Fig. 4: Effects of 5-Aza treatment and LRRC2 on LUAD cell malignant phenotype and tumor growth.

A Design of the amplified sequence for the LRRC2 gene promoter CpG island used in the BSP experiment, highlighting the target region for methylation analysis. B Expression levels of LRRC2 in demethylated H1975 cell lines treated with various concentrations of 5-Aza show a significant increase compared to the DMSO-treated control group. Results were showed as the mean ± standard deviation (N = 3). C BSP dot plot showing reduced methylation levels in the 5-Aza treatment group compared to the DMSO group. Empty dots indicate unmethylated CpG sites, while filled dots represent methylated CpG sites. D BSP line graph further confirming that the 5-Aza group exhibits lower methylation levels than the DMSO group. E Cell proliferation assay conducted on H1975 LUAD cells after transfection with Lv-LRRC2 or control vector (Lv-NC). The results indicate that LRRC2 overexpression significantly inhibits cell proliferation compared to the control group. Results were showed as the mean ± standard deviation (N = 3). F Transwell migration assay assessing the migratory capacity of H1975 cells following transfection with either Lv-LRRC2 or Lv-NC. The number of migrated cells was quantified and statistically analyzed using a paired t-test, demonstrating that LRRC2 overexpression markedly reduces H1975 cell migration. Each experiment was performed in triplicate (N = 3). G Schematic diagram of the xenograft tumor model used for in vivo studies. Nude mice were subcutaneously injected with H1975 cells transfected with either Lv-LRRC2 or Lv-NC, followed by observation of tumor growth over time. H Macroscopic view of tumors in nude mice. I Quantitative comparison of tumor growth between the Lv-NC group (N = 8) and the Lv-LRRC2 group (N = 8). Tumor volumes were measured regularly during the experiment and final tumor weights were recorded at the endpoint. Results are expressed as mean ± standard deviation, indicating that LRRC2 overexpression significantly inhibits tumor growth in vivo. J Ratio of tumor weight to body weight at the endpoint of the xenograft experiment for mice in the Lv-NC (N = 8) and Lv-LRRC2 (N = 8) groups. Data are shown as mean ± standard deviation, further supporting the tumor-suppressive role of LRRC2 in vivo. 5-Aza 5-Aza-2’-deoxycytidine, BSP bisulfite sequencing PCR. Significance levels: ^*P < 0.05, ^**P < 0.01, ^***P < 0.001.