| Objective: According to Globocan data released by the World Health Organization in2020,the incidence of lung cancer morbidity is the second,and mortality of lung cancer is the first all over the world.Current studies have confirmed that dysregulation of insulin-like growth factor 2 m RNA-binding protein 2 is associated with tumor occurrence and progression.Advances in lung cancer treatment and prevention focus on blocking metastasis and delaying the emergence of chemoresistance.IGF2BP2 is associated with the proliferation,migration,invasion,stemness and chemoresistance of malignant tumors,and is indirectly or directly involved in the metastasis and chemoresistance of a variety of malignant tumors.And IGF2BP2 has been highly expressed in many malignant tumors.However,the relevant research of IGF2BP2 in lung cancer is still shallow,and its related mechanism of action is not clear.The purpose of this study was to explore the role of IGF2BP2 in the development and progression of lung cancer and analyze its related pathways.To explore the effects of IGF2BP2 on the mesenchymal phenotype,migration,invasion ability,stemness and chemoresistance of lung cancer cells.It is expected to provide another effective treatment for the precise treatment of lung cancer.Methods: 1.The Kaplan-Meier plotter website was used to analyze the expression of IGF2BP2 in lung cancer tissues and its correlation with the prognosis of lung cancer patients.The expression of IGF2BP2 in clinical tissue samples of lung cancer was detected: 27 clinical samples and 13 cases of adjacent tissue samples confirmed by histopathology were selected.The expression of IGF2BP2 in the sample was detected by immunohistochemistry.2.Refer to the CCLE database to analyze the expression level of IGF2BP2 in different lung cancer cell lines,and select A549,H1299 and LK2 as follow-up experiments.3.Lentiviral vectors were used to construct stable transfected cell lines of A549 and H1299 with IGF2BP2 knockdown,and the gene knockdown rate was confirmed by Real-time PCR and Western blot.IGF2BP2 overexpression plasmid was used to transfect LK2 cells,and the overexpression effect was confirmed by Western blot.4.To explore the effect of IGF2BP2 on the mesenchymal phenotype of lung cancer cells: the correlation between IGF2BP2 and mesenchymal phenotype-related indicators Vimentin(VIM),Fibronectin(FN1),TGFB1(TGFβ1)and SMAD3 was analyzed by CCLE database;The cell morphology of IGF2BP2 knockdown cell line and IGF2BP2 overexpression cell line were observed by phalloidin staining.The expression of Vimentin,Fibronectin and α-SMA(ACTA2)was detected by immunofluorescence.Real-time PCR assays to detect VIM,FN1,and ACTA2 m RNA levels.5.Study the effect of IGF2BP2 on the migration and invasion ability of lung cancer cells: the cell scratch test and Transwell experiment were used to verify the changes in cell migration and invasion ability in IGF2BP2 knockdown cell lines and IGF2BP2 overexpression cell lines.6.In animal experiments,IGF2BP2 knockdown group cells and control group cells were injected into the tail vein to construct a metastasis model of mice to further verify the lung metastasis promoting effect of IGF2BP2 in vivo.7.To explore the stemness effect of IGF2BP2 on lung cancer cells: the correlation between IGF2BP2 and stem-related indicators CD44,ITGA6,IL6 and IL6 ST was analyzed in the CCLE database;The expression of stem-related indexes CD44,ITGA6 protein and m RNA in IGF2BP2 knockdown cell lines was detected by Western blot and Real-time PCR.The expression of stem-related indexes p-STAT3 and STAT3 proteins in IGF2BP2 knockdown cell lines was detected by Western blot.The expression of stem-related indexes CD44,ITGA6,p-STAT3 and STAT3 proteins in cell lines was detected by Western blot.The spheroidic ability of IGF2BP2 on tumor cells was detected to judge its effect on tumor cell stemness.8.To explore the effect of IGF2BP2 on chemoresistance of lung cancer cells: cells treated with different concentrations of cisplatin(CIS: 0,10μM)were detected with flow cytometry,to check the apoptosis levels in the IGF2BP2 knockdown cell line;Cells were treated with different concentrations of CIS(0,5,10,15,20μM),and the OD values of both the IGF2BP2 overexpression and knockdown cell lines were detected by CCK8,to judge the effect of IGF2BP2 on chemoresistance of lung cancer cells.Cells were treated with different concentrations of CIS(0,10μM),and spheroid experiments were used to detect the effect of IGF2BP2 on the spheroid ability of tumor cells in both the IGF2BP2 overexpression and knockdown cell lines,to judge its effect on tumor cell chemoresistance.9.The correlation between IGF2BP2 and SMAD3 was analyzed by CCLE database,and the expression of SMAD3 in IGF2BP2 knockdown cell lines was detected by Real-time PCR and Western blot.Real-time PCR was used to detect the expression levels of SMAD3 m RNA in 0,3 and 6h in transfected cells treated with actinomycin D.The effect of IGF2BP2 on the attenuation of SMAD3 m RNA was evaluated.RIP was used to detect the relationship between SMAD3 m RNA and IGF2BP2.10.To further explore the correlation between IGF2BP2 and SMAD3,SMAD3 overexpression plasmids were used to transfect knockdown strands of IGF2BP2 knockdown cell lines,and Western blot was used to detect whether the transfection was successful.Through phalloidin staining,it was observed whether the morphology of cells was recovered.Through scratch experiments and Transwell experiments,it was observed whether the migration and invasion ability of cells were recovered.11.After overexpression of SMAD3 in IGF2BP2 knockdown cell lines,cells were treated with different concentrations of CIS(0,5,10,15,20μM),and CCK8 experiment was used to determine whether the chemoresistance of cells after overexpression of SMAD3 was recovered.Cells were treated with different concentrations of CIS(0,10μM),and spheroid experiments were used to detect the changes in spheroidic ability of cells after overexpression of SMAD3,to further judge the correlation between SMAD3 and IGF2 BP and its effect on chemoresistance of tumor cells.12.The correlation between IGF2BP2 and SMAD3,SMAD3 and IL6 ST were analyzed by CCLE database,the expression level of IL6 ST in IGF2BP2 knockdown A549 and H1299 cell lines was detected by Real-time PCR and Western blot,and the expression level of IL6 ST in cells after overexpression of SMAD3 and LK2 cell lines was detected.13.IL6 ST overexpression plasmid was used to transfect knockdown chains in IGF2BP2 knockdown cell lines.Western blot was used to detect whether the transfection was successful,and phalloidin staining was used to detect whether the morphology of cells recovered.Through scratch experiments and Transwell experiments,it was observed whether the migration and invasion ability of cells were recovered.14.After overexpressing IL6 ST in the IGF2BP2 knockdown cell line,cells were treated with different concentrations of CIS(0,5,10,15,20μM),and CCK8 experiment was used to determine whether the chemoresistance of cells was recovered.Cells were treated with different concentrations of CIS(0,10μM),and spheroid experiments were used to detect the changes in spheroidic ability of cells after overexpression of IL6 ST to judge the correlation between IL6 ST and IGF2 BP and its effect on chemoresistance of tumor cells.Results:1.Through the analysis of CCLE database,it was found that the overall survival(OS),first progression(FP)and post progression survival(PPS)of lung cancer patients with IGF2BP2 high expression were significantly shortened.2.The expression level of IGF2BP2 in cancer tissues was significantly higher than that of normal tissues.By immunohistochemical detection method,27 lung cancer tissue samples and 13 normal lung tissues were detected,and it was found that the expression of IGF2BP2 in the two groups of samples was significantly different,and the expression of IGF2BP2 in lung cancer tissues was higher than that in normal tissues.3.Western blot and Real-time PCR were used to verify that IGF2BP2 knockdown A549 and H1299 transfection were effective,and the transfection efficiency reached70～80%;Western blot were used to verify that IGF2BP2 overexpression LK2 cell lines were transfected effectively.4.IGF2BP2 maintains the mesenchymal phenotype of lung cancer cells.The results of CCLE database analysis showed that IGF2BP2 was positively correlated with mesenchymal phenotypic indexes such as VIM,FN1,TGFB1 and SMAD3 in a series of lung cancer cell lines.In Real-time PCR and immunofluorescence staining of the transfected A549 and H1299 cell lines,the expression of Vimentin,Fibronectin andα-SMA of the knockdown group were decreased,the mesenchymal characteristics of cells were decreased.Via phalloidin staining it was found that the morphology of cells showed epithelialization.In the LK2 cell line with IGF2BP2 overexpressed,the expression of Vimentin in immunofluorescence was increased,and the phalloidin staining showed mesenchymal changes in cell morphology.5.In IGF2BP2 knockdown A549 and H1299 cell lines,scratch experiments and Transwell experiments showed that the migration and invasion ability of cells in the knockdown group were weakened compared with those in the control group.In the LK2 cell line with IGF2BP2 overexpressed,scratch experiments and Transwell experiments showed that the migration and invasion ability of cells in the overexpressed group were enhanced compared with those in the control group.6.In animal experiments,by constructing a metastasis model of mice,the number of lung metastases in the IGF2BP2 knockdown group was significantly lower than that in the control group,which further suggested that IGF2BP2 could induce changes in mesenchymal properties and had the effect of promoting lung metastasis.7.IGF2BP2 positively regulates stem-related factors in different lung cancer cell lines.Western blot showed the expression of stem-related factors CD44,ITGA6 and p-STAT3 in the knockdown group were reduced;In the spheroid experiment,the spheroidic ability of cells in the IGF2BP2 knockdown group was decreased,indicating that the stemness of the cells were weakened.In the LK2 cell line overexpressed by IGF2BP2,the expression of stem-related factors CD44,ITGA6 and p-STAT3 were increased.In the spheroidic experiment,the spheroidic ability of cells in the IGF2BP2 overexpressed group was increased,indicating that the stemness of cells were enhanced after overexpression of IGF2BP2.8.CCK8 experiment showed the cell viability of the knockdown group was significantly reduced compared with that of the control group,and the sensitivity to drugs was enhanced.In the IGF2BP2 overexpressed LK2 cell line,the cell viability of the overexpressed group was significantly higher than that of the control group,and the sensitivity of cells to drugs was decreased.After treated with different concentrations of CIS,the apoptosis level of tumor cells was detected by flow cytometry technology,and it was found that the apoptosis level of cells in the IGF2BP2 knockdown group was significantly higher than that of the control group.In the spheroidic resistance experiment,after treated with different concentrations of CIS,the spheroidic ability of the knockdown group was significantly lower than that of the control group.In the IGF2BP2 overexpressed LK2 cell line,the spheroidic ability of the overexpressed group was significantly higher than that of the control group.Those experiments showed that IGF2BP2 can promote the development of chemoresistance in cancer cells.9.In the CCLE database,IGF2BP2 and SMAD3 were positively correlated.The results of Western blot and Real-time PCR showed that the expression of SMAD3 protein and m RNA in the cells of the knockdown group were decreased.Real-time PCR was used to detect the levels of SMAD3 m RNA at 0,3 and 6 h after treated with Actinomycin D,and the results showed that the SMAD3 m RNA decay rate of IGF2BP2 knockdown group was significantly faster than that of the control group.And in the RIP experiments,it was confirmed that IGF2BP2 works by binding to SMAD3 m RNA.10.SMAD3 overexpression plasmid was used to transfect the knockdown chain of A549 cells.Through phalloidin staining it was found that the cells transformed from the epithelial state into mesenchymal state;Through scratch experiments and Transwell experiments,it was found that the migration and invasion ability of cells were enhanced.11.After the overexpression of SMAD3 in sh IGF2BP2 A549 cells,the results of CCK8 experiments showed that the chemoresistance of cells in the overexpressed group was enhanced.In the spheroidic chemoresistance experiment,the spheroidic ability of cells in the overexpressed SMAD3 group was also recovered.12.Through CCLE database analysis,IGF2BP2 was positively correlated with SMAD3 and IL6 ST,and the expression of SMAD3 and IL6 ST in the knockdown group were decreased.However,after the overexpression of SMAD3 in sh IGF2BP2A549 and LK2 cell lines,IL6 ST was elevated to a certain extent.13.IL6 ST overexpression plasmid was used to transfect the knockdown chain of A549 cells,and through phalloidin staining it was found that the cells in the epithelial state were recovered to the mesenchymal state;Through scratch experiments and Transwell experiments,it was found that the migration and invasion ability of cells were enhanced.14.After overexpression of IL6 ST in sh IGF2BP2 A549 cells,the results of CCK8 experiments showed that the chemoresistance of cells in the overexpressed group was recovered.In the spheroidic chemoresistance experiment,the spheroidic ability of cells overexpressing IL6 ST group was also recovered. |
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