Functional Characterization of ZFC3H1 and DYNLT3 in A375 Melanoma Cells Using CRISPR Knockout 2266763

Abstract

Melanoma progression is driven by transcriptional and cytoskeletal regulators that influence cell proliferation, migration, and immune evasion. ZFC3H1, a zinc finger RNA-binding protein involved in nuclear RNA decay, and DYNLT3, a dynein light-chain component implicated in intracellular transport and signaling, are both aberrantly expressed in melanoma according to public transcriptomic datasets. This study investigates the functional roles of ZFC3H1 and DYNLT3 in A375 melanoma cells to determine whether their dysregulation contributes to tumor growth or survival. Differential expression analysis of RNA-seq data from melanoma and normal skin samples identified ZFC3H1 and DYNLT3 as upregulated genes. CRISPR-Cas9 knockouts were generated in A375 cells to assess loss-of-function effects. RNA extraction and cDNA synthesis confirmed knockout efficiency by qPCR. Cell viability and proliferation were evaluated using trypan blue exclusion and standard colorimetric assays. Ongoing assays are examining changes in morphology and expression of stress-response genes following knockout. Preliminary results confirmed robust expression of ZFC3H1 and DYNLT3 in wild-type A375 cells and successful CRISPR knockout in target lines. Early viability assays suggest reduced proliferation in DYNLT3-deficient cells, while ZFC3H1 knockout shows altered morphology consistent with cytoskeletal disruption. Additional analyses are underway to quantify transcriptional and phenotypic effects relative to control cells. Loss-of-function analysis of ZFC3H1 and DYNLT3 in A375 melanoma cells provides new insight into their potential roles in tumor cell growth and regulation. These findings may help define RNA-processing and transport mechanisms that support melanoma progression and identify novel targets for therapeutic intervention. n/a Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)