Studies On The Expression Of HNRNPC And Its Possible Mechanisms In Non-small Cell Lung Cancer

Objectives:We discussed the expression of HNRNPC and its malignant biological behavior and its possible molecular mechanism in non-small cell lung cancer.Methods:1.Investigated the expression of RBP4 in patients with non-small cell lung cancer.(1)Bioinformatics analysis: Using the TCGA database,analyze the expression of HNRNPC in non-small lung cancer and its relationship with survival prognosis.(2)Tumor tissues and paired normal tissues of 44 NSCLC patients(22 LUAD patients and 22 LUSC patients)were collected,the expression levels of mRNA and protein of HNRNPC in lung cancer patients were detected by RT-qPCR and Western Blot experiments,and the expression position of HNRNPC in lung tissue cells was determined by IHC and the correlation between HNRNPC expression and clinicopathological features of NSCLC patients was analyzed.2.Investigated the effect of knockdown HNRNPC on the level of total RNA m6 A methylation in NSCLC cells.(1)Western Blot and colorimetric method were used to detect the expression of HNRNPC protein and the methylation level of total RNA m6 A in different lung adenocarcinoma cells,lung squamous cell carcinoma cells and normal lung epithelial cells,and compared with normal lung epithelial cells,cell lines with significantly elevated expression of HNRNPC protein and total RNA m6 A methylation levels were screened;(2)After lung cancer cells were transfected with interfering plasmids,the interference efficiency of the plasmid was clarified by Western Blot and RT-qPCR,and the changes in the methylation level of total RNA m6 A of cells were detected by colorimetric method.3.Investigated the biological behaviors of non-small cell lung cancer cells such as proliferation,migration,invasion,and cell cycle after downregulation of HNRNPC,and analyzed their molecular mechanism.(1)Using the CCK8 assays and the plate cloning experiments to detect the proliferation ability of cells after downregulation HNRNPC;(2)Transwell experiment was used to detect the invasion ability of cells after downregulation HNRNPC;(3)Scratch experiment was used to detect the migration ability of cells after downregulation HNRNPC;(4)Flow cytometry was used to detect the cell cycle of cells after downregulation HNRNPC;(5)Western Blot was used to detect the protein expression levels of Cyclin D1,CDK2,CDK6,PI3 K,AKT,and p-AKT downregulation HNRNPC.4.Statistical methodsThe data of this study were statistically analyzed using SPSS 25.0,the chi-square test was used to analyze the correlation between HNRNPC expression and the clinicopathological characteristics of NSCLC patients,and the results of RT-qPCR and functional experiments were analyzed by independent sample t-test,and the bilateral P<0.05 was considered to be statistically significant.Results:1.HNRNPC is highly expressed in non-small cell lung cancer tumor tissues(1)TCGA database analysis showed that HNRNPC was highly expressed in lung adenocarcinoma tissues,and the abnormally high expression of HNRNPC was negatively correlated with the overall survival of patients;(2)The results of RT-qPCR showed that the mRNA level of HNRNPC was abnormally elevated in tumor tissues of 22 LUAD and 22 LUSC patients(P<0.05),and the abnormal expression of HNRNA was correlated with the 5-year survival status of patients(P<0.05).Western Blot experiments proved that HNRNPC protein glands are highly expressed in both lung cancer and lung squamous cell carcinoma tissues(P<0.05),and IHC results proved that HNRNPC is expressed in the nucleus of lung tissue.2.Knocking down HNRNPC does not affect the total RNA m6 A methylation level of NSCLC cells(1)Western Blot experiment and colorimetric detection screened that the expression of HNRNPC protein and the methylation level of total RNA m6 A in lung adenocarcinoma cells SPCA1 and lung squamous cell carcinoma SK-MES-1were significantly higher than those in normal lung epithelial cells(P<0.05);(2)Western Blot and RT-qPCR experiments proved that after HNRNPC was successfully knocked down,colorimetric detection showed no significant change in the methylation level of total RNA m6 A in SPCA1 and SK-MES-1 cells.3.Relationship between HNRNPC and lung adenocarcinoma whitening SPCA1 and biological behavior of lung squamous cell SK-MES-1 cells(1)The results of CCK8 experimental results showed that compared with the negative control group,the OD values of SPCA1 and SK-MES-1 cells were reduced at 24 h and 48 h(P<0.05)after knocking down HNRNPC.The results analysis of plate cloning experiments showed that compared with the negative control group,the cloning ability of HNRNPC in SPCA1 and SK-MES-1 cells was weakened(P<0.05),that is,knocking down HNRNPC could inhibit the proliferation ability of SPCA1 and SK-MES-1 cells.(2)The results of Transwell invasion experiment showed that compared with the negative control group,the number of membrane penetrations of cells after knocking down HNRNPC in SPCA1 and SK-MES-1 cells was significantly reduced(P<0.05),that is,knocking down HNRNPC could inhibit the invasion ability of SPCA1 and SK-MES-1 cells;(3)The results of scratch experiments showed that the healing area of SPCA1 and SK-MES-1 cells at 24 h and 48 h after knocking down HNRNPC was lower than that of the negative control group(P<0.05),that is,knocking down HNRNPC could inhibit the migration ability of SPCA1 and SK-MES-1 cells;(4)The flow cytometry analysis cycle found that compared with the negative control group,the proportion of G1 phase cells after knocking down HNRNPC in SPCA1 and SK-MES-1 cells increased significantly(P<0.05),that is,knocking down HNRNPC could block the cell cycle of SPCA1 and SK-MES-1 in G1 phase;(5)Western Blot experiments showed that compared with the negative control group,the expression of cyclin Cyclin D1,CDK2 and CDK6 after knocking down HNRNPC in SPCA1 and SK-MES-1 cells decreased significantly(P<0.05),the expression of pathway protein AKT did not change significantly,while the expression of p-AKT and PI3 K decreased significantly(P<0.05).Conclusions:1.HNRNPC is a nuclear expression protein,which is highly expressed in the tumor tissues of NSCLC patients,and HNRNPC expression is negatively correlated with the overall survival of NSCLC patients;2.HNRNPC is not a key factor affecting the methylation of SPCA1 in lung adenocarcinoma cells and SK-MES-1 total RNA m6 A in lung squamous cell carcinoma cells;3.Knockdown HNRNPC may block cells in the G1 phase by downregulating the expression of the proteins Cyclin D1,CDK2 and CDK6,and then reduce the proliferation capacity of cells,thereby inhibiting the malignant biological behavior of SPCA1 and SK-MES-1 cells.