| Objective: Head and neck squamous cell carcinoma( HNSCC) is the common clinical malignant tumor. HNSCC especially nasopharyngeal carcinoma(NPC) 98% belong to low differentiated squamous cell carcinoma, preferred radiotherapy. However, for patients with the first treatment failure, local recurrence and distant metastasis, simple radiotherapy usually has not a good effect. High specificity, targeted radiotherapy sensitization method need to research. Our previous studies have shown that FANCD2 sh RNA interference can improve the cervical lymph node metastasis cell HSC-4 of HNSCC to cisplatin sensitivity by reducing the expression of FANCD2 gene. But the regulation about the FANCD2 gene involved in the radiosensitivity of HNSCC still need to research. In this study, we used HNSCC cervical lymph node metastasis cell HSC-4 which FANCD2 silenced by sh RNA interference as research object, investigate proliferation inhibition effect, apoptosis, cell cycle distribution, survival rate and related protein expression of HSC-4 cell after silencing FANCD2 and radiotherapy, to investigate the effect of expression of FANCD2 in HNSCC cervical lymph node metastasis cell HSC-4 to radiosensitivity and the possible mechanism. Methods: The HNSCC neck lymph node metastasis HSC-4 cell line, which FANCD2 has been silenced bysh RNA interference in early experiment and the silencing effect is stably. experimental cells were divided into three groups, experimental group(FANCD2-sh RNA, HSC-4 cells transfected effective interference sequence and silencing effect of FANCD2 stably), control group( HSC-4 cells without transfection), negative control group(FANCD2-sh RNA-C, HSC-4 cells transfected invalid FANCD2 interference sequence), three groups cells were cultured conventionally, experimental group and negative control group with puromycin select. The growth and proliferation inhibition effect of cobalt 60 effected on the HSC-4 cells at different time points and different radiotherapy dose was detected by CCK-8 method, in order to research the influence on the radiotherapy sensitivity after FANCD2 sh RNA interference in HSC-4 cell; the changes of apoptosis and cell cycle distribution about HSC-4 cells after radiotherapy was detected by FCM(Annexin V/PI staining method); survival rate of HSC-4 cells to radiotherapy after FANCD2 sh RNA interference detected by Colony formation assay; the expression of Bax, Bcl-2, P-38 and p-p-38 protein after FANCD2 silencing and radiotherapy by cobalt 60 detected by Western Blot, in order to investigate the potential mechanisms about the radiosensitivity of HSC-4 Cells by silencing FNACD2 expression. Results:(1) CCK 8 testing results showed that as the radiation dose increased cell proliferation inhibition of three group were improved, compared with the control group the proliferation inhibition effect of the experimental group in every irradiation dose obviously(P < 0.05), which when radiation dose was 8Gy the experimental group OD value(0.256 ± 0.027) significantly less than the negative control group(0.362 ± 0.008) and blank control group(0.382 ± 0.0213), the difference between groups was statistically significant(F = 103.835, P < 0.05); Exposure dose of 5 Gy irradiation, after 24, 48, 72 hours the proliferation level of three groups were extended with the time, at 24 hours(F=6.307, P<0.05), 48 hours(F=9.007, P<0.05) and 72hours(F=22.914, P<0.05) the difference among three groups was statistically significant, compared with the control group the proliferation inhibition of the experimental group obviously(P<0.05).(2) Flow cytometry analysis showed that after 8Gy irradiation and cultured for 48 hours, the apoptosis rate(34.074±0.061)% of the experimental groups significantly increased than the control group(14.241±0.047)% and negative control group(5.926±0.038)%, the difference was statistically significant(F=261.786, P<0.05).(3) Cultured for 48 after radiotherapy the G2/M phase ratio had no obvious changes(F=2.664, P>0.05) for the experimental group, S phase proportion increased(F=160.778, P<0.05), and the G0/G1 phase decreased(F=224.974, P<0.05), compared with negative control group and blank control group the difference was statistically significant(P<0.05).(4)Clone formation experiment results showed that each group cell survival rate decreased with the radiation dose increasing(P<0.05). when radiation dose was 8Gy, the survival rate(0.007±0.021) of sh RNA interference group cell, compared with blank control group(0.039±0.003) and the negative control group(0.037±0.016) significantly decreased, the differencewas statistically significant(F=10.671, P<0.05).(5)Western Blot results showed that the expression of Bax(F=931.591, P<0.05)and p-p38(F=685.425, P<0.05) increased in the experimental group, the expression of p-38(F=1954.140, P<0.05)and Bcl-2(F=3219.791, P<0.05) decreased in the experimental group compared with control groups; after radiotherapy, Bax(F=429.591, P<0.05)and p-p38(F=214.974, P<0.05) expression in the experimental group were also increased compared with control groups, p-38(F=1027.974, P<0.05)and Bcl-2(F=285.974, P<0.05) expression were lower compared with control groups. Conclusions:(1)FANCD2 sh RNA interference could increase the proliferation inhibition effect of the HNSCC neck lymph node metastasis HSC-4 cell line induced by radiotherapy, increase HSC-4 cells apoptosis after irradiation, causes cell cycle distribution changed and decrease cell survival rate after radiotherapy, suggesting that FACND2 sh RNA interference could improve the radiosensitivity of HNSCC neck lymph node metastasis HSC-4 cell.(2)Silencing FANCD2 by sh RNA interference may be involved in regulating Bax/Bcl-2 apoptosis pathway by up-regulating the expression of Bax, decreasing the expression of Bcl-2; enhanced activation p-38 MAPK pathway, jointing the up-regulation of p-p-38 protein expression, might combined with sh RNA interference FANCD2 to regulate the proliferation, apoptosis and cell cycle distribution of HSC-4 cells, thus enhanced radiosensitivity of HNSCC cervical lymph node metastasis HSC-4 cell lines.(3)This suggested that sh RNA interference FANCD2 can enhance theradiosensitivity of HNSCC cervical lymph node metastasis HSC-4 cell lines, which bring the treatment hope for the advanced HNSCC patients especially with cervical lymph node metastasis. it may provide a new idea for HNSCC target treatment. |
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