Based On The P53 Regulatory Point, The Molecular Mechanism Of Neo-gambogic Acid-induced A549/Cis Cell Cycle Arrest And Cisplatin Resistance In NSCLC Was Explored

Purpose:We investigated the effects of GNA on the cell cycle,apoptosis and cisplatin resistance of cisplatin-resistant non-small cell lung cancer cell line A549/Cis through in vitro methords.High throughput sequencing(RNA-seq)was used to analyze the regulation of GNA treated A549/Cis transcription,and the expression and function of the differentially expressed genes,to explore and verify the important clues of GNA impacted A549/Cis biological effects,and to study the potential mechanism.Finally,the roles of MTCYB and TP53 in GNA-mediated growth inhibition were studied by overexpressing MTCYB and silencing TP53 in A549/Cis cells to elucidate the potential regulatory targets of GNA affecting on the cisplatin resistace in A549/Cis cells,and to provide theoretical evidences for its clinical application in the treatment of cisplatin-resistant NSCLCMaterial and method:Thesis 1MTT assay was used to identify the cisplatin resistance of A549/Cis cells.A549 cells and A549/Cis cells were treated with different concentrations of cisplatin.After 24,48 and 72h,20 μl/well MTT(5mg/ml)was added for further incubation at 37℃ for 4 h.The supernatant was then discarded and the formazan product was dissolved in DMSO.The optical density was measured at 490 nm and the experimental results were recorded.The inhibitory effect of GNA on A549 cells and A549/Cis cells growth was determined by MTT assay.The effect of GNA on the morphology of A549/Cis cells was detected by staining with Hoechst 33342 and fluorescence microscopy.Cells were treated with GNA at 37℃ for 24 and 48 h.At the end of the various treatments,the cells were stained with Hoechst 33342(10μg/ml)at 37℃ for 20 minutes,and then changes in the cell morphology were observed by fluorescence microscope The effects of cell cycle distribution and apoptosis of GNA on A54/Cis cells were analyzed via flow cytometry.Cells were washed and fixed in precooled 70%alcohol overnight at-20℃,then incubated with RNaseA solution and stained with PI,the cell cycle analysis was performed using a BD FACSCelesta Flow Cytometer.Cell apoptosis was analyzed using an Annexin V-APC/7-AAD Apoptosis Detection kit,according to the manufacturer’s protocol.The stained cells were analyzed using a BD FACSCelesta Flow Cytometer and the data were analyzed using FlowJo 7.6.1 software.Thesis 2Transcriptomic profiling of A549/Cis cells carried out before and after GNA treatment.The total RNA of A549/Cis cells treated with 4 μM GNA for 24 h was extracted using TRIzol(?)reagent following the manufacturer’s protocol.RNA-seq was performed using an Illumina X10 sequencing platform following the manufacturer’s protocol.Stringtie was used to analyze the expression levels for mRNAs.The differentially expressed mRNAs and genes were selected with |log2 fold change|≥1 and with statistical significance(P-value<0.05)using the R package-Ballgown.For the GO and KEGG pathway enrichment analysis,the Database for Annotation,Visualization and Integrated Discovery web server(http://david.ncifcrf.gov/)was used.The differential gene expression was identified by real-time fluorescence quantitative PCR.The total RNA was extracted from A549/Cis cells using the Gene JET RNA Purification kit according to the manufacturer’s protocol.First Strand cDNA Synthesis kit was used for cDNA synthesis.qPCR was subsequently performed using the SYBR(?)Select Master Mix kit and a QuantStudio 3 Real-Time PCR system.Quantification was performed using the comparative 2-△△CT method.Western blot was used to detect the expression of cell cycle-related and apoptosis-related proteins.Total protein was extracted from cells using RIP A lysis buffer.Nuclear proteins were prepared using a Nuclear and Cytoplasmic Protein Extraction kit according to the manufacturer’s protocol.The protein concentration was determined by a BCA protein analysis kit.The proteins were loaded onto 10%gels or 12%gels separated by SDS-PAGE and transferred to PVDF membranes.The PVDF membrane was blocked,then incubated with the specific primary antibodies.Subsequent to washing three times with TBS T,the membranes were incubated with the following HRP-conjugated secondary antibodies.The immunoblots were visualized using an enhanced chemiluminescent substrate kit.Expression levels were quantified using ImageJ version 1.52 soffware.Thesis 3MTCYB fusion gene overexpressed retroviral vector,MSCV-PGK-MTCYB-2a-zsGreen was constructed and transfected into 293 T cells.A549/Cis cells were infected with the filtered virus-containing cell supernatants of 293T cells.The cells with positive zsGreen expression(green fluorescence)were sorted by flow cytometry and identified by confocal laser scanning microscopy.qRT-PCR was performed to identify the mRNA expression of MTCYB in selected cells.Finally,the MTT assay was used for the measurement of GNA sensitivity in established cell lines as well as primary cells.Human TP53 shRNA knockdown retroviral vector,MSCV-U6-p53shRNA-EF1-BFP were constructed and transfected into 293T cells A549/Cis cells were infected with the filtered virus-containing cell supernatants of 293T cells The cells with positive BFP expression(blue fluorescence)were sorted by flow cytometry and identified by laser confocal microscope.Then the expression of p53 protein in the selected cells was determined by Western blot to identify the efficiency of p53 silencing Finally,the MTT method was used to detect the GNA sensitivity in established cell lines as well as primary cells.Results:Thesis 11.GNA inhibited the growth of A549 and A549/Cis cells and induced apoptotic morphological changes in A549/Cis cellsThe MTT assay demonstrated that A549/Cis cells were significantly more resistant to cisplatin compared with the parental cells(P<0.001).The cytotoxic effect of GNA on A549 and A549/Cis cells was determined.GNA significantly decreased the viability of A549 and A549/Cis cells compared with the untreated group(P<0.001).GNA induced a high degree of cell death at a concentration of 6 μM only after 24 h.Accordingly,2 and 4 μM GNA was used in the subsequent experiments.Hoechst 33342 staining further demonstrated the inhibitory effect of GNA in A549/Cis cells.Compared with the untreated cells,the cells treated with GNA had inhibited proliferation and exhibited morphological alterations.Furthermore,the nuclear condensation of GNA-treated cells was also observed2.GNA induced cell cycle Gi phase arrest and apoptosis in A549/Cis cellsTo investigate the cellular process responsible for the inhibited proliferation by GNA treatment,the cell cycle and apoptosis were examined by flow cytometry in A549/Cis cells The results showed that the cell cycle of A549/Cis cells was significantly arrested at the G1 phase following GNA treatment for 24 and 48 h compared with the untreated group(P<0.05)There was a significantly higher sub-G1 population in the cells treated with 4 μGNA for 48 h compared with the untreated group(P<0.001).Cell cycle arrest may induce cell death,which was measured using flow cytometry.The Annexin V-APC/7-AAD double staining assay revealed that the apoptosis rate was significantly increased compared with the control group when A549/Cis cells were treated with 4 μGNA for 48 h(P<0.001)Thesis 21.Differential gene expression and enrichment analysis in A549/Cis cells treated with GNATo understand how GNA inhibits cell growth and promotes cell death in A549/Cis cells,an RNA-seq assay was performed using samples from the control and GNA-treated cells Data analysis indicated that GNA treatment induced a global gene expression change.All genes whose threshold was restricted with a P-value<0.05,|log2 fold change|≥1 were identified as differentially expressed genes(DEGs).Compared with the untreated cells,there were 353 up-regulated DEGs and 425 down-regulated DEGs in GNA-treated cells.To further investigate the function of DEGs,GO and KEGG pathway enrichment analysis were carried out.It was identified that protein binding,cytosol and cell cycle were mostly enriched in GO enrichment analysis,and there were 26 significantly enriched pathways in KEGG signal pathway enrichment analysis.After sorting the above KEGG signal pathways according to significant differences,it was found that DEGs were mainly enriched in Protein processing in endoplasmic reticulum,Proteasome,Cell cycle,Epstein-Barr virus infection,Estrogen signaling pathway,p53 signaling pathway,DNA replication.Cell cycle,DNA replication and p53 signal pathway are closely related to tumor proliferation.2.Validating RNA-Seq results with qPCR.To validate the RNA-seq results,14 DEGs were selected for RT-qPCR analysis.The results showed that there were significant differences in the expression of all genes in 4 μM GNA group compared with untreated cells.Among these genes,4 were verified to be upregulated in GNA-treated A549/Cis cells whereas the other 10 were identified as downregulated.In total,ten genes(GADD45A,cyclin D3(CCND3),cyclin B1(CCNB1),CDC20,CDC25B,PCNA,PLK1,MCM2,MCM3 and MCM 7)were involved in cell cycle,six genes(GADD45A,CCND3,CCNB1,SERPINE1,THBS1 and TNFRSF10B)were associated with p53 signal pathway,and two genes(CASPASE7 and TNFRSF10B)are related to apoptosis.The results of qRT-PCR were consistent with the RNA-seq analysis3.Expression of cell cycle and apoptosis-associated proteins in A549/Cis cells treated with GNA.In order to further study the potential mechanisms of cell cycle arrest and apoptosis induced by GNA,a Western blot assay was performed to detected the expression level of proteins related to cell cycle checkpoint and apoptosis.The data revealed that the protein levels of cyclin D1,cyclin D3,CDK4 and CDK6 were significantly downregulated(P<0.05),while the expression of p21,GADD45A,p53 and nuclear p53 were significantly upregulated in A549/Cis cells following GNA treatment compared with the untreated group(P<0.05)Furthermore,the regulators of apoptosis were additionally detected subsequent to GNA treatment.The results showed that compared with the control group,GNA was able to significantly increase the protein levels of the precursor forms of caspase 3/7 and their active forms as well(P<0.05).The hallmark of apoptosis,PARP,which is associated with DNA repair,was revealed to be significantly upregulated(P<0.05),and its cleavage was significantly enhanced by GNA treatment in A549/Cis cells(P<0.05)Thesis 31.The GNA sensitivity of MTCYB overexpressed A549/Cis cells did not change significantlyIn this experiment,the synthesized MTCYB fusion gene was successfully connected into the specific site of double-enzyme-digestion linearized expression vector,which was correct according to the sequencing result.The virion-packaging elements and the MTCYB overexpressing vector was co-transfected into 293 T cells to produce infective retroviral particles,the efficiency of the retroviral vetor transduction was high confirmed by fluorescence observation.Then the culture medium was used to infect A549/Cis cells.It was confirmed that A549Cis cells were successfully infected with the retroviral particles by fluorescence observation.The cells with zsGreen positive expression were sorted by flow cytometry and the rate of positive cells was confirmed by laser confocal microscope.The mRNA expression of MTCYB was detected by qRT-PCR.Compared with the control group and A549/Cis-NC group,the expression of MTCYB in A549/Cis-MTCYB group was higher(P<0.05).We used MTT method to determine the effect of GNA on the proliferation and viability of A549/Cis cells with MTCYB overexpression.GNA had obvious inhibitory effect on the three group cells,but there was no significant difference among them.2.The GNA sensitivity of p53-deficient A549/Cis cells was decreasedThe p53 interfering segment we had designed and synthesized in this experiment was successfully connected into the specific site of double-enzyme-digestion linearized expression vector,and the sequencing results showed that the connection was correct.The virion-packaging elements and the shRN A-expressing vector was co-transfected into 293T cells to produce infective retroviral particles,the efficiency of the retroviral vetor transduction was high confirmed by fluorescence observation.Then the culture medium was used to infect A549/Cis cells.It was confirmed that A549Cis cells were successfully infected with the retroviral particles by fluorescence observation.Then the BFP positive expression cells were sorted by flow cytometry and the positive cell rate was confirmed by laser confocal microscope.The expression of p53 protein was detected by Western blot.The results showed that the level of p53 protein in A549/Cis-p53shRNA1,2 and 3 cells significantly decreased compared with negative control cells and A549/Cis-shRNA-NC cells(P<0.05),and the decrease was most significant in A549/Cis-p53-shRNA2 cells(P<0.001).MTT assay was used to determine the effect of GNA on the proliferation and viability of A549/Cis cells with p53 silencing.The growth inhibition of A549/Cis-p53shRNA2 cells was significantly decreased compared with the control cells and A549/Cis-shRNA-NC cells(P<0.05),and its sensitivity to GNA was decreasedConclusion:1.GNA could inhibit the growth of cisplatin-resistant non-small cell lung cancer A549/Cis cells by mediating Gi phase cell cycle arrest and inducing apoptosis2.Transcriptomic studies had shown that GNA can significantly down-regulate the expression of MTCYB in A549/Cis cells,and mediate A549/Cis cell cycle G1 phase arrest by regulating p53/p21/cyclinD-CDK4/6 and induced apoptosis through activation of caspase3/73.p53 is a key regulator in the mechanisms of the growth inhibition induced by GNA in A549/Cis cells.