Dynamic altruistic cooperation within breast tumors.

BACKGROUND: Social behaviors such as altruism, where one self-sacrifices for collective benefits, critically influence an organism's survival and responses to the environment. Such behaviors are widely exemplified in nature but have been underexplored in cancer cells which are conventionally seen as selfish competitive players. This multidisciplinary study explores altruism and its mechanism in breast cancer cells and its contribution to chemoresistance. METHODS: MicroRNA profiling was performed on circulating tumor cells collected from the blood of treated breast cancer patients. Cancer cell lines ectopically expressing candidate miRNA were used in co-culture experiments and treated with docetaxel. Ecological parameters like relative survival and relative fitness were assessed using flow cytometry. Functional studies and characterization performed in vitro and in vivo include proliferation, iTRAQ-mass spectrometry, RNA sequencing, inhibition by small molecules and antibodies, siRNA knockdown, CRISPR/dCas9 inhibition and fluorescence imaging of promoter reporter-expressing cells. Mathematical modeling based on evolutionary game theory was performed to simulate spatial organization of cancer cells. RESULTS: Opposing cancer processes underlie altruism: an oncogenic process involving secretion of IGFBP2 and CCL28 by the altruists to induce survival benefits in neighboring cells under taxane exposure, and a self-sacrificial tumor suppressive process impeding proliferation of altruists via cell cycle arrest. Both processes are regulated concurrently in the altruists by miR-125b, via differential NF-κB signaling specifically through IKKß. Altruistic cells persist in the tumor despite their self-sacrifice, as they can regenerate epigenetically from non-altruists via a KLF2/PCAF-mediated mechanism. The altruists maintain a sparse spatial organization by inhibiting surrounding cells from adopting the altruistic fate via a lateral inhibition mechanism involving a GAB1-PI3K-AKT-miR-125b signaling circuit. CONCLUSIONS: Our data reveal molecular mechanisms underlying manifestation, persistence and spatial spread of cancer cell altruism. A minor population behave altruistically at a cost to itself producing a collective benefit for the tumor, suggesting tumors to be dynamic social systems governed by the same rules of cooperation in social organisms. Understanding cancer cell altruism may lead to more holistic models of tumor evolution and drug response, as well as therapeutic paradigms that account for social interactions. Cancer cells constitute tractable experimental models for fields beyond oncology, like evolutionary ecology and game theory.

Advances in quantifying circulatory microRNA for early disease detection.

Precision preventive healthcare aims to improve patient health by integrating preventive measures with early disease detection for timely intervention with precision medicine. Key to the delivery of preventive healthcare is the clinical adoption of novel assays that enable early disease detection. Such assays, typically based on biomarkers such as microRNAs (miRNAs) from liquid biopsy or excreta, are entering clinical practice after years of clinical development and validation. In this review, we discuss the clinical utility and validation of miRNA-based molecular diagnostics for early disease detection through large-cohort studies and key considerations for developing multi-analyte clinical assays. We also highlight recent advances in the ongoing development of integrated PCR-free miRNA detection systems for point-of-care testing.

Development and validation of a circulating microRNA panel for the early detection of breast cancer.

BACKGROUND: Mammography is widely used for breast cancer screening but suffers from a high false-positive rate. Here, we perform the largest comprehensive, multi-center study to date involving diverse ethnic groups, for the identification of circulating miRNAs for breast cancer screening. METHODS: This study had a discovery phase (n = 289) and two validation phases (n = 374 and n = 379). Quantitative PCR profiling of 324 miRNAs was performed on serum samples from breast cancer (all stages) and healthy subjects to identify miRNA biomarkers. Two-fold cross-validation was used for building and optimising breast cancer-associated miRNA panels. An optimal panel was validated in cohorts with Caucasian and Asian samples. Diagnostic ability was evaluated using area under the curve (AUC) analysis. RESULTS: The study identified and validated 30 miRNAs dysregulated in breast cancer. An optimised eight-miRNA panel showed consistent performance in all cohorts and was successfully validated with AUC, accuracy, sensitivity, and specificity of 0.915, 82.3%, 72.2% and 91.5%, respectively. The prediction model detected breast cancer in both Caucasian and Asian populations with AUCs ranging from 0.880 to 0.973, including pre-malignant lesions (stage 0; AUC of 0.831) and early-stage (stages I-II) cancers (AUC of 0.916). CONCLUSIONS: Our panel can potentially be used for breast cancer screening, in conjunction with mammography.

The Application of Magnetic-Controlled Capsule Gastroscopy in Patients Refusing C-EGD: A Single-Center 5-Year Observational Study.

BACKGROUND AND AIMS: Screening for gastric diseases in symptomatic outpatients with conventional esophagogastroduodenoscopy (C-EGD) is expensive and has poor compliance. We aimed to explore the efficiency and safety of magnetic-controlled capsule gastroscopy (MCCG) in symptomatic outpatients who refused C-EGD. METHODS: We performed a retrospective study of 76794 consecutive symptomatic outpatients from January 2014 to October 2019. A total of 2318 adults (F/M = 1064/1254) in the MCCG group who refused C-EGD were matched with adults in the C-EGD group using propensity-score matching (PSM). The detection rates of abnormalities were analyzed to explore the application of MCCG in symptomatic patients. RESULTS: Our study demonstrated a prevalence of gastric ulcers (GUs) in patients with functional dyspepsia- (FD-) like symptoms of 8.14%. The detection rate of esophagitis and Barrett's esophagus was higher in patients with typical gastroesophageal reflux disease (GERD) symptoms than in patients in the other four groups (P < 0.01). The detection rates of gastric ulcers in the five groups (abdominal pain, bloating, heartburn, follow-up, and bleeding) were significantly different (P = 0.015). The total detection rate of gastric ulcers in symptomatic patients was 9.7%. A total of 7 advanced carcinomas were detected by MCCG and confirmed by endoscopic or surgical biopsy. The advanced gastric cancer detection rate was not significantly different between the MCCG group and the C-EGD matched group in terms of nonhematemesis GI bleeding (2 vs. 2, P = 1.00). In addition, the overall focal lesion detection rate in the MCCG group was superior to that in the C-EGD matched group (224 vs. 184, P = 0.038). MCCG gained a clinically meaningful small bowel diagnostic yield of 54.8% (17/31) out of 31 cases of suspected small bowel bleeding. No patient reported capsule retention at the two-week follow-up. CONCLUSION: MCCG is well tolerated, safe, and technically feasible and has a considerable diagnostic yield. The overall gastric diagnostic yield of gastric focal lesions with MCCG was comparable to that with C-EGD. MCCG offered a supplementary diagnosis in patients who had a previously undiagnostic C-EGD, indicating that MCCG could play an important role in the routine monitoring and follow-up of outpatient. MCCG shows its safety and efficiency in symptomatic outpatient applications.

Development of a microRNA Panel for Classification of Abnormal Mammograms for Breast Cancer.

Mammography is extensively used for breast cancer screening but has high false-positive rates. Here, prospectively collected blood samples were used to identify circulating microRNA (miRNA) biomarkers to discriminate between malignant and benign breast lesions among women with abnormal mammograms. The Discovery cohort comprised 72 patients with breast cancer and 197 patients with benign breast lesions, while the Validation cohort had 73 and 196 cancer and benign cases, respectively. Absolute expression levels of 324 miRNAs were determined using RT-qPCR. miRNA biomarker panels were identified by: (1) determining differential expression between malignant and benign breast lesions, (2) focusing on top differentially expressed miRNAs, and (3) building panels from an unbiased search among all expressed miRNAs. Two-fold cross-validation incorporating a feature selection algorithm and logistic regression was performed. A six-miRNA biomarker panel identified by the third strategy, had an area under the curve (AUC) of 0.785 and 0.774 in the Discovery and Validation cohorts, respectively, and an AUC of 0.881 when differentiating between cases versus those with benign lesions or healthy individuals with normal mammograms. Biomarker panel scores increased with tumor size, stage and number of lymph nodes involved. Our work demonstrates that circulating miRNA signatures can potentially be used with mammography to differentiate between patients with malignant and benign breast lesions.

Multiomics analyses of cytokines, genes, miRNA, and regulatory networks in human mesenchymal stem cells expanded in stirred microcarrier-spinner cultures.

Mesenchymal stem cells (MSCs) are of great clinical interest as a form of allogenic therapy due to their excellent regenerative and immunomodulatory effects for various therapeutic indications. Stirred suspension bioreactors using microcarriers (MC) have been used for large-scale production of MSCs compared to planar cultivation systems. Previously, we have demonstrated that expansion of MSCs in MC-spinner cultures improved chondrogenic, osteogenic, and cell migration potentials as compared to monolayer-static cultures. In this study, we sought to address this by analyzing global gene expression patterns, miRNA profiles and secretome under both monolayer-static and MC-spinner cultures in serum-free medium at different growth phases. The datasets revealed differential expression patterns that correlated with potentially improved MSC properties in cells from MC-spinner cultures compared to those of monolayer-static cultures. Transcriptome analysis identified a unique expression signature for cells from MC-spinner cultures, which correlated well with miRNA expression, and cytokine secretion involved in key MSC functions. Importantly, MC-spinner cultures and conditioned medium showed increased expression of factors that possibly enhance pathways of extracellular matrix dynamics, cellular metabolism, differentiation potential, immunoregulatory function, and wound healing. This systematic analysis provides insights for the efficient optimization of stem cell bioprocessing and infers that MC-based bioprocess manufacturing could improve post-expansion cellular properties for stem cell therapies.

Development and validation of a serum microRNA biomarker panel for detecting gastric cancer in a high-risk population.

OBJECTIVE: An unmet need exists for a non-invasive biomarker assay to aid gastric cancer diagnosis. We aimed to develop a serum microRNA (miRNA) panel for identifying patients with all stages of gastric cancer from a high-risk population. DESIGN: We conducted a three-phase, multicentre study comprising 5248 subjects from Singapore and Korea. Biomarker discovery and verification phases were done through comprehensive serum miRNA profiling and multivariant analysis of 578 miRNA candidates in retrospective cohorts of 682 subjects. A clinical assay was developed and validated in a prospective cohort of 4566 symptomatic subjects who underwent endoscopy. Assay performance was confirmed with histological diagnosis and compared with Helicobacter pylori (HP) serology, serum pepsinogens (PGs), 'ABC' method, carcinoembryonic antigen (CEA) and cancer antigen 19-9 (CA19-9). Cost-effectiveness was analysed using a Markov decision model. RESULTS: We developed a clinical assay for detection of gastric cancer based on a 12-miRNA biomarker panel. The 12-miRNA panel had area under the curve (AUC)=0.93 (95% CI 0.90 to 0.95) and AUC=0.92 (95% CI 0.88 to 0.96) in the discovery and verification cohorts, respectively. In the prospective study, overall sensitivity was 87.0% (95% CI 79.4% to 92.5%) at specificity of 68.4% (95% CI 67.0% to 69.8%). AUC was 0.848 (95% CI 0.81 to 0.88), higher than HP serology (0.635), PG 1/2 ratio (0.641), PG index (0.576), ABC method (0.647), CEA (0.576) and CA19-9 (0.595). The number needed to screen is 489 annually. It is cost-effective for mass screening relative to current practice (incremental cost-effectiveness ratio=US$44 531/quality-of-life year). CONCLUSION: We developed and validated a serum 12-miRNA biomarker assay, which may be a cost-effective risk assessment for gastric cancer. TRIAL REGISTRATION NUMBER: This study is registered with ClinicalTrials.gov (Registration number: NCT04329299).

Molecular exploration of paediatric intracranial germinomas from multi-ethnic Singapore.

BACKGROUND: Germinomas (IG) account for up to 50% of all intracranial germ cell tumours. These tumours are reputed to be more prevalent in Oriental populations in comparison to Western cohorts. Biological characteristics of IG in other ethnic groups are unknown. Singapore is a multi-ethnic country with diverse cultures. Owing to inter-racial heterogeneity, the authors hypothesize there are molecular differences between paediatric IG patients in our local population. The aims of this study are exploratory: firstly, to identify molecular characteristics in this tumour type and circulating CSF unique to different racial cohorts; and next, to corroborate our findings with published literature. METHODS: This is a single-institution, retrospective study of prospectively collected data. Inclusion criteria encompass all paediatric patients with histologically confirmed IG. Excess CSF and brain tumour tissues are collected for molecular analysis. Tumour tissues are subjected to a next generation sequencing (NGS) targeted panel for KIT and PDGRA. All CSF samples are profiled via a high-throughput miRNA multiplexed workflow. Results are then corroborated with existing literature and public databases. RESULTS: In our cohort of 14 patients, there are KIT exon variants in the tumour tissues and CSF miRNAs corroborative with published studies. Separately, there are also KIT exon variants and miRNAs not previously highlighted in IG. A subgroup analysis demonstrates differential CSF miRNAs between Chinese and Malay IG patients. CONCLUSION: This is the first in-depth molecular study of a mixed ethnic population of paediatric IGs from a Southeast Asian cohort. Validation studies are required to assess the relevance of novel findings in our study.

Development of a serum miRNA panel for detection of early stage non-small cell lung cancer.

Minimally invasive testing for early detection of lung cancer to improve patient survival is a major unmet clinical need. This study aimed to develop and validate a serum multi-microRNA (multimiR) panel as a minimally invasive test for early detection of nonsmall cell lung cancer (NSCLC) regardless of smoking status, gender, and ethnicity. Our study included 744 NSCLC cases and 944 matched controls, including smokers and nonsmokers, male and female, with Asian and Caucasian subjects. Using RT-qPCR and a tightly controlled workflow, we quantified the absolute expression of 520 circulating microRNAs (miRNAs) in a Chinese cohort of 180 early stage NSCLC cases and 216 healthy controls (male smokers). Candidate biomarkers were verified in two case-control cohorts of 432 Chinese and 218 Caucasians, respectively (including females and nonsmokers). A multimiR panel for NSCLC detection was developed using a twofold cross-validation and validated in three additional Asian cohorts comprising 642 subjects. We discovered 35 candidate miRNA biomarkers, verified 22 of them, and developed a five-miR panel that detected NSCLC with area under curve (AUC) of 0.936-0.984 in the discovery and verification cohorts. The panel was validated in three independent cohorts with AUCs of 0.973, 0.916, and 0.917. The sensitivity of five-miR test was 81.3% for all stages, 82.9% for stages I and II, and 83.0% for stage I NSCLC, when the specificity is at 90.7%. We developed a minimally invasive five-miR serum test for detecting early stage NSCLC and validated its performance in multiple patient cohorts independent of smoking status, gender, and ethnicity.

Nano-topology guided neurite outgrowth in PC12 cells is mediated by miRNAs.

MicroRNAs (miRNAs) are master regulators of gene expression at post-transcriptional level. The present study investigated the involvement of miRNAs in topological guidance of neurite outgrowth in an NGF treated PC12 cell model cultured on nano-patterned polyethylene terephthalate (PET) substrates fabricated with interference lithography. The expressions of 38 neuronal miRNAs were measured and 3 were found to be differentially regulated during topological guidance of neurite outgrowth. Altering the intracellular levels of these miRNAs disrupted the orderly growth of neurite along nano-patterned substrate. Our results showed miRNAs to be versatile regulators and their involvement should be thoroughly investigated for better understanding of biological processes. FROM THE CLINICAL EDITOR: In this basic science study, strong evidence was found that topological guidance is only one factor, and miRNA-s regulate axonal outgrowth from neurites. Nano-patterned polyethylene terephthalate substrates were used for the study, fabricated using interference lithography. Further studies of this biologically relevant process may pave the way to clinically useful axonal regrowth and axonal guidance methods.

c-Jun N-terminal kinase in synergistic neurite outgrowth in PC12 cells mediated through P90RSK.

BACKGROUND: Synergistic multi-ligand treatments that can induce neuronal differentiation offer valuable strategies to regulate and modulate neurite outgrowth. Whereas the signaling pathways mediating single ligand-induced neurite outgrowth, such as Akt, extracellular signal-regulated kinase (Erk), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (P38), have been extensively studied, the mechanisms underlying multi-ligand synergistic neurite outgrowth are poorly understood. In an attempt to gain insight into synergistic neurite outgrowth, PC12 cells were treated with one of three combinations: pituitary adenylate cyclase-activating peptide (PACAP) with epidermal growth factor (EP), basic fibroblast growth factor (FP), or nerve growth factor (NP) and then challenged with the appropriate kinase inhibitors to assess the signaling pathways involved in the process. RESULTS: Response surface analyses indicated that synergistic neurite outgrowth was regulated by distinct pathways in these systems. Synergistic increases in the phosphorylation of Erk and JNK, but not Akt or P38, were observed with the three growth factor-PACAP combinations. Unexpectedly, we identified a synergistic increase in JNK phosphorylation, which was involved in neurite outgrowth in the NP and FP, but not EP, systems. Inhibition of JNK using the SP600125 inhibitor reduced phosphorylation of 90 kDa ribosomal S6 kinase (P90RSK) in the NP and FP, but not EP, systems. This suggested the involvement of P90RSK in mediating the differential effects of JNK in synergistic neurite outgrowth. CONCLUSIONS: Taken together, these findings reveal the involvement of distinct signaling pathways in regulating neurite outgrowth in response to different synergistic growth factor-PACAP treatments. Our findings demonstrate a hitherto unrecognized mechanism of JNK-P90RSK in mediating synergistic neurite outgrowth induced by the co-treatment of growth factors and PACAP.

GDNF family ligand dependent STAT3 activation is mediated by specific alternatively spliced isoforms of GFRα2 and RET.

Neurturin (NRTN), a member of the GDNF family of ligands (GFL), is currently investigated in a series of clinical trials for Parkinson's disease. NRTN signals through its cognate receptor GFRα2 and co-receptor RET to induce neurite outgrowth, but the underlying mechanism remains to be better understood. STAT3 was previously shown to be activated by oncogenic RET, independent of ligand and GFRα. In this study, we demonstrated that NRTN induced serine(727) but not tyrosine(705) phosphorylation of STAT3 in primary cortical neuron and neuronal cell lines. Remarkably, STAT3 phosphorylation was found to be mediated specifically by GFRα2c and RET9 isoforms. Furthermore, serine but not tyrosine dominant negative mutant of STAT3 impaired NRTN induced neurite outgrowth, indicative of the role of STAT3 as a downstream mediator of NRTN function. Similar to NGF, the NRTN induced P-Ser-STAT3 was localized to the mitochondria but not to the nucleus. Mitochondrial STAT3 was further found to be intimately involved in NRTN induced neurite outgrowth. Collectively, these findings demonstrated the hitherto unrecognized and novel role of specific GFRα2 and RET isoforms in mediating NRTN activation of STAT3 and the transcription independent mechanism whereby the mitochondria localized P-Ser-STAT3 mediated NRTN induced neurite outgrowth.

Identification of MET genomic amplification, protein expression and alternative splice isoforms in neuroblastomas.

BACKGROUND: Crizotinib, a dual anaplastic lymphoma kinase (ALK) and mesenchymal-epithelial transition (MET) tyrosine kinase inhibitor, is currently being evaluated for the treatment of neuroblastoma. Its effects are thought to be mediated mainly via its activity against ALK. Although MET genomic/protein expression status might conceivably affect crizotinib efficacy, this issue has hitherto not received attention in neuroblastomas. AIMS/METHODS: MET genomic and protein expression status was characterised by silver in situ hybridisation and immunohistochemistry (IHC) respectively, in a cohort of 54 neuroblastoma samples. MET splice isoforms were characterised in 15 of these samples by quantitative PCR. RESULTS: One case (1/54; prevalence 1.85%) displayed MET genomic amplification, while another case (1/54; prevalence 1.85%) displayed strong membranous MET protein expression (IHC score 3+). Alternative exon 10-deleted and exon 14-deleted MET splice isoforms were identified. CONCLUSIONS: MET amplification and protein expression, although low in prevalence, are present in neuroblastomas. This has implications when crizotinib is employed as a therapeutic agent in neuroblastomas. Additionally, the existence of alternatively spliced MET isoforms may have clinical and biological implications in neuroblastomas.

Cyclic AMP signalling through PKA but not Epac is essential for neurturin-induced biphasic ERK1/2 activation and neurite outgrowths through GFRα2 isoforms.

Cyclic AMP (cAMP) and neurotrophic factors are known to interact closely to promote neurite outgrowth and neuronal regeneration. Glial cell line-derived neurotrophic factor (GDNF) and its family member neurturin (NTN) transduce signal through a multi-component receptor complex consisting of GDNF family receptor alpha 2 (GFRα2) and Ret receptor tyrosine kinase. Neurons from GFRα2-deficient mice do not promote axonal initiation when stimulated by NTN, consistent with the role of GFRα2 in neuronal outgrowth. Multiple alternatively spliced isoforms of GFRα2 are known to be expressed in the nervous system. GFRα2a and GFRα2c but not GFRα2b promoted neurite outgrowth. It is currently unknown if cAMP signalling is differentially regulated by these isoforms. In this study, NTN activation of GFRα2a and GFRα2c but not GFRα2b induced biphasic ERK1/2 activation and phosphorylation of the major cAMP target CREB. Interestingly, inhibition of cAMP signalling significantly impaired GFRα2a and GFRα2c-mediated neurite outgrowth while cAMP agonists cooperated with GFRα2b to induce neurite outgrowth. Importantly, the specific cAMP effector PKA but not Epac was essential for NTN-induced neurite outgrowth, through transcription and translation-dependent activation of late phase ERK1/2. Taken together, these results not only demonstrated the essential role of cAMP-PKA signalling in NTN-induced biphasic ERK1/2 activation and neurite outgrowth, but also suggested cAMP-PKA signalling as a hitherto unrecognized underlying mechanism contributing to the differential neuritogenic activities of GFRα2 isoforms.

Mitochondrial localized STAT3 is involved in NGF induced neurite outgrowth.

BACKGROUND: Signal transducer and activator of transcription 3 (STAT3) plays critical roles in neural development and is increasingly recognized as a major mediator of injury response in the nervous system. Cytokines and growth factors are known to phosphorylate STAT3 at tyrosine(705) with or without the concomitant phosphorylation at serine(727), resulting in the nuclear localization of STAT3 and subsequent transcriptional activation of genes. Recent evidence suggests that STAT3 may control cell function via alternative mechanisms independent of its transcriptional activity. Currently, the involvement of STAT3 mono-phosphorylated at residue serine(727) (P-Ser-STAT3) in neurite outgrowth and the underlying mechanism is largely unknown. PRINCIPAL FINDINGS: In this study, we investigated the role of nerve growth factor (NGF) induced P-Ser-STAT3 in mediating neurite outgrowth. NGF induced the phosphorylation of residue serine(727) but not tyrosine(705) of STAT3 in PC12 and primary cortical neuronal cells. In PC12 cells, serine but not tyrosine dominant negative mutant of STAT3 was found to impair NGF induced neurite outgrowth. Unexpectedly, NGF induced P-Ser-STAT3 was localized to the mitochondria but not in the nucleus. Mitochondrial STAT3 was further found to be intimately involved in NGF induced neurite outgrowth and the production of reactive oxygen species (ROS). CONCLUSION: Taken together, the findings herein demonstrated a hitherto unrecognized novel transcription independent mechanism whereby the mitochondria localized P-Ser-STAT3 is involved in NGF induced neurite outgrowth.

Normalization with genes encoding ribosomal proteins but not GAPDH provides an accurate quantification of gene expressions in neuronal differentiation of PC12 cells.

BACKGROUND: Gene regulation at transcript level can provide a good indication of the complex signaling mechanisms underlying physiological and pathological processes. Transcriptomic methods such as microarray and quantitative real-time PCR require stable reference genes for accurate normalization of gene expression. Some but not all studies have shown that housekeeping genes (HGKs), beta-actin (ACTB) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which are routinely used for normalization, may vary significantly depending on the cell/tissue type and experimental conditions. It is currently unclear if these genes are stably expressed in cells undergoing drastic morphological changes during neuronal differentiation. Recent meta-analysis of microarray datasets showed that some but not all of the ribosomal protein genes are stably expressed. To test the hypothesis that some ribosomal protein genes can serve as reference genes for neuronal differentiation, a genome-wide analysis was performed and putative reference genes were identified based on stability of expressions. The stabilities of these potential reference genes were then analyzed by reverse transcription quantitative real-time PCR in six differentiation conditions. RESULTS: Twenty stably expressed genes, including thirteen ribosomal protein genes, were selected from microarray analysis of the gene expression profiles of GDNF and NGF induced differentiation of PC12 cells. The expression levels of these candidate genes as well as ACTB and GAPDH were further analyzed by reverse transcription quantitative real-time PCR in PC12 cells differentiated with a variety of stimuli including NGF, GDNF, Forskolin, KCl and ROCK inhibitor, Y27632. The performances of these candidate genes as stable reference genes were evaluated with two independent statistical approaches, geNorm and NormFinder. CONCLUSIONS: The ribosomal protein genes, RPL19 and RPL29, were identified as suitable reference genes during neuronal differentiation of PC12 cells, regardless of the type of differentiation conditions. The combination of these two novel reference genes, but not the commonly used HKG, GAPDH, allows robust and accurate normalization of differentially expressed genes during PC12 differentiation.