| ?Objective?In order to clarify the relationship between Cofilin-2 and radioresistance in nasopharyngeal carcinoma,a stable cell model of Cofilin-2 gene silencing CNE-2R was established using RNA interference technology.To determine whether Cofilin-2 can serve as a protein marker and potential therapeutic target for predicting radiotherapy response in nasopharyngeal carcinoma,the effect of silencing Cofilin-2 on the radiosensitivity of CNE-2R cells was explored through functional studies in vitro and in vivo.At the same time,we further analyzed the gene expression profile of CNE-2R cells after silencing of Cofilin-2 by human gene expression microarray,then preliminary explored the mechanism and pathway of Cofilin-2 in radioresistance of nasopharyngeal carcinoma.?Method?1.Three interference targets were designed and synthesized for Cofilin-2,and the best silence fragments were screened for lentiviral packaging and transfection of CNE-2R cells.The CNE-2R cells model which stably silenced Cofilin-2 gene was obtained by sorting.The qRT-PCR and Western-blot assays were used to detect the expression of mRNA and protein levels to verify the interference effect.2.The Cofilin2-RNAi-LV3 cell line with the best transfection effect was selected as the experimental group.To investigate the relationship between Cofilin-2 and the radiosensitivity of nasopharyngeal carcinoma,the CCK-8 assay,clone formation assay,and flow cytometry were used to observe the proliferation,cycle distribution,and apoptosis of nasopharyngeal carcinoma CNE-2R cells after silencing of Cofilin-2 gene.3.The tumorigenicity in nude mice was used to investigate the relationship between in vivo silencing of Cofilin-2 and radiation-resistance cell CNE-2R in human nasopharyngeal carcinoma.4.The expression of Cofilin-2 in human nasopharyngeal carcinoma tissues and blood was detected by ELISA and immunohistochemistry.5.Analysis of gene expression profiles of CNE-2R cells after silencing of Cofilin-2 by human gene expression chip sequencing,furthermore qRT-PCR and WB were used to preliminarily verify the related pathway of Cofilin-2 involved in radioresistance.?Results?1.After transfection of CNE-2R cells with lentivirus shRNA vector,the infectivity of each group was observed to be above 90% under inverted fluorescence microscope.The best interference target of Cofilin2-RNAi-LV3 among three targets was screened by qRT-PCR and Western blot.2.The results of CCK-8 showed that the survival rate of Cofilin2-RNAi-LV3 cells at doses of 2,4,6,8,10 Gy was lower than that of non-transfected CNE-2R and negative control NC group(F=105.641~153.373,p<0.05),suggesting that silencing Cofilin-2 enhances the inhibitory effect of radiation on the proliferation of CNE-2R cells.Clone formation experiments showed that the survival curves of Cofilin2-RNAi-LV3 group were lower than those of non-transfected CNE-2R group and negative control NC group at 2,4,6,8,10 Gy;The Dq and SF2 value of Cofilin2-RNAi-LV3 group were lower than that of the untransfected CNE-2R group and the negative control NC group(p<0.05).It was suggested that silencing Cofilin2 could increase the sensitivity of nasopharyngeal carcinoma CNE-2R compared to radiation.The cell cycle results showed that the distribution of cells in the untransfected CNE-2R group,negative control NC group,and Cofilin2-RNAi-LV3 group was(11.40±1.82)%,(11.91±2.12)%,and(39.69±2.89)% in the G2/M phase,respectively.The percentage of cells in the Cofilin2-RNAi-LV3 group at the G2/M phase was significantly higher than that of the non-transfected CNE-2R group and the negative control NC group(F=146.016,p<0.05).The results of apoptosis experiments showed that the apoptosis rates of Cofilin2-RNAi-LV3 group,untransfected CNE-2R group,and negative control NC group were(15.90±0.17)% and(10.60±1.71)%(10.60±1.71)% at 0 Gy irradiation,respectively.The apoptotic rates were(34.00±0.89)%,(20.53±1.89)%,and(20.60±1.21)% when irradiated with 10 Gy.The apoptotic rate of the Cofilin2-RNAi-LV3 group was higher than that of the non-transfected CNE-2R group and the negative control NC group(F=26.061,92.496,p<0.05)under irradiation of 0 and 10 Gy.It was showed that silencing of Cofilin2 could promote apoptosis in CNE-2R cells at the same dose.3.In vivo experiments showed that the volume of transplanted tumors in the Cofilin-2 silencing group after irradiation with 10 Gy rays was 241.00±53.02 less than that in the non-irradiated group 509.80±55.57,and the difference was statistically significant(F=20.341,p<0.05).Furthermore,the growth of transplanted tumors in the Cofilin-2 silencing group was also significantly slower than that in the Control and NC groups.In addition,we also used WB and IHC to detect the positive expression of Cofilin-2 protein in all groups of nude mice.It was found that the Cofilin-2 protein was negative in the Cofilin2-RNAi-LV3 group compared with the non-transfected CNE-2R group and the negative control(NC)group.The results were consistent with the in vitro functional verification,suggesting that the lentivirus-mediated Cofilin-2 gene RNAi interference vector can still be stably inherited in vivo and expressed efficiently.4.ELISA and IHC were used to detect the expression of Cofilin-2 protein in the tissues and serum of 35 patients with radiosensitivity and radioresistance.Cofilin-2 protein was found to be highly expressed in serum and tissues of human nasopharyngeal carcinoma radioresistant group,suggesting that it is a radioresistant protein of nasopharyngeal carcinoma and can be used as a molecular marker for predicting radiotherapy response of nasopharyngeal carcinoma.5.The human gene expression microarray(Affymetrix)was used to sequence and analyze changes in the gene expression profile of CNE-2R cells after silencing of Cofilin-2,and we found that there were 874 differentially expressed genes,of which 503 were up-regulated and 371 were down-regulated.Through enrichment and clustering,the differentially expressed genes were mainly related to RhoA Signaling,EMT Pathway,and Tp53 Signaling pathways.Furthermore,WB was used to detect pathway-associated protein expression.It was found that compared with NC group,silencing of Cofilin-2 in CNE-2R cells can inhibit the expression of RhoA pathway related proteins RhoA,Limk1,RND3,and PKN1,and promote the up-regulation of EMT-related pathway protein CDH1 then inhibit the expression of CDH2,VAV2,SNAI2,AKT1,EGFR,at the same time inhibit the expression of TP53 related pathway proteins TP53,MDM2,BCL2,THBS1,HIF1 A.?Conclusion?In this study,it was found that silencing Cofilin-2 can inhibit the proliferation of CNE-2R cells and enhance their radiosensitivity after silencing the expression of Cofilin-2 in CNE-2R cells.It may be related to the apoptosis of CNE-2R cells induced by irradiation of cells and the induction of G2/M phase arrest.In addition,tumor tissue growth in nude mice inoculated with CNE-2R cells that were silenced with Cofilin-2 was also inhibited after irradiation.The high expression of Cofilin-2 protein was detected in serum and tissues of human nasopharyngeal carcinoma radioresistant group.In addition,gene expression profiling of CNE-2R cells after silencing of Cofilin-2 was analyzed by human gene expression chip sequencing,and differential genes were found to be associated with the RhoA?EMT?TP53 pathway.Therefore,we believe that Cofilin-2 may be used as a protein marker and potential therapeutic target for predicting radiotherapy response in nasopharyngeal carcinoma,and it is speculated that Cofilin-2 may activate RhoA?EMT?TP53 pathway,which is involved in the radioresistance of nasopharyngeal carcinoma CNE-2R cells. |
PDF Full Text Request