OBJECTIVETo develop an optimal sequencing system which can improve the resolution of sequencing G-C rich DNA with abundant trinucleotide repeats by applying concentration gradients of betaine to the Sanger sequencing system.

METHODSConcentration gradients of betaine were introduced into the sequencing system by taking the 5' terminal of Nogo-B cDNA (Am-Nogo-B) (G-C%=72%, without trinucleotide repeats) and 5' terminal of Huntingtin cDNA (Am-HTT) (G-C%=74%, with abundant CAG and CCG repeats) the results of sequencing were compared.

RESULTSThe optimum concentration of betaine for sequencing Am-Nogo-B has differed from that for Am-HTT. Result of sequencing Am-Nogo-B has achieved the best quality when the concentration of betaine was at 0.8-1.2 mol/L, whereas the result of sequencing Am-HTT obtained the best quality when the concentration of betaine was at 1.6 -2.4 mol/L. The results were reproducible.

CONCLUSIONG-C rich DNA with similar G-C% required different concentrations of betaine in the sequencing system due to base pair compositions. The sequencing system developed for improving the resolution of sequencing of G-C rich DNA with abundant trinucleotide repeats can be used as a reference for similar studies.

Base Sequence ; Betaine ; pharmacology ; Huntingtin Protein ; Molecular Sequence Data ; Nerve Tissue Proteins ; genetics ; Sequence Analysis, DNA ; methods ; Trinucleotide Repeats

Objective To investigate the effect of apatinib on the migration ability of nasopharyngeal carcinoma NPC cells after X-ray irradiation and involved protein expressions. Methods The migration abilities of human immortalized nasopharyngeal epithelial cells ( NP69) and nasopharyngeal carcinoma cells ( CNE-1, CNE-2 ) treated with different concentrations of apatinib ( 0, 5, 10 and 15 μmol/L) were compared by wound healing assay. The effect of apatinib on the activity of NPC cells was detected by CCK-8 for determining the suitable intervention concentration of apatinib. Then NPC cells were divided into control group, apatinib group (15 μmol/L), X-ray irradiation group and apatinib combined with X-ray irradiation group, and the migration ability of each group was compared by wound healing assay. The expressions of pVEGFR2, pSTAT3, STAT3, MMP-9 and EMT related proteins were detected by western blot. Results Compared with the NP69, the migration abilities of CNE-1 and CNE-2 were significantly enhanced ( t=-5. 759, -16. 578, P<0. 05) . Compared with the control group ( 0 μmol/L) , the migration ability of NPC cells after treatment with apatinib(5, 10 and 15 μmol/L) was significantly decreased in a concentration dependent manner ( t=2. 804-13. 362, P<0. 05) . Compared with the X-ray irradiation group, the wound healing rate of NPC cells in the apatinib combined with X-ray irradiation group was decreased ( t=5. 932, 2. 791, P<0. 05) , indicating that apatinib can significantly inhibit the migration of NPC cells after X-ray irradiation. Western blot assay showed that the expressions of pVEGFR2 and pSTAT3 were significantly decreased in NPC cells treated with apatinib, meanwhile, the expression of MMP-9 protein was significantly decreased, and the EMT-related protein was changed. Conclusions Apatinib inhibits migration of X-ray irradiated NPC cells by inhibiting EMT through down-regulating VEGFR2/STAT3/MMP-9 signaling pathway.