| Background and purpose:According to GLOBOCAN 2020 data,it is known that lung cancer ranks second（11.4%）in incidence and first（18%）in mortality,and non-small cell lung cancer（NSCLC）accounts for the majority of newly diagnosed lung cancer cases,about 85%.It is a heavy burden and problem in healthcare worldwide.The concept of precision oncology drugs is gradually taking shape,and the development of new targets has been a hot topic in lung cancer research.Chloride ions are widely present in various tissues and organs of the human body.Chloride Intracellular Channels（CLICs）,with six subtypes,CLIC1-CLIC6,have been identified to play a role in tumor biology,cardiovascular,inflammation&immunity and neurodegeneration.Chloride Intracellular Channel 4（CLIC4）has been more intensively studied in the CLICs family and plays a role in membrane electrical homeostasis,intra-and extracellular ion homeostasis and transmembrane transport.It has also been reported to be involved in various pathways,which can be regulated by TGF-β,P53,Myc and TNFα.The expression and role of CLIC4 vary in various cancers,and it has been reported to be a tumor-suppressor protein in gastric cancer,skin cancer,prostate cancer,but as a tumor-promoting protein in pancreatic cancer,ovarian cancer,breast cancer and other cancer types.Because of the controversial role of CLIC4 in cancer,its performance is inconsistent among different cancer types,and its mechanism is not fully elucidated,there are few studies on CLIC4 in NSCLC.Based on the above considerations,This study first assessed the difference in CLIC4 expression in NSCLC versus normal tissues adjacent to the cancer and the prognostic impact of CLIC4 on patients with NSCLC using bioinformatic analysis.The results of bioinformatic analysis of decreased CLIC4 expression in NSCLC were validated both in cell lines and carcinoma tissues,Immunofluorescence and exosome extraction were also applied to verify the distribution of CLIC4 in NSCLC.Stable cell lines with overexpression and knockdown of CLIC4 were successfully constructed,and their effects on the proliferation,migration and invasion ability of NSCLC cell lines were verified using CCK8,clonal plate,migration assay and matrigel invasion assay.We also used RNA-seq technology to sequence the transcriptome of CLIC4 overexpression and CLIC4 knockdown cell lines for GO,KEGG,PPI and other analytical methods.The findings related to transcriptome sequencing were verified and explored.The relationship between CLIC4 and the apoptotic signaling pathway was also explored.This study provides a theoretical basis for the pathogenesis of CLIC4 in NSCLC,it may act as a diagnostic indicator and potential therapeutic target.Chapter 1:CLIC4 expression and distribution in NSCLCObjective:1.To explore the expression and distribution of CLIC4 in NSCLC and its potential correlation with patient survival.Methods:1.Differential analysis of CLIC4 in lung cancer data samples using databases TIMER2.0,UALCAN and HPA.2.The association between high and low CLIC4 expression levels and NSCLC patient survival was investigated using the Kaplan-Meier Plotter database.3.Tissue specimens from patients with NSCLC were collected and cultured by culturing normal alveolar epithelial cells（BEAS-2B）,NSCLC cell lines（H1975,H1299,HCC827,PC9,H358,H460,A549）.Total RNA and protein were extracted for q-PCR and western blot to verify the expression of CLIC4,and selected pathological samples of in situ and invasive adenocarcinoma were stained for immunohistochemistry.4.Immunofluorescence staining was performed to observe the protein distribution of CLIC4 in BEAS-2B,PC9 cells.5.Electron microscopy verified the successful extraction of exosomes from H1299,PC9 cells.and the expression of CLIC4 was verified using western blot in combination with exosomal markers CD9,CD63.Results:1.TCGA source database suggested that CLIC4 was lower in NSCLC tissues than in paraneoplastic tissues at the m RNA level.CPTAC source database analyzed LUAD samples and CLIC4 was low expressed in LUAD at the protein level.HPA database concluded that CLIC4 was stronger in normal lung tissues than in lung cancer tissues by immunohistochemical staining.2.In the Kaplan-Meier Plotter database analysis of 1925 lung cancer patients,patients with high CLIC4 expression had a better prognosis HR=0.86 95%CI（0.75-0.97）and a longer median survival,which was statistically different after the log-rank test（P<0.05）.In subgroup analysis in LUAD,patients with high expression of CLIC4 also had better survival（P=0.0032）.3.CLIC4 transcript and protein levels were lower in NSCLC cell lines and cancer tissues.4.Immunohistochemistry and immunofluorescence suggested that CLIC4 was mainly distributed in the cytoplasm outside the nucleus,but could also enter the nucleus.5.Western blot verified the presence of CLIC4 expression in the exosomes from H1299,PC9 cells.Conclusion:1.CLIC4 expression was reduced in NSCLC both at the cellular level and tissue level.High expression of CLIC4 suggested better survival in lung cancer patients.2.CLIC4 is mainly distributed outside the nucleus.It can be present in the exosomes of NSCLC cell lines H1299,PC9.Chapter 2 Effect of CLIC4 on proliferation,migration and invasion ability of NSCLC cellsObjective:1.To investigate the effect of overexpression of CLIC4 and knockdown of CLIC4 on the biological behavior of NSCLC.Methods:1.H358 and PC9 cell lines were selected for CLIC4 overexpression,transfected with CLIC4 overexpression plasmid and corresponding empty plasmid respectively,and then performed resistance screening,picked out monoclonal culture after western blot to verify the degree of CLIC4 overexpression.2.H1299 and PC9 cell lines were selected for CLIC4 knockdown,transfected with sh NC,sh CLIC4#1,sh CLIC4#2 plasmids,resistance screening was performed,and the monoclonal cultures were picked out to verify the degree of CLIC4knockdown by western blot.CLIC4 knockdown plasmids carrying GFP can also be observed under the microscope for transfection efficiency.3.The successfully constructed cell lines were subjected to CCK8,clone formation,migration assay and matrigel invasion assay.Results:1.CCK8 experiment showed that overexpression of CLIC4 restricted the growth of H358 with statistical difference（P<0.01）,and the clone formation ability was also reduced.In the migration and matrigel invasion assays,the H358 CLIC4overexpression group showed significantly reduced migration and invasion cells compared to the control group.Similar results were observed for PC9 cells with CLIC4 overexpression.2.CCK8 assay showed that knockdown of CLIC4 had no significant effect on cell proliferation（P>0.05）,and clone formation also supported this conclusion.In migration and matrigel invasion assays,H1299 sh CLIC4#2 migration and invasion cells were significantly higher than the control group.A similar conclusion was obtained for PC9 cells with knockdown CLIC4.Conclusion:1.Stable overexpression of CLIC4 inhibits the proliferation,clone formation,migration and invasion ability of H358 and PC9 cells.2.Stable knockdown of CLIC4 promoted the migration and invasion abilities of H1299 and PC9 cells.While no significant effect on cell proliferation was observed.Chapter 3 Transcriptome sequencing bioinformatics of stable CLIC4 overexpression and knockdown cell linesObjective:1.To investigate the effect of overexpression and knockdown of CLIC4 on the transcriptome of stable cell lines and to search for associated pathway changes.Methods:1.Three biological replicates of stable cell lines of CLIC4 overexpression and knockdown,as well as their respective control cell lines,were constructed and sent for sequencing.2.The sequencing data was cleaned and subjected to quality control,followed by analysis using R software.The analysis included the generation of volcano plots,heat maps of differentially expressed genes,GO and KEGG pathway analyses.Results:1.The volcano plot for CLIC4 overexpression versus its control（PC9 CLIC4 vs PC9 EM）had 602 upregulated genes and 618 downregulated genes.the GO classification suggested enrichment in inflammatory response,plasma membrane,extracellular region,calcium binding function.the KEGG analysis differential genes were enriched in Ras,MAPK,Rap1 signaling pathway,rheumatoid arthritis and inflammation regulation.2.The volcano plot for CLIC4 knockdown versus its control（PC9 sh CLIC4 vs PC9 sh NC）showed differential expression of 295 upregulated genes and 283downregulated genes.GO analysis revealed enrichment in biological processes related to extracellular matrix and epithelial differentiation function.KEGG analysis showed enrichment in cancer pathways.Conclusion:1.The number of differential genes caused by overexpression of CLIC4（1220）was significantly more than that caused by downregulation of CLIC4（578）.The differential genes were found to be enriched in pathways involved in MAPK,Ras and inflammation.Chapter 4:Mechanistic study of the effect of CLIC4 on the biological function of NSCLCObjective:1.To validate the pathway changes identified in transcriptome sequencing and to explore additional underlying mechanisms.2.To explore the relationship between CLIC4 and apoptosis.Methods:1.The effect of stable CLIC4 overexpression in H358 and PC9 cells on related molecules in the MAPK and other pathways was verified using western blot analysis.2.Immunoprecipitation（IP）protein samples from PC9 cells were analyzed using mass spectrometry with both Ig G and CLIC4 antibodies to explore potential protein interactions.3.Western blot analysis was conducted to investigate whether changes in CLIC4expression affect the epithelial-mesenchymal transition process,and FITC Phalloidin staining was used to observe changes in the cytoskeleton of PC9 cells following CLIC4 overexpression.4.The role of CLIC4 in the onset of apoptosis activation was assessed by inducing apoptosis with different gradients of staurosporine（STS）and hydrogen peroxide.Flow cytometry was used to measure apoptosis under relevant conditions,and western blot analysis was performed to verify changes in the apoptotic pathway in PC9 cells following CLIC4 overexpression and changes in the apoptotic pathway following apoptosis induction.Results:1.Overexpression of CLIC4 in H358 and PC9 cell lines could reduce the expression levels of p-Raf1,p-MEK1/2,p-ERK1/2.while also inhibiting p-AKT.There was no direct interaction observed between CLIC4 and Ras.2.Overexpression of CLIC4 in H358 and knockdown of CLIC4 in H1299 did not cause significant changes in the epithelial-mesenchymal transition（EMT）indexes.No significant morphological changes were observed between PC9 CLIC4 and its control group PC9 EM after FITC Phalloidin staining.3.The cell viability gradually decreased with increasing staurosporine dose,and PC9 CLIC4 was statistically lower compared with the control group under staurosporine 0.1μM,0.25μM,0.5μM and 1μM intervention conditions.Similarly,the inhibition of cell viability by H2O2was concentration-dependent,and the cell viability of PC9 CLIC4 was lower than that of the control at all the removed 50μM concentrations.4.Flow analysis showed that overexpression of CLIC4 had a significant effect on early apoptosis.Overexpression of CLIC4 increased early apoptosis,with an even more pronounced increase after the addition of staurosporine.5.In PC9 cell line,after overexpression of CLIC4,Bax expression increased while Bcl-2 expression decreased.The cleaved PARP was upregulated relative to total PARP,indicating changes in the apoptotic pathway.After staurosporine induction,cleaved PARP increased further compared to total PARP,and cleaved Caspase3 was also elevated relative to the control group.Conclusion:1.Overexpression of CLIC4 in NSCLC inhibits the MAPK pathway by reducing the phosphorylation levels of Raf1,MEK1/2 and ERK1/2,and inhibiting AKT phosphorylation.Although CLIC4 does not directly interact with Ras,it has significant downstream effects on the pathway.Moreover,the alteration of migration and invasion ability in NSCLC by CLIC4 is independent of the traditional EMT changes.2.High expression of CLIC4 induced apoptotic pathway changes in NSCLC,and in the case of apoptosis induction,apoptosis was further increased after overexpression of CLIC4 compared to control. |
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