应用慢病毒shRNA文库结合二代测序筛选口腔鳞癌放疗抵抗相关分子

Screening for regulation genes of radioresistance of oral squamous cells using lentiviral shRNA library combined with next generation sequencing

目的 用高通量方法筛选口腔鳞癌放疗抵抗相关的激酶基因,为口腔鳞癌的临床治疗决策提供理论基础。方法 以慢病毒构建的短发夹核糖核酸(shRNA)激酶文库(含4 675个不同shRNA序列,覆盖709个人激酶基因)感染舌鳞癌Cal-27细胞,嘌呤霉素筛选去除未成功感染的细胞后,以光子射线进行照射(0、10和15 cGy),然后继续培养3 d,以增加不同射线抗性细胞的丰度差异。提取细胞基因组DNA,利用PCR获得完整shRNA序列,同时每组引入不同标签序列,利用illumina平台进行二代测序,找出丰度发生显著变化的shRNA,其对应基因即为影响射线抗性的基因。结果 本次实验共筛选到5个影响口腔鳞癌放射线抗性的激酶基因,其中,PKLR和IPMK敲减后会增加射线抵抗,AURKB、ITPKB和DLG2敲减后会导致放射敏感,其高表达会导致患者对放疗耐受。结论 本研究利用shRNA慢病毒文库结合二代测序方法筛选到3个口腔鳞癌放射抵抗基因,但具体作用机制尚需进一步探究。

Objective To screen the kinase genes related to radioresistance by high-throughput method and provide a theoretical basis for clinical treatment strategy of oral squamous cell carcinoma. Methods The tongue squamous carcinoma cell line Cal-27 was infected with lentivirus shRNA kinase library that contains 4 675 different shRNAs regulating 709 human kinase genes. The uninfected cells were removed by puromycin screening. Then, the cells were irradiated with different doses (0, 10, 15 cGy) and continued to culture for 3 d to enrich the differences among groups. Afterwards, the cell genomic DNA was extracted and the complete shRNA sequences were obtained by PCR.Different tags were labeled in shRNAs of each group. An illumina platform was used for next generation sequencing to acquire the shRNA abundance, and then the differentiated expressed genes among different groups were identified. Results A total of 5 kinase genes (PKLR, IPMK, AURKB, ITPKB and DLG2) were screened out from irradiated cells. Knockdown of PKLR and IPMK increased radiation resistance, while knockdown of AURKB, ITPKB and DLG2 increased radiation sensitivity, and the high expression of these genes would lead to radiotherapy tolerance. Conclusions In this study, 3 genes relative to radioresistance of oral squamous cell carcinoma were identified by using shRNA lentivirus library combined with next generation sequencing, but the underlying mechanism requires further exploration.