Hydroxyapatite Nanoparticles Mediated Human Telomerase Reverse Transcriptase RNA Interference Of Lung Cancer In Vitro

Experimentâ… Transformation, extraction and identification of siRNA expressionplasmid targeting human telomerase reverse transcriptaseObjective: To prepare the siRNA expression plasmid targeting human telomerase reversetranscriptase for gene transfection.Methods: An interference target sequence GCATTCCTGCTCAAGCTGA was designed,which aimed at hTERT gene, and then two strands of oligonucleotides were synthesizedwhich including the sequence above.The sequence of the oligonucleotides contained theparts as follow: the BamHâ… site, positive-sense sequence, loop sequence, antisensesequence, the terminator of RNA polymeraseâ…¢, the Sa1â… site and the Hindâ…¢site.Thetwo strands of oligonucleotides formed a double-strand DNA after annealing.ThepGenesil-1 plasmid was digested with BamHâ… and Hindâ…¢, and then purified and retrievedthe 4800bp linear plasmid by agarose electrophoresis separation.The double-strand DNAwas inserted downstream from the U6 promoter of the linear plasmid.The siRNAexpression vector pGenesil-hTERT was constructed.The susceptible E.coli DH5a whichwas prepared by CaCl₂ method.Then, the recombinant plasmid was transfected into thesusceptible E.coli DH5a, and the positive colonies were chosen.The extracted recombinantwas digested by Sa1â… and Pstâ… respectively, and analyzing the result by agarose gelelectrophoresis and DNA sequencing.With alkaline lysis method, plasmids were extractedon a large scale and measured for purity and quantity with spectrophotometer.Results: A linear plasmid vector strip about 4800bp could been seen through agarose gelelectrophoresis analysis after the pGenesil-1 plasmid had been digested with BamHâ… andHindâ…¢.The two plasmids were digested by Sa1â… and Pstâ… respectively.Electrophoresis showed that the recombinant plasmid could be digested by Sa1â… and yield a nearly 400bpfragment but could not be digested by Pstâ… .The negative Control recombinant plasmidcould not be digested by Sa1â… but could be digested by Pstâ… and yield a nearly 400bpfragment.The sequencing identification proved that the designed sequence had insertedinto the predicted site precisely.The OD260/280 ratios of all the extracted plasmids rangedfrom 1.8 to 2.0 and the concentration from 1.30μg/μl to 1.77μg/μl.Conclusions: The synthesized shRNA interference sequence cloned to pGenesil-1expression plasmid precisely, and the siRNA expression plasmid that aimed at hTERT wasconstructed successfully.The pGenesil-hTERT can provide new method to the research ofeffect of telomerase in tumor cell.Purity and quantity of pGenesil-hTERT extracted withalkaline lysis method meet the requirement of gene transfection.Experimentâ…¡Surface modification and binding with DNA of hydroxyapatitenanoparticlesObjective: To synthesize hydroxyapatite nanoparticles (nHAP) modified withpoly-L-lysine (PLL), and investigate the feasibility of surface modification of nHAP andthe capability of nHAP in combining and protecting DNA.Methods: The nHAP were synthesized by the ultrasound-assisted homogeneousprecipitation method.The structure of the nanoparticles was observed under transmissionelectron microscope.The resulting nHAP were modified with PLL in various PH solution.Zeta potential of nHAP and nHAP modified with poly-L-lysine (nHAP-PLL) was detectedrespectively.The nHAP-PLL were tested for the capacity of combining DNA by measuringDNA concentration in supernatant after centrifugation.The capacity of protecting DNA of nHAP-PLL was tested by the experiment of DNA degradation.Results: The synthesized hydroxyapatite particles presented well dispersed particles withevenly distributed sizes of (15～20)nm×(60～80)nm under transmission electronmicroscope.Pretreated by Na₂CO₃, nHAP were effectively modified with PLL in PH7.4and PH7.6 solution.The Zeta potential of nHAP was (-42.4±7.5)mV.The Zeta potentialof nHAP-PLL was (8.5±1.5)mV.The Zeta potential of nHAP-PLL was significantlyhigher than that of nHAP (P<0.01).DNA binding test showed that nHAP-PLL couldcompletely combine DNA when the weight/weight ratio of nHAP-PLL and DNA was morethan 20:1.The nHAP-PLL/DNA mixtures with weight/weight ratio upwards of 20:1 couldeffectively protect DNA against being digested by DNaseâ… .Conclusions: Through ultrasound-assisted homogeneous precipitation method, nHAP canbe synthesized with well dispersed particles and evenly distributed sizes.The Na₂CO₃pretreated nHAP can be well surface-modified with Poly-L-lysine and effectively combineand protect DNA.Experimentâ…¢Hydroxyapatite nanoparticles mediated gene transfection to A549human lung cancer cells in vitroObjective: To investigate the capability of nHAP-PLL as a carrier for gene transfection toA549 human lung cancer cells in vitro.Methods: The nanoplexes of nHAP-PLL-DNA were made according to methods ofexperiment tow.The transfection of pGenesil-hTERT into A549 was divided into threegroups as follows: nHAP-PLL group mediated by hydroxyapatite nanoparticles modifiedwith poly-L-lysine (nHAP-PLL), liposome group mediated by Lipofectamine and control group.After 48 hours transfection, the expression of EGFP was observed with fluorescentmicroscope.Flow cytometry was used to detect the transfection rate of each group.Results: under fluorescent microscope, the expression of EGFP was detected in A549 cellsof nHAP-PLL group and liposome group while no expression of EGFP was seen in A549cells of control group at 48 hours after transfection.Flow cytometry analyses showed that,the transfection rate of A549 cells was (31.8±1.9)% of nHAP-PLL group and (33.4±3.7)% of liposome group.Compared with the control group, there was significantdifference respectively (P<0.01), but there was no significant difference of transfection ratebetween the nHAP-PLL group and liposome group (P>0.01).Conclusion: The nHAP modified with poly-L-lysine can mediate the transfection ofplasmid into A549 human lung cancer cells in vitro and may be used as a potential genecarrier.Experimentâ…£Hydroxyapatite nanoparticles mediated human telomerase reversetranscriptase RNA interference of A549 human lung cancer cells in vitroObjective: To investigate the effect of nHAP mediated human telomerase reversetranscriptase RNA interference of A549 human lung cancer cells in vitroMethods: The nanoplexes of nHAP-PLL-DNA were made according to methods ofexperiment tow.The transfection of pGenesil-hTERT into A549 was divided into fourgroups as follows: nHAP-PLL group mediated by hydroxyapatite nanoparticles modifiedwith poly-L-lysine (nHAP-PLL), liposome group mediated by Lipofectamine, nHAP groupmediated by hydroxyapatite nanoparticles and control group.The growth ability of cellswas assayed with methyl thiazolyl tetrazolium method.The level of hTERT mRNA was examined by RT-PCR.The expression level of hTERT protein was analyzed by Westernblotting.Telomerase activity was detected by TRAP-ELISA.Flow cytometry was used todetect the apoptosis ratio of A549.Results: The proliferation of A549 cells of nHAP-PLL group, liposome group and nHAPgroup were obviously inhibited as compared with the control group (P<0.05).Theinhibition rate of nHAP-PLL group was more than the other groups.There was a significantdifference of inhibition rate between the nHAP-PLL group compared with the liposomegroup and nHAP group (P<0.05).The level of hTERT mRNA, hTERT protein andtelomerase activity had similar varietal tendencies with the results of proliferation of eachgroup.Flow cytometry showed the apoptosis ratio of nHAP-PLL group, liposome group,nHAP group and control group was (28.1±1.4)%, (19.2±1.3)%, (10.9±1.2)% and(0.3±0.2)% respectively.There was a significant difference of apoptosis ratio between thenHAP-PLL group, liposome group and nHAP group compared with the control group(P<0.05).In contrast to the nHAP-PLL group, the liposome group and nHAP groupdemonstrated a significantly lower apoptosis rate (P<0.05).Conclusion: Hydroxyapatite nanoparticles can induce apoptosis of A549 human lungcancer cells in vitro.A549 cells overexpress human telomerase reverse transcriptase,hTERT may be a target for inhibiting proliferation of A549.Hydroxyapatite nanoparticleshave some characters as follow: inhibiting proliferation of A549, combining and protectingDNA and mediating hTERT RNA interference of A549.Therefore, hydroxyapatitenanoparticles may be a useful material in the field of lung cancer gene therapy.