Mechanism Studies On The Effects Of Radiation-induced LNC CRYBG3 On The Progression Of NSCLC

Objective:Lung cancer is a cancer with high morbidity and mortality.China accounts for one third of newly diagnosed with lung cancer of the global population each year.Radiotherapy alone or in combination is a common treatment for lung cancer patients,but radiation resistance is one of the factors that make it difficult to further improve the efficacy of conventional radiotherapy.Due to its unique inverted depth dose distribution,higher relative biological effects,and lower oxygen effects,heavy ions have better therapeutic effects than conventional radiation.Compared with conventional radiotherapy,the local tumor control rate of heavy ion therapy is increased,and the better safety and shorter treatment course are also achieved.However,the exact radiobiological mechanism of carbon ion therapy has not been fully elucidated.Therefore,the study of effector molecules with different changes after carbon ion and X-ray radiation in lung cancer cells may have potential value for the clinical application of carbon ion-induced effector biological macromoleculesMethods:The Squamous cancer cell line and melanoma cell line were used as cell models for the research on the sensitivity of cancer cells to ionizing radiation.The radiation type included X-rays and carbon ion beams,The experiment was divided into different dose groups.The cell survival rate after X-ray and carbon ion irradiation was measured by the clone-formation assay.The total RNA was extracted after 4 Gy of X-ray and carbon ion radiation for IncRNA detection with a chip,and Q-PCR was performed to verify the results.Gene expression detected with qRT-PCR,cell proliferation quantified with cell counting,and cell cycle distribution monitored with flow cytometry were performed to study on the relationship between LNC CRYBG3 and lung cancer progression.LncRNA overexpression in lung cancer cells was achieved with an adenovirus overexpression vector.LncRNA knockdown was achieved with a Lentiviral overexpression shRNA vector.In the mechanism study section,the effect of LNC CRYBG3 on cytoskeletal microfilaments was studied using immunofluorescence.Target RNA pull-down experiments were conducted to reveal the target proteins that interact with LNC CRYBG3.Protein expression was verified by Western Blot.The interaction between the lncRNA and its target protein was verified by RNA immunoprecipitation(RIP).In the animal experiment part,we constructed a cell line of LNC CRYBG3 over-expression and knock-down expression,then the constructed cell line was injected into the lower groin of mice's bilateral rear limbs to construct a tumor-bearing mouse model.After the transplantation tumor model was established,The LNC CRYBG3 adenovirus overexpression vector or Lentiviral overexpression shRNA vector was injected to study the effect of LNC CRYBG3 on tumors.The expression of LNC CRYBG3 and Actin polymerization in mouse tumors were detected by FISH.And Immunohistochemistry was used to detect the tumor differentiation.In vivo lung cancer cell metastasis model study,the constructed cell lines were injected through the tail vein of mice,and the homing and proliferation status of lung cancer cells in the lungs of mice were observed with a live animal imager.Results:(1)Sensitivity of cancer cells to heavy ion radiation.Squamous cell carcinoma cells were more sensitive to X-rays than melanoma cells,however,both melanoma cells and squamous cell carcinomas were sensitive to carbon ion beams.Squamous carcinoma cells were more sensitive to carbon ions than to x-rays.(2)Radiation-related lncRNA expression profiles.Chip data proves that both X-ray radiation and carbon ion radiation caused in a wide range of long-chain noncoding RNA expression.These changes are closely related to cytoskeleton dynamics regulation,cell cycle regulation,cell mitosis regulation,and apoptosis regulation.The expression of LNC CRYBG3 was up-regulated by both-X-ray and carbon ion irradiation,and the expression of LNC CRYBG3 induced by irradiation with carbon ion was significantly higher than by X-ray irradiation.The results suggest that LNC CRYBG3 may be related to radiosensitivity to carbon ion radiation.(3)LNC CRYBG3 expression in clinical lung cancer tissues and normal tissues.LNC CRYBG3 expression in lung normal tissues was significantly lower than that in lung cancer tissues.The expression of LNC CRYBG3 in lung cancer tissues of patients with lymph node metastasis was significantly higher than that of patients without lymph node metastasis.The results show that LNC CRYBG3 has a positive correlation with the malignancy of lung cancer.(4)Biological effects of LNC CRYBG3.Overexpression of LNC CRYBG3 can significantly caused G2/M nhase arrest Further phosphorvlated H3 assay revealed that the cells mainly exhibited M-phase arrest.After LNC CRYBG3 overexpression,a large number of binuclear cells appeared.The fate of M phase arrest cells was apoptosis and significantly reduced cell proliferation.(5)Effects of LNC CRYBG3 on cytoskeleton dynamics.Immunofluorescence experiments revealed that LNC CRYBG3 overexpression had no effects on the microtubule structure but microfilament in three lung cancer cells A549,H1299,and Calu-1 and human normal lung embryo fibroblasts MRC-5.LNC CRYBG3 inhibited the polymerization of globular actin into filamentous actin,and then increased the ratio of G-actin and F-actin in the cell.The destruction of the microfilament structure lead to Cyto skeletal destruction and the failure of the constricted ring at the end of mitosis.Co-immunoprecipitation of RNA,RNA Pull Down experiments and G-actin polymerization experiment in an in vitro cell-free system proved that LNC CRYBG3 interacted with G-actin directly.The truncated and mutant designs of LNC CRYBG3 further clarified that the spatial structure of LNC CRYBG3 is related to its function.Subsequent construction of amino acid mutants proved that the Serine at actin 14 may be one of the important sites that affect the binding to LNC CRYBG3 or the other function of actin.(6)LNC CRYBG3 could inhibit the growth of xenografts tumors.The volume and mass of tumors produced by cells overexpressing LNC CRYBG3 was significantly reduced.The tumor volume of cells with LNC CRYBG3 knockdown increased significantly.This phenomenon indicated that high expression of LNC CRYBG3 inhibited tumor growth.But the degree of tumor differentiation increased when LNC CRYBG3 was knocked down.While it decreased when LNC CRYBG3 was overexpressed.Conclusion:This study focused on the effects and mechanisms of carbon ion radiation on lung cancer cells and lung cancer tissues.The conclusions are as follows.1?Ionizing radiation,including X-ray and carbon ion radiation,significantly increased the expression of LNC CRYBG3,However,the expression of LNC CRYBG3 increased more drastical in carbon ion irradiation.2?Overexpressing LNC CRYBG3 caused cells apoptosis and cell cycle M phase arrest,which suggests that the change of LNC CRYBG3 expression is one of the mechanisms of biological effects of carbon ion radiation and X-ray for cells apoptosis.It also suggests that LNC CRYBG3 maybe related to the mechanisms why the cell is more radiosensitivity to carbon ions than to X-rays.3?LNC CRYBG3 inhibited the growth and metastasis of xenografts tumors in mice This may also be related to the fact that LNC CRYBG3 can lead to M phase arrest and apoptosis.4?LNC CRYBG3 can inhibit the polymerization of G-actin into F-actin,which caused the collapse of cytoskeleton.So,at the end of mitosis,the constricted ring couldn't be properly assembled and cytokinesis was not completed,which may be one of the reasons for the final apoptosis of cells.5?LNC CRYBG3 inhibitsed G-actin polymerization by its direct binding to G-actin.F-actin couldn't be assembled,resulting in cytoskeleton collapse.Then,binuclear cells were generated.So the cell atypia was increased.All these suggest that LNC CRYBG3 can affect the development of lung cancer by distructing the cytoskeleton.6?LNC CRYBG3 is highly expressed in lung cancer cells and lung cancer tissues,The degree of tumor differentiation increased when LNC CRYBG3 was suppressed.While it decreased when LNC CRYBG3 was overexpressed.All these suggested LNC CRYBG3 may promote the malignant differentiation of lung cancer.Further study is needed to investigate whether LNC CRYBG3 plays different roles in the development and progression of lung cancer.7?LNC CRYBG3 may serve as a novel entry point for the study of the occurrence,progression and treatment of lung cancer.