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Construction of PTEN Eukaryotic Expression Vector and Its Effect on Human Tongue Cancer Cell Line SCC-4

Author: DongXiaoZuo
Tutor: ZhangBin
School: Liaoning Medical
Course: Clinical Stomatology
Keywords: Tongue cancer PTEN gene Plasmid construction Gene transfection Cell proliferation
CLC: R739.86
Type: Master's thesis
Year: 2013
Downloads: 4
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Abstract


ObjectivesTo construct and identify wild-type PTEN gene eukaryotic expression vector,and transfect into human tongue cancer cell line scc-4, observed its inhibition ontongue cancer cell in vitro and discussed its related mechanism, which may laya foundation for research the relationship between tongue cancer and itsbiological behavior, at the same time provide experimental basis for genetherapy of tongue cancer.Methods1. The total RNA that extracted from hela cells was reversed transcription tocDNA,The products of PCR were directly cloned into the pMD18-T Vector, therecombinant plasmid was digested by endonuclease and the positive plasmidscontaining the PTEN gene was acquired by screening. Both pEGFP-N1emptyVector and pT-PTEN Vector were digested by double-endonuclease, thefragments obtained were connected by Taq DNA Polymerase, the recombinantplasmids were identified by double-endonuclease and sequencing.2. Recombinant plasmid mediated by lipofection was transfected intohuman tongue cancer SCC-4cell lines, Fluorescence microscope was used toobserve the expression of green fluorescent protein after transfected24、48and72h， respectively, The experiment was divided into three groups:pEGFP-PTNE-SCC-4group, pEGFP-N1-SCC-4group and SCC-4group. 3. Positive clones was obtained by G418screening. RT-PCR and Westernblotting were used to analysis the expression of PTEN gene mRNA and protein,4. MTT method was used to determine cell proliferation rate, and cellgrowth curve was drawed to observe the inhibition effects of PTEN gene in vitroon tumor.Results1. The results of double-endonuclease and sequencing: A1200bp bandwas obtained by double-endonuclease and the sequence results were fullyaccordant with that published in GeneBank,which declared that pEGFP-PTENwas constructed successfully.2. The results of fluorescence microscopy: After transfected24h，pEGFP-PTEN-SCC-4and pEGFP-N1-SCC-4group gave out greenfluorescence, but there is no green fluorescence expression in SCC-4group.After transfected24、48、72h，fluorescence microscopy was used to observedthe expression of green fluorescence of pEGFP-PTEN-SCC-4andpEGFP-N1-SCC-4cell lines， the result showed the expression of aftertransfected48h was the most obvious.3. The results of RT-PCR and Western blotting: a1200bp specific bandwas acquired in every group, but the pEGFP-PTEN-SCC-4group is significantlybrighten and thincken, the expression of PTEN mRNA in pEGFP-PTEN–SCC-4was0.95±0.04, there was significant difference compared withpEGFP-N1–SCC-4(0.26±0.02) and SCC-4(0.24±0.03)(P<0.05). A55kDprotein band was observed in every group, but the pEGFP-PTEN-SCC-4groupis significantly brighten and thincken, the expression of PTEN protein inpEGFP-PTEN–SCC-4was1.45±0.04, there was significant differencecompared with pEGFP-N1–SCC-4(0.17±0.02) and SCC-4(0.15±0.03)(P<0.05).4. The results of MTT: the growth rate of pEGFP-PTEN-SCC-4is obviously slower than pEGFP-N1-SCC-4and SCC-4. Since the third day, the inhibitionrate of pEGFP-PTEN-SCC-4is significantly increased than control group (P <0.05), whereas there was no significant difference between pEGFP-N1-SCC-4group and SCC-4group(P>0.05).Conclusions1. The eukaryotic cell expression vector of pEGFP-PTEN was constructedsuccessfully.2. The exogenous PTEN gene was transfected into Tongue cancer cell lineSCC-4successfully.3. The exogenous PTEN gene can inhibit growth of Tongue cancer cell.4. The PTEN gene was expected to become a new target of gene therapyin tongue cancer.

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CLC: > Medicine, health > Oncology > Oral cavity, maxillofacial tumors > Tongue cancer, sublingual tumor
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