Objective To investigate the role and mechanism of the C/D box small nucleolar RNA 3 (SNORD3) gene family in the proliferation and radiosensitivity of HeLa cells.

Methods Bioinformatics techniques were used to analyze the sequence and structural conservation of the SNORD3 gene family (SNORD3A, SNORD3B, SNORD3C, SNORD3D), and pan-cancer analysis was performed to predict the impact of SNORD3A gene expression on tumor prognosis. HeLa cells were infected with LV-SNORD3A, LV-SNORD3B, LV-SNORD3C, and LV-SNORD3D overexpression lentiviruses, respectively, to establish overexpression cell lines, namely, the LV-SNORD3A, LV-SNORD3B, LV-SNORD3C, and LV-SNORD3D groups, with LV-NC-infected cells as the negative control group. HeLa cells were infected with sg-SNORD3A/C and sg-SNORD3B/D knockdown lentiviruses, respectively, to establish cell lines with targeted knockdown of SNORD3A/C and SNORD3B/D, namely, the sg-SNORD3A/C and sg-SNORD3B/D groups; cells were co-infected with sg-SNORD3A/C and sg-SNORD3B/D knockdown lentiviruses to establish a cell line with simultaneous knockdown of SNORD3A/C and SNORD3B/D, namely, the sg-SNORD3A/B/C/D group; sg-NC-infected cells were used as the negative control group. Cell counting kit-8 (CCK-8) assay and cell colony formation assay were used to analyze the effects of the SNORD3 gene family on cell proliferation and colony formation. Cells were irradiated with ⁶⁰Co γ-rays at doses of 0, 1, 2, 4, and 6 Gy to analyze the effect of the SNORD3 gene family on cellular radiosensitivity. The single-hit multitarget model was used to fit cell survival curves and calculate the sensitization enhancement ratio (SER). Indirect immunofluorescence assay was used to detect phosphorylated histone H2AX (γ-H2AX) foci and analyze the effect of the SNORD3 gene family on radiation-induced DNA double-strand breakage (DSB). Measurement data between two groups were compared using independent-samples t-test. The Kaplan-Meier method was used to calculate survival rates and draw survival curves, and the log-rank test was used for comparison between two groups.

Results Bioinformatics analysis showed that the gene sequences and secondary structures of SNORD3A, SNORD3B, SNORD3C, and SNORD3D were highly conserved. Pan-cancer analysis revealed that high expression of SNORD3A significantly shortened the overall survival (χ²=60.73, P<0.001) and disease-free survival (χ²=44.60, P<0.001) of cancer patients. CCK-8 assay results showed that overexpression of SNORD3A, SNORD3B, SNORD3C, and SNORD3D promoted cell proliferation. On days 2, 3, and 4 of the experiment, the absorbance values of the LV-SNORD3A, LV-SNORD3B, LV-SNORD3C, and LV-SNORD3D groups were significantly higher than those of the LV-NC group, with statistically significant differences (t=3.734–11.440, all P<0.05). By contrast, knockdown of SNORD3A/C, SNORD3B/D, and SNORD3A/B/C/D inhibited cell proliferation. On days 3 and 4 of the experiment, the absorbance values of the sg-SNORD3A/C, sg-SNORD3B/D, and sg-SNORD3A/B/C/D groups were significantly lower than those of the sg-NC group, with statistically significant differences (t=6.221–12.710, all P<0.05). Colony formation assay results showed that the colony formation rate and colony area of the LV-SNORD3A, LV-SNORD3B, LV-SNORD3C, and LV-SNORD3D groups were significantly higher than those of the LV-NC group, with statistically significant differences (t=3.620–8.792, all P<0.05). Conversely, the colony formation rate and colony area of the sg-SNORD3A/C, sg-SNORD3B/D, and sg-SNORD3A/B/C/D groups were significantly lower than those of the sg-NC group, with statistically significant differences (t=3.399–22.250, all P<0.05). Radiosensitivity analysis showed that overexpression of SNORD3A, SNORD3B, SNORD3C, and SNORD3D enhanced cellular radioresistance (SER=0.487, 0.580, 0.716, 0.780, respectively), whereas knockdown of SNORD3A/C, SNORD3B/D, and SNORD3A/B/C/D enhanced cellular radiosensitivity (SER=2.106, 1.612, 1.558, respectively). Indirect immunofluorescence assay showed that compared with the respective negative control groups, overexpression of SNORD3A, SNORD3B, SNORD3C, and SNORD3D reduced radiation-induced DNA DSB (t=2.715–9.660, all P<0.05), whereas knockdown of SNORD3A/C, SNORD3B/D, and SNORD3A/B/C/D resulted in a significant increase in DNA DSB (t=2.899–15.600, all P<0.05).

Conclusions The SNORD3 gene family regulates cell proliferation and radiosensitivity, and the modulation of DNA DSB repair is an important mechanism underlying its effect on the radiosensitivity of HeLa cells. This finding suggests that the SNORD3 gene family can serve as a candidate target for radiosensitivity intervention.