| Part I: Construction and Identification of Vectors andStably Transfected Cell LinesObjective: To construct and verify three eukaryotic expressional vectors that can beexpressed in human glioma cell lines, which are pcDNA3.1(+)-hIDH1(mu)/3×Flag/IRES/eGFP, pcDNA3.1(+)-hIDH1(wt)/3×Flag/IRES/eGFP, and pcDNA3.1(+)-eGFP.To establish three glioma cell lines U87/mu, U87/wt, and U87/vector, stably transfectedwith pcDNA3.1(+)-hIDH1(mu)/3×Flag/IRES/eGFP, pcDNA3.1(+)-hIDH1(wt)/3×Flag/IRES/eGFP, or pcDNA3.1(+)-eGFP using G418selection, the former two cell linesexpressing mutational (U87/mu) or wide type (U87/wt) isocitrate dehydrogenase1(IDH1).Methods: Three objective DNA fragments hIDH1(mu)/3×Flag/IRES/eGFP,hIDH1(wt)/3×Flag/IRES/eGFP and eGFP were amplified by PCR, introducing a NheⅠ siteto the5’-terminals and a XhoⅠ site to the3’-terminals. The purified objective DNAfragments were digested with two restriction enzymes and forced cloned into the plasmidframe pcDNA3.1(+). The Stbl3E.coli was transformed with the constructed vectors.Plasmids extracted from the positive E.coli clones were screened by PCR and DNAsequencing finally. U87cell lines were transfected with the three verified vectorsrespectively. To establish stably transfected cell lines, G418was used for selection. Invertfluorescent microscope and Western blot were employed for verification of the stablytransfected cell lines.Results: Three vectors pcDNA3.1(+)-hIDH1(mu)/3×Flag/IRES/eGFP, pcDNA3.1(+)-hIDH1(wt)/3×Flag/IRES/eGFP, and pcDNA3.1(+)-eGFP were constructed. PCR andDNA sequencing proved the vectors correct. Three stably transfected cell lines wereestablished by tranfecting U87with the three verified vectors respectively using G418selection. Photos taken by invert fluorescent microscope showed that green fluorescentprotein was expressed in100%of the stably transfected cell lines. Western blot using an anibody against Flag demonstrated that Flag tagged foreign IDH1protein was expressed inU87/mu and U87/wt.Conclusions: Three vectors pcDNA3.1(+)-hIDH1(mu)/3×Flag/IRES/eGFP,pcDNA3.1(+)-hIDH1(wt)/3×Flag/IRES/eGFP, and pcDNA3.1(+)-eGFP were constructedsuccessfully. Stably transfected U87/mu, U87/wt, and U87/vector were established. Theseworks lays a foundation for better comprehension of the role of the R132H mutation inglioma.Part Ⅱ: The Effect of R132H Mutated IDH1on the Growth,Proliferation, Apoptosis and Migrationof Human Glioma Cell LineObjective: To study the effect of R132H mutated IDH1on the growth, proliferation,apoptosis and migration of human glioma cell line.Methods: RNA interference technique was used for down-regulating the expressionof IDH1. The transfection efficacy was screened by invert fluorescent microscope. RNAinterference efficiency was validated by real-time PCR and Western blot. The viability ofcells of U87, U87/vector, U87/mu, U87/wt, U87/siRNA and U87/siRNA control wasdetermined by sulforhodamine B (SRB) colorimetric assay at different time point. Growthcurves were drawn. The cell proliferation was measured by Click-iT EdU (Invitrogen)assay at24h. The apoptosis of the cells and the cell cycles were measured by flowcytometry. The migration was determined by wound healing assay.Results:(1) The transfection efficacy of the siRNA was nearly100%. The mRNA ofthe U87/siRNA group was down-regulated compared with the control group (P<0.05). Theexpression of the IDH1protein of the U87/siRNA group was inhibited significantly(P<0.05).(2) The U87/mu group exhibited higher degree of cell growth compared with thecontrol group (P <0.05at2,3,4and5day). The U87/siRNA group showed lower degreeof cell growth compared with the control group (P <0.05at2,3,4and5day). TheU87/vector, U87/wt and U87/siRNA control groups manifested no significant differencecompared with the control group (P<0.05).(3) EdU assay showed the EdU positive cells ofthe U87/mu group were higher than the control group (P<0.05). The EdU positive cells ofthe U87/vector, U87/wt, U87/siRNA and U87/siRNA control groups exhibited nodifference with the control group (P>0.05).(4) The percentage of the G0/G1phase cells of the U87/mu group was lower, and the S/G2phase higher than the control group (P<0.05).The percentage of the G0/G1phase and S/G2phase cells of the U87/vector, U87/wt,U87/siRNA and U87/siRNA control groups showed no difference with the control group(P>0.05). The G0/G1phase apoptosis cells of the U87/siRNA group was higher than thecontrol group (P<0.05). The U87/vector, U87/wt, U87/mu, U87/siRNA control groupsexhibited no difference of the G0/G1phase apoptosis cells compared with the controlgroup (P>0.05). The S/G2phase apoptosis cells of the U87/mu and the U87/siRNA groupswere higher than the control group (P<0.05). The U87/vector, U87/wt, U87/siRNA controlgroups exhibited no difference of the S/G2phase apoptosis cells compared with the controlgroup (P>0.05). The total apoptosis cells of the U87/mu and the U87/siRNA groups werehigher than the control group (P<0.05), while the U87/vector, U87/wt, U87/siRNA controlgroups exhibited no difference of the total apoptosis cells compared with the control group(P>0.05).(5) The number of cells migrating onto the scratched area of the U87/mu groupwas higher, and that of the U87/siRNA group lower, than that of the control group(P<0.05). The U87/vector, U87/wt, U87/siRNA control groups exhibited no difference ofthe number of cells migrating onto the scratched area compared with the control group(P>0.05).Conclusions: Expressing R132H mutational foreign IDH1in glioma cells can elevatethe proliferation, growth and capacity of migration of the cells. Introduce R132Hmutational foreign IDH1to glioma cells can induce the S/G2phase apoptosis, while has noeffect on the G0/G1phase apoptosis. Down-regulating the expression of IDH1in gliomacells by RNA interference can inhibit the growth and migration of the cells, and induceboth G0/G1phase and S/G2phase apoptosis, while has now effect on the proliferation ofthe cells.Part Ⅱ: Preliminary Exploration of Mechanistic Link betweenthe R132H mutated IDH1and GliomaObjective: To explore the mechanistic link between the R132H mutated IDH1andglioma preliminarily from a view angle of lipid metabolism.Methods: Western blot was employed to detect the expression of the sterol regulatoryelement-binding protein (SREBP)1and SREBP2in the cells of the U87, U87/vector,U87/mu, U87/wt, U87/siRNA and U87/siRNA control groups at72h. The mRNA levels of SREBP1a, SREBP1c, SREBP2, HMGCS, HMGCR and FASN were measured byreal-time PCR at48h. Clinical glioma specimens were collected and screened by DNAsequencing for specific IDH1/2mutations. The expression of SREBP1, SREBP2, MMP2and MMP9proteins in clinical glioma specimens harboring R132H mutational IDH1wasdetermined by immunohistochemistry.Results:(1) The expression of the precursor and mature forms of SREBP1andSREBP2in the cells of U87/mu and U87/siRNA groups was higher than that in the controlgroup cells. No changes of the levels of these proteins in the cells of U87/vector, U87/wtand U87/siRNA control groups compared with those in the control group cells.(2) Theexpression levels of the mRNA of SREBP1a, SREBP2, HMGCS and HMGCR in the cellsof U87/mu and U87/siRNA groups were higher than those of the control group (P <0.05).No significant changes of the mRNA levels of SREBP1c and FASN in the cells of U87/muand U87/siRNA groups were observed (P>0.05). No significant changes of the mRNAlevels of SREBP1a, SREBP1c, SREBP2, HMGCS, HMGCR and FASN in the cells ofU87/vector, U87/wt and U87/siRNA control groups compared with those in the cells ofU87group (P>0.05).(3) Thirty eight clinical glioma specimens were collected, one ofwhich was destroyed and lost in the processing of it. R132H mutation of IDH1wasdetected in20(54.05%) specimens. R172K mutation of IDH2was observed in1(2.7%)specimen. All the mutations were observed in the WHO Ⅱ-Ⅱ grade gliomas. Nomutations were detected in the WHO Ⅰ gliomas or the glioblastomas. The one observedR172K mutation of IDH2was detect in an oligodendroglioma specimen.(4) The SREBP1and SREBP2were expressed in the cell nucleus, while MMP2and MMP9were expressedin the cytoplasm. The expression levels of SREBP1, SREBP2, MMP2and MMP9proteinsin the glioma specimens harboring R132H mutational IDH1were higher than those in thespecimens having the same pathological diagnosis, which did not carrying any IDHmutations (P <0.05).Conclusions: Expressing R132H mutational foreign IDH1or down-regulating theexpression of IDH1in glioma cells by RNA interference can induce the expression ofSREBP1a and SREBP2and their down-stream genes HMGCS and HMGCR. Theexpression levels of SREBP1, SREBP2, MMP2and MMP9proteins in the gliomaspecimens harboring R132H mutational IDH1were higher. SREBPs regulated lipidsynthesis may contribute to the formation and development of gliomas. Our study may give a fresh perspective from the view angle of lipid metabolism to cure gliomas. |
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