Identification of Importin 8 (IPO8) as the most accurate reference gene for the clinicopathological analysis of lung specimens

Nguewa, Paul A.; Agorreta, Jackeline; Blanco, David; Lozano, Marı́a D.; Gómez‐Román, Javier; Sanchez, Blas A; Valles, Iñaki; Pajares, María J.; Pı́o, Rubén; Rodrí­guez, Marí­a José; Montuenga, Luis M.; Calvo, Alfonso

doi:10.1186/1471-2199-9-103

Cited by 41 publications

(47 citation statements)

References 33 publications

(43 reference statements)

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“…Previous studies have shown that GAPDH expression is increased in other human tumors and tumor-derived cell lines from colon, kidney, liver, lung and pancreas (Nguewa et al, 2008;Lau et al, 2000;Gong et al, 1996;Chang et al, 2011;Revillion et al, 2000;Yamagata et al, 1998), which complies with our results. Because of the variability of expression of GAPDH in osteosarcomas, GAPDH expression should not be used as housekeeping genes in analyses on mRNA level and protein level in osteosarcomas studies.…”

Section: Discussionsupporting

confidence: 82%

“…GAPDH might play a role in cancer pathogenesis and translocate to nucleus in response to several anti-cancer agents (Kim et al, 1999). The accumulated results indicated that GAPDH was differentially expressed in prostate, breast, lung, and cervical carcinomas under certain conditions (Nguewa et al, 2008;Lau et al, 2000;Gong et al, 1996;Chang et al, 2011;Revillion et al, 2000;Yamagata et al, 1998;Rienzo et al, 2012). However, the alteration of GAPDH gene expression has little report in osteosarcoma.…”

Section: Introductionmentioning

confidence: 93%

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Expression and significance of glyceraldehyde-3-phosphate dehydrogenase in patients with osteosarcoma

Fei¹

2012

Afr. J. Pharm. Pharmacol.

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To observe the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in patients with osteosarcoma and its relationship with surgical stage, 60 patients with osteosarcoma, including 20 cases in Stage I, 17 cases in Stage II and 23 cases in Stage III were sequentially enrolled in this study. The expression of GAPDH in osteosarcoma and osteochondroma tissues on mRNA and protein levels were measured by fluorescent quantitative polymerase chain reaction (qPCR) and immunohistochemical stain, respectively. It was found that GAPDH was significantly up-regulated in osteosarcoma in comparison to the healthy control on mRNA and protein levels (P < 0. 05). The positive rate of GAPDH osteosarcomas in Stage III were significantly higher than those in Stages I and II (P < 0. 05). These data demonstrated that the expression of GAPDH correlates with the clinical stage of osteosarcomas, which implied that over-expressions of GAPDH play an important role in the development of osteosarcoma.

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Section: Discussionsupporting

confidence: 82%

Section: Introductionmentioning

confidence: 93%

Expression and significance of glyceraldehyde-3-phosphate dehydrogenase in patients with osteosarcoma

Fei¹

2012

Afr. J. Pharm. Pharmacol.

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“…Survivin and c-Jun mRNAs were analyzed by semiquantitative PCR, using TaqMan Gene Expression Assays (Applied Biosystems). IPO8 mRNA expression was used as the endogenous control (24).…”

Section: Reverse Transcriptase-pcrmentioning

confidence: 99%

The Oncoprotein SF2/ASF Promotes Non–Small Cell Lung Cancer Survival by Enhancing Survivin Expression

Ezponda

Pajares²,

Agorreta³

et al. 2010

Clinical Cancer Research

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Purpose: SF2/ASF is a splicing factor recently described as an oncoprotein. In the present work, we examined the role of SF2/ASF in human non-small cell lung cancer (NSCLC) and analyzed the molecular mechanisms involved in SF2/ASF-related carcinogenesis.Experimental Design: SF2/ASF protein levels were analyzed in 81 NSCLC patients by immunohistochemistry. SF2/ASF downregulation cellular models were generated using small interfering RNAs, and the effects on proliferation and apoptosis were evaluated. Survivin and SF2/ASF expression in lung tumors was analyzed by Western blot and immunohistochemistry. Survival curves and log-rank test were used to identify the association between the expression of the proteins and time to progression.Results: Overexpression of SF2/ASF was found in most human primary NSCLC tumors. In vitro downregulation of SF2/ASF induced apoptosis in NSCLC cell lines. This effect was associated with a reduction in the expression of survivin, an antiapoptotic protein widely upregulated in cancer. In fact, SF2/ASF specifically bound survivin mRNA and enhanced its translation, via a mammalian target of rapamycin complex 1 (mTORC1) pathway-dependent mechanism, through the phosphorylation and inactivation of the translational repressor 4E-BP1. Moreover, SF2/ASF promoted the stability of survivin mRNA. A strong correlation was observed between the expression of SF2/ASF and survivin in tumor biopsies from NSCLC patients, supporting the concept that survivin expression levels are controlled by SF2/ASF. Furthermore, combined expression of these proteins was associated with prognosis.Conclusion: This study provides novel data on the mTORC1-and survivin-dependent mechanisms of SF2/ASF-related carcinogenic potential, and shows that SF2/ASF and survivin expression is involved in NSCLC progression. Clin Cancer Res; 16(16); 4113-25. ©2010 AACR.Regulation of mRNA metabolism (processing, export, localization, surveillance, translation, and decay) contributes more relevantly than previously suspected to the genesis of a great variety of diseases, including cancer (1-7). Modifications in the levels of mRNA processing proteins can elicit global changes in the profile of mRNA expression, affecting a number of cancer-associated genes. In lung cancer, the leading cause of cancer mortality (8), several studies have shown changes in the expression of members of two families of mRNA processing proteins: heterogeneous nuclear ribonucleoproteins (hnRNP) and serine/arginine (SR)-rich proteins (9-11). Elevated expression of hnRNP A1 and the SR protein SF2/ASF has been related to the presence of metastasis-associated alternative splicing isoforms of CD44 in a mouse model of lung cancer (10). SF2/ASF has been recently found to be upregulated in several human neoplasias, and has been described as an oncoprotein with roles in the establishment and maintenance of transformation (12). SF2/ASF plays a role in several key features of RNA metabolism, such as mRNA constitutive and alternative splicing, nuclear export, stability...

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“…A total of 15 commonly used reference genes were evaluated against these criteria, namely: ACTB, beta-2-microglobulin (B2M), GAPDH, betaglucuronidase (GUSB), hydroxymethylbilane synthase (HMBS), hypoxanthine phosphoribosyltransferase 1 (HPRT1), importin 8 (IPO8), phosphoglycerate kinase 1 (PGK1), polymerase RNA II DNA directed polypeptide A 220 kDa (POLR2A), peptidylprolyl isomerase A (PPIA), ribosomal protein large P0 (RPLP0), TATA box binding protein (TBP), transferrin receptor (TFRC), ubiquitin C (UBC), and tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta polypeptide (YWHAZ) (Fig. 1 and Table 1) [13][14][15][16][17][18][19][20][21]. The full name, NCBI Gene ID, function, and location of these genes can be found in Supplementary Table S1.…”

Section: Resultsmentioning

confidence: 99%

Identification of reference genes for qRT-PCR in human lung squamous-cell carcinoma by RNA-Seq

Zhan

Zhang

et al. 2014

ABBS

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Although the accuracy of quantitative real-time polymerase chain reaction (qRT-PCR) is highly dependent on the reliable reference genes, many commonly used reference genes are not stably expressed and as such are not suitable for quantification and normalization of qRT-PCR data. The aim of this study was to identify novel reliable reference genes in lung squamous-cell carcinoma. We used RNA sequencing (RNA-Seq) to survey the whole genome expression in 5 lung normal samples and 44 lung squamous-cell carcinoma samples. We evaluated the expression profiles of 15 commonly used reference genes and identified five additional candidate reference genes. To validate the RNA-Seq dataset, we used qRT-PCR to verify the expression levels of these 20 genes in a separate set of 100 pairs of normal lung tissue and lung squamous-cell carcinoma samples, and then analyzed these results using geNorm and NormFinder. With respect to 14 of the 15 common reference genes (B2M, GAPDH, GUSB, HMBS, HPRT1, IPO8, PGK1, POLR2A, PPIA, RPLP0, TBP, TFRC, UBC, and YWHAZ), the expression levels were either too low to be easily detected, or exhibited a high degree of variability either between lung normal and squamous-cell carcinoma samples, or even among samples of the same tissue type. In contrast, 1 of the 15 common reference genes (ACTB) and the 5 additional candidate reference genes (EEF1A1, FAU, RPS9, RPS11, and RPS14) were stably and constitutively expressed at high levels in all the samples tested. ACTB, EEF1A1, FAU, RPS9, RPS11, and RPS14 are ideal reference genes for qRT-PCR analysis of lung squamous-cell carcinoma, while 14 commonly used qRT-PCR reference genes are less appropriate in this context.

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Identification of Importin 8 (IPO8) as the most accurate reference gene for the clinicopathological analysis of lung specimens

Cited by 41 publications

References 33 publications

Expression and significance of glyceraldehyde-3-phosphate dehydrogenase in patients with osteosarcoma

Expression and significance of glyceraldehyde-3-phosphate dehydrogenase in patients with osteosarcoma

The Oncoprotein SF2/ASF Promotes Non–Small Cell Lung Cancer Survival by Enhancing Survivin Expression

Identification of reference genes for qRT-PCR in human lung squamous-cell carcinoma by RNA-Seq

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