LPAR3 Is Associated with Migration of NIH 3T3 Cells and Progression to Malignant Cell Transformation during Carcinogenesis

Abstract

We have previously identified that the LPAR3 gene is commonly hypermethylated in foci transformed by two non‐genotoxic carcinogens (NGTxC). In this study, we validated the down‐regulation of LPAR3 genes using qPCR analysis in both NGTxC‐induced transformed foci and another v‐Ha‐ras‐transformed NIH 3T3 cells (Ras‐NIH 3T3). We revealed that LPAR3 overexpression significantly increased migration of Ras‐NIH 3T3 whose migration capacity was much lower than that of their parental cells. However, LPAR3 overexpression in Ras‐NIH 3T3 cells resulted in the inhibition of cell survival concomitant with G2/M arrest. On the other hand, inhibition of LPAR3 signaling with Ki16425 had little effect on cell survival of Ras‐NIH 3T3 cells with low level of LPAR3 expression, whereas inhibiting cell survival of NIH 3T3 cells. These results imply that LPAR3 plays a positive role in proliferation of NIH 3T3 cells but not in that of its ras‐transformed cells. To further analyze the role of LPAR3 during carcinogenesis, LPAR3 knockout (KO) in NIH 3T3 cells was generated using the CRISPR/Cas9 genome editing. Four clones from nine initial clones were confirmed with sequencing for genomic editing at the target site. From this initial screen, one clone with biallelic homozygous fragments deletions was selected for further analysis. WST‐1 assay showed that LPAR3 KO greatly inhibited cell survival of NIH 3T3 cells. LPAR3 KO also reduced migration of NIH 3T3 cells. Taken together, our results indicate that LPAR3 might be functionally involved in malignant cell transformation. Further research will be required to understand the intrinsic relationships between cell survival and migration.Support or Funding InformationThis work was supported by the National Research Foundation of Korea (NRF) grant funded by the Ministry of Science and ICT (MIST) (NRF‐2019R1F1A1048733).