Screening Of Differentially Expressed MiRNAs In A549 Cells Induced By 17-AAG-M And Its Effect On Radio-sensitivity

PurposeTo screen differentially expressed miRNAs in 17-AAG-M pretreated human non-small cell lung cancer A549 cells under X-ray,to analyze the miRNA that can be combined with 17-AAG-M,X-ray to treat NSCLC.To study its roles in A549 cells and its effects on radiation response ability,to evaluate its potential applicability in combination with 17-AAG-M and X-ray for NSCLC,and to analyze its effects on DNA damage response induced by ionizing radiationMethodsSolvent dialysis was used to prepare 17-AAG-M.Transmission electron microscope was used to observe the morphological characteristics and distribution of micelles.Nanometer particle size and zeta potential analyzer were used to determine the size and dispersion of micelles.UV spectrophotometry was used to determine the concentration.Microarray experiments were used to screen 17-AAG-M pretreated A549 cells for differentially expressed miRNAs at 24h after 4Gy radiation.The TCGA database was used to analyze the expression of miRNAs in tumors which may be involved in the radiation response,and their relationship with clinical factors,and to predict the miRNA that can be combined with 17-AAG-M,X-ray to treat NSCLC.The miRbase database was used to predict the target genes of hsa-miR-30a-3p and to perform GO and KEGG analysis,qPCR was used to detect the expression of hsa-miR-30a-3p in A549 cells.MTT experiment and clone formation experiment were used to detect the effects of over-expressing hsa-miR-30a-3p on the viability and proliferation of A549 cells,and the changes after combined with X-ray and 17-AAG-M;multitarget-single hitting model was used to analyze the effect of hsa-miR-30a-3p overexpression on the radiosensitivity of A549 cells,and the change in radioactivity after combined with 17-AAG-M.After confirming the role of hsa-miR-30a-3p in the radiation response of A549 cells,γ-H2AX immunofluorescence staining experiment and cell cycle experiment were used to detect the effects of hsa-miR-30a-3p overexpression on the DNA damage response.Results(1)The solvent evaporation method was successfully used to prepare nano-polymer micelles 17-AAG-M with small particle size,good dispersibility,and stability:the average particle size was(66.96±0.61)nM,and the polydispersity coefficient(PDI)was 0.149,the transmission electron microscope results confirmed that 17-AAG-M was round,approximately spherical,and uniformly distributed.(2)Microarray experiment confirmed that ionizing radiation affected miRNA expression and altered the expression profile of miRNA.20 miRNAs that were differentially expressed after irradiation were screened from 17-AAG-M pretreated A549 cells.(3)TCGA showed that hsa-miR-30a-3p was significantly under-expressed in most human tumors,especially lung cancer,and was related to the pathological T stage and gender of LUAD patients.(4)GO and KEGG analysis showed that hsa-miR-30a-3p participated in 50 biological processes including cell proliferation.And it affected cancer signaling pathways,hsa04010 MAPK signaling pathway and cell cycle.(5)The qPCR results confirmed that whether or not A549 cells were pretreated with 17-AAG-M,the relative expression level of hsa-miR-30a-3p decreased after 4Gy radiation(Control:1.00;X-ray:0.72;17-AAG-M:2.42;17-AAG-M+X-ray:0.16).(6)MTT results showed that up-regulating the expression of hsa-miR-30a-3p could inhibit the cell viability of A549 cells(survival rate:78.52%),and was more toxic to A549 cells under X-ray and 17-AAG-M.(7)Clone formation experiment confirmed that overexpression of hsa-miR-30a-3p could inhibit the proliferation of tumor cells(p<0.05),and could improve the radiosensitivity of human non-small cell lung cancer A549 cells.After combined with 17-AAG-M,it had more obvious tumor suppressive effect and radiation sensitization effect,the radiation sensitization ratio was 1.48.(8)Overexpression of hsa-miR-30a-3p or using 17-AAG-M could significantly increase the number of DNA double-strand breakpoints in A549 cells induced by ionizing radiation,causing delayed DNA damage repair.The effect of the combined group was more significant(9)17-AAG-M combined with X-ray could cause significant G2/M phase arrest,and overexpression of hsa-miR-30a-3p could also interfere with the cell cycle processConclusionshsa-miR-30a-3p was underexpressed in non-small cell lung cancer and differentially expressed under the action of X-ray and/or 17-AAG-M.Overexpression of hsa-miR-30a-3p could inhibit the vitality and proliferation ability of human non-small cell lung cancer A549 cells,and could enhance the radiation response ability of A549 cells,with radiation sensitization effect;after combined with 17-AAG-M,the effects were more significant.These could be partially attribute to the facts that hsa-miR-30a-3p and/or 17-AAG-M combined with X-ray caused a more obvious delay of DNA damage repair and interfered with the cell cycle process.