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Objective To analyze and detect the whole genome sequence of human mitochondrial DNA (mtDNA) by Ion Torrent PGMTM platform and to study the differences of mtDNA sequence in different tissues.Methods Samples were collected from 6 unrelated individuals by forensic postmortem examination,including chest blood,hair,costicartilage,nail,skeletal muscle and oral epithelium.Amplification of whole genome sequence of mtDNA was performed by 4 pairs of primer.Libraries were constructed with Ion ShearTM Plus Reagents kit and Ion Plus Fragment Library kit.Whole genome sequencing of mtDNA was performed using Ion Torrent PGMTM platform.Sanger sequencing was used to determine the heteroplasmy positions and the mutation positions on HV Ⅰ region.Results The whole genome sequence of mtDNA from all samples were amplified successfully.Six unrelated individuals belonged to 6 different haplotypes.Different tissues in one individual had heteroplasmy difference.The heteroplasmy positions and the mutation positions on HV I region were verified by Sanger sequencing.After a consistency check by the Kappa method,it was found that the results of mtDNA sequence had a high consistency in different tissues.Conclusion The testing method used in present study for sequencing the whole genome sequence of human mtDNA can detect the heteroplasmy difference in different tissues,which have good consistency.The results provide guidance for the further applications of mtDNA in forensic science.
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AIM:To investigate the mechanism of microRNA-138-5p (miR-138-5p) inhibiting the proliferation,migration and invasion abilities of lung cancer cells.METHODS:The lung cancer A549 and H460 cells were transfected with miR-NC (control group) or miR-138-5p (experimental group).The bioinformatic analysis was performed to predict the target genes of miR-138-5p.The expression levels of miR-138-5p,forkhead box protein C1 (FOXC1) mRNA and vimentin mRNA were detected by RT-qPCR.The protein expression of FOXC1,vimentin,E-cadhcrin,N-cadherin and β-catenin was determined by Western blot.MTS method and colony formation assay were used to detect cell viability and proliferation ability.Wound healing assay and Transwell assay were used to detect cell migration and invasion ability.RESULTS:Over-expression of miR-138-5p significantly reduced the expression of FOXC1 and vimentin at mRNA and protein levels (P ＜ 0.05).The expression of E-cadherin and β-catenin were up-regulated and the expression of N-cadherin was down-regulated.The proliferation,migration and invasion abilities of the lung cancer cells were inhibited by the over-expression of miR-138-5p.CONCLUSION:miR-138-5p inhibits the proliferation,migration and invasion abilities of lung cancer cells by targeting FOXC1 and vimentin.It may be a potential target for lung cancer gene therapy.
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AIM:To investigate the mechanism of microRNA-138-5p (miR-138-5p) inhibiting the proliferation,migration and invasion abilities of lung cancer cells.METHODS:The lung cancer A549 and H460 cells were transfected with miR-NC (control group) or miR-138-5p (experimental group).The bioinformatic analysis was performed to predict the target genes of miR-138-5p.The expression levels of miR-138-5p,forkhead box protein C1 (FOXC1) mRNA and vimentin mRNA were detected by RT-qPCR.The protein expression of FOXC1,vimentin,E-cadhcrin,N-cadherin and β-catenin was determined by Western blot.MTS method and colony formation assay were used to detect cell viability and proliferation ability.Wound healing assay and Transwell assay were used to detect cell migration and invasion ability.RESULTS:Over-expression of miR-138-5p significantly reduced the expression of FOXC1 and vimentin at mRNA and protein levels (P ＜ 0.05).The expression of E-cadherin and β-catenin were up-regulated and the expression of N-cadherin was down-regulated.The proliferation,migration and invasion abilities of the lung cancer cells were inhibited by the over-expression of miR-138-5p.CONCLUSION:miR-138-5p inhibits the proliferation,migration and invasion abilities of lung cancer cells by targeting FOXC1 and vimentin.It may be a potential target for lung cancer gene therapy.