Abstract:
Objective To explore the expression patterns of small nucleolar RNA (snoRNA) SNORA72 gene in various cancers, particularly in colorectal cancer (CRC), and its effect on the growth and radiosensitivity of CRC cells.
Methods The expression of SNORA72 in different cancer and CRC tissues was analyzed using open cancer databases. The CRC cell line HT29, overexpressing or knocking down SNORA72, was constructed, dividing HT29 cells into the overexpressing SNORA72 group (LV-SNORA72) and its negative control group (LV-NC), as well as the SNORA72 knockdown group (ASO-SNORA72) and its negative control group (ASO-NC). The expression of SNORA72 in HT29 cells was detected by real-time fluorescence quantitative polymerase chain reaction (qRT-PCR). The effects of SNORA72 overexpression or knockdown on cell proliferation, colony formation, apoptosis, and cell cycle were evaluated. The HT29 cells from the LV-SNORA72 and LV-NC groups were irradiated with different doses of 60Co γ-rays, and the survival fraction (SF) and apoptosis rate of cells in each group were assessed. Transcriptomic analysis was employed to explore the potential mechanisms by which SNORA72 affects HT29 cell growth. An independent sample t-test was used for comparisons between two groups.
Results Analysis of cancer databases revealed that SNORA72 is overexpressed in various cancer tissues, including CRC, the difference were statistically significant (all P<0.05). qRT-PCR results indicated that the relative expression of SNORA72 in the LV-SNORA72 group was significantly higher than that in the LV-NC group ((2.68±0.06) vs. (1.00±0.17)), and the difference was statistically significant (t=16.570, P<0.001). Conversely, the relative expression of SNORA72 in the ASO-SNORA72 group was significantly lower than that in the ASO-NC group ((0.61±0.08) vs. (1.00±0.13)), and the difference was statistically significant (t=4.355, P<0.05). Cell proliferation assay results showed that the absorbance values of the LV-SNORA72 group were significantly higher than those of the LV-NC group ((0.79±0.05) vs. (0.51±0.09), (1.78±0.04) vs. (1.22±0.05), and (3.30±0.05) vs. (2.19±0.06)) on the 3rd, 4th, and 5th day of the experiment, and the difference were statistically significant (t=8.582, 16.400, 31.200; all P<0.001). Conversely, the absorbance values of the ASO-SNORA72 group were significantly lower than those of the ASO-NC group ((0.42±0.07) vs. (0.55±0.05), (1.04±0.08) vs. (1.25±0.05), and (1.46±0.09) vs. (1.74±0.08)), and the difference were statistically significant (t=3.957, 6.147, 8.471; all P<0.01). Colony-formation assay results indicated that the colony formation rate of the LV-SNORA72 group was significantly higher than that of the LV-NC group((40.87±1.70)% vs. (26.60±0.40)%), and the difference was statistically significant (t=14.140, P<0.001). Conversely, the colony formation rate of the ASO-SNORA72 group was significantly lower than that of the ASO-NC group ((9.60±0.40)% vs. (12.43±0.38)%), and the difference was statistically significant (t=8.910, P<0.001). Apoptosis assay results showed that the apoptosis rate of the LV-SNORA72 group was significantly lower than that of the LV-NC group ((1.89±0.1)% vs. (2.64±0.15)%), and the difference was statistically significant (t=6.115, P<0.01). Conversely, the apoptosis rate of the ASO-SNORA72 group was significantly higher than that of the ASO-NC group((6.44±0.54)% vs. (3.92±0.37)%), and the difference was statistically significant (t=6.644, P<0.01). Western blot results demonstrated that compared with the ASO-NC group, the ASO-SNORA72 group promoted the cleavage activation of apoptosis proteins PARP and Caspase3, increased the expression of Bax protein, and inhibited the expression levels of anti-apoptotic proteins Survivin and Bcl-2. The results of radiosensitivity analysis through colony formation assay post-radiation showed that after exposure to 1, 2, 4, and 6 Gy of γ-rays, the SF of the LV-SNORA72 group increased compared with that of the LV-NC group ((0.89±0.05) vs. (0.81±0.03), (0.64±0.10) vs. (0.47±0.01), (0.16±0.04) vs. (0.09±0.01), and (0.04±0.01) vs. (0.02±0.01)), the difference were statistically significant (t=4.063, 8.802, 4.045, 2.937; all P<0.05). Radiation-induced apoptosis results showed that 48 h and 72 h after 4 Gy irradiation, the apoptosis rate in the LV-SNORA72 group was significantly lower than that in the LV-NC group ((8.14±0.12)% vs. (9.86±0.22)% and (11.26±0.52)% vs. (15.83±1.54%)), and the difference were statistically significant (t=3.470, 9.208; both P<0.05). After 8 Gy irradiation at 48 h and 72 h, the apoptosis rate in the LV-SNORA72 group was significantly lower than that in the LV-NC group ((13.29±0.17)% vs. (14.88±0.58)% and (19.82±0.56)% vs. (23.7±0.6)%), and the difference were statistically significant (t=3.201, 7.819; both P<0.05). After 12 Gy irradiation at 48 h and 72 h, the apoptosis rate in the LV-SNORA72 group was significantly lower than that in the LV-NC group ((14.06±0.32)% vs. (18.56±1.08)%) and (22.19±0.02)% vs. (26.84±0.66)%), the difference were statistically significant (t=9.054, 9.369; both P<0.001). Transcriptomic analysis results showed that overexpression of SNORA72 affects biological processes, such as cell activation, cell adhesion, and immune and inflammatory responses, cell migration, and cell proliferation.
Conclusions SNORA72 is specifically overexpressed in CRC tissues and associated with poor prognosis in patients. It promotes CRC cell growth and proliferation and increases cellular radio-resistance.