Comprehensive analysis of PTEN-related ceRNA network revealing the key pathways WDFY3-AS2 - miR-21-5p/miR-221-3p/miR-222-3p - TIMP3 as potential biomarker in tumorigenesis and prognosis of kidney renal clear cell carcinoma.

Kidney renal clear cell carcinoma (KIRC) is one of the most common malignancies, and there is still a lack of effective biomarkers for early detection and prognostic prediction. In here, we compared the characteristics of RNA sequencing data sets of KIRC samples based on the tumor suppressor gene phosphatase and tensin homolog (PTEN). The 1016 long noncoding RNAs, 48 microRNAs (miRNAs), and 2104 messenger RNAs associated with PTEN were identified and these genes were differentially expressed between tumor and paracancerous tissues. The most relevant pathway was found to be WDFY3-AS2 - miR-21-5p/miR-221-3p/miR-222-3p - TIMP3 according to the rules of competing endogenous RNA (ceRNA) regulation. WDFY3-AS2 and TIMP3 expression were positively correlated and reduced in KIRC samples, while miR-21-5p, miR-221-3p, and miR-222-3p were relatively highly expressed. The relatively low expression of WDFY3-AS2 and TIMP3 in KIRC were associated with poor prognosis in KIRC patients, while higher expression of miR-21-5p, miR-221-3p, and miR-222-3p predicted reduced survival (p < 0.05). Univariate and multivariate Cox regression analysis showed that lower expression of WDFY3-AS2 and TIMP3 was significantly related to tumor grade, tumor size, lymph node metastasis, distant metastasis, and TNM stage. The expression of TIMP3 in KIRC tissues was also verified by immunohistochemistry, and the results were consistent with our analytical data. In summary, this study constructed a new model with clinical predictive value and identified the WDFY3-AS2/TIMP3 pathway that was closely associated with the prognosis of KIRC, which could serve as a promising biomarker for the diagnosis and treatment of KIRC.

Establishment of New Genetic Markers and Methods for Sex Determination of Mouse and Human Cells using Polymerase Chain Reactions and Crude DNA Samples.

Background: The currently available methods for sexing human or mouse cells have weaknesses. Therefore, it is necessary to establish new methods. Methods: We used bioinformatics approach to identify genes that have alleles on both the X and Y chromosomes of mouse and human genomes and have a region showing a significant difference between the X and Y alleles. We then used polymerase chain reactions (PCR) followed by visualization of the PCR amplicons in agarose gels to establish these genomic regions as genetic sex markers. Results: Our bioinformatics analyses identified eight mouse sex markers and 56 human sex markers that are new, i.e. are previously unreported. Six of the eight mouse markers and 14 of the 56 human markers were verified using PCR and ensuing visualization of the PCR amplicons in agarose gels. Most of the tested and untested sex markers possess significant differences in the molecular weight between the X- and Y-derived PCR amplicons and are thus much better than most, if not all, previously-reported genetic sex markers. We also established several simple and essentially cost-free methods for extraction of crude genomic DNA from cultured cells, blood samples, and tissues that could be used as template for PCR amplification. Conclusion: We have established new sex genetic markers and methods for extracting genomic DNA and for sexing human and mouse cells. Our work may also lend some methodological strategies to the identification of new genetic sex markers for other organismal species.