Abstract 5436: Identification of early lung cancer miRNA biomarkers using qRT-PCR and novel label-free nanoarray technology
Authors: Santos Julia M. , Joiakim Aby , Putt David A. , Herrera-Fierro Pilar , Ray Vishva , Dombokowski Alan , Chen Guoan , Beer David G. , Kim Hyesook 
Published: July 2017

Abstract
Whereas over 75% of lung cancer patients are diagnosed at an advanced or metastatic stage and their 5-year survival rate is &lt;5%, patients diagnosed at an early stage, Stage I, have ~70% 5-year survival rate. In the present study, Stage I lung cancer miRNA biomarkers were identified using qRT-PCR and novel label-free facile nanoarray technology for early lung cancer detection. Total RNA from human A549 lung and ACHN kidney cancer cell lysates and media were isolated and qRT-PCR/Taqman® analyses (two-step RT-PCR) were carried out for 8 miRNA lung cancer biomarker candidates. The 8 miRNA levels were normalized by the miR16 level (no change reported in lung cancer). Ratios of miR486/miR16 and miR29c/miR16 increased ~10-fold in A549 lung cancer cell lysates and media compared with those in ACHN kidney cells. Total RNA fractions were isolated from 11 healthy subjects, 21 Stage I and 19 Stage II/III adenocarcinoma and 5 squamous lung cancer patients and levels of the 8 miRNAs were measured by qRT-PCR/Taqman® analysis (in total, 1,026 analyses) and normalized by the miR16 level. miR486 levels were up-regulated (p=0.02) and miR203 (p=0.0005) and miR205 (p=0.041) levels were down-regulated in Stages I and II/III adenocarcinoma. Whereas miR122 and miR29c were not detected in serum samples from the majority of healthy subjects, the miRNAs were expressed in 82% and 71%, respectively, of Stage I and 90% and 100%, respectively, of Stages II/III lung cancer patients. A novel label-free facile 90 nm (diameter) miRNA nanowell technology was developed. Electrochemical analyses of the early lung cancer biomarker candidates, miR486 and miR29c, and, an internal control, miR16, were carried out using various concentrations of miRNA standards (0 to 100 fM) with biotinylated cDNA captured by streptavidin coated on the nanogold surface. The Nyquist plots showed a dose-dependent increase in impedance (-Z” kohm) and sensitivity of the miRNA nanowell electrochemical technology was ≤1 fM. The impedance level obtained with miR29c in A549 lung cancer cell media was ~2.8-fold higher (mean value ± SD, 163.9 ± 32.8 kohm) compared with ACHN kidney cancer cell media (60.8 ± 33.1 kohm) by 90 nm nanowell analysis (p=0.000019). Electrochemical analyses of a serum sample obtained from a lung cancer patient revealed that miR486 and miR29c levels were ~2-fold (p=0.0031) and 9-fold (p=0.0006) higher, respectively, compared to the pooled human control sera. This result agreed with the result obtained by qRT-PCR/Taqman® analysis of human serum samples. Our results suggest that miR486 and miR203 levels up- and down-regulated, respectively, in lung cancer serum samples are biomarkers for early (Stage I) lung cancer diagnosis. Supported by NCI SBIR Phase I contract, N261201500040C (Topic 337).
Citation Format: Julia M. Santos, Aby Joiakim, David A. Putt, Pilar Herrera-Fierro, Vishva Ray, Alan Dombokowski, Guoan Chen, David G. Beer, Hyesook Kim. Identification of early lung cancer miRNA biomarkers using qRT-PCR and novel label-free nanoarray technology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5436. doi:10.1158/1538-7445.AM2017-5436