Reference Genes for Addressing Gene Expression of Bladder Cancer Cell Models under Hypoxia: A Step Towards Transcriptomic Studies

Abstract

Highly aggressive, rapidly growing tumors contain significant areas of hypoxia or anoxia as a consequence of inadequate and/or irregular blood supply. During oxygen deprivation, tumor cells withstand a panoply of adaptive responses, including a shift towards anaerobic metabolism and the reprogramming of the transcriptome. One of the major mediators of the transcriptional hypoxic response is the hypoxia-inducible factor 1 (HIF-1), whose stabilization under hypoxia acts as an oncogenic stimulus contributing to chemotherapy resistance, invasion and metastasis. Gene expression analysis by qRT-PCR is a powerful tool for cancer cells phenotypic characterization. Nevertheless, as cells undergo a severe transcriptome remodeling.in response to oxygen deficit, the precise identification of reference genes poses a significant challenge for hypoxic studies. Herein, we aim to establish the best reference genes for studying the effects of hypoxia on bladder cancer cells. Accordingly, three bladder cancer cell lines (T24, 5637, and HT1376) representative of two distinct carcinogenesis pathways to invasive cancer (FGFR3/CCND1 and E2F3/RB1) were used. Additionally, we have explored the most suitable control gene when addressing the influence of Deferoxamine Mesilate salt (DFX), an iron chelator often used to avoid the proteasomal degradation of HIF-1α, acting as an hypoxia-mimetic agent. Using bioinformatics tools (GeNorm and NormFinder), we have elected B2M and HPRT as the most stable genes for all cell lines and experimental conditions out of a panel of seven putative candidates (HPRT, ACTB, 18S, GAPDH, TBP, B2M, and SDHA). These observations set the molecular basis for future studies addressing the effect of hypoxia and particularly HIF-1α in bladder cancer cells.

Wnt11 regulates cardiac chamber development and disease during perinatal maturation

“…Rb1, a well-established suppressor of cell cycle entry, has been implicated in controlling terminal differentiation in many tissues, including the heart (38-40). Importantly, Rb1 is known to be regulated by hypoxia in cancer cells (44). Here we demonstrated that hypoxia modulates Rb1 activity, leading to chamber-specific induction of cell cycle genes in neonatal heart.…”

Posttranscriptional Regulation of LOXL1 Expression Via Alternative Splicing and Nonsense-Mediated mRNA Decay as an Adaptive Stress Response

“…Because GAPDH has been reported to be induced by stress factors, such as oxidative and mechanical stress, 44 we validated the effect of oxidative stress, UV-B radiation, and mechanical stress on the expression of LOXL1-a and LOXL1 in hTCF and hTMC using HPRT as a stable reference gene. 45 The results were, however, not different from those using GAPDH as reference gene ( Supplementary Fig. S4).…”

Tissue Morphology and Gene Expression Characterisation of Transplantable Adenocarcinoma Bearing Mice Exposed to Fluorodeoxyglucose-Conjugated Magnetic Nanoparticles

“…33 34 In addition, studies are now also defining appropriate reference gene selection within tumour tissue and cancer cell lines. [35][36][37] Therefore, the aim of the present study was to define the impact of our FDG-MNPs in their non-magnetic stimulated state on tumour and non-tumour tissue in mice bearing MAC16 adenocarcinomas in a non-magnetic field environment. Here, we establish the expression profile of commonly used reference genes in a mouse adenocarcinoma model exposed to FDG-MNPs.…”