ABSTRACT
The continuing discoveries of novel classes of RNA modifications in various organisms have raised the need for improving sensitive, convenient, and reliable methods for quantifying RNA modifications. In particular, a subset of small RNAs, including microRNAs (miRNAs) and Piwi-interacting RNAs (piRNAs), are modified at their 3'-terminal nucleotides via 2'-O-methylation. However, quantifying the levels of these small RNAs is difficult because 2'-O-methylation at the RNA 3'-terminus inhibits the activity of polyadenylate polymerase and T4 RNA ligase. These two enzymes are indispensable for RNA labeling or ligation in conventional miRNA quantification assays. In this study, we profiled 3'-terminal 2'-O-methyl plant miRNAs in the livers of rice-fed mice by oxidative deep sequencing and detected increasing amounts of plant miRNAs with prolonged oxidation treatment. We further compared the efficiency of stem-loop and poly(A)-tailed RT-qPCR in quantifying plant miRNAs in animal tissues and identified stem-loop RT-qPCR as the only suitable approach. Likewise, stem-loop RT-qPCR was superior to poly(A)-tailed RT-qPCR in quantifying 3'-terminal 2'-O-methyl piRNAs in human seminal plasma. In summary, this study established a standard procedure for quantifying the levels of 3'-terminal 2'-O-methyl miRNAs in plants and piRNAs. Accurate measurement of the 3'-terminal 2'-O-methylation of small RNAs has profound implications for understanding their pathophysiologic roles in biological systems.
Subject(s)
Animals , Humans , Mice , High-Throughput Nucleotide Sequencing , Methylation , MicroRNAs/genetics , Oxidative Stress , RNA, Small Interfering/metabolism , Real-Time Polymerase Chain ReactionABSTRACT
Purpose@#Long non-coding RNAs (lncRNAs) may act as oncogenes in small-cell lung cancer (SCLC). Exosomes containing lncRNAs released from cancer-associated fibroblasts (CAF) accelerate tumorigenesis and confer chemoresistance. This study aimed to explore the action mechanism of the CAF-derived lncRNA maternally expressed gene 3 (MEG3) on cisplatin (DDP) chemoresistance and cell processes in SCLC. @*Materials and Methods@#Quantitative real-time PCR was conducted to determine the expression levels of MEG3, miR-15a-5p, and CCNE1. Cell viability and metastasis were measured by 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-h-tetrazolium bromide and invasion assays, respectively. A xenograft tumor model was developed to confirm the effect of MEG3 overexpression on SCLC progression in vivo. Relationships between miR-15a-5p and MEG3/CCNE1 were predicted using StarBase software and validated by dual luciferase reporter assay. Western blotting was used to determine protein levels. A co-culture model was established to explore the effects of exosomes on MEG3 expression in SCLC cell lines. @*Results@#MEG3 was overexpressed in SCLC tissues and cells. MEG3 silencing significantly repressed cell viability and metastasis in SCLC. High expression of MEG3 was observed in CAF-derived conditioned medium (CM) and exosomes, and promoted chemoresistance and cancer progression. Additionally, MEG3 was found to serve as a sponge of miR-15a-5p to mediate CCNE1 expression. Overexpression of miR-15a-5p and knockout of CCNE1 reversed the effects of MEG3 overexpression on cell viability and metastasis. @*Conclusion@#MEG3 lncRNA released from CAF-derived exosomes promotes DDP chemoresistance via regulation of a miR-15a-5p/CCNE1 axis. These findings may provide insight into SCLC therapy.
ABSTRACT
Objective To explore effect of 5 -aminolevulinic acid -photodynamic therapy combined with thymopentin -5 on IL -17,IL -23 expression of recurrent condylomata acuminata patients.Methods 140 patients with recurrent condylomata acuminata were randomly divided into 3 groups.53 cases in observation group were treated by 5 -aminolevulinic acid -photodynamic therapy combined with thymopentin -5,42 cases in control group 1 were treated by 5 -aminolevulinic acid -photodynamic therapy,and 45 cases in control group 2 were treated by thymopen-tin -5.24 healthy subjects were served as normal controls.IL -17,IL -23 levels were determined by enzyme linked immunosorbent assay before and after the clinical therapy.Results IL -17,IL -23 levels in the patients with recur-rent condylomata acuminata were significantly lower than those in healthy subjects(t =28.10,P <0.01;t =11.10, P <0.01).There were significant differences in IL -17,IL -23 between recurrent condylomata acuminata patients and healthy persons before treatment.There was significant difference after treatment(t =61.17,P <0.01;t =28.02, P <0.01).Conclusion 5 -aminolevulinic acid -photodynamic therapy combined with thymopentin -5 in the treat-ment of recurrent condylomata acuminata inhibited IL -17,IL -23 expression,so as to achieve therapeutic effect.