| Purpose:Lung cancer is a serious threat to human health.So many studies in China have shown that the morbidity and mortality of lung cancer are the highest in malignant tumors.Basing on histopathology,lung cancer includes small cell lung cancer and non-small cell lung cancer,of which about 80%are non-small cell lung cancer.At present,the main treatments of lung cancer include traditional surgery,radiotherapy and chemotherapy,and some newly emerging methods such as molecular targeting,immunotherapy,and three-dimensional implantation of radioactive iodine-125.The treatments of lung cancer are still being updated and improved constantly,so that the life quality and survival of patients continue to be improved.However,when many patients are diagnosed clinically,the cancer cells have already metastasized,resulting in poor therapeutic outcome.Therefore,it is urgent to reveal novel therapeutic targets of lung cancer at the gene and molecular levels and screen potential biomarkers to monitor the metastasis of lung cancer cells,so as to provide more accurate treatment schemes to improve the treatment efficiency and survival rate of patients.In recent years,lots of studies have revealed that long non-coding RNA(lncRNA)is associated with the growth of many cancers and plays an important regulatory role in tumor cell proliferation,death,metastasis,and metabolism.Based on the analysis of clinical samples,this work finds that LNC CRYBG3 is highly expressed in lung cancer tissues,especially in metastasizing cancer cells,and is associated with malignant degree of cancer.So,the functions of LNC CRYBG3 in lung cancer metastasis and radiation sensitivity should be confirmed in further research and LNC CRYBG3 will be expected to be a therapeutic target in clinical lung cancer treatment.Methods:(1)We cultured four lung cancer cell lines including A549,HCC827,H1299,and H460,and two normal lung cell lines lincluding pulmonary bronchial epithelial cell lines BEAS-2B and pulmonary small airway epithelial cell lines HSAEC1-KT,then extracted total RNA of these cell lines for reversing transcription into cDNA.The expression of LNC CRYBG3 in these cells was detected by real-time fluorescent quantitative PCR.LNC CRYBG3 was knocked down in A549 and H1299 lung cancer cells using two lentiviral vectors containing LNC CRYBG3 shRNA to build cell models with stably suppressed expression of LNC CRYBG3.The proliferation of A549 and H1299 lung cancer cells with LNC CRYBG3 knockdown was observed by crystal violet staining.Transwell assay was used to analyze the migration and invasion abilitives of A549,HCC827,H1299,and H460.Basing on migration ability,A549 was considered as a high metastasis lung cancer cell model,and HCC827 was a low metastasis cell model.Using shRNA of LNC CRYBG3 to build a stable A549 cell lines with suppressed expression of LNC CRYBG3(A549-shLNC CRYBG3 cells).Using a transfection adenovirus vector containing LNC CRYBG3 gene to establish a LNC CRYBG3 over expression HCC827 cell line(HCC827-shLNC CRYBG3 cells).Using Transwell and Wound healing assay to analysis migration and invasion abilitives of the two cell lines with LNC CRYBG3 expression changes.The effect of LNC CRYBG3 on the expression levels of migration-related proteins Vimentin,Snail,N-cadherin and E-cadherin was detected by Western Blot.(2)In order to conform the effect of LNC CRYBG3 on metastasis of lung cancer cells in vivo,we established an animal model to determine the regulatory effect of LNC CRYBG3 on metastasis in vivo using SPF NOD/SCID mice injected with 2×106 of luciferase expressing A549 and HCC827 cells through tail veins.Twenty-four days after injection,the generation of bioluminescence in mouse body was detected by small animal imaging system,and the location and proliferation of metasta tumors were monitored.After the completion of imaging,the lung tissues of the mice were taken out to observe the pathological morphology of pulmonary nodules by HE staining and the immunohistochemical staining of the tumor proliferation marker Ki67.Immunohistochemical experiments were also carried out to detect the expression of metastasis-related proteins,Vimentin,Snail,N-cadherin,and E-cadherin in the lung tumors of each group.(3)The interaction between LNC CRYBG3 and eEF1A1 was analyzed by mass spectrometry and RNA pull down technique.In vitro experiments,the influence of LNC CRYBG3 on the expression levels of eEF1 A1 and MDM2 was detected by Western Blot.A plasmid containing mdm2 promoter region or promoter mutation sequence,and luciferase reporter gene was designed and transfected into LNC CRYBG3 knockdown and overexpressed cell model to verify the regulatory effect of LNC CRYBG3 on mdm2 gene transcription.After transfection of the plasmid for 48 h,the activity of luciferase in the cell lysate was detected.In order to further verify whether LNC CRYBG3 affects mdm2 transcription through eEF1A1,we used a small interfering RNA(siRNA)of eEF1A1 in the above experiments to study the difference in the expression activity of plasmids containing the normal mdm2 promoter sequence compared with the negative control group and the mutant group in LNC CRYBG3 overexpressed HCC827 cells.To further confirm that LNC CRYBG3 affects the metastasis of lung cancer cells through interacting with eEF1A1 and MDM2,transwell assay was used to detect the effects of eEF1A1 and MDM2 siRNA on the invasion and migration of HCC827-LNC CRYBG3 cells,and Western Blot was used to detect the effects of eEF1A1 and MDM2 siRNA on the expression of migration-related proteins in HCC827-LNC CRYBG3 cells.In order to explore the effect of LNC CRYBG3 on the co-localization of MDM2 and MDM2 binding protein(MTBP),as well as the co-localization of ACTN4 and MTBP,the interaction of MDM2,ACTN4,and MTBP was detected by immunoprecipitation and immunofluorescence techniques.(4)The expression of LNC CRYBG3 in lung cancer cells A549 and H1299 were detected at 12 h and 24 h after 4 Gy X-ray irradiation.In order to verify the effect of LNC CRYBG3 on the radio-sensitivity of lung cancer cells,A549 and H1229 cells knocked down of LNC CRYBG3,and irradiated by X-rays of 0,2,4,6,and 8 Gy,respectively.Cell colony formation was used to detect the clone formation rate and survival rate of the cells after X-ray irradiation.Flow cytometry analysis with 7-AAD/AnnexinV staining,TUNEL detection,yH2AX generation,and Western Blot were used to determine whether the decreased expression of LNC CRYBG3 affected the apoptosis of lung cancer cells after X-ray irradiation.Results:(1)The expression levels of LNC CRYBG3 in lung cancer cell lines A549,HCC827,H1299,and H460 were significantly higher than human lung bronchial epithelial cell line BEAS-2B and human lung small airway epithelial cell line HSAEC1-KT,and more than five times higher than HSAEC1-KT(p<0.001).After LNC CRYBG3 was knocked down,the proliferative abilities of both A549 cells and H1299 cells were significantly reduced,which were significantly different from the control cells with normal expression of LNC CRYBG3(p<0.05,p<0.05).It was found that the migration and invasion ability of HCC827 was significantly weaker than and other three cell lines,and the expression level of LNC CRYBG3 in HCC827 was also significantly lower than other three cell lines(p<0.001).Therefore,we chose A549 cells as high metastatic lung cancer cell model,and HCC827 as low metastatic lung cancer cell model,then using shRNAs to establish a stable LNC CRYBG3-low expression A549 cell line,using LNC CRYBG3 gene overexpression adenovirus vector to build a LNC CRYBG3-overexpression HCC827 cell line.After LNC CRYBG3 expression decreased,the migration and invasion ability of A549 was significantly reduced(p<0.001).On the contrary,after overexpressing LNC CRYBG3 in HCC827 with weak migration ability,its migration ability was significantly enhanced(p<0.001).Similarly,in the scratch repair experiment,the wound healing rate of A549 shLNC CRYBG3 cells within 48 h was significantly lower than the shNC group(p<0.05).In HCC827,after LNC CRYBG3 overexpression,both cell migration ability and wound healing speed were improved(p<0.05).After LNC CRYBG3 was knocked down in A549,the expression of cell migration-promoting proteins Vimentin and Snail was decreased,while in HCC827 with LNC CRYBG3 overexpression,the expression of cell migration-promoting proteins Vimentin and Snail was increased.In addition,increasing the expression of LNC CRYBG3 promoted a significant increasse in mesenchymal cell marker N-cadherin in the HCC827 cells,while the opposite effect was found in A549 after LNC CRYBG3 was knocked down.A549-shLNC CRYBG3 and HCC827-LNC CRYBG3 cells with luciferase expression were injected into the tail vein of NOD/SCID mice to establish an animal model for observing the metastasis of lung cancer cells in vivo.It was found that the number of A549-shLNC CRYBG3 cells migrating to mouse lung was significantly less than shNC,but more HCC827-LNC CRYBG3 cells migrated to lung.Secondly,the anatomical observation of these mouse lungs showed that the number of solid tumor nodules in the mouse lungs injected with A549-shLNC CRYBG3 cells was less compared to control group,while the number of solid tumors in the mice with HCC827-LNC CRYBG3 cells was more than control group(p<0.001).HE staining was used to observe the pathological morphology of pulmonary nodules and the immune-histochemical staining of the tumor proliferation marker Ki67,which further confirmed that these solid nodules in the mouse lungs were generated by injected tumor cells.In addition,immunohistochemical detection showed that the expression of Vimentin,Snail,and N-cadherin significantly increased in LNC CRYBG3 overexpression group.(2)Mass spectrometric analysis and RNA pulldown showed that LNC CRYBG3 could bind to eEF1A1 and promote the translocation of eEF1A1 into the nucleus.After entering the nucleus,eEF1A1 may act as a molecular chaperone of a transcription factor and participate in the regulation of MDM2 expression.To test this hypothesis,we designed a plasmid containing the mdm2 promoter region or promoter mutation sequence and luciferase reporter gene,and transfected it into the LNC CRYBG3 knockdown and overexpressed cell models to verify the regulatory effect of eEF1A1 on mdm2 gene transcription.After the plasmid was transfected for 48 h,luciferase activity was detected to determine the expression activity of the plasmid.It was found that in the HCC827-LNC CRYBG3 cells,the activity of the plasmid containing normal mdm2 promoter sequence was significantly activiated compared to control group and the mutant group(p<0.01).In contrast,in the LNC CRYBG3 knockdown cells,the expression activity of plasmids containing the normal mdm2 promoter sequence was significantly lower than the control group(p<0.05),and there was no significant change in the mutant group.Therefore,we learned that overexpressed LNC CRYBG3 promoted mdm2 transcription.In order to make sure the relationship between LNC CRYBG3 and eEF1A1 in regulating mdm2 transcription,we treated these cells with an eEF1A1 siRNA in the above experiments and found that the activity of normal mdm2 plasmid was inhibited in LNC CRYBG3 overexpressed HCC827 cell with no difference compared with negative control group and mutation group.Further studies showed that the increased expression of MDM2 induced by LNC CRYBG3 could bind to its downstream protein MTBP to reduce the binding of MTBP and ACTN4.Then,the free ACTN4 could promote the formation of cell pseudopod for increasing cell migration ability.These results fully demonstrated that LNC CRYBG3 promoted the expression of MDM2 through eEF1A1,increased the binding of MDM2 to MTBP,decreased the binding of MTBP to ACTN4,and promoted cell migration.(3)The expression level of LNC CRYBG3 in lung cancer cell lines A549 and H1299 were detected after 4 Gy X-ray irradiation.The LNC CRYBG3 expression levels in this two cell lines were significantly increased at 12 h and 24 hours after irradiation(p<0.01).To verify the effect of LNCCRYBG3 on the radiosensitivity of lung cancer cells,we knocked down the expression of LNC CRYBG3 in A549 and H1299.Cell colony formation assay was used to detect the survival rate of the A549-shLNC CRYBG3 and H1299-shLNC CRYBG3 cells after X-ray irradiation.It was found that the survival fraction of A549-shLNC CRYBG3 cells ware statistically different from A549-shNC cells with 2,4,6 and 8 Gy irradiation(p<0.01),but there was no effect on H1299 cells.Using flow cytometry assay with 7-AAD/AnnexinV staining shown that A549-shLNC CRYBG3 apoptotic rate was higher than A549-shNC.TUNEL staining showed that the intensity of green fluorescence in A549-shLNC CRYBG3 was more intense than A549-shNC after 4 Gy X-ray irradiation,suggesting the DNA damage was more serious with LNC CRYBG3 reduced.Similarly,the expression of yH2AX was seriously inecreased in A549-shLNC CRYBG3 related to A549-shNC by Western Blot assay,also the number of yH2AX positive foci were more in A549-shLNC CRYBG3 using immunofluorescence staining.Anti-apoptotic protein Bcl-2,apoptosis-promoting protein Bax and p53 were detected using Western Blot assay at 48 h after 4 Gy X-ray irradiation.We found that Bax and p53 was seriously increased while Bcl-2 was decreased in A549-shLNC CRYBG3 compared to shNC.However,low expression of LNC CRYBG3 had no effect on radiation-induced apoptosis of H1299 cells.Conclusion:(1)The expression of LNC CRYBG3 in lung cancer cells is significant upregulation compared to that in normal lung cells,and the increased expression of LNC CRYBG3 promotes the proliferation and metastasis of lung cancer.(2)The direct interaction of LNC CRYBG3 with eEF1A1 promotes eEF1A1 translocation into nucleus and binding to the promoter region of mdm2 gene to enhance MDM2 expression,then modulating the metastasis of lung cancer cells through MDM2/MTBP/ACTN4 axis.(3)LNC CRYBG3 is related to the radiation resistance of lung cancer cells,and inhibiting LNC CRYBG3 expression can enhance radiosensitivity of the lung cancer cells.The function of LNC CRYBG3 in radiosensitivity is associated with MDM2/P53 pathway. |
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