促癌基因SNORA72对结直肠癌细胞放射敏感性的作用研究

张文成; 邓佳荣; 刘鑫; 张宏; 王治东; 沈丽萍

doi:10.3760/cma.j.cn121381-202310003-00398

促癌基因SNORA72对结直肠癌细胞放射敏感性的作用研究

Oncogene SNORA72 enhances radioresistance in colorectal cancer cells

摘要

摘要:
目的探索核仁小RNA（snoRNA）SNORA72基因在不同癌症特别是结直肠癌（CRC）中的表达模式及其对CRC细胞的生长及放射敏感性的影响。
方法应用开放的癌症数据库分析SNORA72在不同癌症组织和CRC组织中的表达水平。构建过表达或敲低SNORA72的CRC细胞株HT29，将HT29细胞株分为过表达 SNORA72组（LV-SNORA72）及其阴性对照组（LV-NC）、敲低SNORA72表达组（ASO-SNORA72）及其阴性对照组（ASO-NC）。采用实时荧光定量聚合酶链反应（qRT-PCR）检测HT29细胞中SNORA72表达情况。分别检测在体外过表达或敲低SNORA72后，对细胞增殖、细胞克隆形成、细胞凋亡、细胞周期的影响。对LV-SNORA72组及LV-NC组的HT29细胞进行不同剂量⁶⁰Co γ射线照射，检测各组细胞的存活分数（SF）和凋亡率。采用转录组学分析法探讨SNORA72影响HT29细胞生长可能的作用机制。两组间的比较采用独立样本t检验。
结果癌症数据库分析发现SNORA72在包括CRC在内的多种癌症组织中高表达，且差异均有统计学意义（均P<0.05）。qRT-PCR 检测结果显示，与LV-NC组相比，LV-SNORA72组SNORA72的相对表达水平明显升高（2.68±0.06）对（1.00±0.17），且差异有统计学意义（t=16.570，P<0.001）。另外，与ASO-NC组相比，ASO-SNORA72组SNORA72的相对表达水平明显降低（0.61±0.08）对（1.00±0.13），且差异有统计学意义（t=4.355，P<0.05）。细胞增殖检测结果显示，在实验的第3、4、5天，LV-SNORA72组的吸光度值明显高于LV-NC组（0.79±0.05）对（0.51±0.09）、（1.78±0.04）对（1.22±0.05）、（3.30±0.05）对（2.19±0.06），且差异均有统计学意义（t=8.582、16.400、31.200，均P<0.001）。相反，ASO-SNORA72组的吸光度值明显低于ASO-NC组（0.42±0.07）对（0.55±0.05）、（1.04±0.08）对（1.25±0.05）、（1.46±0.09）对（1.74±0.08），且差异均有统计学意义（t=3.957、6.147、8.471，均P<0.01）。细胞克隆形成实验结果显示，LV-SNORA72组的克隆形成率明显高于LV-NC组（40.87±1.70）%对（26.60±0.40）%，且差异有统计学意义（t=14.140，P<0.001）。相反，ASO-SNORA72组的克隆形成率明显低于ASO-NC组（9.60±0.40）%对（12.43±0.38）%，且差异有统计学意义（t=8.910，P<0.001）。细胞凋亡检测结果显示，LV-SNORA72组的细胞凋亡率明显低于LV-NC组（1.89±0.16）%对（2.64±0.15）%，且差异有统计学意义（t=6.115，P<0.01）。相反，ASO-SNORA72组的细胞凋亡率明显高于ASO-NC组（6.44±0.54）%对（3.92±0.37）%，且差异有统计学意义（t=6.644，P<0.01）。Western blot结果显示，与ASO-NC组相比，ASO-SNORA72组可以促进凋亡蛋白PARP和Caspase3发生剪切活化，Bax蛋白表达水平升高，同时抑制抗凋亡蛋白Survivin和Bcl-2的表达。放射敏感性分析的细胞克隆形成实验结果显示，在经1、2、4、6 Gy γ射线照射后，LV-SNORA72组细胞的SF均较LV-NC组增加（0.89±0.05）对（0.81±0.03）、（0.64±0.10）对（0.47±0.01）、（0.16±0.04）对（0.09±0.01）、（0.04±0.01）对（0.02±0.01），且差异均有统计学意义（t=4.063、8.802、4.045、2.937，均P<0.05）。放射诱导的细胞凋亡结果显示，在4 Gy照射后48 h和72 h，与LV-NC组相比，LV-SNORA72组细胞凋亡率明显降低（8.14±0.12）%对（9.86±0.22）%、（11.26±0.52）%对（15.83±1.54）%，且差异均有统计学意义（t=3.470、9.208，均P<0.05）；在8 Gy照射后48 h和72 h，与LV-NC组相比，LV-SNORA72组细胞凋亡率明显降低（13.29±0.17）%对（14.88±0.58）%、（19.82±0.56）%对（23.7±0.6）%，且差异均有统计学意义（t=3.201、7.819，均P<0.05）；在12 Gy照射后48 h和72 h，与LV-NC组相比，LV-SNORA72组细胞凋亡率明显降低（14.06±0.32）%对（18.56±1.08）%、（22.19±0.02）%对（26.84±0.66）%，且差异均有统计学意义（t=9.054、9.369，均P<0.001）。转录组学分析结果显示，SNORA72过表达影响细胞活化、细胞黏附、免疫和炎症反应，以及细胞迁移和增殖等生物学过程。
结论 SNORA72在CRC组织中特异性高表达且与患者不良预后相关，过表达SNORA72促进CRC细胞的生长和增殖，增加细胞放射抵抗性。

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.

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