| Chapter one:The expression and clinical significance of Kpnβ1 in non-small cell lung cancerObjective:To study the expression a of Kpnβ1 in non-small cell lung cancer(NSCLC),and its relationship with prognosis,to explore the application prospects of Kpnβ1 in the diagnosis and treatment of NSCLC.Methods:154 cases tissue specimens were collected,including 8 cases of fresh NSCLC cancer tissues and adjacent normal lung tissue,and 146 cases of cancer tissues and corresponding adjacent tissue paraffin blocks.Western Blot method was used to detect the expression of Kpnβ1 and PCNA in fresh NSCLC and adjacent tissues.Immunohistochemical staining was performed to detect the expression of Kpnβ1 protein on 146 paraffin-embedded tissues and corresponding adjacent tissues.The relationship between the expression of Kpnβ1 protein and the clinicopathological parameters of 146 NSCLC patients was analyzed statistically.COX multivariate analysis and Kaplan-Meier survival curve were used to analyze the correlation between the expression of Kpnβ1 protein and the prognosis of NSCLC patients.Results:(1)The expression of Kpnβ1 protein in fresh NSCLC tissues was significantly higher than that in adjacent tissues,and the difference was statistically significant(P<0.05):At the same time,Western Blot results also showed that the expression of PCNA protein in fresh NSCLC was significantly higher than that in adjacent tissues,which was consistent with the expression trend of Kpnβ1 protein.(2)Immunohistochemical results showed that Kpnβ1 and ki-67 proteins were mainly expressed in the nucleus,and the positive expressions were yellow or brown-yellow.The positive expression of Kpnβ1 and ki-67 in NSCLC tissues was significantly higher than that in normal lung tissues,and it was related to the degree of differentiation of NSCLC.The lower the degree of differentiation,the higher the expression of Kpn 1 and ki-67 in the samples.(3)Statistical analysis showed that the high expression of Kpnβl was correlated with the patient’s age(p=0.038<0.05),clinical stage(p<0.001),tissue differentiation degree(p=0.017<0.05),tumor size(p=0.028<0.05),lymph node metastasis(p<0.001),and ki-67 expression level(p=0.013<0.05),the difference was statistically significant.However,there was no significant relationship between gender,histological type and smoking status(P<0.05).In multivariate analysis,high expression of Kpnβ1 protein(HR,1.911,95%ci,1.015-3.598;P=0.045)and the degree of tissue differentiation(HR,3.226,95%ci,2.026-5.136).P<0.001)was an independent factor affecting the prognosis of NSCLC.Kaplan-Meier survival curve indicated that the survival rate of Kpnβ1 high expression group was worse than that of Kpnβ1 low expression group,the difference was statistically significant(P<0.001).Conclusion:Kpnβ1 was highly expressed in NSCLC tissues,and was closely related to patient’s age,clinical stage,tissue differentiation,tumor size,lymph node metastasis,and ki-67 expression.The prognosis of patients with high expression of Kpnβ1 was worse than that of patients with low expression of Kpnβ1.It was an independent prognostic factor of NSCLC.Chapter two:The Effect of Kpnβlon proliferation of non-small cell lung cancer through the PI3K/AKT pathwayObjective:The changes of proliferation ability and chemosensitivity of lung cancer cells after the silencing of Kpnβ1 gene were detected by molecular biology method,and the effects on the expression of PI3K/AKT signaling pathway protein were also detected.To investigate the possible mechanism of Kpnβ1 affecting the biological characteristics of non-small cell lung cancer through the PI3K/AKT signaling pathway.Methods:Western blot was used to detect the expression of Kpnβ1 protein in NSCLC cell lines(A549,H1299 and spca-1),the highest expression of A549 cell line was screened out.Then the cycle changes of A549 cells were detected by flow cytometry,at the same time,the expressions of Kpnβ1,PCNA and Cyclin D1 protein in different phases were detected by Western Blot.Kpnβ1 short hairpin RNA(shRNA)was used to knock down the expression of Kpnβ1 in A549 cells,and then cck-8,plate clonal formation assay and 5-ethynyl-2 ’deoxyuridine nucleoside kit(EdU)were used to detect the proliferation of A549 cells after the down-regulation of Kpnβ1 gene expression.The effect of the cell cycle progression of A549 cells was detected by Flow cytometry after the down-regulation of Kpnβ1 gene.In addition,the sensitivity of A549 cells to CDDP after Kpnβ1 gene silencing was detected by cck-8,the expressions of Kpnβ1,PI3K,p-akt(Ser473)and p-akt(Thr308)proteins after Kpnβ1 silencing and cisplatin treatment,as well as the expression of cleaved-parp,an indicator of cell apoptosis,were detected by Western blot.Results:(1)Kpnβ1 protein was expressed in A549,H1299 and SPCA-1,but its expression in A549 was higher than that in the other two cell lines.Therefore,we selected A549 cells for subsequent experiments.(2)Flow cytometry detection of A549 cells showed that the proliferation ability of A549 cells was significantly inhibited in serum starvation culture,and the cell cycle was blocked in G0/G1 phase;after the restoration of serum supply,cell proliferation was restored and the cells rapidly entered the S phase from the G0/G1 phase,the proportion of G0/G1 phase decreased from 72.32%to 35.08%,and the cells in the S phase increased from 22.55%to 56.62%;at the same time,the expression level of Kpnβ1 protein was significantly decreased after serum starvation,and the expression of Kpnβ1 protein was also gradually increased with the increase of cell proliferation after the restoration of serum supply culture,reaching a peak at 48h;the expression level of Kpnβ1 protein was consistent with the trend of Cyclin D1 and PCNAprotein;however,the expression of Cyclin A2,Cyclin D1 and PCNA protein decreased significantly after Kpnβ1 gene silencing compared with the control group,the difference was statistically significant(P<0.05).(3)CCK8 method,plate cloning experiment and EdU kit detection all found that the cell proliferation ability of the interference group was significantly reduced after the Kpnβ1 gene silencing,compared with the control group,and the difference was statistically significant(P<0.05).(4)Flow cytometry showed that the number of G0/G1 phase A549 cells increased(from 65.31%to 77.77%)and the number of S phase cells decreased(from 25.99%to 17.94%)after the silencing of Kpnβ1 gene,and the difference was statistically significant(P<0.05).(5)the results of cisplatin sensitivity test showed that the proliferation of cells in the Kpnβ1 gene interference group and the control group both decreased after treatment with different concentrations of cisplatin.The decrease was most significant in the Kpnβ1 silencing group,which was positively correlated with CDDP dose,and was most significant at the concentration of CDDP:20 mol/L,the difference with statistically significant difference(P<0.05).(6)Western Blot results showed that PI3K/AKT pathway-related proteins:PI3K,p-akt(Ser473),p-akt(Thr308)and PCNA protein were significantly decreased after Kpnβ1 gene silencing compared with the control group.(7)To further investigate the synergistic effect of Kpnβ1 gene and cisplatin,Western Blot assay showed that the expressions of PI3K,p-akt(Ser473)and p-akt(Thr308)proteins were significantly decreased after the treatment with Kpnβ1 interference and cisplatin.In addition,compared with the control group,the expression of cleaved-parp was increased in the other three groups,especially in the combined group.Conclusion:Silencing Kpnβ1 gene can inhibit the proliferation of NSCLC cells and increase the sensitivity of NSCLC cells to CDDP.Kpnβ1 May play its role by regulating the PI3K/AKT signaling pathway.This study investigated the expression of Kpnβ1 in NSCLC and its possible mechanism,providing a laboratory basis and theoretical basis for further clinical application. |
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