Ionizing Radiation Induced LncRNA CRYBG3 Regulates Lung Cancer Growth And Metastasis Through Mechanotransduction

Purpose:Radiotherapy is an essential component of comprehensive oncology treatment and it is reported that 65～75%cancer patients need it during their treatment process.Ionizing radiation poses killing effect on tumours through causing DNA lesions directly or reactive oxygen species(ROS)induced biological macromolecular damage indirectly.However,the infuence of ionizing radiation on tumor tissue’s mechanical properties is not well documented.Cells receive external mechanical signals through mechanoreceptors and generates the corresponding internal mechanical and signal transmission by the cytoskeleton system.It has been well recognized that mis-regulation of mechanotransduction leads to diseases or even cancer.Recent studies have shown that the alteration in mechanotransduction pathways in tumor tissues are also an immportant contributor to tumor migration.Therefore,the link between mechanotransduction pathways and cancer radiotherapy needs to be addressed.In the previous work,we found that the lncRNA CRYBG3 induced by ionizing radiation can directly bind to G-actin to inhibit its polymerization,leading to cell cycle M-phase block and functioning as an oncogene.In mouse experiments,it was confirmed that lncRNA CRYBG3 knockdown contributed to tumor progression.LncRNA CRYBG3 binds to G-actin and prevents its formation of actin filaments,so,does lncRNA CRYBG3 regulate mechanotransduction pathways through the cytoskeleton system?In this project,we used lung tumor cells and mouse xenograft models to study the effect of lncRNA CRYBG3,an ionizing radiationsensitive responser,on mechanotransduction pathways in order to elucidate whether or not lncRNA CRYBG3 acts as a bond between ionizing radiation and mechanotransduction pathway to regulate lung tumor proliferation,invasion,and metastasis by regulating the activity of mechanotransduction pathway effector molecules YAP/TAZ.Methods:(1)Mouse model bearing tumors was established.After precise X-ray irradiation of the tumor area guided by CT,the stiffness of the tumor tissue,collagen fiber content,and changes in p-MLC2 were detected after ionizing radiation using atomic force microscopy(AFM),Sirius red staining,and immunofluorescence experiments,respectively,to evaluate the changes in mechanical characteristics of the tumor tissues induced by ionizing radiation;(2)An A549-lncRNA CRYBG3 shRNA xenograft model or a lncRNA CRYBG3 overexpression xenograft model was constructed,and the stiffness of tumor tissues and the change of p-MLC2 were detected using atomic force microscopy and immunofluorescence experiments,respectively,to evaluate the impact of lncRNA CRYBG3 on the change in mechanical characteristics of tumor tissues;and the changes in Young’s modulus as well as p-MLC2 after overexpression of lncRNA CRYBG3 in A549 cells were examined using atomic force microscopy experiments and immunofluorescence assays,respectively,to assess the mechanical properties of the tumor cells themselves;(3)High-throughput sequencing of eukaryotic transcriptome was used to detect the downstream pathways regulated by lncRNA CRYBG3;Lung cancer cell lines A549 and Calu-1 were used to study the effects of lncRNA CRYBG3 on YAP/TAZ,key effector molecules in mechanotransduction pathway.The expression of lncRNA CRYBG3 in tumor cells and the effect of lncRNA CRYBG3 on the transcriptional activity of YAP/TAZ were evaluated using immunofluorescence,real-time fluorescent quantitative PCR,immunoblotting,and luciferase reporter assays;(4)The biological effects of lncRNA CRYBG3 on lung tumors were studied mainly by constructing lncRNA CRYBG3 and TAZ knockdown A549 cell line,and by using colony-formation assays,Transwell invasion assays,and scratch assays;We established tumor-bearing model and in vivo metastasis model,by using tumor volume monitoring,immunohistochemistry,and animal imaging experiments to detect the growth of subcutaneous lung tumors,changes in Ki67 expression,and metastasis of lung tumor cells in each organ,respectively;(5)and(6)To investigate whether lncRNA CRYBG3 regulates the transcriptional activity of the effector molecule YAP/TAZ by inhibiting the formation of cytoskeletal microfilaments,we used both exogenous drug Jasplakinolide treatment and interference with the expression of the endogenous molecule ADF/CFL1 to inhibit the depolymerization of actin microfilaments in A549 and Calu-1 cell lines.Atomic force microscopy,immunofluorescence,real-time fluorescence quantitative PCR,and luciferase reporter assays were used to examine whether inhibition of cytoskeletal depolymerization caused by lncRNA CRYBG3 could restore the Young’s modulus of cells,protein localization of YAP/TAZ,expression and transcriptional activity levels of downstream target genes,respectively;(7)Finally,to investigate how the cytoskeleton regulates YAP/TAZ,we examined the phosphorylation level of LATS1,a key kinase of the Hippo pathway,using immunoblotting assays,while immunofluorescence assays,real-time fluorescence quantitative PCR assays and luciferase reporter assays were used to examine whether combined treatment with LATS1/2 interference and lncRNA CRYBG3 overexpression could restore cytoskeletal microfilament depolymerization and the transcriptional activity level of YAP/TAZ,respectively.Results:(1)After irradiation,Young’s modulus of tumor tissue was significantly decreased.Meanwhile Sirius red staining revealed that X-rays was able to reduce the collagen fiber content in tumor tissues.And immunofluorescence revealed that X-rays reduced the level of p-MLC2;(2)We also found that overexpression of lncRNA CRYBG3 in tumor tissues significantly reduced their Young’s modulus and p-MLC2 expression levels,which was consistent with the phenotype obtained from transplanted tumors irradiated with X-rays,while knockdown of lncRNA CRYBG3 increased Young’s modulus and pMLC2 expression levels in tumor tissues.Overexpression of lncRNA CRYBG3 also significantly reduced Young’s modulus and p-MLC2 expression levels in in vitro cultured tumor cells;(3)We found that the expression levels of major YAP/TAZ target gene were significantly suppressed in A549 cells overexpressing lncRNA CRYBG3 by gene-set enrichment analysis of high-throughput sequencing data.Immunofluorescence experiments revealed that overexpression of lncRNA CRYBG3 significantly inhibited the formation of actin microfilaments,while YAP/TAZ was also retained in the cytoplasm.To further demonstrate YAP/TAZ out of the nucleus,we did cell subcomponent isolation experiments and found that YAP protein expression was significantly increased in the cytoplasm and decreased in the nucleus after overexpression of lncRNA CRYBG3.Real-time fluorescent quantitative PCR assays revealed that the mRNA expression levels of classical target genes of YAP/TAZ,such as CTGF,CYR61 and ANKRD1,were significantly reduced after overexpression of lncRNA CRYBG3.Simultaneous,using a luciferase reporter vector containing a YAP/TEAD target promoter revealed that lncRNA CRYBG3 significantly reduced the transcriptional activity of endogenous YAP/TAZ.In contrast,knockdown of lncRNA CRYBG3 significantly enhanced the transcriptional activity of YAP/TAZ;(4)Next,we examined the biological effects of lncRNA CRYBG3 on lung tumors.The results of colony-formation assays showed that knockdown of lncRNA CRYBG3 significantly increased the clonogenic ability of lung tumor cells,and this enhancement could be restored by TAZ inhibition.In the xenograft model,deletion of TAZ restored the accelerated tumor growth,increased tumor volume and increased Ki67 expression caused by lncRNA CRYBG3 knockdown.Also,using Transwell invasion assays and cell scratching assays,it was found that lncRNA CRYBG3 could promote the invasion and metastasis of lung tumor cells and could be recovered by TAZ inhibition.In addition,an in vivo metastasis mouse model constructed by tail vein injection revealed that lncRNA CRYBG3 promoted lung metastasis of tumor cells,and this increase in organismal metastatic capacity could be restored by knocking down TAZ;(5)and(6)Finally,we explored the potential molecular mechanism of lncRNA regulation of YAP/TAZ.We found that the reduction in cellular Young’s modulus induced by lncRNA CRYBG3 was restored after inhibition of F-actin depolymerization using Jasplakinolide,which promotes excessive cytoskeletal polymerization,or by interfering with the expression of ADF/CFL1.Meanwhile,inhibition of cytoskeletal depolymerization could restore the reduced YAP/TAZ transcriptional activity caused by lncRNA CRYBG3;(7)We further explored the mechanism through which the cytoskeleton regulates YAP/TAZ transcriptional activity and found that LATS1/2,a key kinase of the Hippo pathway,plays an important role.LncRNA CRYBG3 was able to significantly increase the expression of phosphorylated LATS1,which in turn led to the phosphorylation of YAP and eventually resulted in the retention and degradation of YAP in the cytoplasm.It was also found by immunofluorescence experiments that knockdown of LATS1/2 could restore the reduced YAP/TAZ transcriptional activity caused by lncRNA CRYBG3,but not the cytoskeletal depolymerization caused by lncRNA CRYBG3.Conclusion:Ionizing radiation-induced lncRNA CRYBG3 can attenuate the transcriptional activity of YAP/TAZ by interfering with the mechanotransduction pathway,thereby limiting the proliferation,invasion and metastasis of lung cancer cells.In mice,we found that deletion of lncRNA CRYBG3 enhanced lung tumor proliferation and metastasis,but these effects were abolished by concomitant interference with TAZ.At the molecular level,lncRNA CRYBG3 was able to inhibit actin microfilaments and promote phosphorylation of LATS 1/2,leading to phosphorylation and retention of YAP/TAZ in the cytoplasm,ultimately resulting in reduced transcriptional activity of YAP/TAZ.Our study proposes that lncRNA CRYBG3 can control lung tumor growth and metastasis by controlling mechanotransduction and linking YAP/TAZ transcriptional activity in tumor tissues,thus playing an important role in tumour radiotherapy.