Study of Hypericin effect on Long noncoding RNAs Expression pattern in MCF-۷ Breast Cancer Cells Using RNA Sequencing

Background and Aim: Long non-coding RNAs or LncRNAs (over ۲۰۰ bp) are a type of non-coding RNAs. They play an important role in central cellular processes such as transcriptional regulation and post-translational processes. In addition, LncRNAs represent potential effects on tumor suppressor pathways. On the other hand, Breast cancer is one of the most common perilous cancers among women accompanied with high mortality rates which threatens life of women in all over the world.Moreover, Hypericin as an antibiotic and antiviral agent has apoptotic, cytotoxic and suppressive effects on tumor cell lines. Therefore, this study was planned to investigate the effects of Hypericin apoptosis and toxicity on lncRNAs on MCF-۷ cell line as a human breast cell line.Methods: MCF-۷ cells cultured in DMEM medium were treated with different concentrations of hypericin for ۲۴ to ۴۸ hours. To obtain appropriate concentration of cancer cells, MTT kit was used , then cell viability was measured ELIZA reader in wavelength ۵۷۰ nm. To determine apoptosis, Annexin V/Propidium Iodide assay was applied. Purification of RNA using TRIZOL reagent was done which followed by next generation sequencing of lncRNA; using rRNA-depletion approach and the data was analyzed for lncRNAs expression by multiple softwares and paths.Results: Over ۲۰ million reads were sequenced for each sample. DE (Differential Gene Expression) analysis showed significant differential expression in ۸۰ lncRNAs between the control and treated samples. This study showed some of the most important pathways for overexpressing lncRNAs XBP۱(S) activating chaperone genes and pre-NOTCH process with DNAJC۳, SSR۱ and POGLUT۱ target genes. In addition, it was indicated that SMAD Signaling and Effects of PIP۲ hydrolysis with TGIF۱ and PRKCQ target genes were the most well-known pathways for downregulated lncRNAs.Conclusion: All of these factors increased apoptosis and decreased the cell cycle, resulting in the removal of the cancer cells.

Keywords:

Breast cancer , Hypericin , LncRNAs , MCF-۷ , RNA Sequencing , NGS.