endometrial lesions, endometrial hyperplasia without atypia, and adenomyosis. In contrast, YTHDF2 was upregulated in EAH/EIN and EMAC. These results indicate that YTHDF2 immunostaining may be a useful tool to distinguish EAH/EIN from EHWA. Finally, YTHDF2 expression can accurately assess the depth of myometrial invasion (DMI) in EMAC when EMAC coexists with adenomyosis.

CircVPS13C promotes pituitary adenoma growth by decreasing the stability of IFITM1 mRNA via interacting with RRBP1.

CircRNAs play important roles in a variety of biological processes by acting as microRNA sponges and protein scaffolds or by encoding functional proteins. However, their functions and underlying mechanisms remain largely unknown. Distinctive circRNA patterns were explored by comparing nonfunctioning pituitary adenomas (NFPAs) and normal pituitary tissues with a circRNA array. The biological functions of selected circRNAs were determined in vitro and in vivo. RNA-seq and circRNA pulldown assays were applied to investigate the underlying mechanisms. The circRNA profile of NFPAs is tremendously different from that of normal pituitary tissues. CircVPS13C is significantly upregulated in NFPA samples and cell lines. Gain- and loss-of-function experiments demonstrate that silencing circVPS13C inhibits the proliferation of pituitary tumor cells in vitro and in vivo. Mechanistically, circVPS13C silencing increases the expression of IFITM1 and subsequently activates its downstream genes involved in MAPK- and apoptosis-associated signaling pathways. Rescue experiments show that IFITM1 overexpression partly reverses the biological effects of circVPS13C. Further studies reveal that circVPS13C inhibits IFITM1 expression through a novel mechanism mainly by competitively interacting with RRBP1, a ribosome-binding protein of the endoplasmic reticulum membrane, and thereby alleviating the stability of IFITM1 mRNA. Clinically, circVPS13C expression is markedly higher in high-risk NFPA samples and is downregulated in patient serum 7 days post-transsphenoidal adenoma resection. Our findings suggest that circVPS13C is a critical regulator in the proliferation and development of NFPAs through a novel mechanism, whereby regulating mRNA stability via interacting with ribosome-binding proteins on the endoplasmic reticulum membrane.

Artesunate exerts anti-prolactinoma activity by inhibiting mitochondrial metabolism and inducing apoptosis.

BACKGROUND: Prolactinoma is the most common hormone-secreting pituitary adenoma. Dopamine receptor agonists (DAs) are effective in reducing prolactin levels and tumor mass, but some prolactinoma patients are resistant to DAs. Treating patients with DA-resistant prolactinoma is challenging. In this study, we examined the anti-prolactinoma effect of artesunate (ART), a potential new treatment option for prolactinoma, and its mechanism of action. METHODS: Cell Counting Kit-8 (CCK8) and flow cytometry were used to detect the effect of ART on the proliferation, cycle, and apoptosis of rat pituitary adenoma cell line MMQ. The subcellular localization of ART was observed using confocal fluorescence microscopy. The JC-1 mitochondrial membrane potential (MMP) detection and Seahorse assays were used to detect the effect of ART on mitochondrial function. Real-time quantitative polymerase chain reaction (RT-qPCR) and Western blot analysis were used to detect the effect of ART on the expression of prolactin (PRL) and apoptosis-related proteins. A mouse xenograft model of prolactinoma was used to detect the inhibitory effect of ART on MMQ in vivo. RESULTS: ART specifically inhibited MMQ proliferation and PRL synthesis, induced G0/G1 phase arrest and apoptosis in vitro. ART accumulated in the mitochondria of MMQ cells, inhibiting mitochondrial respiratory function and mediating apoptosis through the mitochondrial pathway. ART also inhibited proliferation and activated the apoptosis of MMQ cells in vivo. CONCLUSIONS: ART has a strong inhibitory effect on prolactinoma both in vitro and in vivo, and its effects rely on high MMP to inhibit mitochondrial metabolism and induce apoptosis. Our results provide evidence for ART as a candidate drug for the treatment of prolactinoma.