Screening And Function Study Of Long Non-coding RNA Related To Radiotherapy Resistance In Lung Cancer Cell Line

Background:Radiotherapy as an effective local tumor cure way,however,radioresistance has been an obstacle for the success radiotherapy in lung cancer.It has been reported that long non-coding RNAs(lnc RNAs)play a role in radioresistance.In this study,we aimed to research the potential significance of lnc RNAs in the radioresistance of lung cancer.Methods:1.Establishment of radioresistant cells H460 R and A549 R.H460 cells were irradiated 3 times with the dose of 4 Gy,then 4 times with the dose of 6 Gy and 3 times with the dose of 8 Gy.The cells are cultured for 1-2 weeks after each irradiation.At last,The processes were repeated 10 times over a period of 6 months,for a total dose of 60 Gy.The irradiated strains were named H460 R and A549 R.2.Using CCK-8 assay to detection of radiosensitivity.Cells were seeded in ninety six-well plates and incubated for 12 hours,and then irradiated with a gradient of X-ray dose(0,2,4,6,8,12 and 16 Gy).After 48 hours,radiotherapy resistance was detected by CCK-8 assay.3.Using flow cytometry to detect the cell cycle distribution and using transwell assay to test migration behavior.4.Using high-throughput lnc RNA microarray assays to investigate differences in lnc RNA expression profiles between radiotherapy resistant strains H460 R and parental cell line H460.A candidate Lnc RNA MIR4453 was selected for radiotherapy resistance.5.Detected the biological effects of down regulating lnc RNA MIR4453 expression in radiotherapy resistant strains H460 R.Results:The radiotherapy resistant strains H460 R and A549 R demonstrated a significantly increased survival rate compared with the parental cells after 0,2,4,6,8,12,16 Gy radiation,respectively.Cell cycle analysis showed that H460 R and A549 R cells showed a statistically significant decrease in G1 phase and increase in S and G2/M phase.Transwellassay indicated that H460 R and A549 R cells significantly enhanced migration behavior.H460 and H460 R cells were detected by high-throughput sequencing and there were 71 differentially expressed lnc RNA(36 up-and 35 down-regulated,p<0.05).Down regulation MIR4453 expression can improve radioresistance sensitivity,reduce cell migration ability.Conclusions:1、The H460 R and A549 R radioresistant cell lines could be acquired by repeated exposure to radiation.2、Compared with the H460 and A549,radioresistant cell lines H460 R and A549 R showed great changes in biological characteristics.Cell cycle analysis showed that H460 R and A549 R cells decrease in G1 phase and increase in S and G2/M phase.Transwell assay indicated that H460 R and A549 R cells significantly enhanced migration behavior.3、LncRNAs of radiotherapy-resistant strains H460 R showed significant changes compared with H460.4、Lnc RNA MIR4453 is one of LncRNAs associated with radiotherapy resistance in lung cancer cells.Down regulation MIR4453 expression can improve radioresistance sensitivity,reduce cell migration ability.