| Objective:TPX2(targeting protein for xenopus kinesin-like protein2), amicrotubule-associated protein, plays important roles in mitotic spindleformation.The abnormal expression of TPX2in various types of malignanttumors has been reported, but less is known for medullary thyroid cancer(MTC). In this work, we studied the relationship between TPX2expressionand the biological behavior of MTC, then investigated the effects of inhibitedTPX2expression on tumor proliferation, apoptosis and cell cycles diffusion ofMTC derived cell line TT. We sought to explore the potential of TPX2as atherapeutic target.Methods:1Expression of TPX2in MTC. Immunohistochemical SP method wasused to analyze the expression of TPX2in32cases of MTC tissues and8cases of normal thyroid tissues, and then we analyzed the relation betweenTPX2expressions with clinicocharactors.2Four pairs of TPX2-siRNA and a pair of randomly negative-controlledsiRNA were synthesized. SiRNA was transfected into the TT cells byLipofectamine2000. The transfection efficiency was evaluated by calculatingthe ratio of fluorescent cells to total cells tested by Laser confocol scanmicroscoPe. TPX2mRNA was determined by Real-time PCR to selecte thebest silencing effect of TPX2-siRNA.3The role of TPX2down-regulated by RNAi in proliferation，cellularcycle, and expressions of Aurora-A，CyclinB1and P53in TT cells. MTTassay and flow cytometry (FCM) were used to observe the biologicalbehaviors of the TT cells. Western blot was used to detect the expression ofTPX2, Aurora-A, CyclinB1and P53in TT cells treated with TPX2-siRNA. 4Statistical analysis. SPSS16.0statistical software was used for statist-ical analyses. Count data were campared with Chi-square. Quantitative datawere presented as mean±SD. The single-factor analysis of variance (One-wayANOVA) was used to compare multiple samples. A P-value of <0.05wasconsidered as statistically significant.Results：1The expression of TPX2protein was detected in different thyroidtissues. The positive rate of TPX2protein in MTC tissues was62.5%, whichwas significantly higher than that in normal thyroid tissue(P<0.05). Thepositive rate of TPX2protein in MTC was correlated with clinical stage,tumor size, and lymph node metastasis (P＜0.05). The positive rate of TPX2had no correlation with patients’ age，gender and existence of thyroiditis(P>0.05).2The result of transfection, when the ratio between siRNA and lipo2000was2vs1, the transfection efficiency was the highest [(96.23±2.53)%]. Theexpression of TPX2mRNA in TT cells transfected with TPX2-SiRNA2wasdecreased [(87.7±6.16)%] with the most silencing effect.3MTT assay indicated that the proliferation of TT cells was obviouslyinhibited in TPX2-siRNA2-transfected group, and the growth inhibition ratewas26.7%relative to blank control (P <0.05) at24h after transfection.4FCM analysis showed that,the apoptosis rate of TPX2-siRNA2groupswas significantly higher than that in blank control groups [(14.96±2.69)%vs(2.24±0.62)%](P＜0.05). There was no statistical difference between blankcontrol groups and negative control groups (P>0.05).The percentages of G2-Phase cells in TPX2-siRNA2transfected groupswas higher than that in blank control groups and negative siRNA controlgroups (both, P<0.05), the percentage of s-phase cells in TPX2-siRNA2transfected group was lower than that in blank control group and negativesiRNA control group (both, P<0.05).Therefore, TPX2silencing may arrest thecell cycle at the G1and G2Phases. 5Western blot analysis showed that the expression level of TPX2proteinwas significantly lower than that in blank control and negtive control(0.124±0.017vs0.407±0.022,0.393±0.020)(P＜0.05), there was no statisticaldifference between blank control groups and negative control groups (P>0.05).The expression levels of Aurora-A and CyclinB1proteins weredown-regulated whereas the expression level of P53protein was up-regulatedin TT cells after transfection with TPX2-siRNA2.Conclusions:1We observed that firstly, the positive rate of TPX2protein in MTCtissues was higher than that in in normal thyroid tissues. The positive rate ofTPX2in MTC was correlated with clinical grading, tumor size and lymphnode metastasis.2Overexpression of TPX2can promote cell proliferation and prevent cellapoptosis.3TPX2RNAi in thyroid medullary cancer cells cause thedown-regulation of Aurora-A and CyclinB1protein expression, theup-regulation of P53, and arrest the cell cycle at the G2/M and G1/S phase,inhibite cell proliferation and induce apoptosis of TT cells. |
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