Proteomics Study Of Cell Proliferaton

BackgroundAdenosine deaminase acting on RNA (ADAR) is a family of double-stranded RNA (dsRNA) editing enzymes that specifically converts adenosine to inosine in dsRNA. Homologous genes occur in organisms from unicellular protozoa to humans, indicating conservation of dsRNA editing in evolution. In mammals, ADAR1, ADAR2 and ADAR3 have been identified, each of which is endowed with dsRNA binding and dsRNA editing domains. ADAR3 is only expressed in brain. ADAR1 and ADAR2 are ubiquitously expressed in many tissues. The ADAR1 is the protein on which the most attention is paid. The ADAR1 includes two subtypes, the p150 with 150kd and the p110 with 110kd. The p150 is a full-length ADAR1, which can perform nucleocytoplasmic shuttling. The p110 stays in cell’s nucleus. The full-length ADAR1 is ubiquitously expressed in several organs and exhibits several unique features such as two putative Z-DNA binding domains, three dsRNA binding repeats, an adenosine deaminase domain and nuclear localization signal (NLS-c). A nuclear export sequence (NES) is at N terminal of the full-length ADAR1.In this article, the ADAR1 refers to the full-length ADAR1.Notably, ADAR1 is embryonically lethal, and ADAR 1-deficient embryonic stem cells do not contribute to the development of the liver, bone marrow, spleen, thymus and blood in adult chimeric mice. Widespread apoptosis has been detected in many tissues in mice homozygous for an ADAR1 null mutation. Thus, ADAR1 is essential for mammalian development likely due to its dsRNA editing activity. ADAR1 is ubiquitously expressed in eukaryotes and participate in various cellular processes such as differentiation, proliferation and immune responses. The fact that downregulation of ADAR1 inhibits cell growth leads to the hypothesis that overexpression of ADAR1 would promote cell proliferation. We report here a new proteomics study of ADAR1 overexpressing promoting cell proliferation.Objectives1. Proteomics study of ADAR1 affecting the protein expression of cells2. To find the evidence of ADAR1 promoting cell proliferation by label-free quantification of proteomics.Materials and Methods1. Plasmid constructionThe full-length mouse ADAR1 cDNA sequence was cloned into the pEGFP-N1 vector, and the construct was named N1_ADAR1. A mouse mutant ADAR1 cDNA sequence without a deaminase domain was cloned into the pEGFP-N1 vector, and the construct was named N1_mutant.2. Cell culture and transfectionThe human kidney cell line HEK293T was cultured in Dulbecco’s modified Eagle’s medium (10% FBS) and maintained at 37℃ with 5% CO2. DNA transfection was performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s recommendation.3. Protein extraction for in vitro RNA editing and quantitative proteomicsFull-length and mutant ADAR-expressing plasmids were transfected into human HEK293T cells. After 48 h, the cells transfected with either full-length or truncated ADAR1 were harvested. The cells were suspended in 1 ml of chilled RIPA lysis buffer containing a protease inhibitor cocktail and extracted. The protein concentration was determined using a bicinchoninic acid (BCA) assay.4. Preparation of dsRNA substrate and in vitro editing assayFor dsRNA editing assay,1 fmol/ml dsRNA substrate was incubated with 0.5 ml of cell lysates expressing the full-length or mutant ADAR1 in editing buffer at 37℃ for 2 h. The dsRNA was recovered by phenol/chloroform extraction and ethanol precipitation. The recovered dsRNA was reverse-transcribed and the cDNA was amplified by PCR. The PCR products were sequenced.5. In-solution trypsin digestionFor quantitative proteomics analysis, cell lysates (200μg) were subjected to trypsin digestion. Cell lysate was reduced with 20 mM dithiothreitol (DTT; 56℃,30 min), alkylated with 50 mM iodoacetamide (26℃,20 min in the dark) and digested with sequencing-grade modified trypsin (1:50 w/w) at 37℃ for 24 h. The solution was filtered by centrifugation at 15,000xg for 30 min in a 10k Ultra filter. The filtered peptides were then desalted using ZipTip C18 columns. The desalted peptides were collected, dried in a SpeedVac and stored at-20℃ until further analysis.6. LC-MS/MS analysisThe peptides were separated by high pressure liquid chromatography (HPLC, Thermo EASY-nLC System) and analyzed using a hybrid linear ion trap-Orbitrap mass spectrometer (Thermo LTQ Orbitrap Velos Elite, Thermo Scientific). For each analysis, the sample was trapped on a C18 precolumn and then on a C18 reversed-phase analytical column. The peptides were eluted from the column with a linear solvent gradient to wash the column. Peptides were analyzed in positive ion mode. MS spectra were acquired in profile mode using the Orbitrap analyzer in the m/z range between 300 and 1800 at 60,000 resolutions. MS/MS analysis was performed in the collision-induced dissociation (CID) mode. Every sample was run in triplicate with the same methods.7. Data analysis and peptide identificationCollision-induced dissociation (CID) data for each sample was searched using Proteome Discoverer 1.4 (Thermo Fisher Scientific) against the Swiss-Prot human database.8. Label-free quantificationLabel-free quantification of all six runs was analyzed using Progenesis LC-MS software (version 4.1; Nonlinear Dynamics, UK). Among the statistically significant proteins detected by the ANOVA test (p<0.05), protein abundances that changed less than 1.5-fold (ADAR1/mutant) were discarded.9. Bioinformatics analysis of the differentially abundant proteinsFor the ontological analysis, the lists of regulated proteins from the progenesis LC-MS analysis were submitted to the PANTHER (http://www.pantherdb.org/) database with their UniProt accession number to analyze protein functions that were regulated upon ADAR1 overexpression. For the protein network analysis, the lists of identified proteins were submitted to the STRING (http://www.string-db.org/) database with their UniProt accession numbers from HUMAN.10. Immunoblot analysisWestern blot analyses were performed on cell extracts from ADAR1-and mutant-transfected cells. The proteins were lysed and extracted in ice-cold RIPA lysis buffer. The protein extracts (50μg) were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and were electrotransfered to nitrocellulose (NC) membranes. After blocking with 5% BSA in PBS, the membranes were probed with the first antibodies and the second antibody. Quantification of the western blot results was performed using ImageJ software.11. Cell proliferation assay11.1 EDU cell proliferation assayHEK293T cells and A549 cells (2500 cells/well) were seeded in triplicate in 24-well plates and were incubated in DMEM with 10% bovine serum for 12 h at 37℃. Four groups of cells (two groups of HEK293T cells and two groups of A549 cells; three wells per group) were transfected with either ADAR1 or mutant plasmids for 36 h. The EDU cell proliferation assay is performed by using an EdU DNA Cell Proliferation Kit. EDU labels the proliferated cells (Red) and the Hoechst 33342 labels all cells (Blue). Observation is performed by using an inverted fluorescence microscope with 200× magnification. Image-Pro Plus 6.0 software (IPP 6.0) was used to calculate the percentage of EdU-positive cells in all cells.11.2 MTT cell proliferation assayCell proliferation was tested using a 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay. For the assay, HEK293T cells and A549 cells were seeded into 96-well plates at a density of 3x103 cells per well in 100ul of DMEM (10% FBS) in five replicates (20 wells for ADAR1 group and Mutant group) and incubated for 24h, respectively. DNA transfection was performed using Lipofectamine 2000 according to the manufacturer’s recommendation. After 48h of incubation,10μl of MTT solution was added per well. After 4h of incubation, the media were carefully discarded and the formazan crystals were dissolved with 100μl of DMSO. Optical density (O.D.) was measured using varioskan flash at 570 nm and 630 nm. The absorbance at 570 nm was measured with the absorbance at 630 nm subtracted to account for the plastic well and cellular debris. The value from the subtracted absorbance was analyzed.12. Statistical analysisANOVA t test is used for statistical analysis of proteomic quantification. p<0.05 is regarded as significant. Other quantificational analysis is performed by Student’s t test. p<0.05 is regarded as significant. The data is showed by mean±standard error.Results1. ADAR1 overexpression in HEK293T cells increased the editing activity on dsRNA.2. Proteins regulated by ADAR1 overexpression in HEK293 cellsAs a result, the levels of 370 proteins in ADAR1 overexpresssion group were up-or down-regulated for at least 1.5-fold in comparison with the mutant group. Among the ADAR1-regulated proteins,211 were up-regulated and 159 were down-regulated. Thus, ADAR1 over-expression resulted in a significant proteomic change in HEK293T cells.3. Gene ontology analysis of ADAR1-regulated proteins showed the evidence of overexpressing ADAR1 promoting cell proliferation at the molecular level.4. ADAR1 overexpression mediated protein translation and cell cycle networksThe STRING analysis showed ADAR1 overexpression mediated protein translation and cell cycle networks. The protein translation network included 4 ribosomal protein small units (RPS5, RPS3, RPS13, and RPS10),7 ribosomal protein large units (RPL7, RPL10, RPL22, RPL28, RPL35, RPL36A, and MRPL49),6 eukaryotic translation initiation factors (EIF3A, EIF3G, EIF3I, EIF3L, EIF4G2 and EIF5B), and 2 eukaryotic translation elongation factors (EEF1A1 and EEF1A2). The cell cycle network was centered at proliferating cell nuclear antigen (PCNA).5. ADAR1 overexpression mediated PCNA interaction networkSequence analysis revealed that all proteins in the PCNA interaction network contained the PCNA interaction domains, either the KA boxes and/or PIP-boxes. The upregulated proteins in the network can promote cell proliferation. The upregulated proteins, which interacted with PCNA by either the KA boxes and/or PIP-boxes, included pre-mRNA processing factor 19(PRPF19) and X-ray repair cross-complementing protein 5(XRCC5).6. Western blot analysis confirmed that over-expressing ADAR1 up-regulated some proteins related to cell proliferationConsistent with label-free quantification, western blot analysis showed PCNA expression increased 2.0-fold (p<0.01, n=3) when ADAR1 was overexpressed by using ImageJ software. Similarly, western blot analysis demonstrated that PRPF19 increased 1.76-fold (p<0.01, n=3) and that XRCC5 increased 1.6-fold (p<0.01, n=3). Notably, Exportin-5 was shown to mediate nuclear exportation of cellular components necessary for protein translation, including Elongation Factor 1-alpha 1(eEF1A), tRNA and signal recognition particle RNA (SRP RNA). Western blot analysis showed Exportin-5 expression increased 1.56-fold (p<0.01, n=3) when ADAR1 was overexpressed.7. Cell proliferation assay showed that overexpressing ADAR1 increased the proliferation rate of HEK293T cells and A549 cellsTo support the results from quantitative proteomics, 5-Ethynyl-2’deoxyuridine (EDU) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) were used to measure the proliferation of HEK293T cells and A549 cells with or without ADAR1 overexpression. The cell proliferation ratio of HEK293T cells between EDU and Hoechst fluorescent signals was 0.58±0.08 with and 0.26±0.04 without ADAR1 overexpression; the cell proliferation ratio of A549 cells between EDU and Hoechst fluorescent signals was 0.24±0.04 with and 0.15±0.01 without ADAR1 overexpression. In consistent with the conclusion from quantitative proteomics, ADAR1 overexpression upregulated cell proliferation of HEK293T cells for 2.2-fold (p<0.01, n=3) and cell proliferation of A549 cells for 1.6-fold (p<0.01, n=3). The results of MTT cell proliferation assay showed that the proliferation index (PI) of HEK293T cells was 1.59±0.20 with and 0.79±0.10 without ADAR1 overexpression; the PI of A549 cells was 0.96±0.14 with and 0.59±0.14 without ADAR1 overexpression. With MTT assay, ADAR1 overexpression upregulated PI of HEK293T cells for 2.0-fold (p<0.001, n=5) and PI of A549 cells for 1.6-fold (p<0.001,n=5).Conclusion1. With LC-MS/MS and label free quantificational proteomics analysis, we believed that overexpressing ADAR1 upregulated many genes of gene transcription, protein translation, protein transport and cell cycle. Overexpressing ADAR1 contributed to cell proliferation by upregulating these genes.2. Overexpressing ADAR1 upregulated protein translation interacting network and cell cycle interacting network. Overexpressing ADAR1 also upregulated PCNA-proteins interacting network centered by PCNA. Overexpressing ADAR1 promoted cell proliferation by upregulating the three interacting network in the proteomic molecular level.3. Cell proliferation assay with EDU and MTT all showed that overexpressing ADAR1 increased the cell proliferation ratio of HEK293T cells and A549 cells in cell level. The conclusion of overexpressing ADAR1 promoting cell proliferation was supported both in protein molecular level and in cell level.BackgroundSome pseudogenes are thought to be active through the formation of long non-coding RNAs (IncRNAs) upon their transcription. However, little attention has been paid to the HIST1H2APS4 pseudogene, commonly known as H2A/K, and mutated versions of this pseudogene have not been studied.Results from our laboratory have shown that 7 of 10 kidney cancer patients carried a mutant H2A/K pseudogene:four kidney cancer patients expressed a mutant H2A/K IncRNA carrying the C228A mutation; two kidney cancer patients expressed a mutant H2A/K IncRNA carrying the C290T mutation; and one kidney cancer patient expressed a mutant H2A/K IncRNA carrying the A45G mutation. However, no mutations were found in the H2A/K IncRNAs in the 12 healthy persons in the control group. This result was significant (Fisher’s exact test, p= 0.0007036), and we were interested in the relationship between mutant H2A/K IncRNAs and cell proliferation. Therefore, we used shotgun and label-free proteomic methods to study whether the mutant H2A/K lncRNAs affected cell proliferation.Objectives1. To demonstrate the activity of the mutant H2A/K pseudogene.2. To prove the function of the mutant H2A/K pseudogene promoting cell proliferation.Materials and Methods1. PCR analysis of H2A/K IncRNA-cDNA from kidney cancer tissuesKidney cancer tissues (30 mg) were ground in liquid nitrogen and added to 1ml Trizol and 0.2ml chloroform and vortexed in a 1.5ml RNAse-free EP tube. Each lysate was centrifuged at 12,000 g for 15 min at 4℃, and 0.1ml of the supernatant was collected in a 1.5ml tube treated by 0.1% DEPC water, followed by precipitation with 0.1ml isopropanol. The purified RNA templates were reverse-transcribed using a reverse transcriptase kit and the primer PF (5’-GGGATCCATGCAGGGCAGCA-3’). Substrate-specific amplification was performed using the primers PF 5’-GGGATCCATGCAGGGCAGCA-3’ and PR 5’-AGAATTCTCACTTGTATAGA-3’, and the PCR amplicons were subcloned into pCDNA3.0 and sequenced.2. Cell culture and transfectionHEK293T cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 4.5 g/l glucose and 10% fetal bovine serum at 37℃,5% CO2 and 95% humidity.Four plasmids (expressing the normal H2A/K IncRNA and the mutant C290T, C228A and A45G H2A/K IncRNAs) were constructed by ligating cDNA sequences from kidney cancer patients and healthy controls. DNA transfections were carried out using Lipofectamine 2000. Briefly, DNA was mixed with Lipofectamine 2000 at a ratio of 1μg/1μl and the mixture was incubated in the serum-free medium for 15 min at room temperature. HEK293T cells plated at 60-70% confluency were exposed to the transfection complex at a concentration 8μg DNA per ml of culture medium for 48 h.3. Protein extractionFour plasmids (expressing the normal H2A/K IncRNA and the mutant C290T, C228A and A45G H2A/K IncRNAs) were transfected into human HEK293T cells in 16x100-mm dishes (4 x100-mm dishes/group) using Lipofectamine 2000 reagent according to the manufacturer’s instructions. After 48 h,1 ml of chilled RIPA lysis buffer containing protease inhibitors (Cocktail) was added into the transfected cells in a 100-mm dish. The cells in the 100-mm dish were blown with a pipette and transferred into a 1.5ml cool EP tube. After dissociating in ice for 20 min, each lysate was centrifuged at 13,500 g for 30 min at 4℃, and the supernatant was collected in a new 1.5ml cool EP tube. Protein concentrations were determined using the BCA assay4. In-solution trypsin digestion and peptide pre-fractionationProtein extracts (200μg) were subjected to trypsin digestion. Briefly, the proteins were treated with 20 mM DTT to remove disulfide bonds (56℃,30 min), alkylated with 50 mM iodoacetamide (26℃,20 min in the dark) and digested with sequencing-grade modified trypsin (1:30 w/w) at 37℃ for 20 h. The solution was then filtered by centrifugation at 15,000 g for 30 min using a 10k Amicon Ultra-0.5 filter. The filtered peptides were then desalted using ZipTip C18 columns.5. LC-MS/MS analysisThe dry peptides were dissolved by 20ul 0.1% formic acid before analysis. The peptides were separated on a Thermo Fisher LC System and analyzed online using an electrospray ion trap. For each analysis, the sample was trapped on a C18 pre-column and then on a C18 reverse-phase analytical column. The peptides were eluted from the column with a linear solvent gradient (A:0.1% FA in water; B:100% acetonitrile/0.1% FA) for 120 min at a flow rate of 200 nl/min. MS spectra were acquired in profile mode using the Orbitrap analyzer in the m/z range of 300-1800 at a resolution of 60,000. MS/MS analysis was performed in the collision-induced dissociation (CID) mode. Four groups of peptides samples (Q1, M_C290T, M_C228A and M_A45G) were analyzed by the LC-MS/MS for four times, respectively.6. Data analysis and peptide identification for histone h2a without H2A/KCollision-induced dissociation (CID) data for each sample was searched using Proteome Discoverer 1.4 (Thermo Fisher Scientific) against the Swiss-Prot human database (01/03/2013).7. MaxQuant quantification analysis16 raw files (MS data) were processed using the famous quantification software MaxQuant (http://www.maxquant.org/) to quantify the proteins expressions from the 4 groups of cells (control Q1, M_C290T, M_C228A and M_A45G). MaxQuant was used with the UniProt Human (01/03/2013) protein database. MS/MS spectra were searched using the Andromeda search engine. Those proteins (control Q1, M_C290T, M_C228A and M_A45G) that were quantified in four biological replicate experiments were subjected to a Welch’s t test. Proteins whose abundances exhibited a less than 2-fold difference (Mutant/Q1) were discarded.8. Bioinformatics analysisFor the ontological analysis, the lists of regulated proteins (control Q1, M_C290T, M_C228A and M_A45G) from the MaxQuant analysis were submitted to the PANTHER (http://www.pantherdb.org/) database to analyze which protein functions were regulated upon mutant H2A/K IncRNAs. For the PCNA-protein network analysis, the lists of regulated proteins (control Q1, M_C290T, M_C228A and M_A45G) were submitted to the STRING (http://www.string-db.org/) software to analyze which proteins in the network were regulated upon mutant H2A/K IncRNAs.9. Immunoblot analysisWestern blot analyses were performed on cell extracts from the control Q1 and mutant cell groups (M_C290T, M_C228A and M_A45G). The proteins were lysed and extracted in ice-cold RIPA lysis buffer. The protein extracts (50 μg) were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and were electrotransfered to PVDF membranes. After blocking with 5% BSA in PBS, the membranes were probed with the first antibodies and the second antibody. The figures were obtained by using Odyssey image analyzer. Quantification of the western blot results was performed using ImageJ software.10. Cell proliferation assaysHEK293T cells (2500 cells/well) were seeded in triplicate in 24-well plates and incubated overnight. Four groups of cells (three wells per group) were transfected with plasmids expressing control H2A/K IncRNA or H2A/K IncRNAs with the C290T, C228A, or A45G mutation for 36 h. The EDU cell proliferation is performed by using an EdU DNA Cell Proliferation Kit. EDU labels the proliferated cells (Red) and the Hoechst 33342 labels all cells (Blue). Observation is performed by using an inverted fluorescence microscope with 200x magnification. Image-Pro Plus 6.0 software (IPP 6.0) was used to calculate the percentage of EdU-positive cells in all cells.11. Statistical analysisFisher’s exact test is used for statistical analysis of the mutant H2A/K IncRNAs among10 kidney cancer patients and 12 healthy persons. p<0.05 is regarded as significant. Welch’s t test is used for statistical analysis of proteomic quantification. p<0.05 is regarded as significant. Other quantificational analysis is performed by Student’s t test. p<0.05 is regarded as significant. The data is showed by mean±standard error.Results1. The H2A/K pseudogene can only be transcribed into an IncRNAThe H2A/K pseudogene is not a normal histone H2A gene due to a one-base frameshift toward the 5’end that generates premature stop codons. However, the H2A/K pseudogene is transcribed into IncRNA and can be found by PCR analysis in some samples.2. Expressing mutant H2A/K IncRNAs in cellsWe individually transfected 4 plasmids (expressing different IncRNAs with Q1, C290T, C228A and A45G) into HEK293T cells.After 48 h, the transfected HEK293T cells were collected and submitted to RNA extraction and cDNA reverse transcription. Sequencing was then performed using the primer sequence "CATGGGAGACTACAAGGAC", which showed that the control and mutant H2A/K IncRNAs were expressed. No H2A/K proteins were found in the transfected cells by mass spectrometry analysis.3. Shotgun and MaxQuant proteomic methods to analyze mutant IncRNAsPeptide mixtures of quadruplicate cell samples overexpressing H2A/K control or mutant IncRNAs were prepared and processed by LC-MS/MS. The raw data from the shotgun proteomics method were analyzed to produce quantitative proteomic profiling using MaxQuant. The analyses of HEK293T cells expressing control or mutant H2A/K IncRNAs quantified 403 (Q1),320 (M_C290T),259 (M_C228A) and 309 (M_A45G) proteins in the control and mutant groups. The numbers of differentially expressed proteins (Welch’s t test, p value< 0.05) in the control and mutant groups were 296 (Q1),193 (M_C290T),191 (M_C228A) and 177 (M_A45G). The numbers of downregulated proteins (>2-fold) in the mutant groups were 28 (M_C290T),29 (M_C228A) and 37 (M_A45G), and the numbers of upregulated proteins (>2-fold) in the mutant groups were 147 (M_C290T),142 (M_C228A) and 108 (M_A45G).4. Gene Ontology/category analysis of the quantified proteins shows that mutant H2A/K IncRNAs promote cell proliferationIn the Gene Ontology/category analysis, the regulated chromatin binding proteins in mutant groups were consistent with the finding that IncRNAs can induce chromatin remodeling.The only co-downregulated chromatin binding protein was cohesin subunit SA-2 (STAG2), a clinically significant tumor suppressor, in three mutant groups. This agreed with previous findings that SNPs of some IncRNAs have been linked to carcinogenesis. The vast majority of transcription factors, translation activators and cell proliferation interacting (STRING) proteins, whose expression were altered in mutant groups, were upregulated. Furthermore, many proteins involved in biosynthetic processes, transporter activity, cellular processes and primary metabolic processes were upregulated in three mutant groups according to the Gene Ontology/category analysis. In view of this analysis, it is suggested that the mutant H2A/K IncRNAs can promote cell proliferation.5. Analysis of the PCNA-protein interaction network in three mutant groups show that mutant H2A/K IncRNAs promote cell proliferationPathway analysis was performed to characterize the functional interaction network linking the differentially expressed proteins in the Q1 control and mutation groups (M_C290T, M_C228A and M_A45G) using the Search Tool for the Retrieval of Interacting Genes (STRING) 9.0 database. The pathway analysis revealed a PCNA-protein network with PCNA in the center.The resulting network showed significant connections among many upregulated proteins that interacted with PCNA. The intriguing point is that most of the proteins in the PCNA interaction networks of mutant groups contribute to carcinogenesis.Four carcinogenesis proteins, i.e., PCNA, DNA-binding protein A(CSDA), Heat shock cognate 71 kDa protein (HSPA8) and probable ATP-dependent RNA helicase DDX17 (DDX17), were co-upregulated in three PCNA interaction networks. Nine carcinogenesis-related proteins, i.e., Catenin beta-1 (CTNNB1), Small ubiquitin-related modifier 2 (SUM02), Lamin-B1 (LMNB1), Alpha-enolase (ENO1),78 kDa glucose-regulated protein (HSPA5), Heat shock 70 kDa protein 1A (HSPA1A),60 kDa heat shock protein in mitochondrial (HSPD1), the apoptosis regulator BAX (BAX) and ATP-dependent RNA helicase A (DHX9), were co-upregulated in two PCNA interaction networks. Four carcinogenesis-related proteins, namely, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Importin subunit alpha-2 (KPNA2), Nucleolin (NCL) and the RNA-binding protein FUS (FUS), were regulated in one PCNA interaction network.According to the analysis of the PCNA-protein interaction network in STRING, most of the up/downregulated proteins in the networks centered by PCNA were related to carcinogenesis. The above information showed that the PCNA-protein interaction network generated from the mutant H2A/K IncRNAs in three mutant groups may promote cell proliferation.6. Confirmation of the upregulation of PCNA in the mutant groups (M_C290T, M_C228A and M_A45G) by western blottingWestern blot analyses were performed on cell extracts from the control Q1 and each mutant group (M_C290T, M_C228A and M_A45G).The results showed that PCNA was upregulated in cells expressing a mutant H2A/K IncRNA (C290T, C228A and A45G).7. Cell proliferation assays reveal that mutant H2A/K IncRNAs (C290T, C228A and A45G) promote cell proliferationThe proliferating cells were labeled with EdU of the Cell Proliferation Kit (Guangzhou Ribobio Co., Ltd., China), and Hoechst 33342 was used to label the DNA of all cells. The stained cells were imaged with a fluorescence microscope, and the percentage of EdU-positive cells was calculated using Image-Pro Plus 6.0 software. The percentage of EdU-positive cells represented the cell proliferation ratio.The cell proliferation ratios of the Q1 control, M_C290T, M_C228A and M_A45G groups were 0.134±0.003,0.453±0.086,0.398±0.092, and 0.388±0.070, respectively. The results are shown as the means±standard deviations, and the significance of the differences was determined using Student’s t-test; p-value<0.001 (n=3). The cell proliferation assays also proved that the mutant H2A/K IncRNAs (C290T, C228A and A45G) promoted cell proliferation.Discussion1. Mutant H2A/K IncRNAs increase the general transcriptional activity of the cell to promote cell processes and cell proliferation by regulating chromatin-binding proteins and DEAD-box RNA helicases.2. The H2A/K pseudogene cannot be expressed as protein, but it can be transcribed into an IncRNA.3. We used shotgun and label-free proteomic methods to study the mutant IncRNAs of the H2A/K pseudogene. Quantitative proteomic analysis indicated that the mutant IncRNAs upregulated many oncogenes and downregulated some inhibitors of cancers compared to the normal H2A/K IncRNA. Gene Ontology/category analysis using bioinformatics tools showed that the mutant IncRNAs upregulated multiple transcription activators, translation activators, biosynthetic process proteins and transporter activity proteins to enhance cellular processes and proliferation. Further analysis showed that most of the carcinogenesis-related proteins in the PCNA-protein interaction networks centered on PCNA that are up-/downregulated in cells expressing mutant H2A/K IncRNAs contribute to cell proliferation.4. The role of mutant H2A/K IncRNAs (C290T, C228A and A45G) in promoting cell proliferation was confirmed by cell proliferation assays and was supported at the protein expression level.Conclusion1. The H2A/K pseudogene is active2. The role of mutant H2A/K IncRNAs (C290T, C228A and A45G) in promoting cell proliferation was supported at the protein expression level and was confirmed by cell proliferation assays.