Objective To investigate the effects of cysteine-rich motor neuron protein 1 (CRIM1) on radiosensitivity and metastasis of non-small cell lung cancer (NSCLC) and its possible mechanisms.

Methods Short hairpin RNA (shRNA) was used to knock down the expression level of CRIM1 in NSCLC cells H460 and H358, and H460 and H358 cells were divided into three groups respectively: H460, H460-shCRIM1, H460-shNC, and H358, H358-shCRIM1, H358-shNC, where shCRIM1 indicates the knockdown of CRIM1 expression level by shRNA and shNC indicates the negative control. Small interfering RNA was used to knock down the expression of CRIM1 in H358 cells, and the cells were divided into two groups: H358-siNC and H358-siCRIM1. Clone formation assay (irradiation dose of 0, 1, 2, and 4 Gy), comet assay (irradiation dose of 8 Gy), and cellular immunofluorescence assay (irradiation dose of 6, 8, and 12 Gy) were used in observing the effect of CRIM1 on the radiosensitivity of H460 cells. Transwell and cell adhesion assays were used in observing the effect of CRIM1 on cell metastasis in vitro. A mouse in situ tumor model was constructed, and the metastasis of tumors inoculated in situ into nude mouse lungs was assessed using histopathological examination, and transcriptomic analysis was used in exploring the possible effects of CRIM1 on the mechanism of NSCLC cell metastasis. Independent samples t-test was used in comparing groups.

Results Clone formation assay showed statistically significant differences between the clone formation rates of H460-shNC and H460-shCRIM1 after 1 and 2 Gy of irradiation ((1 Gy: (87.04±8.04)% vs. (58.01±4.39)%, t=4.48, P<0.05; 2 Gy: (48.23±1.22)% vs. (31.43±0.08)%, t=19.50, P<0.05)). The results of comet assay showed that the olive tail moment of H460-shCRIM1 cells after 8 Gy of irradiation was longer than that of H460-shNC cells, and the difference was statistically significant (1.27±0.54 vs. 1.05±0.42, t=2.14, P<0.05). The results of cell immunofluorescence experiments showed that the number of phosphorylated histone H2AX foci was higher in H460-shCRIM1 cells than in H460-shNC after irradiation (6 Gy: 14.33±2.81 vs. 11.00±3.92; 8 Gy: 34.00±11.14 vs. 21.17±6.15; 12 Gy: 25.80±3.96 vs. 20.17±3.31), and the difference was statistically significant (t=2.45, 5.52, 2.47; all P<0.05). Transwell assay showed that the mobility rates of H460-shCRIM1 and H358-shCRIM1 were significantly higher than those of H460-shNC and H358-shNC, respectively, and the differences were statistically significant (t=4.73, 10.19; both P<0.05). The results of cell adhesion assay showed that the adhesion ability of H460-shCRIM1 and H358-shCRIM1 decreased relative to that of H460-shNC and H358-shNC, respectively, and the difference was statistically significant (t=2.86, 3.66; both P<0.05). Histopathological examination results showed the in situ inoculation of H460-shCRIM1 into nude mice. Intrapulmonary metastases of H460-shCRIM1 were more extensive than those in H460-shNC. Transcriptomic analysis showed that the expression levels of the screened cell adhesion-related genes junctional adhesion molecule 2 (JAM2), nectin cell adhesion molecule 3 (NECTIN3), and claudin 4 (CLDN4) decreased in H460-shCRIM1 and H358-siCRIM1, and this result was verified in in vitro experiments. The expression levels in H460-shCRIM1 decreased by 86.66% (JAM2), 49.35% (NECTIN3), and 30.27% (CLDN4) compared with those in H460-shNC (t=47.52, 7.47, 18.98; all P<0.05), and in H358-siCRIM1 decreased by 36.60% (JAM2), 31.70% (NECTIN3), and 50.00% (CLDN4) compared with those in H358-siNC (t=7.40, 7.10, 16.56; all P<0.05).

Conclusion The inhibition of CRIM1 enhances the radiosensitivity of NSCLC cells H460, and CRIM1 may influence NSCLC metastasis by affecting tumor cell junctions.