The Effect Of RNAi Scilience Of PTP4A1Gene In Tongue Squamous Cancernomia TCA8113Cells

PARTⅠEXPRESSION OF PTP4A1IN PATIENTS WITH TONGUESQUAMOUS CELL CARCINOMAObjective：To investigate PTP4A1gene expressions in human tonguesquamous cancer tissues and TCA8113cell line and to explore therelationship between PTP4A1expressions and clinicopathological featuresof human tongue squamous cancer.Methods: Expressions of PTP4A1in30cases of human tonguesquamous cell carcinoma and15cases of normal tongue mucosaparaffin-embedded specimens were examined by immunohistochemicaltechnology and in15freshly-taken tongue squamous tissues and TCA8113cells by real-time PCR technology.Results: Immunoreactive staining was located in the cytoplasm andcell nucleus of human tongue squamous cancer. Positive rate of PTP4A1inhuman tongue squamous cancer was83percentage was remarkably higherthan that in normal tongue tissue (P<0.05), with significant differences.Expression of PTP4A1was closely related with lymphatic metastases(P<0.05)，but had no relationship with age,gender,tumor size,and clinicalstage. Result of real-time PCR also showed that PTP4A1mRNA level washigher in human tongue squamous cancer tissues and TCA8113cells than in para-carcinoma and normal tissues (P<0.05).Conclusions: Both PTP4A1mRNA and protein expressions are higherin human tongue squamous cancer tissues.PTP4A1expression may play asignificant role in human tongue squamous cancer development andmetastases. PART ⅡCONSTRUCTION AND IDENTIFICATION OF LENTIVIRALVECTOR OF PTP4A1GENEObiective: To construct a lentiviral vector of RNA interference(RNAi)of PTP4A1gene and to study the inhibit effect on PTP4A1expression andto select stable transfected cells of human tongue squamous cancer cell lineTCA8113.Methods: Fiver target sequence of oligonucleotides were designedaccording to PTP4A1gene sequence,the complementary DNA containedboth sense and antisense strands were designed and synthesized. After theoligonucleotiedes were inserted into the plasmid expression system GV248containing GFP sequence and puromycin. The resulting lentiviral vectorcontaining PTP4A1shRNA was named siRNA-PTP4A1, and it wasconfirmed by PCR and sequencing. The five plasmids of lentiviral vectorwere contransfected to293T cells,By the Western-Blot to tested theexpression of GPF in293T cells,and to confirmed the most effective targetsequence. Then,293T cell were con-transfected with lentiviral vetorsiRNA-PTP4A, pHeleper1.0and pHelper2.0to produce the lentiviral vectors. The titer of virus was tested according to the expression level ofGFP. Further we used this lentiviral vector to transfected TCA8113cells,and detected the PTP4a1expression level by Realtime-PCR andWestern blot.Results: By PCR and DNA sequencing and Western-blot demonstratedthat the lentiviral RNAi vector of PTP4A1was constructed successfully.The titer of virus tested by expression level of GFP was7.0E+8TU/ml.After infected of lentivral vector siRNA-PTP4A1,silence thePTP4A1mRNA and protein level were almost55%and60%(P<0.05)Conlusions: The lentiviral RNAi vector of PTP4A1has beensuccessfully constructed;stable transfected TCA8113cells were slectedsuccessfully to next study. PART ⅢINFLUENCE OF SILENCING OF PTP4A1GENE BYLENTIVIRUS VECTOR SIRNA ON BIOLOGICAL OFTCA8113CELLS IN VITRO AND IN VIVOObjective:To study the effect of silence PTP4A1gene expressionby lentiviral vector mediated siRNA and its effect on biologicalbehavior in human tongue squamous cancer cell line TCA8113in vitroand in vivo.Methods:MTT analysis was used to detect the inhibition of cellproliferation. Flow cytometry was proformed to detect the cell apoptosisand cell cycle. Bcl-2and bax protein expression levels were detected byWestern blot analysis.Realtime PCR was performed to detec the MMP2and MMP9mRNA expression level.Clone formation ability was measured by plate clone forming test. Transwell test performed to examined theTCA8113cell invasion ability.further,to observed the effect of PTP4A1down regulated on the growth and volumes of human tongue squamouscarcinoma cell line TCA8113xenograft in nude mice.Results: down regulated PTP4A1expression reduce the cellproliferation in Tca8113, and increase the apoptosis rate by(17.48±0.70)%(P<0.01), and arrested the cell cycle at G1/G0and Sphase(P<0.05), and the Western blot analysis showed that bcl-2proteinexpression level was down-regulated (P<0.01) but the bax proteinexpression level was increased (P<0.01),and the realtime-PCRanalysis showed that MMP2and MMP9mRNA expression level wasdown regulated about27.4%(P<0.05)and35.2%(P<0.05), and reducethe clone formation ability (P<0.01) in vitro and reduce the invasionability（P<0.01）,Both the xenograft tumor weight and volume werefound to be dramatically deduced in siRNA PTP4A1groups than innegative control groups and in non-infected groups.Conclusions:Silence PTP4A1expression were effective in inhibitingtumor biological behavior in human tongue squamous cancer cell lineTCA8113in vitro and in vivo.PTP4A1may play an important role inhuman tongue squamous cell carcinoma development.