Single-Cell RNA Sequencing Reveals Multiple Pathways and the Tumor Microenvironment Could Lead to Chemotherapy Resistance in Cervical Cancer.

BACKGROUND: Cervical cancer is one of the most common gynecological cancers worldwide. The tumor microenvironment significantly influences the therapeutic response and clinical outcome. However, the complex tumor microenvironment of cervical cancer and the molecular mechanisms underlying chemotherapy resistance are not well studied. This study aimed to comprehensively analyze cells from pretreated and chemoresistant cervical cancer tissues to generate a molecular census of cell populations. METHODS: Biopsy tissues collected from patients with cervical squamous cell carcinoma, cervical adenocarcinoma, and chronic cervicitis were subjected to single-cell RNA sequencing using the 10× Genomics platform. Unsupervised clustering analysis of cells was performed to identify the main cell types, and important cell clusters were reclustered into subpopulations. Gene expression profiles and functional enrichment analysis were used to explore gene expression and functional differences between cell subpopulations in cervicitis and cervical cancer samples and between chemoresistant and chemosensitive samples. RESULTS: A total of 24,371 cells were clustered into nine separate cell types, including immune and non-immune cells. Differentially expressed genes between chemoresistant and chemosensitive patients enriched in the phosphoinositide 3-kinase (PI3K)/AKT pathway were involved in tumor development, progression, and apoptosis, which might lead to chemotherapy resistance. CONCLUSIONS: Our study provides a comprehensive overview of the cancer microenvironment landscape and characterizes its gene expression and functional difference in chemotherapy resistance. Consequently, our study deepens the insights into cervical cancer biology through the identification of gene markers for diagnosis, prognosis, and therapy.

Oxidative stress-induced mitophagy is suppressed by the miR-106b-93-25 cluster in a protective manner.

Increased reactive oxygen species levels in the mitochondrial matrix can induce Parkin-dependent mitophagy, which selectively degrades dysfunctional mitochondria via the autolysosome pathway. Phosphorylated mitofusin-2 (MFN2), a receptor of parkin RBR E3 ubiquitin-protein ligase (Parkin), interacts with Parkin to promote the ubiquitination of mitochondrial proteins; meanwhile, the mitophagy receptors Optineurin (OPTN) and nuclear dot protein 52 (NDP52) are recruited to damaged mitochondria to promote mitophagy. However, previous studies have not investigated changes in the levels of OPTN, MFN2, and NDP52 during Parkin-mediated mitophagy. Here, we show that mild and sustained hydrogen peroxide (H2O2) stimulation induces Parkin-dependent mitophagy accompanied by downregulation of the mitophagy-associated proteins OPTN, NDP52, and MFN2. We further demonstrate that H2O2 promotes the expression of the miR-106b-93-25 cluster and that miR-106b and miR-93 synergistically inhibit the translation of OPTN, NDP52, and MFN2 by targeting their 3' untranslated regions. We further reveal that compromised phosphorylation of MYC proto-oncogene protein (c-Myc) at threonine 58 (T58) (producing an unstable form of c-Myc) caused by reduced nuclear glycogen synthase kinase-3 beta (GSK3ß) levels contributes to the promotion of miR-106b-93-25 cluster expression upon H2O2 induction. Furthermore, miR-106b-mediated and miR-93-mediated inhibition of mitophagy-associated proteins (OPTN, MFN2, and NDP52) restrains cell death by controlling excessive mitophagy. Our data suggest that microRNAs (miRNAs) targeting mitophagy-associated proteins maintain cell survival, which is a novel mechanism of mitophagy control. Thus, our findings provide mechanistic insight into how miRNA-mediated regulation alters the biological process of mitophagy.

Advantages of a 21-loci short tandem repeat method for detection of cross-contamination in human cell lines.

Cross-contamination of cell lines is a highly relevant and pervasive problem. The analysis of short tandem repeats (STR) is a simple and commercially available technique to authenticate cell lines for more than two decades. At present, STR multiple amplification kits have been developed up to 21 loci while the current STR databases only provide 9-loci STR profiles. Here, we compared the advantages of 21-loci STR methodology using the same algorithm as 9-loci method. The 21-loci method reduced the uncertainty ratio for authentications by 97.5% relative to the 9-loci method and exclude effectively false positive. We show that the additional 12 loci helped to greatly reduce sample-site marker specificity arising from genetic isolation and the occurrence of null alleles, suggesting that inclusion of additional loci in these databases will ultimately improve the efficiency and accuracy of authentication of cell lines. Taken together, we demonstrate the utility of a 21-loci method in human cells, providing a novel marker panel for use as a valuable alternative to 9-loci analyses to minimize cell line authentication errors and reduce costs due to erroneous experiments.

Advances in CRISPR-Cas systems for RNA targeting, tracking and editing.

Clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) systems, especially type II (Cas9) systems, have been widely used in gene/genome targeting. Modifications of Cas9 enable these systems to become platforms for precise DNA manipulations. However, the utilization of CRISPR-Cas systems in RNA targeting remains preliminary. The discovery of type VI CRISPR-Cas systems (Cas13) shed light on RNA-guided RNA targeting. Cas13d, the smallest Cas13 protein, with a length of only ~930 amino acids, is a promising platform for RNA targeting compatible with viral delivery systems. Much effort has also been made to develop Cas9, Cas13a and Cas13b applications for RNA-guided RNA targeting. The discovery of new RNA-targeting CRISPR-Cas systems as well as the development of RNA-targeting platforms with Cas9 and Cas13 will promote RNA-targeting technology substantially. Here, we review new advances in RNA-targeting CRISPR-Cas systems as well as advances in applications of these systems in RNA targeting, tracking and editing. We also compare these Cas protein-based technologies with traditional technologies for RNA targeting, tracking and editing. Finally, we discuss remaining questions and prospects for the future.