CRISPR/Cas9-Based Screening for RNA Modification-Related Genes Influencing Bone Metastasis in Non-Small Cell Lung Cancer
DU Fenglin1, REN Yizhe2, ZHANG Lingfei3, CHENG Yaling1, ZHAO Mingna2, ZHANG Xianzhou2, LIU Chang2, LOU Jiatao1,2,4*
This study employed a CRISPR/Cas9-based systemic screening approach to investigate the role of RNA modification-related genes in bone metastasis of NSCLC (non-small cell lung cancer) and to explore their underlying mechanisms. A CRISPR/Cas9 knockout library targeting 118 RNA modification-related genes was constructed and introduced into human NSCLC H460 BM (H460 bone-metastatic) cells via lentiviral infection. An in vivo bone metastasis model was established via external iliac artery injection in nude mice for functional screening. The distribution of sgRNAs (single guide RNAs) in cells after in vivo selection was analyzed by high-throughput sequencing, and key genes were identified using the MAGeCK-RRA algorithm. Knockdown of METTL3 in H460 BM cells using siRNA—verified by qRT-PCR and Western blot, effectively downregulated the expression of EMT (epithelial-mesenchymal transition)-related markers. Furthermore, knockdown and rescue experiments combined with qRT-PCR, Western blot and Transwell invasion assay verified the function of EML4 in mediating the EMT process downstream of METTL3. The expression and prognostic value of METTL3 in lung adenocarcinoma tissues were analyzed using databases including TIMER and PanCanSurvPlot. Results showed that METTL3 ranked first in negative selection, with its sgRNAs being significantly depleted in the in vivo model. Knockdown of METTL3 effectively inhibited the migratory ability of H460 BM cells, downregulated the protein expression of EMT mesenchymal markers N-cadherin and Vimentin, and upregulated the expression of epithelial marker E-cadherin. Moreover, further studies demonstrated that METTL3 drives the EMT process by positively regulating the expression of its downstream target gene EML4. Bioinformatics analysis showed that METTL3 expression was significantly elevated in lung adenocarcinoma tissues compared with normal tissues, and its high expression was associated with poorer overall survival in patients. This study demonstrates that CRISPR/Cas9-mediated systematic screening can effectively identify functional genes involved in NSCLC bone metastasis, and highlights the key role of the RNA modification-related gene METTL3 in promoting bone metastasis by upregulating EML4 expression to regulate the EMT process.
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