| Background: Lung cancer is the world’s second largest malignant tumor after breast cancer in terms of incidence rate.Among its various histopathological subtypes,adenocarcinoma accounts for the highest proportion,about 40%.Lung adenocarcinoma has a high degree of invasion and metastasis,and most patients are diagnosed in advanced stage,resulting in poor prognosis with an average survival period of less than 5 years.Therefore,it is very important to develop new methods for the diagnosis and treatment of lung cancer.Cold-inducible RNA-binding protein(CIRBP)is named after its involvement in stress responses induced by low-temperature stimulation.In recent years,a large number of studies have shown that CIRBP is also involved in the occurrence and development of various tumors,and its expression level can predict the prognosis of some tumor patients.However,as of now,little is known about the role of CIRBP in lung adenocarcinoma.Objective: To construct a lung adenocarcinoma cell line overexpressing the CIRBP gene through in vitro cell experiments,detect the CIRBP level in lung adenocarcinoma tissue through immunohistochemistry(IHC)assay,and detect the serum CIRBP level in lung adenocarcinoma patients through enzyme-linked immunosorbent assay(ELISA).To observe the effect of overexpressing the CIRBP gene on the apoptosis,invasion,and proliferation function of lung adenocarcinoma cells,understand its histological and serological characteristics,explore the role of CIRBP in lung adenocarcinoma,and provide theoretical basis for its clinical application in early diagnosis and precise treatment of lung adenocarcinoma.Methods:1.Quantitative real-time polymerase chain reaction(q PCR)was used to detect the m RNA expression abundance of CIRBP gene in human lung adenocarcinoma PC-9,H125,NCI-H1975,A549,and Calu-3 cells.2.Human lung adenocarcinoma A549 and PC-9 cells were divided into negative control(NC)group and overexpression(OE)group,and transfected with negative control virus and Lentivirus overexpressing CIRBP gene respectively.72 hours later,q PCR was used to detect the expression of CIRBP gene.3.The effects of overexpression of CIRBP gene on the apoptosis,invasion,and proliferation of lung adenocarcinoma cells were observed through Annexin V-APC single staining cell apoptosis assay,in vitro invasion(Transwell)assay,and CCK-8 cell counting(Cell Counting Kit-8)assay.4.IHC assay was used to detect the expression of CIRBP in tumor tissue and corresponding normal tissue samples of 24 lung adenocarcinoma patients preserved in the Pathology Department of Lanzhou University Second Hospital.5.Collect the blood samples and relevant clinical data of 64 patients with lung adenocarcinoma admitted to the thoracic surgery of the Second Hospital of Lanzhou University,and recruit 24 healthy volunteers as the healthy control group at the same time.Apply ELISA assay to detect the level of CIRBP in the serum samples of subjects.Analyze and describe the general and clinical pathological data of 88 subjects,grouped by disease,gender,age,TNM staging,clinical staging,site of onset,tumor markers,etc.Analyze the relationship between serum CIRBP levels and various clinical pathological features,as well as the diagnostic value in lung adenocarcinoma.Results: 1.The q PCR detection results showed that CIRBP gene m RNA was highly expressed in human lung adenocarcinoma PC-9,H125,NCI-H1975,A549,and Calu-3 cells.2.After 72 hours of infection with human lung adenocarcinoma A549 and PC-9 cells,it was observed under a microscope that both the NC group and OE group cells were in good condition,without significant death.The infection rate reached 80% and can be used for subsequent experiments.The q PCR detection results showed that the expression abundance of the CIRBP gene in the OE group was 3.260 times higher in A549 cells than in the NC group(P<0.05),and 4.248 times higher in PC-9 cells than in the NC group(P < 0.05).3.The results of the cell apoptosis experiment showed that in A549 and PC-9 cells,the apoptosis rate of the OE group was significantly lower than that of the NC group(P<0.05);Transwell experiment showed that in A549 and PC-9 cells,the number of cell migration and change rate in OE group were significantly higher than those in NC group(P<0.05);The CCK-8cell counting experiment results showed that in A549 and PC-9 cells,the cell count and proliferation multiple of the OE group were significantly higher than those of the NC group(P<0.05).4.The IHC assay results showed that the expression level of CIRBP in lung adenocarcinoma tissue samples was higher than that in normal tissues(P=0.05).CIRBP is mainly present in the cytoplasm of lung adenocarcinoma cells.The expression level of CIRBP is higher in lung adenocarcinoma cells with poorer differentiation.5.The ELISA results showed that the serum CIRBP concentration of lung adenocarcinoma patients was higher than that of normal individuals(P<0.05);The sensitivity,specificity,and accuracy of using serum CIRBP concentration to diagnose lung adenocarcinoma were 40.6%,95.8%,and 56.8%,respectively;The serum CIRBP concentration in male patients was higher than that in female patients(P < 0.05);The difference in serum CIRBP concentration was not statistically significant(P > 0.05)due to age.The difference in serum CIRBP concentration between TNM stages was statistically significant(P<0.05);The clinical staging had a statistically significant impact on the difference in serum CIRBP concentration(P<0.05);The difference in serum CIRBP concentration was not statistically significant(P>0.05)due to the location of the disease;There is a positive correlation between CEA concentration and serum CIRBP concentration(P<0.05),while the differences in serum CIRBP concentration between NSE,CYFRA21-1,Pro GRP,and SCC concentrations are not statistically significant(P>0.05).Conclusion:1.CIRBP gene m RNA is highly expressed in human lung adenocarcinoma PC-9,H125,NCI-H1975,A549,and Calu-3 cells.2.Lentivirus transfection is a feasible method to construct lung adenocarcinoma cell lines with overexpression of CIRBP gene.3.In vitro,the increased expression level of CIRBP gene in human lung adenocarcinoma A549 and PC-9 cells can lead to an increase in tumor cell proliferation and invasion ability,as well as a decrease in apoptosis rate.4.CIRBP is highly expressed in the cytoplasm of human lung adenocarcinoma cells,but low expressed in the nucleus and normal tissues.Its increased expression is related to the malignancy of lung adenocarcinoma.5.High levels of serum CIRBP are expected to become biomarkers for the diagnosis of lung adenocarcinoma.Gender,TNM staging,clinical staging,and CEA concentration are influencing factors for serum CIRBP concentration in lung adenocarcinoma patients,while age,NSE,CYFRA21-1,Pro GRP,and SCC concentration are not influencing factors for serum CIRBP concentration in lung adenocarcinoma patients. |
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