| Background and objectiveGlioblastoma(GBM) is the most common and malignant intracranial primary tumor, according to the report of stupp in 2005, the prognosis of glioblastoma is still very poor in general and the mean overall survival is 14.6 months. The resistance to standard chemo-radiotherapy in GBM cells is a major cause of poor prognosis. Previous study indicated that O6-methylguanine-DNA-methyltransferase (MGMT), as a DNA repair en2yme, could transfer the increased alkyl in guanine O6 of DNA molecular to position 145 cysteine residues in MGMT itself, to make guanine structure restoration and DNA molecular recovery, to protect GBM cells from damage of temozolomide(TMZ). It is reported that MGMT silence after MGMT promoter methylation is associated with better prognosis for GBM patients because of more sensitivity to TMZ. Even though, it is still some patients with low or none MGMT expression whose tumor cells is strongly TMZ-resistance. So we speculate that there are some other TMZ-resistance mechanism except MGMT DNA repair way. Dynein-cytoplasmic 2-heavy chain 1, which is called DYNC2H1 or DHC2, get involved in chromosome segregation in U87 cell lines mitosis, and reverse transport by microtubules of intracellular vesicles and various membrane-bound organelles. In previous study by our laboratory, we found that cell-cycle of U87cell lines was blocked at G2/M when stimulated with TMZ, a lot of cells were induced into death. But there were a few cells still alive with decreased sensitivity to TMZ and some typically replicable cell morphological changes, these changes such as large cell body, large nuclear body, tenuous and slender pseudopodium and cytoskeleton rearrangement. We believe the strongly TMZ-resistance is due to representative cytoskeleton rearrangement, we adopted some proteomics methods and found some up-regulated proteins related to cellular cytoskeleton, DHC2 was one of those and brought to our attention. The up-regulated DHC2 expression was induced by TMZ stimulation with some typically replicable cell morphological changes described above, even in the early stage of stimulation(1 week). Disruption of cytoskeleton rearrangement through dync2hl-siRNA interfered or Cytochalasin D (CCD) could increased sensitivity of GBM cells to TMZ, CCD is known as Cytoskeletal disrupting drugs. These results suggest that we can use sensitizer which targeted important molecules of the DHC2 pathway to increase sensitivity of GBM cells to TMZ and enhance the curative effect of TMZ. However, how does TMZ work through up-regulation of DHC2 which associated cytoskeleton to decrease GBM cells sensitivity to TMZ is not very clear, the regulation mechnism of DHC2 is still needed to research.The basic program of eukaryotic gene transcriptional regulation including the chromatin structure remodeling, regulation of transcription initiation, regulation of transcription elongation, regulation of transcription termination, regulation of post-transcription processing, regulation of mRNA decorating and regulation of cytoplasmic localization. Every link were undered strict control, these regulations were not worked by one single factor, but worked by various factors that influenced each other, formed a huge and complicated regulatory networks. Among these regulation described above, regulation of transcription initiation which was regulated at gene promoter level is most important stage. It actually is interaction between DNA and protein or interaction between protein and protein. DNA pull down assay based on the Biotin streptavidin technology and magnetic beads separated technology is the most appropriate way to research the regulation of gene expression at transcription level, especially promoter expression regulation level.In this study, we used DNA pull down assay based on the Biotin streptavidin technology and magnetic beads separated technology to search for transcription factors binding to dync2hl promotor sequence, and then, we used mass spectrometry and bioinformatics analysis to lock the main transcription factor, and next, we verified its real regulatory situation about dync2hl gene expression and the influence to TMZ-sensitivity in U87 cell lines, in order to provide a candidate target for GBM treatment in the future.Contents and methods1. Preparationof nuclear extract and 5’-biotin dync2hl promotor sequence probe200uM TMZ(0.125% DMSO) medium was continued applicated into dishes of U87 cell lines. Nucleocytoplasmic separation was adopted after typically replicable cell morphological changes appeared which was induced by TMZ stimulation. The nuclear extract replaced for the appropriate buffer by ultrafiltration. The same processes were adopted in control group which used culture medium(0.125% DMSO). The effect of nucleocytoplasmic separation was verified by western blot experiment. Searching for dync2hl promotor sequence in NCBI, found out upstream of initiation codon ATG(almost 1688bp), designing upstream and downstream primer, amplifying the whole promotor sequence as dync2h1-biotin probe by Polymerase Chain Reaction (PCR).2. Screening for proteins binding with dync2hl promotor probeDNA pull down assay based on the Biotin streptavidin technology and magnetic beads separated technology was adopted to mixed nuclear e xtract and 5’-biotin dync2hl promotor probe, then eluted proteins with different concentration of salt ions. Collected the protein eluent to performed gel silver staining, and found out differential bands, cutting staining bands by a clean blade. Sending differential protein bands to mass spectrometry, using Mascot Distiller database (http://www.matrixscience.com, MatrixSicence Ltd., London, UK)to search Peptide Mass Fingerpriming (PMF) which got from mass spectrometry, confirming protein name. The bioinformatics analysis was adopted Uniprot database to comfirmed related molecular biological function of each protein, to provide theoretical basis for the next step of functional verification. Transcription factors is often not a single action, but rely on a network interacted by a number of transcription factors,, so we also used the STRING database to research protein-protein interaction between each protein.3. Investigation of each protein mRNA level after TMZ stimulation in U87 cells200uM TMZ(0.125% DMSO) medium was continued applicated into dishes of U87 cell lines. Subsequently, using Trizol method to extracted total RNA of U87-TMZ stimulation group after typically replicable cell morphological changes appeared which was induced by TMZ stimulation. The same processes were adopted in control group which used culture medium(0.125% DMSO). Reverse transcription PCR (RT-PCR) and real-time fluorescence quantitative PCR (qPCR) were adopted to detect all dync2hl promotor binding proteins expression levels then. The expression levels of protein binding to dync2hl promotor does not meet the common gene levels of a transcription factor were excluded.4. Investigation of dync2hl mRNA level after interference of target protein in U87 cellsIdentifying Potential target transcription factors according to the imformation we got through bioinformatics analysis, subsequently, small interference RNA (siRNA) were used to specifically and transiently knock down the expression of potential target protein in U87 cell lines. The efficiency of siRNA was comfirmed by RT-PCR and qPCR. The most efficient siRNA was selected for further analysis. Then, the mRNA levels of dync2hl were measured by RT-PCR and qPCR.5. Cell proliferation and TMZ-sensitivity analysis after interference of target protein in U87 cellsSmall interference RNA (siRNA) were used to specifically and transiently knock down the expression of potential target protein in U87 cell lines. The efficiency of siRNA was comfirmed by RT-PCR and qPCR. If the efficiency of siRNA meet the requirements of the next step experiment, CCK8 analysis was performed to determine the function of target peotein on tumor growth and TMZ-sensitivity in vivo.Results1. Preparation of nuclear extract and 5’-biotin dync2hl promoter sequence probe.After 200uM TMZ(0.125% DMSO) medium was continued applicated into dishes of U87 cell lines for 1 week, the typically replicable cell morphological changes were successfully induced, then maintain 200uM TMZ dose till 2 weeks to make sure the cell morphological changes more typical. The nuclear extract replaced for the appropriate buffer by ultrafiltration. The same processes were adopted in control group which used culture medium(0.125% DMSO). The protein quantitative results as follow:TMZ-nuclear extract 2.1 ug/ul; TMZ-cytoplasm extract 4.8 ug/ul; control-nuclear extract 1.7 ug/ul; control-cytoplasm extract 3.2 ug/ul. Moreover, the effect of nucleocytoplasmic separation was verified by western blot experiment, cytoplasmic protein (GAPDH,146KD) pollution in the extract of nuclear protein were in acceptable range, similarly, neclear protein pollution(H3,15KD) in the extract of cytoplasmic protein were also in acceptable range. Additionally, we performed gel silver staining to analysis the effect of nucleocytoplasmic separation, our data revealed that the main bands of between nuclear extract and cytoplasm extract were significantly different, and the main bands of between TMZ-group and control group were also significantly different. The effect of nucleocytoplasmic separation can meet the requirements of the next experiment.Genomic DNA was extracted from U87 cells by phenol chloroform method as template for polymerase chain reaction (PCR),1% agarose gel electrophoresis of PCR products showed a slightly larger than 10 KB specific bands and no tail degradation. The effect of genomic DNA extraction can can be further used for PCR reaction to amplified biotin labeled dync2hl promoter probe,1% agarose gel electrophoresis of promotor probe showed a specific band in the marker position of 1600bp, results of amplification were consistent with our expectations.2. Screening for proteins binding with dync2hl promotor probe.After DNA pull down assay, beads which combined probe-protein mix was eluted by different salt concentration resepctively,12% SDS-PAGE gel electrophoresis and gel silver staining was performed. The data indicated that 13 different protein bands between TMZ-group and DMSO control group in low salt concentration eluent (0.25mol/L), and 6 different bands between TMZ-group and DMSO control group in high salt concentration eluent (1 mol/L), among these bands, 4 protein bands were in coincidence between low salt concentration eluent (0.25mol/L) and high salt concentration eluent (1 mol/L). In general, there were 17 different protein bands at all,2 bands more than 180KD,2 bands in 75KD-135KD,1 band in 63KD-75KD,3 bands in 48KD-63KD,3 bands in 35KD-48KD,1 bands in 25KD-35KD,3 bands in 17KD-25KD,2 bands in 11KD-17KD. cutting staining bands with a clean blade. Sending differential protein bands to mass spectrometry, using Mascot Distiller database to search Peptide Mass Fingerpriming (PMF), protein with low score were exclude. According to the bioinformatics analysis, proteins binding with dync2hl promotor region were identified as follow:Heat shock cognate 71 kDa protein (HSPA8), Pyruvate kinase iso2ymes M1/M2 (PKM2), Heterogeneous nuclear ribonucleoprotein K (HNRNPK), Putative annexin A2-like protein (ANXA2P2), Annexin A2 (ANXA2), Heterogeneous nuclear ribonucleoproteins A2/B1 (HNRNPA2B1), Triosephosphate isomerase (TPI1), Peroxiredoxin-1 (PRDX1), Peptidylprolyl cis-trans isomerase A-like 4B (PPIAL4B), Peptidyl-prolyl cis-trans isomerase A (PPIA), Galectin-1(LGALS1).3. HNRNPK, HNRNPA2B1, PKM2 might be transcription-regulating protein of DHC2 according to bioinformatics analysis.The mRNA level of proteins binding with dync2hl promotor region and dync2hl is verified by qPCR in U87 cells, our data showed that HSPA8, PRDX1 and TPI1 were at very low expression level no matter in TMZ group or DMSO control group(2-△Ct, X±s, ,PRDX1-TMZ:18.31±0.87;PRDX1-DMSO:17.95±0.71;HSPA8-TMZ:19.52±0.18; HSPA8-DMSO:28.39±0.67;TPI1-TMZ:15.17±0.37;TPI1-DMSO:14.90±0.91), obviously not in conformity with the common transcription factor expression level of gene transcription activation within the cell. The mRNA levels of HNRNPK were up-regulated(2-△Ct, X±s, HNRNPK-TMZ:0.1309±0.0174; HNRNPK-DMSO: 0.0626±0.0377, p=0.046), but there was significant difference between two groups; The mRNA levels of HNRNPA2B1 were down-regulated (2-△Ct, X±s, HNRNPA2B1-TMZ:0.0372±0.0007;HNRNPA2B1-DMSO:0.0597±0.0170, p=0.084), but there was no significant difference between two groups; The mRNA levels of PKM2 were down-regulated (2-△Ct, X±s, PKM2-TMZ:0.0013±0.00002; PKM2-DMSO:0.0025±0.00006, p=0.046), there was significant difference between two groups. We paid close attention to HNRNPK, HNRNPA2B1, PKM2 according to related gene transcription biological functions and DNA binding capacity reported in previous literatures via Uniprot database. The STRING database also indicated that there were explicit interaction among HNRNPK, HNRNPA2B1, PKM2.4. Knockdown of HNRNPK attenuate the mRNA levels of dync2hl, and knockdown of PKM2 show opposite result, but knockdown of HNRNPA2B1 didn’t show any changes of dync2hl.The mRNA level of dync2hl is verified by qPCR in U87 cells after knockdown of HNRNPK, HNRNPA2B1 and PKM2, the results showed that the mRNA level of dync2hl is obviously down-regulated after knockdown of HNRNPK (2-△Ct, X±s, HNRNPK-KN:0.002642±0.000367, HNRNPK-NC:0.004644±0.000143, P< 0.001); and the mRNA level of dync2hl is obviously up-regulated after knockdown of PKM2 by PKM2 2# (2-△Ct, X±s, PKM2-KN 2#:0.004657±0.000607, PKM2-NC: 0.002637±0.000414, P=0.009); but both HNRNPA2B1 2# (2-△Ct, X±s,, HNRNPA2B1-KN:0.003234±0.001297, HNRNP A2B1-NC:0.003342±0.001368, P > 0.05) and PKM21# (2-△Ct, X±s, , PKM2-KN 1#:0.003099±0.000119, PKM2-NC: 0.002637±0.000414, P> 0.05) were not showed any siginificant regulatory functions to DHC2. Knockdown of HNRNPK attenuate the mRNA levels of dync2hl, and Knockdown of PKM2 show opposite result, but knockdown of HNRNPA2B1 didn’t show regulatory function to dync2hl.5. Knockdown of HNRNPK attenuate proliferation and TMZ-resistance in U87 cells, and Knockdown of PKM2, HNRNPA2B1 show opposite result.To evaluate the cell proliferation after HNRNPK interference for 7 days via CCK8, the results showed that cell proliferation in HNRNPK knockdown group was slower than NC control group from 24 hours to 6 days (P＜0.001), which means that the knockdown of HNRNPK could reduce the proliferation of U87 cells. Meanwhile, the cell proliferation in knockdown group under TMZ-stimulation was slower than NC group under TMZ-stimulation(P<0.001), which indicates that knockdown of HNRNPK could reduce the resistance to TMZ in U87 cells, and make U87 cells more easier to death under TMZ stimulation.And after PKM2 interference, the cell proliferation in PKM2 knockdown group was slower than NC control group from 24 hours to 6 days (P＜0.001), which means that the knockdown of PKM2 could reduce the proliferation of U87 cells. Meanwhile, the cell proliferation in knockdown group under TMZ-stimulation wasslower than NC group under TMZ-stimulation too(P＜0.001), which indicates that knockdown of PKM2 could reduce the resistance to TMZ in U87 cells, and make U87 cells more easier to death under TMZ stimulation, but it doesn’t meet the trend of DHC2 induced TMZ-resistance that mentioned above. According to the above content, HNRNPK maybe the possible transcription factor which get involved in dync2hl induced TMZ-resistance in U87 cell lines.ConclusionIn this study, we used DNA pull down assay based on the Biotin streptavidin technology and magnetic beads separated technology to search for transcription factors binding to dync2hl promotor sequence, and then, mass spectrometry and bioinformatics analysis was performed to screen the possible transcription factor that might be HNRNPK, HNRNA2B1, PKM2. According to Preliminary analysis of cell function, we believe that HNRNPK may likely participate in dync2hl promotor expression regulation. After knockdown of HNRNPK was found that dync2hl expression significantly decreased in U87 cells, the proliferation of U87 cells also decreased significantly. And the sensitivity of U87 cells to TMZ was enhanced by HNRNPK interference, that was to say, knockdown of HNRNPK make U87 cells more easier to death under TMZ stimulation. After knockdown of PKM2 was found that dync2hl expression significantly increased in U87 cells, but the proliferation of U87 cells decreased significantly. And the sensitivity of U87 cells to TMZ was enhanced by PKM2 interference, that doesn’t meet the trend of dync2hl induced TMZ-resistance that mentioned above. Under the continuous stimulation of TMZ, HNRNPK may be up-regulate its expression, resulting in the ability binding with dync2hl promoter enhancement, and promote the DHC2 up-regulated, so as to induce typical morphological changes, cell cytoskeleton rearrangement and TMZ-resistance in U87 cell lines. Combining with bioinformatics analysis and cell functional results, so we believe that the major transcription factor involved in DHC2 mediated process of TMZ resistant in U87 cell lines maybe HNRNPK. |
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