Project description:①Background:Tuberculosis is mainly a respiratory tract infection caused by mycobacterium tuberculosis and one of the leading causes of death worldwide. According to the Global Tuberculosis Report in 2021, About a quarter of the world's population is infected with Mycobacterium tuberculosis and China is the second highest burden of TB. Although TB diagnosis and prevention techniques have become more mature, the number of TB cases is still increasing, mainly due to: the prevalence of drug-resistant tuberculosis bacteria, tuberculosis and HIV co-infection, long incubation time of mycobacterium tuberculosis difficult to early diagnosis and so on. Therefore, it is of great significance to study the pathogenesis of mycobacterium tuberculosis infection.②Method: THP-1 cells were treated with 50ng/ml PMA for 24 hours, so that THP-1 cell can be induced into macrophages. After that THP-1 macrophages were infected with mycobacterium tuberculosis H37Rv(MOI=1), which were collected and applied to RNA-sequencing. The constructed sequencing library was sequenced using an Illumina Novaseq 6000 system.
Project description:Perturbation of host physiology by intracellular Mycobacterium tuberculosis (H37Rv) can be reflected by changes in gene expression pattern of host genes. Total RNA was isolated from PMA differentiated uninfected cells or cells infected with H37Rv for 16, 48 or 90 hours and gene expression profile was obtained. There are in all 8 samples, two replcates of each 4. Two samples, namely 1D,1E (replicates of one) are control (PMA differentiated Thp-1 cells Uninfected controls). Cells were infected with H37Rv at an MOI of 1:10 and samples were collected at 16 hours, 48 hours and 90 hours post-infection.
Project description:Differentially regulated miRNA candidates in H37Rv infected THP-1 cells were analysed with respect to uninfected THP-1 reference samples. THP-1 cells are monocytes differentiated to macrophages after treatment with PMA for 48 hrs. Total RNA was isolated from infected THP-1 cells after 24 hrs of infection, cDNA was synthesized for TLDA real time PCR reaction using TaqMan MicroRNA Reverse Transcription kit and Megaplex Human Pool A and Pool B stem loop RT primers (version 3.0) as per manufacturer’s protocol. Further real time reaction was performed on QuantStudio 12K Flex Real-Time PCR System (Applied Biosystems) by using cDNA (without pre-amplification) on TLDA card A and card B (version 3.0).
Project description:To compare gene expression changes induced by infection with Mycobacterium tuberculosis (Mtb) with changes induced by purified Mtb products, we infected THP-1 cells with Mtb strain H37Rv or treated with purified Mtb products, then performed RNAseq.
Project description:Perturbation of host physiology by intracellular Mycobacterium tuberculosis (H37Rv) can be reflected by changes in gene expression pattern of host genes. Total RNA was isolated from PMA differentiated uninfected cells or cells infected with H37Rv for 16, 48 or 90 hours and gene expression profile was obtained.
Project description:Purpose: The goals of this study are to obtain the NGS-derived transcriptome profiling (RNA-seq) for THP-1 macrophages response to Mycobacterium tuberculosis (H37Rv and H37Ra) Methods: mRNA and long noncoding RNA profiles of THP-1 macrophages infected with H37Rv and H37Ra for 1, 4, 12, 24, 48 hours were generated by deep sequencing, using Illumina Hiseq3000. The sequence reads that passed quality filters were first mapped to the latest UCSC transcript set using Bowtie2 (version 2.1.0). Then the gene expression level was estimated using RSEM (RNA-Seq by Expectation Maximization, v1.2.15), for lncRNA analysis, reads were mapped to lncRNA transcript set from LNCipedia.org. The sequence reads were normalized with TMM (trimmed mean of M-values) to identify differentially expressed genes (DEGs) using the edgeR package edgeR. qRT–PCR validation was performed using SYBR Green assays. Results: Using an optimized data analysis workflow, we mapped about 20 million sequence reads per sample to the human genome (GRCh38/hg38) and identified 25,343 mRNA and 47877 long non-coding RNA transcripts. Conclusions: Our study represents the detailed analysis of transcriptomes for THP-1 macrophages response to H37Rv and H37Ra, generated by RNA-seq technology.
Project description:This study aims to identify the specific miRNA of mycobacterium tuberculosis (M.tb) infected THP-1 by next-generation sequencing, and further to explore the role of miRNA in innate immunity against M.tb infection.Comprehensive analysis of the next-generation sequencing results showed that the expression of miR-99a-5p was significantly lower in the MTB infected THP-1 cells.
Project description:Circular RNAs (circRNAs) play a critical role in pathological mechanisms of Mycobacterium tuberculosis (Mtb) and can be used as a new biomarker for active tuberculosis (ATB) diagnosis. Therefore, we identified significantly dysregulated circRNAs in ATB patients and healthy controls (HC) and explored its molecular mechanism. We found that hsa_circ_0002371 was significantly up-regulated in PBMCs of ATB patients and H37Rv- or BCG-infected THP-1 human macrophages. Functional experiments demonstrated that hsa_circ_0002371 inhibited autophagy of BCG-infected THP-1 human macrophages and promoted intracellular BCG survival rate. Mechanistically, hsa_circ_0002371 promoted the expression of hsa-miR-502-5p, and hsa_circ_0002371 overexpression-induced protective effects in BCG-infected THP-1 human macrophages was largely overturned by the inhibition of hsa-miR-502-5p. Notably, hsa-miR-502-5p inhibited autophagy via suppressing autophagy related 16 like 1 (ATG16L1) in BCG-infected macrophages and thus promoting intracellular BCG growth. In summation, hsa_circ_0002371 increased the suppression of hsa-miR-502-5p on ATG16L1 and inhibited autophagy to promote Mtb growth in macrophages. In Conclusion, our data suggested that hsa_circ_0002371 was significantly up-regulated in the PBMCs of ATB patients compared with HC. The hsa_circ_0002371/hsa-miR-502-5p/ATG16L1 axis promoted the survival of intracellular Mtb and inhibited autophagy in macrophages. Our findings suggested hsa_circ_0002371 could act as a potential diagnostic biomarker and therapeutic target.
Project description:THP-1 cells were differentiated into macrophages with 0,1 µM PMA overnight then infected for 2 days with either H37Rv, M. marinum or heat killed H37Ra. Then RNA was collected in order to quantify the inflammatory response transcription as compared with the relative uninfected controls.
Project description:Primary human monocytes were isolated from four healthy donors. Monocytes were differentiated into macrophages, infected with virulent Mycobacterium tuberculosis (Mtb), strain H37Rv, for 24 or 48 hours, at a multiplicity of infection of 5 or 10. Following infection, infected cells and time-matched uninfected controls were harvested, total RNA including small RNAs was isolated and used for next-generation small RNA sequencing. Small RNA sequencing data was processed using miRge2.0, including a novel miRge2.0-based tRF detection tool. Processed data was used to determine differential expression of microRNAs and differential production of tRNA-derived fragments (tRFs) during infection with Mtb.
