Background – Pulmonary fibrosis is a rare disease characterised by scarring of the lung. While new anti-fibrotic drugs offer hope of slowing disease progression, lung transplant remains the only effective cure. In pulmonary fibrosis, type II alveolar epithelial (AT2) cells play a key role in driving the disease pathogenesis, however the exact mechanisms whereby injured AT2 cells initiate and maintain the fibrotic process are poorly understood. Non-coding RNAs, and in particular microRNAs (miRNAs), are thought to be involved in disease pathogenesis but little is known about their precise role.
Hypothesis – We hypothesize that microRNAs (miRNAs) contribute to AT2 injury and that functional studies on reliable AT2 human in vitro models should be carried out to explore their exact role and therapeutic potential for pulmonary fibrosis.
Aim – With this project, we aim to optimise a transfection method for small RNA molecules (such as miRNA mimic) in advanced in vitro models of AT2 cells, with the ultimate goal to study the functional role of selected miRNAs in AT2 injury.
Experimental plan – In our lab, we are currently working on two advanced in vitro models of human AT2, i.e. lung adenocarcinoma H441 cell line (grown at air-liquid interface, ALI) and induced pluripotent stem cell-derived AT2 (i.e. the 3D alveolospheres ‘iAT2’). H441 cells will be grown in ALI culture and transfected with miRNA mimic using transfection reagents with different formulation and we will assess (i) transfection efficiency via fluorescent microscope and flow cytometry and (ii) their effects on cytotoxicity and cell viability. The most efficient and less cytotoxic transfection method will be validated in the iAT2 model. While optimising a method for transfection of small RNA molecules in AT2 models, this project will also provide a baseline for future experiments where more novel delivery strategies will be employed.
