Preliminary Study On The Abnormal Expression Of CREB3L4 Gene Affecting The Development Of Non-small Cell Lung Cancer

Objective:Lung cancer is a serious threat to human health and life diseases.In China the incidence and mortality of lung cancer have risen rapidly,and it has become a malignant tumor with high morbidity and mortality in China.Yunnan Xuanwei is an area that attracts the attention of researchers around the world.Because Xuanwei is the region with the highest incidence of lung cancer among non-smokers in the country,the mortality rate of female lung cancer is the highest in the country.Therefore,the potential molecular markers of Xuanwei lung cancer were screened out,and the mechanism of lung cancer development and development in Yunnan Xuanwei was further explored to find new targets for diagnosis and treatment.The previous study of this subject found that the expression of CREB3L4 gene in cancer tissues of Xuanwei lung cancer patients in Yunnan is significantly higher than that in adjacent tissues,and is associated with TNM staging and lymph node metastasis,suggesting that CREB3L4 gene may be involved in the development of lung cancer in Xuanwei,Yunnan.Important role,and may be a potential oncogene in Xuanwei lung cancer.In the previous study,the lung cancer cell JT and human lung adenocarcinoma cell A549 cell model were used in Yunnan,and the effects of up-regulation and down-regulation of CREB3L4 on the biological behavior of Xuanwei lung cancer cell line and human lung adenocarcinoma cell line were observed in vitro.CREB3L4 gene was found.It promotes the development of lung cancer cells.This study aimed to construct a tumor model of nude mice,and explore the role of CREB3L4 in animal models by detecting tumor growth and metastasis.Methods:1.The plasmids that were successfully constructed and sequenced correctly were transfected,packaged with lentivirus,and harvested with lentivirus and concentrated.Lentiviral titers were determined by RT-qPCR.The collected overexpression and its control,interference group and its control lentiviral concentrate were stably transfected into Yunnan Xuanwei lung cancer cell line JT,and the stably infected cell lines and their corresponding control cell lines were screened.CREB3L4 was performed by Western Blot.Determination of protein content.The migration ability of the overexpressing group and the interfering group cells was examined by Wound Healing test.2.JT of Yunnan Xuanwei lung cancer cells were cultured in large quantities,and nude mice,SCID mice and B-NDG mice were injected subcutaneously to construct an animal model.Human lung adenocarcinoma A549 was cultured in large quantities,subcutaneously injected into nude mice,and an animal model was constructed.The tumor volume was measured every three days after the cells were injected.After the tumor grew to 28 days,the tumor tissues and lung tissues were taken out for HE staining.Immunofluorescence detection of CREB3L4 antibody.A549 cells were cultured in large numbers for subcutaneous tumor formation in nude mice.After 20 days of injection,the cells were overexpressed and interfered with lentivirus and the corresponding control lentivirus.The day after injection of the lentivirus,the tumor was injected with the corresponding lentivirus.Volume,after 5 consecutive injections of lentivirus,subcutaneous xenografts and mouse lung tissues were removed,and the mRNA expression of corresponding CREB3L4 was detected by RT-qPCR after lentivirus injection.The pathological changes of tumor tissue and lung tissue were observed by HE staining.Immunofluorescence was used to observe the expression and localization of CREB3L4 protein in tumor tissue and lung tissue.Results:1.Western Blot was used to detect the overexpression of stable transfected lentivirus and its control,and the expression of CREB3L4 protein in the interference group and its control group showed that the overexpression and its control,the stable transfected cell line of the interference group and its control were successfully constructed.Prove that the packaged lentivirus has a significant biological effect.In the Wound Healing test,the overexpression of CREB3L4 was significantly decreased after 48 h compared with the control group,suggesting that overexpression of CREB3L4 gene may promote tumor development.According to the stably transfected overexpression group and the interference group Western Blot,the expression of CREB3L4 protein was detected.The interference efficiency of siCREB3L4-32 was significantly higher than that of siCREB3L4-31.Therefore,siCREB3L4-32 was selected for migration ability detection and subsequent animal experiments.After 48h,the width of the scratched area of siCREB3L4-32 was slightly wider than that of the control group,and there was a certain difference,suggesting that knocking down CREB3L4 may inhibit the development of tumor.2.A large number of cultured JT Xuanwei lung cancer cells were injected into nude mice,SCID mice and B-NDG mice respectively.The growth of tumors was basically the same.Within 10 days after injection of lung cancer cells,the tumors gradually grew up,but with the increase of time,the tumors gradually shrank until they disappeared.A large number of cultured human lung adenocarcinoma cells A549 were injected into nude mice subcutaneously,and the obvious tumors could be touched about 7 days.The tumors increased gradually with time,and the A549 animal model was successfully constructed.After 28 days of injection,the tumors and lung tissues were taken out for HE staining and immunofluorescence detection of CREB3L4 antibody.After injection of A549 cells,HE staining of transplanted tumors in nude mice was found.Compared with the results of HE staining in normal nude mice,HE staining in nude mice lung tissue showed obvious pathological changes,wider alveolar spacing and infiltration of tumor cells.CREB3L4 immunofluorescence assay in nude mice subcutaneously transplanted tumors showed that CREB3L4 was localized in cytoplasm,which was consistent with previous studies.After subcutaneous injection of A549 cells into nude mice for 20 days,A549 cells were subcutaneously injected with lentiviruses,which expressed in the tumors,interfered with and controlled by lentiviruses.The tumors were injected every other day for 5 times.The volume of the tumors was measured the next day after each injection.Then the tumors were taken out for HE staining,immunofluorescence detection of CREB3L4 antibody and RT-qPCR expression of CREB3L4 gene.RT-q PCR results showed that the expression of CREB3L4 was significantly increased in the overexpression group and decreased in the interference group.HE staining showed that there was no difference between the pathological changes of tumor tissue and lung tissue in mice after injection of lentivirus with over-expression,interference and control,and that in mice after injection of A549.Immunofluorescence results showed that although there was no significant difference in the expression of CREB3L4 in lung tissues of mice after injection of lentivirus,the expression of CREB3L4 in subcutaneous transplanted tumors of mice had significant corresponding changes.Conclusion:The CREB3L4 gene is beneficial to the development of lung cancer cells.It was found that CREB3L4 was localized in the cytoplasm of subcutaneous transplanted tumors in nude mice,which was consistent with previous studies.The exogenous CREB3L4 gene can be stably expressed in nude mice subcutaneously transplanted tumors.CREB3L4 has a certain tendency to promote tumorigenesis and development.This study laid a foundation for further construction of animal models and in vivo study of the biological role of CREB3L4 gene expression changes in the development of non-small cell lung cancer.