| Objective:Study C-C chemokine ligand 5(CCL5)expressed in head and neck squamous cell carcinoma,and by siRNA transfection laryngeal squamous cell carcinoma Hep-2,TU177,explore siRNA interference after laryngeal squamous cell carcinoma on the proliferation,migration,cycle,apoptosis,drug-resistant related biological characteristics research,thus for future CCL5 in laryngeal squamous cell carcinoma of the targeted therapy provides theoretical basis.Methods:TCGA database was mined and analyzed for the expression of CCL5 in patients with head and neck squamous cell carcinoma.Laryngeal squamous cell lines(TU177 and Hep-2)were cultured and RT-PCR was used to detect the knockdown efficiency of CCL5 in different cell lines.The siRNA with the highest knockdown efficiency was screened in different cell lines for subsequent experiments.Each cell line was transfected with siRNA and NC,the proliferation of cell lines in each group was detected by CCK8 method,the migration of cells in each group was detected by cell scratch method,the cell cycle and apoptosis of cells in each group were detected by flow cytometry,and the mRNA and protein levels of drug-resistant genes Bax and MRP2 were detected by RT-PCR and western blot.The results were analyzed by SPSS 25.0 software,and the result pictures were completed by Graph Pad Prism 8.0.The cell scratch and western blot experiments were analyzed by Image J software.The experimental results meet the normal distribution and the differences between the two groups are evaluated by the t-test of two independent samples.One-way analysis of variance(ANOVA)shows statistical differences between multiple groups of samples.If there is statistical significance,LSD-t is used for pair comparison between groups.Measurement data was represented by?x±s.If the experimental results did not meet the normal distribution,rank sum test was used.If the rank sum test among multiple groups was statistically significant,pairwise comparison was conducted between groups.P < 0.05 was considered statistically significant.Results:1.The expression of CCL5 in head and neck squamous cell carcinoma was significantly higher than that in head and neck normal tissues by testing TCGA database(P < 0.001).2.All three siRNAs were transfected into laryngeal carcinoma squamous cells TU177 and Hep-2,and RT-PCR was used to detect their knockdown efficiency.It was found that si-1 had the best knockdown efficiency in TU177 cell line,while si-2 and si-3had higher knockdown efficiency in Hep-2 cell line.Since si-2 had a higher knockdown efficiency,si-2 was selected for the subsequent experiments of Hep-2 cell line.The TU177 cell line was divided into si-1 group and NC group,and the Hep-2 cell line was divided into si-2 group and NC group.Proliferation,migration,cycle,apoptosis and the genes related to drug resistance were detected in the two cell lines.The results showed that the proliferation rate of the si-1 group at 24 h,48h,72 h and 96 h was significantly lower than that of the NC group(P < 0.01),and the mobility rate of the si-1 group at 24 h was significantly lower than that of the NC group(P < 0.05).The late apoptosis of si-1group was higher than that of NC group(P < 0.05),and the relative expression level of Bax in si-1 group was higher than that in NC group(P < 0.05),suggesting that inhibiting the expression of CCL5 in TU177 cell lines can inhibit the proliferation and migration of LSCC,mainly promote its late apoptosis,and improve the sensitivity to cisplatin.In Hep-2 cell lines,the proliferation rate of si-2 group at 24 h,48h,72 h and 96 h was significantly lower than that of NC group(P < 0.01),and the mobility rate of si-2 group at 24 h and 48 h was significantly lower than that of NC group(P < 0.05).The early and late apoptosis in si-2 group were higher than those in NC group(P<0.001),and the relative expression of MRP2 in si-2 group was lower than that in NC group(P < 0.05).The protein expression level of Bax in si-2 group was higher than that in NC group(P <0.001).The results suggest that inhibiting the expression of CCL5 in Hep-2 cell lines can inhibit the proliferation and migration of laryngeal squamous cell carcinoma,promote its apoptosis,and reduce the cisplatin resistance.However,in Hep-2 cell lines,CCL5 was found to inhibit cell cycle by G2/M phase(P < 0.01)in statistical method,but its inhibitory effect was not obvious and did not reach clinical significance.Conclusion:CCL5 is highly expressed in head and neck squamous cell carcinoma,and siRNA interference with CCL5 can inhibit the proliferation,migration and cisplatin resistance of laryngeal carcinoma cells,and promote apoptosis and cisplatin sensitivity. |
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