Role And Mechanism Of MiR-18a On Sensitizing Radiation Response Of Cancer-stem-like Cells In Lung Adenocarcinoma

BackgroundAs the incidence and mortality increased continuously,cancer is the leading cause of death in China.The latest statistical data indicated that an estimated 4,292,000 new cancer cases and 2,814,000 cancer deaths occurred in China in 2015,with lung cancer being the most common incident cancer and the leading cause of cancer death.Radiotherpy is one of the main treatments in lung cancer at present.However,non small cell lung cancer?NSCLC?,especially lung adenocarcinoma is radioresistant which caused poor therapeutic outcomes when treated with radiotherapy.Recentlly,more and more researchs manifest that cancer stem cells?CSCs?are the root of radioresistance.Hence,inorder to target killing the CSCs,it is a new stratege to explore the mechanism of radioresistance caused by CSCs and it would be a new way of gene-radiotherpy in future.microRNAs?miRNAs?,as a small non-coding RNA,engage in tumor generation and development and act as tumor suppressor or tumor promoter.miRNAs are involved in cancer development and play an important role in tumor promotion and tumor suppression.miRNAs are also involved in the regulation of the biological functions of cancer stem cells,including cancer stem cell self-renewal,differentiation and resistantance to chemotherapy and radiotherapy.Our previous study found low expression of miR-18a in CD 133 + cancer stem-like cells,whereas miR-18a can improve the general lung adenocarcinoma cell line?A549?radiosensitivity.However,it is still not known whether miR-18a could regulate cancer stem-like cells' radiosensitivity and whether it could be a molecular target for radiation targeted killing of cancer stem cells.The project is planned to start from miR-18a and explore the relationship between the expression level of miR-18a in CD133 + cancer stem-like cells and the radiosensitivity of CD133 + cancer stem cell-like cells.PurposesTo investigate the role of miR-18a and its regulatory mechanism in lung cancer stem-like cells in radiation resistance and explore the possibility of miR-18a as a radiation sensitizer molecule in targeted killing of cancer stem cells.Methods1.The use of paclitaxel and stem cell culture medium to screen lung adenocarcinoma cell A549 and lung adenocarcinoma stem cells Flow cytometry was used to isolate CD133+ cells.Real-time fluorescence quantitative polymerase chain reaction?Real-time fluorescent quantitative reverse transcription polymerase chain reaction,qRT-PCR?,balling experiment and the clone formation induction assay were used to determine the screened stem cell characteristics.Clone forming ability of irradiated CD133+ and CD133-cells was detected by colony formation assay.The expression of miR-18a in CD133+ and CD133-cells was assayed by qRT-PCR.Construct the miR-18a over expression lentiviral vector?Lentivirus,LV?and transfection system.CD133+ cells were divided into miR-18a lentiviral transfection group?miR-18a-LV?,empty vector transfected group?EV?and blank control Group?blank?.Sternness related genes expression and mir-18a expression.were detected by qRT PCR after transfection and the ball formation ability was aslo tested.Different doses of ray irradiation were used to irradiate the CD133+ cells and LV transfected CD133+ cells.Divided into miR-18a-LV group,EV group and blank group,clonogenic assay was calculated by the number of colonies formed.The cell survival curve was gained and the adiobiological parameters,D0,Dq,and SF2 were calculated according to the survival rate.2.The online target gene prediction softwares pictar?http://pictar.mdc-berlin.de/cgi-bin/PicTar?,targetscan?Http://www.targetscan.org/vert₅0/50RMB/?and Miranda?http://www.microrna.org/?were used to predict the target gene of miR-18a.Downregulation of hypoxia inducible factor 1 alpha?HIF-la?by miR-18a was measured by the dual luciferase system,qRT-PCR and Western blot experiment.The lentiviral vector establish miR-18a overexpression of human lung adenocarcinoma cell line A549 and nude mice transplantation tumor experiment were used to verify miR-18a enhanced radiation on tumor killing effect.qRT-PCR and Western blot was used to detect the expression of miR-18a and HIF-1? in transplanted tumor tissue.3.Plasma samples were collected in patients with NSCLC before radiotherapy.qRT-PCR was used to detect miR-18a expression in these plasma samples.The correlation between expression of mir-18a and radiotherapy in patients was analyzed and the value of best cut-off miR-18a was calculated though receiver operating characteristic?ROC?.Based on miR-18a boundary value,the NSCLC patients were devided into two groups.The response rates?ORR?were compared between the two groups.According to the follow-up data,whether there is difference in progression free survival?PFS?and overall survival?OS?between miR-18a high expression group and miR-18a low expression group were analyzed.The sensitivity and specificity were calculated as miR-18a expression level in NSCLC to predicte the radiosensitivity.Results1.Serum free medium induced lung adenocarcinoma cell clone formation and CD133+cells were obtained after flow cytometry.Stemness experiments show that these cells have characteristic of stem cells.Cloning formation assay results showed that CD 133+ cell colony formation ability were higher than that of CD 133-cells.qRT-PCR results suggested that the level of miR-18a in CD133+ cells was lower than that of CD133-cells.The miR-18a stable overexpression CD 13 3+ cells were obtained by lentiviral transfection.Radiation cloning experiment showed that miR-18a overexpression CD 133+ cells' clone formation ability is lower than the control cells and the radiobiological parameters like D0,Dq,SF2 were lower than the control group?P<0.05?.Indicate that miR-18a increased the radiation sensitivity of CD133+ cells.2.Dual luciferase results showed that miR-18a can combine with the HIF-1? gene'3'-UTR.The results of Western blot suggested that the the expression of HIF-la protein was decreased after up regulate of miR-18a in A549 cells,?P<0.05?.After exposure to radiation,miR-18a in A549 cells declined while the expression level of HIF-1? gene and protein increased.Tumorigenicity and irradiation experiments confirmed that,compared with the vector group and blank control group,the tumor volume and tumor growth decreased in the miR-18a lentivirus transfection group in vitro?P<0.05?.Compared with the vector group and blank control group,the expression of miR-18a increased and HIF-la gene and protein expression decreased in the miR-18a lentivirus transfection group after irradiation in the xenografts.3.The plasma samples of 54 NSCLC patients were collected before radiotherapy.The expression level of miR-18a in plasma was detected by qRT-PCR assay.Correlation analysis suggested that miR-18a expression had significant difference in radiotherapy effective group?CR+PR?and radiotherapy resistant group?SD+PD??P<0.05?.ROC results suggest that the best cut-off value of miR-18a/Cel-39 is 2.28.Divided by the cut-off value,the objective response rate?ORR?of the miR-18a high expression group is higher that of the miR-18a low expression group?87%vs 6%,P<0.05?.Followed the same division,both the median PFS?8.1m vs 6.5m,P>0.05?and OS?18.7m vs 15.5m,P>0.05?are both no significant difference.The sensitivity and the specificity of miR-18a predicting the radio sensitivity was 87%and 95%.Conclusions1.CD133+ stem cell like cells have a radiation resistance effect,its low expression of miR-18a,may be one of the important mechanisms of radiation resistance.2.miR-18a overexpression can enhance the sensitivity of CD133+ stem cell like cells to radiation.3.HIF-1? is a target gene of miR-18a,and miR-18a down regulated HIF-la expression is the molecular mechanism of enhancing the sensitivity of lung adenocarcinoma stem cell like cells to radiation.4.Plasma miR-18a can be used as a biomarker to predict the sensitivity of radiotherapy in patients with NSCLC.