(D-140) Dynamics of lncRNA H19 Transcript Variant Relative to Triple Negative Breast Cancer

Introduction:: Triple negative breast cancer (TNBC) is an extremely aggressive cancer with high growth rates and high rates of metastasis. TNBC is often diagnosed in late stages due to its aggression and the lack of efficient and effective methods of diagnosis. There are currently a limited number of tools (CEA, CA 15-3, and CA 27-29) for monitoring TNBC. We analyzed the RNAseq data deposited at the Cancer Genome Atlas database and showed that although there is no difference in lncRNA transcript abundance in the cancer tissues, lncRNA showed higher accuracy in predicting early-stage breast cancer¹. This prompt us to investigate the dynamics of lncRNA transcript variant, such as H19, which is reported to be an oncogene and a tumor suppressor under different conditions. Using specifically designed primers targeting different regions of H19, we compared the region-specific transcript abundance in normal breast epithelial, ER+ breast cancer, and ER- breast cancer, and stem cell lines. The region-specific transcript abundance highlighted possible roles of these transcript variant in tumorigenesis and transition to and from the cancer stemness.


Materials and Methods:: Human mammary gland epithelial (MCF-12), human mammary gland epithelial adenocarcinoma (MCF-7, surface display estrogen receptor ER(+)), human mammary gland epithelial-like adenocarcinoma cell line (MDS-MB-231, ER(-), a cell model for TNBC) were obtained through ATCC, Inc. Human dental pulp stem cells (DPSC) are obtained through Lonza, Inc. MCF-12, MCF-7, and MDS-MB-231 cells are cultured at 37℃, 5% CO₂ in Dulbecco's Modified Eagle's Medium (DMEM), [+] 4.5 g/L glucose, L-glutamine[-], sodium pyruvate[-], supplemented with fetal bovine serum (FBS), Gibco Antibiotic-Antimycotic, and mammary epithelial cell growth media (MEGM) supplements and growth factors. DPSC are cultured in DPSC media (Lonza, Inc). TNBC tissue samples were obtained through Prisma Health Biorepository. Cells were harvested and extracted for RNA using Zymo quick RNA isolation kit. First strand cDNA synthesis was achieved using the Invitrogen Superscript IV cDNA synthesis kit. Differential transcript abundance was calculated using Pfaffl method using GAPDH as reference gene.


Results, Conclusions, and Discussions:: H19 gene structure is illustrated in Figure 1A. Three known transcript variants are color coded. H19 codes for a non-coding RNA transcript with active biological function, including the generation of microRNA mir675. The mir675 primer set detect the junction of the mir675 precursor and the downstream sequence. Transcript 3 is a short isoform which does not include the mir675 precursor coding region. The transcript 3 primer set detect the junction of exon 2 and a distant 5’ region upstream of exon 1. Figure 1B showed the log2 differential transcript abundance of respective targeting sites in MCF-7, MDA-MB-231 (TNBC), and stem cell (DPSC, dental pulp stem cell) compared to the human mammary gland epithelial cells (MCF-12). Using the specific primer sets targeting different regions of the lncRNA H19, we found that the transcript isoform patterns of the two breast cancer cell lines, MCF-12/ER (+) and TNBC/ER (-) are both decreased at the mir675 precursor regions, indicating mir675 precursor excision occurs in both cancer types. However, the availability of the let-7 binding site in the H19 transcript is distinctly different, indicating that ER (+) and ER (-) cancer types may differ in H19 let-7 binding site. This study also characterized the H19 isoform patterns in the DPSC, a pluripotent stem cell. Our result shows all possible isoforms regions are highly abundant in the DPSCs, implying a unique role of H19 in the stemness. Tracking the H19 isoform patterns may be used as a tool for early mesenchymal to epithelial transition. This isoform pattern is also confirmed in the clinical TNBC tissue samples. This study assists in understanding the role of lncRNA transcript variant in the stemness and aggressiveness of TNBC. Additional studies are indispensable on the functions of lncRNA variants in TNBC prognosis management.


Acknowledgements (Optional): : The authors are partially supported by institutional Development Awards (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant numbers [P20GM121342 and P20GM139769]; Prisma Health Research Seed Grant, Clemson University’s Vice President for Research; Clemson University’s College of Science; Clemson University’s Creative Inquiry, and the South Carolina Governor & Joint Bond Review Committee.


References (Optional): : 1. Cancer Genome Atlas Research Network; Weinstein JN, Collisson EA, Mills GB, Shaw KR, Ozenberger BA, Ellrott K, Shmulevich I, Sander C, Stuart JM. The Cancer Genome Atlas Pan-Cancer analysis project. Nat Genet. 2013 Oct;45(10):1113-20. doi: 10.1038/ng.2764. PMID: 24071849; PMCID: PMC3919969. https://www.cancer.gov/ccg/research/genome-sequencing/tcga