Determination of reference genes for ovine pulmonary adenocarcinoma infected lung tissues using RNA-seq transcriptome profiling

Yang, Hui; Zhang, Liang; Liu, Shuying

doi:10.1016/j.jviromet.2020.113923

Cited by 8 publications

(6 citation statements)

References 44 publications

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“…Beta-actin (ACTB), a highly conserved cytoskeleton structural protein associated with cell growth and cell migration [5], has been considered as an endogenous housekeeping gene and a reference gene in cells and tissues for years [6][7][8]. However, emerging evidence suggests that ACTB is differentially expressed and plays a crucial role in multiple human diseases, especially cancers [9][10][11][12][13][14].…”

Section: Introductionmentioning

confidence: 99%

A pan-cancer analysis of the prognostic and immunological role of β-actin (ACTB) in human cancers

Tang

Wang

et al. 2021

Bioengineered

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Beta-actin (ACTB), a highly conserved cytoskeleton structural protein, has been regarded as a common housekeep gene and used as a reference gene for years. However, accumulating evidence indicates that ACTB is abnormally expressed in multiple cancers and hence changes the cytoskeleton to affect the invasiveness and metastasis of tumors. This study aimed to investigate the function and clinical significance of ACTB in pan-cancer. The role of ACTB for prognosis and immune regulation across 33 tumors was explored based on the datasets of gene expression omnibus and the cancer genome atlas. Differential expression of ACTB was found between cancer and adjacent normal tissues, and significant associations was found between ACTB expression and prognosis of tumor patients. In most cancers, ACTB expression was associated with immune cells infiltration, immune checkpoints and other immune modulators. Relevance between ACTB and metastasis and invasion was identified in various types of cancers by CancerSEA. Moreover, focal adhesion and actin regulation-associated pathways were included in the functional mechanisms of ACTB. The expression of ACTB was verified by quantitative real-time polymerase chain reaction. Knockdown of ACTB inhibited head and neck squamous carcinoma cell migration and invasion by NF-κB and Wnt/β-catenin pathways. Our first pan-cancer study of ACTB offers insight into the prognostic and immunological roles of ACTB across different tumors, indicating ACTB may be a potential biomarker for poor prognosis and immune infiltration in cancers, and the role of ACTB as a reference gene in cancers was challenged.

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Section: Introductionmentioning

confidence: 99%

A pan-cancer analysis of the prognostic and immunological role of β-actin (ACTB) in human cancers

Tang

Wang

et al. 2021

Bioengineered

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“…Transcriptome sequencing is an important research method for gene expression analysis and screening differentially expressed and functional genes. Notably, screening reference genes using transcriptome data is an effective experimental method for screening reference genes in non-model species ( 32 – 34 ). RPS4X and RPS6 are more stable than traditionally used housekeeping genes in the goat rumen ( 35 ), while NCBP3, SDHA , and PTPRA are more stable than traditionally used housekeeping genes in goat skin tissue ( 10 ).…”

Section: Discussionmentioning

confidence: 99%

“…Herein, GABARAP was the most stable reference gene, followed by CLTA. GABARAP has also been reported to be a more stable reference gene in ovine pulmonary adenocarcinoma ( 32 ). At the same time, CLTA is a relatively stable reference gene in melanoma samples and melanoma cell lines ( 37 ).…”

Section: Discussionmentioning

confidence: 99%

Transcriptome-based selection and validation of optimal reference genes in perirenal adipose developing of goat (Capra hircus)

et al. 2022

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Brown adipose tissue (BAT) is mainly present in young mammals and is important for maintaining body temperature in neonatal mammals because of its ability to produce non-shivering thermogenesis. There is usually a large amount of BAT around the kidneys of newborn kids, but the BAT gradually “whiting” after birth. Screening and validating appropriate reference genes is a prerequisite for further studying the mechanism of goat brown adipose tissue “whiting” during the early stages. In this study, the expression stability of 17 candidate reference genes: 12 COPS8, SAP18, IGF2R, PARL, SNRNP200, ACTG1, CLTA, GANAB, GABARAP, PCBP2, CTSB, and CD151) selected based on previous transcriptome data as new candidate reference genes, 3 (PFDN5, CTNNB1, and EIF3M) recommended in previous studies, and 2 traditional reference genes (ACTB and GAPDH) was evaluated. Real-time quantitative PCR (RT-qPCR) technology was used to detect the expression level of candidate reference genes during goat BAT “whiting”. Four algorithms: Normfinder, geNorm, ΔCt method, and BestKeeper, and two comprehensive algorithms: ComprFinder and RefFinder, were used to analyze the stability of each candidate reference genes. GABARAP, CLTA, GAPDH, and ACTB were identified as the most stable reference genes, while CTNNB1, CTSB, and EIF3M were the least stable. Moreover, two randomly selected target genes IDH2 and RBP4, were effectively normalized using the selected most stable reference genes. These findings collectively suggest that GABARAP, CLTA, GAPDH, and ACTB are relatively stable reference genes that can potentially be used for the development of perirenal fat in goats.

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“…In this experiment, there were 3 biological replicates per group. Table 1 shows the primer sequences of selected mRNA transcripts and the reference gene β-actin and GAPDH [23]. qRT-PCR was performed using 7500 Real Time System (version: 2.3.1) (Applied Biosystems, Foster City, CA, USA).…”

Section: Qrt-pcr Validation Of Rna-seq Datamentioning

confidence: 99%

Interaction between envelope protein of Jaagsiekte Sheep Retrovirus and Hippo signaling pathway is inferred from transcriptome analysis of naturally infected Ovine Pulmonary Adenomatosis

Duan

Yang

Zhang

et al. 2021

Preprint

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Background Ovine pulmonary adenomatosis (OPA) is a contagious lung epithelial tumor of sheep caused by jaagsiekte sheep retrovirus (JSRV), which causes severe economic losses for the sheep industry in the world. The specific oncogenic mechanism of JSRV is not yet clarified. Methods In this study, RNA was extracted from lung tissues of 3 naturally infected OPA cases and 3 healthy individuals for transcriptome sequencing (RNA-Seq). Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was used to confirm the sequencing data. Immunohistochemistry (IHC) and western blot (WB) were performed to confirm the signaling pathway enriched by DEGs that was activated in naturally infected OPA cases. Cell viability, wound-healing, transwell and colony formation assays were performed to assess the cell malignant transformation of sheep trophoblast cells (STCs) transformed with JSRV- env lentivirus in vitro, and then WB was performed to confirm the signaling pathway that had been validated in the lung tissues. Results A total of 366 DEGs (154 up-regulated and 212 down-regulated) were identified by RNA-Seq of lung tissues of naturally infected OPA cases and healthy individuals. GO analysis showed that 366 DEGs were significantly enriched in 178 GO terms, including 114 biological processes, 19 cellular components and 45 molecular functions. KEGG analysis showed that the DEGs mainly enriched in cell proliferation, differentiation, apoptosis and migration, such as PI3K/Akt/mTOR, MAPK and Hippo signaling pathway, and Hippo signaling pathway has never been reported in naturally infected OPA cases. qRT-PCR results of 10 DEGs which were selected randomly were consistent with RNA-Seq results. The protein expression of Hippo signaling pathway were up-regulated in naturally infected OPA lung tissues. Cell viability, wound-healing, transwell and colony formation assays confirmed that JSRV- env lentivirus caused malignant transformation of STCs and JSRV Env increased the protein expression of Hippo signaling pathway. Conclusions This research first identified the changes in the transcriptome level of naturally infected OPA lung tissues. These data confirm that the Hippo signaling pathway is involved in the mechanism of OPA, clarify the interaction between Hippo signaling pathway and JSRV Env, provide further evidence for the tumorigenic mechanism of JSRV.

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Determination of reference genes for ovine pulmonary adenocarcinoma infected lung tissues using RNA-seq transcriptome profiling

Cited by 8 publications

References 44 publications

A pan-cancer analysis of the prognostic and immunological role of β-actin (ACTB) in human cancers

A pan-cancer analysis of the prognostic and immunological role of β-actin (ACTB) in human cancers

Transcriptome-based selection and validation of optimal reference genes in perirenal adipose developing of goat (Capra hircus)

Interaction between envelope protein of Jaagsiekte Sheep Retrovirus and Hippo signaling pathway is inferred from transcriptome analysis of naturally infected Ovine Pulmonary Adenomatosis

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