| Objective:Lung cancer is the most common malignancy,and lung adenocarcinoma is the major subtype of lung cancer development.Although encouraging progress has been made in the treatment of lung cancer,the prognosis of lung cancer has not improved significantly.Therefore,an in-depth exploration of the mechanisms of lung cancer development is of great significance to create more effective precision treatments for lung adenocarcinoma.Positive regulatory domain zinc finger protein(PRDM)is a family of transcriptional regulators closely related to human cancer,and has been reported to play important roles in cancer development.However,there are few studies on PRDM6,especially the function and mechanism of action in lung adenocarcinoma are still unclear.DIXDC1(Dishevelled-Axin domain-containing 1),as an oncogene,is involved in the progression of multiple cancers.The role of DIXDC1 in lung cancer development is still not clear.Therefore,this study aims to explore the specific mechanism and downstream effects of PRDM6 regulating the occurrence and development of lung adenocarcinoma through DIXDC1,so as to provide a new research direction for the development of diagnostic markers and therapeutic targets.Method:In the first part of this study,several databases and online tools were integrated,including TCGA database,NCBI GEO database,c Bio Portal database,TIMER2.0,MEXPRESS,Gene MANIA and other online analysis tools,to analyze the important role,expression level and prognostic value of PRDM6 in lung adenocarcinoma from multiple levels.Further,cancer and paracancer tissue specimens from 20 pairs of lung adenocarcinoma patients were collected,and the expression of PRDM6 protein in clinical samples was detected by immunohistochemistry and Western blot,respectively;meanwhile,Western blot analysis was used to compare the expression of PRDM6 in four lung adenocarcinoma cell lines NCI-H1359,H1975,A549,SK-LU-1,and human immortalized lung epithelial cell line HBE135-E6E7,to further validate the aberrant expression of PRDM6 in lung adenocarcinoma at clinical and cellular levels.In the second part of this study,we first constructed stable overexpression and silenced cell lines of PRDM6 by lentivirus system,and detected the effect of PRDM6 on cell proliferation by CCK8 and Ki67 immunofluorescence.The apoptosis rate was detected by flow cytometry,and the expressions of apoptosis-related proteins Bcl-2 and Bax were detected by Western blot to verify the effect of PRDM6 on the apoptosis of lung adenocarcinoma cells.The migration and invasion ability of PRDM6 cells were detected by scratch assay and Transwell assay,respectively,to verify the effect of PRDM6 on the migration and invasion of lung adenocarcinoma cells.The expressions of EMT markers E-cadherin and N-cadherin were detected by Western blot.Prdm6-silenced LUAD was injected into mice and tumor volume was measured.Finally,the expression of nuclearβ-catenin and the protein levels of target genes of Wnt/β-catenin signaling pathway PD-L1,c-MYC,Snail and Cyclin D were detected by Western blot and immunofluorescence,and the effect of PRDM6 on Wnt/β-catenin signaling was detected.In the third part,we first verified the influence of PRDM6 overexpression or silence on DIXDC1 expression,and then detected the influence of PRDM6 on DIXDC1transcription by Ch IP and double luciferase reporting experiments.After that,cells with stable overexpression of PRDM6 were selected and transfected with DIXDC1overexpression vector.CCK8,Transwell and Western blot tests were used to determine whether PRDM6 regulated cell proliferation,invasion and Wnt/β-catenin signal through DIXDC1.The subsequent lung adenocarcinoma cells were cocultured in direct contact with NK92 cells,and then the lysis rate of lung adenocarcinoma cells was detected by CCK8.At the same time,Granzyme B and MIP-1αlevels in co-culture supernatant were detected by ELISA to verify whether PRDM6 regulates the sensitivity of lung adenocarcinoma cells to NK cell killing through DIXDC1.In addition,immunohistochemical labeling CD56 was used to detect the infiltration level of NK cells in clinical lung adenocarcinoma tissues,and the correlation between PRDM6 and DIXDC1 expression levels and NK cell infiltration was analyzed.Results:Part Ⅰ:Bioinformatic database-based analysis showed that the expression level of PRDM6 gene was significantly downregulated in lung adenocarcinoma(LUAD).The distribution of PRDM6 gene expression levels was significantly different among different subgroups of gender,T-stage,grading and smoking history.High PRDM6 expression suggested a better long-term prognosis.Among them,T-stage and N-stage and PRDM6expression levels were independent prognostic correlates.To further validate the results of the public database analysis,we used Western blot and immunohistochemical staining to find that the expression of PRDM6 in lung adenocarcinoma tissues was significantly lower than that in normal tissues adjacent to the cancer;the expression levels of PRDM6protein in four lung adenocarcinoma cells,NCI-H1359,H1975,A549,and SK-LU-1,were significantly lower than that in human immortalized lung epithelial cell line HBE135-E6E7 cells.Part Ⅱ:CCK8 results showed that overexpression of PRDM6 significantly inhibited the proliferation of lung adenocarcinoma cells,while silence of PRDM6 had the opposite effect.Immunofluorescence results showed that overexpression of PRDM6 significantly inhibited Ki67 expression in lung adenocarcinoma.Flow cytometry results showed that overexpression of PRDM6 significantly promoted apoptosis of lung adenocarcinoma cells,while silence of PRDM6 had the opposite effect.Western blot results showed that overexpression of PRDM6 significantly inhibited promoted the expression of Bcl-2 and promoted the expression of Bax,while silence of PRDM6 significantly inhibited the expression of Bax and promoted the expression of Bcl-2.The results of scratch test and Transwell invasion test showed that overexpression of PRDM6 could significantly inhibit the migration and invasion of lung adenocarcinoma,while silence of PRDM6 had the opposite effect.Western blot results showed that overexpression of PRDM6 significantly promoted the expression of E-cadherin and inhibited the expression of N-cadherin,while silence of PRDM6 showed an opposite trend.Tumor formation in nude mice showed that silencing PRDM6 could significantly promote the growth of lung adenocarcinoma cells in vivo.Western blot and immunofluorescence results showed that overexpression of PRDM6 could significantly inhibit the level of nuclearβ-catenin,and inhibited the m RNA and protein levels of Wnt/β-catenin signaling pathway target genes PD-L1,c-MYC,Snail,Cyclin D.Part Ⅲ:Real-time PCR and Western blot results showed that overexpression of PRDM6significantly inhibited DIXDC1 m RNA and protein expression.Immunohistochemical scores showed that PRDM6 and DIXDC1 levels were negatively correlated in lung adenocarcinoma.Ch IP and dual luciferase results showed that PRDM6 could bind to DIXDC1 promoter and inhibit DIXDC1 transcription.Further results showed that restoration of DIXDC1 expression could weaken the effect of PRDM6 overexpression on cell proliferation,invasion and Wnt/β-catenin signal.In addition,in the immune correlation analysis on the basis of public database,we found a significant positive correlation between the proportion of NK cells and PRDM6 expression levels in LUAD samples,suggesting that PRDM6 may be involved in the development of lung adenocarcinoma by influencing NK cell infiltration.Therefore,we further performed molecular experiments to verify these above data.CCK8 results showed that overexpression of PRDM6 could significantly increase the cleavage rate of NK cells to lung adenocarcinoma cells,ELISA results showed that overexpression of PRDM6 could significantly increase the release levels of Granzyme B and MIP-1α.The cleavage rate of PRDM6-OE+DIXDC1-OE group and the levels of Granzyme B and MIP-1αin PRDM6-OE+Vector group were significantly lower than those in PRDM6-OE+vector group,suggesting that restoring DIXDC1 expression could inhibit the effect of PRDM6overexpression on the killing sensitivity of NK cells.IHC PRDM6 score was positively correlated with the number of CD56~+NK cells,while DIXDC1 score was negatively correlated with the number of CD56~+NK cells.Conclusion:1.Bioinformatics-based analysis suggested that PRDM6 expression level was an independent prognostic factor for lung adenocarcinoma.2.The expression of PRDM6 was significantly reduced in lung adenocarcinoma tissues compared with normal tissues.3.PRDM6 expression was significantly reduced in lung adenocarcinoma cell lines compared with normal lung epithelial cells.4.PRDM6 effectively promoted apoptosis and inhibited the malignant phenotype of lung adenocarcinoma cell proliferation,migration,invasion,in vivo growth,EMT process and Wnt/β-catenin signaling.5.PRDM6 can bind to DIXDC1 promoter and inhibit DIXDC1 transcription.6.PRDM6 can regulate the malignant behavior of lung adenocarcinoma cells,Wnt/β-catenin signaling and the sensitivity of lung adenocarcinoma cells to NK cell killing through DIXDC1.7.In clinical lung adenocarcinoma tissues,PRDM6 expression was positively correlated with CD56~+NK cell infiltration,and DIXDC1 expression was negatively correlated with CD56~+NK cell infiltration. |
