| ObjectiveIn many developed countries and some China large and medium-sized cities,lung cancer has become one of the common malignant tumors.The mortality and morbidity rate are higher than other types of malignant tumors,and it shows a trend of increasing year by year.It is one of the main diseases that endanger human health and life.In order to find more effective treatments for lung cancer,the research on lung cancer pathogenesis has become a key research in the treatment of lung cancer.Numerous studies have shown that lung cancer development is not only a multi-factor synthesis process,but also a multi-step process,mainly related to lung tissue aging,genetic susceptibility,telomere shortening,chronic inflammatory response and epigenetic changes.In recent years,the epigenetic modification of RNA has been favored by researchers.With the continuous understanding and exploration of RNA methylation modification,it is found that the imbalance of RNA m6A in the body can cause various diseases,such as Alzheimer's disease,obesity,HIV infection,tumors,etc.The current research results show that m6A is closely related to the development of tumors such as glioma,acute myeloid leukemia,liver cancer,breast cancer and gastric cancer.Since the discovery of the Alk B gene in E.coli variants in 1983,9Alk B homologous proteins,ALKBH1-8 and FTO,have been found in the human genome.This family protein is primarily responsible for the methylation of DNA and RNA.Studies have found that FTO and ALKBH5 mainly catalyze the demethylation of DNA 6m A,which in turn affects the transcription and translation of downstream genes.As a member of the Alk B family,it has been found that ALKBH1 also has the demethylation function of nucleic acids,mainly catalyzing the 3-mec demethylation of ss DNA and RNA.With the research deepening,it has been reported that ALKBH1can regulate the demethylation of t RNA 1m A and affect the translation process of proteins;ALKBH1 affects the development of star glioma by regulating the 6m A demethylation of DNA;silencing the ALKBH1 gene can cause DNA and RNA m6A modification affects the development of liver cancer and gastric cancer.However,the contribution of ALKBH1 to RNA m6A demethylation,the specific catalytic mechanism,and the effect of this catalysis on lung cancer are not well understood.Based on the above research,lung cancer cells,normal lung cells,and urethane-induced mouse lung cancer model,ALKBH1 protein"substrate analog"NOG,ALKBH1 gene silencing and ALKBH1 overexpression were utilized to explore the role of ALKBH1 and m6A in lung cancer.Protein expression,purification,crystallization,structural analysis,SPR assay,EMSA analysis,protein thermal shift assay,and LC-MS/MS were utilized to explore the molecular mechanism of ALKBH1promoting lung cancer.Methods1.The role of ALKBH1 in lung cancerLung epithelial cells BEAS-2B,lung cancer cells A549,H1299 and H1650 were cultured normaly.RT-PCR analysis was performed to detecte ALKBH1 gene level;Western blot assay was utilized to explore ALKBH1protein expression level.2.Effect of ALKBH1 substrate analog NOG on A549 proliferation,migration and invasionThe concentration of NOG was determined by MTT assay.A549 cells were treated with different concentration NOG for 2 days.The expression of ALKBH1gene and protein in A549 cells was detected by RT-PCR assay and Western blot assay,respectively.The level of m6A was detected by RNA dot blot assay.EdU assay and plate clone formation assay were performed to detect the effect of NOG on cell proliferation;Transwell migration assay and Transwell invasion assay were used to examine the effect of NOG on cell migration and invasion.3.Effect of silencing ALKBH1 gene on cell proliferation,migration and invasionALKBH1 gene silencing was performed in A549 cells by siRNA interference technique;ALKBH1 silencing effect was detected by RT-PCR assay and Western blot assay;m6A level was detected by RNA dot blot assay;MTT assay was used to detect cell viability after silencing ALKBH1gene;EdU assay was used to detect the effect of silencing ALKBH1 gene on cell proliferation ability;Wound healing assay was used to detect the effect of silencing ALKBH1 gene on cell migration ability;Effect of silencing ALKBH1 gene on cell invasion ability was detected by Transwell invasion assay4.Effect of ALKBH1 overexpression on cell proliferation,migration and invasionThe hALKBH1-pcDNA3.1-EGFP recombinant plasmid was constructed by molecular cloning technology;the ALKBH1 gene was overexpressed in A549 cells utilizing plasmid transfection;ALKBH1 gene expression was detected by RT-PCR assay;ALKBH1 protein expression was detected by Western blot assay;m6A leve was detected by RNA dot blot assay;MTT assay was utilized to detect A549 cells viability;Wound healing assay was performed to analyze the effect of ALKBH1overexpression on A549 cells migration;Transwell invasion assay detected the effect of ALKBH1 overexpression on cell invasion;Extrame Limiting Dilution Analysis was utilized to detect the effect of ALKBH1 overexpression on cells proliferation ability.5.The effect of ALKBH1 on the development of lung cancer in vivoThe lung cancer was induced by urethane reagent.The pathological characteristics of lung tissue were detected by HE staining.The protein expressions of PCNA and CK7 were detected by immunohistochemistry,and ALKBH1 protein expressions were detected by immunohistochemistry and Western blot.ALKBH1gene leve in mouse lung tissues was detected by RT-PCR assay.The m6A level in mouse lung tissues was detected by RNA dot blot assay.6.mALKBH1 and mALKBH1 mutants protein expression and purificationThe gene sequence encoding mALKBH1(Gene ID:211064)was searched in the NCBI database,and the selection of Nde I and Xho I restriction sites was confirmed by Snap Gene software.Design specific primers,and add protective bases and Nde I and Xho IA restriction sites on both ends of the specific primers.The target gene fragment was amplified by ordinary PCR,and the mALKBH1 gene fragment and p ET-28a(+)vector were subjected to Nde I and Xho I restriction double digestion,respectively,and then ligated with T4 ligase to obtain the recombinant plasmid mALKBH1-p ET-28a(+).An expression vector for the mALKBH1 mutant was constructed using the Quick Change site-directed mutagenesis technique.mALKBH1 and its mutants were expressed in E.coli BL 21-Codon Plus(DE3)cells;Both were purified by two steps:Ni-NTA affinity purification?Hi Trap Q HP(1 m L)anion exchange column.7.mALKBH1 protein structure analysisAfter obtaining the purer mALKBH1 protein,the crystal was first screened and optimized;then the characteristics of the crystal were analyzed by silver staining;finally,the crystal was screened by SSXFR after screening by the indoor X-ray single crystal diffractometer of the School of Pharmacy,Sun Yat-sen University.Collect and use the software HKL2000 for data indexing,normalization and integration.Finally we obtained diffraction data with a resolution of about 3?.Since no homologous protein was found for molecular replacement,in order to solve the phase problem,after various attempts,we finally carried out the expression,purification,crystallization and diffraction of selenoprotein,and obtained a resolution of about 2.8?data.Structural analysis and refinement are done through the software CCP4 and Phenix.8.mALKBH1 protein catalytic activity in vitroSPR assay was used to verify the binding of mALKBH1 to 2OG.Protein thermal shift assay was used to analyze protein stability and binding ability to 2OG between mALKBH1 and its mutants.The binding ability to m6A of mALKBH1 and its mutants was explored by EMSA assay.Finally,the difference among mALKBH1 and its mutants catalyzeing RNA m6A demethylation in vitro was detected by RNA dot blot assay and LC-MS/MS assay.9.Statistical analysisData analysis was performed using software SPSS 20.0,shown as Mean±SD,and using Oneway ANOVA for statistics.If the variance is equal,the LSD statistical method is used.If the variance is not uniform,Dunnett's T3 statistical method is used,and P<0.05 is defined as having statistics.Significant differences in learning,*P<0.05,**P<0.01,***P<0.001.Results1.ALKBH1 and m6A are highly expressed in lung cancer cellsIn this experiment,normal lung epithelial cells BEAS-2B cells were used as control to detect the expression of ALKBH1 in lung cancer cells A549,H1299,and H1650.After culture,the growing well and same cell aging cells were selected.The expression levels of ALKBH1 gene and protein in different cell lines were detected by RT-PCR and Western blot.Western blot results showed that ALKBH1 protei expression in lung cancer cells A549,H1299,and H1650 increased compared with normal lung epithelial cells BEAS-2B.Protein expression increased to 576.72%±85.13%(P<0.01),762.46%±84.01%(P<0.001),621.58%±75.44%(P<0.001).RT-PCR results showed that gene expression of ALKBH1 in lung cancer cells A549,H1299,and H1650 were increased compared with that of BEAS-2B.The gene expression leve increased to 10.9±1.1 folds(P<0.001),4.01±0.17 folds(P<0.001),and 20.80±0.57 folds(P<0.001),respectively.2.NOG significantly inhibited the proliferation,migration and invasion ability of lung cancer cells A549The cells treated with 0.1%DMSO were utilized as a control.After the cells were cultured,A549 cells in a logarithmic growth phase and in good condition were selected,seeded into 6-well plate,and treated with NOG for 2 days.The expression of ALKBH1 gene and protein was detected by RT-PCR assay and Western blot assay.The level of m6A was detected by RNA dot blot assay.The effect of NOG on cell proliferation was detected by EdU assay and plate clone formation assay.Transwell migration assay and Transwell invasion assay were used to examine the effect of NOG on cell migration and invasion.The results of Western blot showed that ALKBH1 protein expression had no significant difference in NOG-treated A549 cells.The expression level(%)of ALKBH1 protein in each group was:100.00%±14.00%,98.29%±14.64%(P>0.05),and 99.56%±13.09%(P>0.05).RT-PCR results showed that there was no significant change ALKBH1 gene expression in different groups.The expression level(fold)of ALKBH1 gene in each group was:1.00±0.04 folds,1.20±0.04(P>0.05),0.95±0.11folds(P>0.05).RNA dot blot results showed that the level of m6A was significantly increased in NOG-treated A549 cells.The m6A levels(%)of each group were:100.00%±31.38%,181.26%±9.78%(P<0.001),and 168.08%±37.69%(P<0.001).EdU results showed that NOG significantly inhibited the proliferation of A549cells in a concentration-dependent manner.The percentage inhibition of A549 cell proliferation by different concentrations of NOG were 81.64%±8.99%(P>0.05),36.19%±1.67%(P<0.01),and 28.79%±1.95%(P<0.01).The results of plate clone formation assay showed that NOG could significantly inhibit the clonality of A549cells.The percentage inhibition of A549 cell proliferation by different concentrations of NOG was 27.33%±6.51%(P<0.001)and 45.67%±4.04%(P<0.001).Transwell migration experiment showed that NOG inhibited the migration ability of A549 cells in a concentration-dependent manner.The absorbances of corresponding crystal violets under different concentrations of NOG were 0.413±0.013,0.405±0.022(P>0.05),0.390±0.005(P>0.05),and 0.385±0.018(P<0.05).At the same time,Transwell invasion experiment showed that NOG inhibited the invasion ability of A549 cells.The absorbances of corresponding crystal violets under different concentrations of NOG were 0.823±0.038,0.753±0.040(P>0.05),0.720±0.017(P<0.05),and 0.640±0.052(P<0.01).3.ALKBH1silence inhibited A549 cells proliferation,migration and invasion.A549 cells in a logarithmic growth phase and in good condition were selected,and the cells were incubated with siRNA for 12 hours,then normally cultured for 48hours,and samples were collected.The silencing effect of ALKBH1 was detected by RT-PCR assay and Western blot assay.The level of m6A was detected by RNA dot blot assay.The cell viability of ALKBH1 was detected by MTT assay.The effect of silencing ALKBH1 on cell proliferation was detected by EdU assay.Wound healing assay and Transwell invasion assay were used to examine the effect of silencing ALKBH1 on cell migration and invasion.Western blot results:siRNA interference effects(%)at different sites were 41.14%±3.32%(P<0.001),32.01%±3.49%(P<0.001),41.11%±4.01%(P<0.001).RT-PCR experiments showed that the siRNA interference effects(folds)at different sites were 0.026±0.003(P<0.001),0.264±0.004(P<0.001),and 0.249±0.020(P<0.001).The RNA dot blot assay showed that siRNA interfered withALKBH1significantly increased m6A levels in A549 cells.The experimental results were semi-quantitative.The m6A levels(%)were:275.52%±0.05%(P<0.001),125.16%±0.25%(P<0.01),384.63%±0.05%(P<0.001).A549 cell viability was detected by MTT assay.The results showed that A549cell viability was significantly inhibited after siRNA interfered with the siRNA.Before the EdU experiment,photographs of A549 cells in the bright field were collected to observe changes in cell density after transfection.The results showed that the siRNA interfered with the expression of ALKBH1 significantly inhibited A549cell proliferation.Wound healing assay was used to detect the migration ability of A549 cells.The results showed that the siRNA interfered with the expression of ALKBH1,and the migration ability of A549 was significantly inhibited.Transwell invasion assay was used to detect the invasive ability of A549 cells.The results showed that the invasion ability of A549 was significantly inhibited.*P<0.05,**P<0.01,***P<0.001.4.Overexpression of ALKBH1 gene promoted lung cancer cell A549proliferation,migration,and invasion.The hALKBH1-pcDNA3.1-EGFP recombinant plasmid was constructed by molecular cloning technology;ALKBH1 gene was overexpressed in A549 cells utilizing plasmid transfection;ALKBH1 gene level was detected by RT-PCR assay;ALKBH1 protein expression level was detected by Western blot assay;m6A leve was detected by RNA dot blot assay;MTT assay was utilized to detect A549 cells viability;Wound healing assay was performed to analyze the effect of ALKBH1 overexpression on A549 cells migration;Transwell invasion assay detected the effect of ALKBH1overexpression on cell invasion;Extrame Limiting Dilution Analysis was utilized to detect the effect of ALKBH1 overexpression on cells proliferation.RT-PCR results showed that ALKBH1 overexpression increased the level of ALKBH1 mRNA by about 2-fold.The results of the two groups were:1±0.06 folds,2.02±0.24 folds(P<0.01).Western blot results showed that overexpression of ALKBH1 gene increased the level of ALKBH1 protein by about 146.5%.The results of the two groups were:100%±6.24%,146.5%±8.77%(P<0.01).RNA dot blot results showed that overexpression of ALKBH1 gene can significantly reduce the level of m6A in A549 cells,and semi-quantify the experimental results(%)were:100%±4.81%,33.73%±5.43%(P<0.001).Wound healing assay results showed that ALKBH1 overexpression increased A549 migration ability to 306.7%±67.23%(P<0.01).Transwell invasion results showed that ALKBH1 overexpression increased the invasive ability of A549 and the invasion rate increased by 246.7%±32.83%(P<0.01).The effect of ALKBH1overexpression on cell self-renewal by Extrame Limiting Dilution Analysis,and the update rate increased from 1/3.56 of the control group to 1/2.29.5.The effect of ALKBH1 gene on the development of lung cancer in vivoThe lung cancer was induced by urethane reagent.The pathological characteristics of lung tissue were detected by HE staining.The protein expressions of PCNA and CK7 were detected by immunohistochemistry.ALKBH1 protein expression was detected by immunohistochemistry and Western blot.ALKBH1 gene leve in mouse lung tissues was detected by RT-PCR assay.The m6A level in mouse lung tissues was detected by RNA dot blot assay.At the end of urethane-induced mouse lung cancer experiment,mouse lung tissues were dissected and the number of nodules in mouse lung tissues was observed.Results showed that compared with the normal tissues,the lung cancer tissues showed macroscopic pulmonary nodules with a tumor-bearing rate of 100%.The results of tissue water content test showed that lung cancer tissues weight and water content were significantly higher than that in mormal lung tissues.During the experiment,the mice in each group grew normally.With the progress of the experiment,the normal group had good activity,normal water intake,soft and shiny hair,and gradually increased body weight.In the lung cancer group,the activity and water intake decreased,showing signs of languidness,dull hair,and a decrease in body weight.At the same time,with the extension of the experimental period,the death rate of mice in the model group gradually increased.HE staining results showed that cells in the lung cancer tissues were disordered,the cell nucleus became larger and the staining was deepened,inflammatory cells infiltrated,and a large number of cancer nest where cells proliferate in large numbers were observed.IHC results demonstrated that the expression of PCNA,CK7 and ALKBH1 in the lung cancer tissues were higher than that in the normal lung tissues.Western blot results showed that ALKBH1 protein level in lung cancer tissues was significantly increased,and the elevated level(%)was 133.4%±6.59%(P<0.01).RT-PCR results showed that ALKBH1 gene level in lung cancer tissues was also significantly increased,and the elevated level was 2.17±0.15 folds(P<0.01).RNA dot blot results showed that the m6A level in the lung cancer tissues was significantly higher than that in normal lung tissues,and the elevated level(%)was 213.4%±40.99%(P<0.001).6.Protein expression and purification of mALKBH1 and its mutantsSDS-PAGE electrophoresis showed that at the molecular weight of 45.4 k Da,a band of induced protein expression was observed.The molecular weight of this band was consistent with the predicted molecular weight of the mALKBH1 protein.The results indicated that the mALKBH1 protein was expressed solubly.In addition,Ni-NTA binding,Ni-NTA buffer A washing to remove non-specific binding protein,Ni-NTA buffer B eluting the target protein,and observed relatively pure protein and the purity wasd more than 85%.Then,the target protein was purified by Hi Trap Q HP(1 m L)anion exchange column.SDS-PAGE electrophoresis result showed that after purification by Hi Trap Q HP,the purity of the protein was over 95%,and the purer protein mainly existed in the section“UB”and section“peak 1”.7.mALKBH1 protein structure analysisThe mALKBH1 crystal structure was obtained preliminarily.The protein structure mainly exists as the form of a 4-mer and an 8-mer,which are formed by folding two?-helices and 10?-sheets.Silver staining experiments showed degradation bands in the crystal of mALKBH1 protein.According to the results of mALKBH1 protein structure and comparison of key amino acids,mALKBH1 protein acids Y184,H231,D233,H287,R338 and R344 are highly conserved in the Alk B family and are key amino acids that binding 2OG and Fe(II).8.ALKBH1 protein catalytic activity in vitroSPR results showed that the KD value of 2OG binding to mALKBH1 protein was 9.16e-5,indicating that the protein mALKBH1 binds well to 2OG.Protein thermal shift assay results showed that the Tm value of mALKBH1 combined with its cofactor 2OG and Fe(II)was significantly changed.That was to say,the stability of mALKBH1 increased after binding with cofactors.The stability of the Y184A,H231A,D233A,H287A,R338A and R344A mutants was explored by protein thermal shift assay.The results showed that the Tm values of the mutants were significantly lower than that of the WT,indicating that mutants stability was worse than that of WT.EMSA results showed that the binding of the mutant protein to m6A was reduced compared to WT.Results of enzyme activity experiments and LC-MS/MS showed that mALKBH1 protein can catalyze the demethylation of RNA m6A,and the catalytic activity of the protein is significantly reduced after mutating the key amino acids described above.ConclusionThis study investigated the role of ALKBH1 in lung cancer development,and proved that ALKBH1 regulated lung cancer development by regulating RNA m6A methylation modification in vitro and in vivo.Silencing ALKBH1 gene in vitro inhibited A549 cell proliferation,migration and invasion,and ALKBH1overexpression significantly enhanced A549 cell proliferation,migration and invasion.The results of urethane-induced mouse lung cancer showed that the ALKBH1 protein and gene levels were increased in lung cancer tissues,indicating that ALKBH1 plays an important role in lung cancer development.At the same time,the mALKBH1protein structure was analyzed.We had proved that mALKBH1 catalyzed the demethylation of RNA m6A in vitro and Y184,H231,D233,H287,R338 and R344played a key role in mALKBH1 protein stability,binding to cofactors and substrates,maintenance catalytic activity.Therefore,this study provides a basis for elucidating the principle of ALKBH1 demethylation,and lays a structural foundation for the search for ALKBH1 inhibitors.It also provides a new idea for the treatment of lung cancer withALKBH1 as a target. |
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