GFP Reporter Cell Line - RKO

Colorectal cancer (CRC) is a highly heterogeneous disease, the pathogenesis of which is attributed to the interplay between genetic and environmental factors. As a frequently mutated gene, p53 plays a pivotal role in the "adenoma-to-carcinoma" transition-a critical stage in the pathological progression of tumors. Here, utilizing high-throughput screening techniques, researchers identified TRIM3 as a tumor-associated gene in colorectal cancer. Cellular experiments revealed that TRIM3 exhibits a dual nature, functioning as both a tumor suppressor and a tumor promoter; its specific role is contingent upon whether the intracellular p53 protein exists in its wild-type (wtp53) or mutant (mutp53) state. TRIM3 directly interacts with the C-terminal region of the p53 protein (amino acid residues 320–393)-notably, a structural domain shared by both wild-type and mutant p53. Furthermore, TRIM3 modulates distinct tumor biological behaviors by sequestering p53 within the cytoplasm, thereby reducing its expression levels within the nucleus; the execution of this regulatory mechanism is dependent upon the specific type of intracellular p53 protein (i.e., wild-type or mutant).

Utilizing High-Content Screening (HCS) technology, researchers designed and constructed an RNA interference library targeting a panel of genes associated with colorectal cancer (CRC). RKO cells-a widely used model for colon cancer-were employed for transfection experiments and for screening cellular proliferation capabilities. Leveraging HCS technology, the researchers monitored the dynamic growth of GFP-labeled RKO cells transfected with either a control vector or TRIM3-shRNA; this monitoring was conducted twice daily over a period of three days (Figure 1a). As illustrated in Figure 1a, compared to the control group, the number of GFP-labeled cells in the TRIM3-shRNA group was significantly reduced, and this reduction exhibited a time-dependent pattern.

Figure 1. RKO cells with TRIM3 knockdown monitored by HCS technology. (Han, Yang, et al., 2023)