| Ionizing radiation(IR)can induce a series of DNA damages,thereby affecting cellular genomic stability and inducing various acute cellular responses,as well as long-term biological effects such as mutation 、transformation 、death and carcinogenesis.In recent years,non-coding RNAs have been extensively reported to involve in regulation of carcinogenese and tumor progression,and therefore their functions attract broadening attention.However,there are few research reports focused on the effects of sno RNAs in IR-induced cancers,thus performing molecular exploration in this aspect will be in favor of deepening our understanding on the mechanism of radiation-related damages.AIM:To identify the sno RNAs which are involved in the cellular responses to IR-induced damage and carcinogesise,and further to investigate their functions and molecular mechanism in these processes.Method:Microarry analysis of transcriptom expression spectrum was performed to screen the differentially expressed sno RNAs between normal human bronchial epithelial cells BEP2 D and its cancerous derivative BERP35T-1 、 BERP35T-4 cells generated by α particle irradiation.Differentially expressed RNAs selected were further validated by real-time quantitative PCR;The exogenous expression vectors and si RNAs were prepared to overexpress or knockdown the expression of targeted RNAs;Flow cytometry and CCK8 assay were applicated to detect apoptosis and proliferation activity respectively after overexpressing or depleting targeted RNAs;Computer software(PLEXY and RNA structure)in combination with NCBI database were applied to predict the functional sequence elements of sno RNA and its targets;Afterwards,RT-q PCR,western blotting and dual luciferase reporter gene assay were used to examine the functional association between sno RNA / sd RNA and its predicted molecular targets;Finally,cell apoptosis and proliferation changes were determined after overexpressing or depleting these targets.Result:Transcriptome expression microarray analysis showed that sno RNAs is generally down-regulated in BERP35T-1 and BERP35-4 cells compared with BEP2 D cells,and most of the down-regulated sno RNAs belonged to the SNORD116 family.The sno RNA that is significantly down-regulated in both BERP35T-1 and BERP35T-4 cells is SNORD116-14.In consistence with results of transcriptome mircoarrays,the expression level of sno RNA SNORD116 family was found decreased in α-particlesinduced cancerous BERP35T-1、BERP35T-4 cells,and human lung cancer A549 and H1299 cells;the expression of the four sd RNAs which are derived from SNORD116-14 is decreased in both BERP35T-1 and BERP35T-4 cells.However,the expression level of these four sd RNAs derived from the same sno RNAs seems different even in the same cell at the unique condition.Overexpression of exogenous SNORD116-14 enhanced apoptosis occurrence and repressed cell proliferation activity of H1299 cells;ZNF280D,TFDP1,CCDC28 B,RPS6KA3,RUNX1,and KALRN were identified as the targets of SNORD116-14 through in silico analysis;the m RNA levels of ZNF280 D,TFDP1 and RPS6KA3 were upregulated in BERP35T-1 and BERP35T-4 cells as compared with the normal BEP2 D cells,and overexpressing SNORD116-14 depressed the m RNA levels of ZNF280 D,TFDP1 and RPS6KA3,as well as the protein level of RPS6KA3;The sd116-14-2,a derivative of SNORD116-14,was found to target the degradation of RPS6KA3,and knockdown of RPS6KA3 could potentiate the induction of apoptosis and suppression of the cell proliferation of He La cells by γ-rays irradiaiton.Conclusion:The expression level of SNORD116 family is depressed in α particle-induced cancerous BERP35T-1 and BERP35T-4 cells as well as human lung cancer A549 and H1299 cells.Furthermore,sd116-14-2 is specifically derived from SNORD116-14,it can drive tumor cells to undergo apoptosis and inhibit cell proliferation,and which could be partilly attributed to it-mediated silencing of RPS6KA3.Our data suggest that SNORD116-14 may act as a tumor suppressor. |
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