Abstract
To explore radiosensitivity-related LncRNA in esophageal cancer stem cell. The Eca109 cells were divided into tumor sphere cells group and adherent cells group by using suspended and adherent culture. Differences in stem cell characteristics of two groups were compared by CD44 positive expression rate, CCK8 cell proliferation, and plate cloning formation. Using Arraystar LncRNA microarray analysis the gene expression differences of CD44+ esophageal cancer stem cells and CD44- esophageal cancer cells were sorted out by flow separation technology. Bioinformatics analysis and real-time PCR were applied to verify the specific LncRNA related to radiation sensitivity of esophageal cancer stem cells. 1. The result of CCK8 cell proliferation experience showed that the absorbance value of the tumor sphere cells group was significantly higher than an adherent cells group after 24h, 48h, and 72h (P< 0.05). The cell clone formation rate of the tumor sphere cells group was higher than adherent cells group (P < 0.05). 2. The plate cloning formation showed that the values of D0, Dq, N, and SF2 were significantly higher in the tumor sphere cells group than adherent cells group, and the radiation sensitization ratio was 1.556. 3. The detection of CD44 showed that the positive rate and expression rate of CD44 in the tumor sphere cells group than adherent cells group (P < 0.05). 4. Arraystar LncRNA chip analysis showed following the criteria that DEGseq at |log2Ratio|≥1和q<0.05, 4961 DEGs including 2517 up-regulated genes and 2444 down-regulated genes were identified. 5. We verified the top 10 difference LncRNA genes (including up-regulated genes CH17-360D5.2, CH17-360D5.3, AC113607.2, RP11-439E19.10, MSTRG.424, MSTRG.64087, MSTRG.116589, MSTRG.15903 and down-regulated genes RP13-895J2.7, MSTRG.73085) by real-time PCR. The results showed that MSTRG. 73085 was downregulated and its expression level on average was 20.7 times (chip was 49.3 times), CH17-360D5.2, RP11-439E19.10, MSTRG.424, MSTRG.64087, and MSTRG.15903 was upregulated and their expression level on average were 2.7, 8.7, 3.7, 5, and 7 (chip were 8.2, 5.2, 78.9, 3, and 4.7 times). We verified the different LncRNA genes by arraystar LncRNA chip and real-time PCR, The results showed that MSTRG. 73085 was downregulated and CH17-360D5.2, RP11-439E19.10, MSTRG.424, MSTRG.64087, and MSTRG.15903 was up-regulated. These LncRNA genes may be related to the radiosensitivity of esophageal cancer stem cells and could be a potential new target for esophageal squamous carcinoma.
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