| Objective:TPX2is microtubμle-associated protein that is a tightly regμlated by the cell cycle and play an important role in mitotic spindle formation process. The researchers found that TPX2is highly expressed in some malignant tumors and resμlt to abnormal centrosome amplification, aneuploidy and cell malignant transformation, which is contribut to cell proliferation, cell apoptosis,and have a relationship with differentiation, metastasis,recurrence of tumor.B locking its expression can inhibit the growth of tumor cells. TPX2is expected to be a new candidate targets of tumor therapy. Cervical cancer is one of the common malignant tumors in the female. Many factors play a role in its occurrence, development and outcomes, whether TPX2play an important role, we have not seen any report at home and abroad.In our research, in order to explore the new targets for the diagnosis and treatment of cervical cancer,we discuss the expression of TPX2in carcinoma of the cervix and the effect on the proliferation and invasion of cervical cancer cell lines when TPX2was silenced.Methods:The research was divided into four parts.The first part was to reseach expression of TPX2in cervical cancer tissue, siha and hela cells lines by RT-PCR and immunohistochemical techniques and the relationship between the expression of TPX2and clinical stage, lymph node metastasis, pathologic differentiation of cervical squamous cell carcinoma. The second part was to research the biological behavior of cervical squamous cancer siha cells when TPX2gene was silenced in vitro. Four pairs specific siRNA of TPX2and a pair of negative control siRNA was designed. lipofectamine2000-mediated siRNA were transfected into siha cell lines in vitro, measured its transfection efficiency by fluorescence microscopy. In this study, all test items are grouped as follows:PBS instead of the transfer agent and siRNA mixture as the blank control group. Negative siRNA as negative control group, TPX2-siRNA group as experimental group. We detect the expression of TPX2before and after transfection and select the most effective siRNA to do follow-up experiments by RT-PCR. The expression of TPX2protein was detected by western blot. The proliferation of siha cell lines was detected by MTT.The cells lines were divied into transfect group, negative control group and blank control group. The experiment was repeated three times each group. The cell lines were seeded in a96-well plate, incubated for24h and the MTT and dimethyl sμlfoxide (DMSO) were added according to the empirical procedure. The OD value of each well was measured using a microplate reader with a test wavelength of570nm at Oh,24h,48h,72h,96h from interference. The changes of the cycle and apoptosis of siha cell lines were detected by Flow cytometry. The invasive ability of siha cell in vitro was detected by Transwell invasion experiment. The cell of each group was Droped into the transwell chambers which contain microporous membrane, cultured24hours,95%ethanol fixation, crystal violet stained and counted the numbers of each group of transmembrane cell under the microscope. In the third part, the influence of TPX2-siRNA on human cervical glands cancer hela cell lines in vitro has been studied. The method was similar to the second part.In order to verify the influence of TPX2-siRNA on the growth of cervical cancer cell lines. The forth part was to reseach the influence to the nude mouse transplant tumor of hela cells.SiRNA was midified by2’ome and incubated with lipo2000,then was transfected into hela cells and injected into transplanted tumor. The nude mice were divided into four groups, the TPX2-siRNA transfection group (hela cells transfected with TPX2-siRNA was inocμlated into nude mouse). Negative siRNA control group (hela cells transfected with negative siRNA was inocμlated into nude mouse). TPX2-siRNA treatment group (hela cells was inoculated into nude mouse, TPX2-siRNA was injected into tumor). The blank control group (hela cells was inocμlated into nude mouse, physiological saline was injected into tumor). By observing the time for tumor formation,the tumor growth curve,the tumor volume and weight, to research the antitumor effects of TPX2-siRNA and TPX2silence effect by using immunohistochemical and RT-PCR method.Resμlt:The first part:①The expression of TPX2mRNA in normal cervical tissue was0.080±0.030, however its relative content in cervical squamose cancer tissue was0.441±0.011,0.467±0.031in Cervical adenocarcinoma tissue,1.923±0.040in hela cell,1.679±0.042in siha cell. Every group compared with normal cervical tissue, There were significant differences (p <0.01).②The TPX2protein was positive expression in the cervical squamose cancer Siha cell lines and the cervical glands cancer hela cell line, locates in the cell nucleus detected by the cellμlar immunity chemistry examination. The TPX2protein was not detected in the normal glands epidermis,but its expression in the cervical adenocarcinoma is positive. There were significant differences in the two groups.The expression of TPX2protein in normal cervical squamous epithelial tissue was not be detected,but the positive expression intensity increased in the CIN and cervical cancer tissue,the difference among the three was significant ((p<0.05).③The expression of TPX2in cervical cancer tissue with clinical stage II was stronger than clinical stage I,low-middle differentiated group was stronger than well differentiated group, lymph node metastasis group was stronger than non-lymph node metastasis group.The second part:①When the transfection volume is600μl, the concentration of siRNA is20μmoL/L,the ratio of siRNA and Lipoectamine2000was2.5:2, the transfection efficiency in siha cells was (97.13±1.82)%after24hours since transfection.②TPX2-siRNA4have the strongest silence effect among the four pairs of TPX2-siRNA.it was (84.9±0.73)%.③After transfected with TPX2-siRNA4, The expression of TPX2was obvious inhibited and inhibititory rate was (62.5±1.26)%.④The proliferation of siha cell lines was determined by MTT method. The proliferation of siha cell lines were slow down when transfected with TPX2-siRNA. The absorbance began decreased when transfected with TPX2-siRNA for48h. The absorbance values in transfected group was significantly lower than that in the blank control group and negative siRNA group at24,48,72and96h after transfection(p<0.05). The absorbance value gradually increased along with the prolonged incubation time in blank control group and negative control group at24,48,72and96h after transfection. There were no statistically significant in these two group(p>0.05).⑤The invasion ability of siha cells was determined by transwell chamber method in vitro. The number of siha cells under transwell chamber in TPX2-siRNA4transfected group (12.33±3.06) was significantly lower than blank control group (50.67±5.52) and negative control group (48.33±4.04)(p<0.05).The number of siha cells for penetrating membranes in the negative control group and blank control group have no significant difference (p>0.05).⑥The effects of TPX2-siRNA4on cell cycle distribution in siha cells was detected by flow cytometry. The cycle distribution of siha was changed in TPX2-siRNA4transfected group compared with the control group.The G0/G1phase cells are decreased (p <0.05), the proportion of G2/M phase of cells increased (p<0.05) in TPX2-siRNA4group compared with the control group. While the negative control group and blank control group have no significant difference in each phase cells (p>0.05). The transfection effect of TPX2-siRNA was detected on apoptosis of siha cells by flow cytometric. After transfected with TPX2-siRNA4, the apoptosis rate in TPX2-siRNA4transfection group, the siRNA negative control group, the control group is10.45±0.63%,3.05±0.28%,2.26±0.46%, respectively. Apoptosis rate of the transfected cells was significantly higher than the negative control group and blank control group, p <0.05), Rates of apoptosis between the negative control group and blank control group have no statistically significant.(P>0.05)The third part:The effect of TPX2-siRNA on the growth of human cervical gland cancer HeLa cells in vitro.①TPX2-siRNA was successful transfectedf into hela cells and the transfection efficiency was (98.33±1.53)%after24h after24hours since transfection.②TPX2-siRNA4have the strongest silence effect among the four pairs of TPX2-siRNA. The inhibitory rate was (82.5±0.43)%③Hela cells was transfected with TPX2-siRNA4.The expression of TPX2was obvious inhibited and the inhibitory rate was (63.4±1.05)%.④The proliferation of heLa cell lines were determined by MTT method. The proliferation of hela cell were slow down when transfected with TPX2-siRNA. The absorbance values began decreased when transfected with TPX2-siRNA for48h. The absorbance values in transfected group was significantly lower than that in the blank control group and negative siRNA group at24,48,72and96h after transfection(p<0.05). The absorbance value gradually increased along with the prolonged incubation time in blank control group and negative control group at24,48,72and96h after transfection. There were no statistically significant between these two group(p>0.05).⑤The invasion ability of hela cells was determined by transwell chamber method in vitro.The number of hela cells under transwell chamber in TPX2-siRNA4transfected group (13.20±1.92) was significantly lower than blank control group (55.20±1.30) and negative control group (55.40±2.07)(p<0.05).⑥The effects of TPX2-siRNA4on cell cycle distribution in hela was detected by flow cytometry. The cycle distribution of hela was changed in TPX2-siRNA4transfected group compared with the control group.The G0/G1phase cells are decreased (p<0.05), the proportion of G2/M phase of cells increased (p<0.05) in TPX2-siRNA4group compared with the control group. While the negative control group and blank control group have no significant difference in each phase cells (p>0.05), Detection the transfection effect of TPX2-siRNA on apoptosis of hela by flow cytometric, the apoptosis rate in TPX2-siRNA4transfection group, the siRNA negative control group, the blank control group is13.47%±0.60%,3.48%±0.55%,2.73%±0.51%, Respectively, the apoptosis rate of transfected cells was significantly higher than the negative control group and blank control group,there is a statistically significant difference (p<0.05),butthe rate of apoptosis between the negative control group and blank control group have no significant difference,(p>0.05).The forth part, The condition of transplanted tumor in nude mice,after 9.37±0.75days since inocμlation we can touch the palpable nodμles of miliary size in transfected group nude mice.The rate of tumor formation was100%. The difference of tumor formation time have statistically significant in transfected group nude mice compared with the other three group,②Volume growth curve of the nude mice subcutaneously transplanted tumor shows that the growth of tumor in the blank group and the negative control group grew faster,while,the transfection and treatment group have been suppressed. Inocμlated30days later, the volume of transplanted tumor of nude mice was measured, the volume of TPX2-siRNA transfection group (252.13±27.45) and TPX2-siRNA treatment group (133.45±7.41) were lower than the blank control group (1364.88±226.73) and siRNA negative control group (1301.13±237.80))(p<0.05).④The comparison of volume and quality of transplanted tumor which is removed from nude mice of each group.30days ago,remved the transplanted tumor from nude mice and then measure the volume and quality.compareing the volume and quality of each group, The final volume and weight of the transplanted tumor in transfected group were (61.88±25.70) mm3and (52.93±18.90) g, The treatment group is (58.62±27.23) mm3and (41.95±12.97) g. The blank control group is (608.25359.38) mm3and (452.7±111.72) g. The negative control group is (569.63329.47) mm3and (407.8±174.34) g. Comparing the final volume and weight between the transfection group and treatment group, between blank control group and negative control group,there were no significant difference (p>0.05). Comparing the final volume and weight between the transfected group and the treatment group with the blank control group and negative control group, there was statistics differenc between them (P<0.05).⑤Expression of TPX2mRNA in each group nude mice transplanted tumor. TPX2-siRNA transfected group, TPX2-siRNA treatment group compared to the blank control group, siRNA-negative control groups,there were statistically significant differences (P<0.05), TPX2-siRNA theraped group compared with the TPX2-siRNA transfected with statistical differences (P<0.05). Blank control group compared to the negative control group, no statistically significant differences (P>05),The expression of TPX2in nude mice transplanted tumor was detected by Immunohistochemical methods.TPX2protein is localized in the nucleus, the expression of TPX2protein in TPX2-siRNA transfection group and treatment group are significantly weaker than which in blank control group and negative siRNA control group (p<0.05). Compared with blank control group and negative siRNA control group, there have no significant difference (p>0.05).Conclusion1TPX2may have been involved in the occurrence and development of carcinoma of the cervix and can be used as a target for the diagnosis and malignancy assessment in cervical cancer.2The proliferation and invasion of siha cell line can be inhibited by TPX2-siRNA,and the cycle distribution and apoptosis also be affected.3The proliferation and invasion of hela cell line can be inhibited by TPX2-siRNA,and the cycle distribution and apoptosis also be affected.4The growth of transplantation tumor of nude mice can be inhibited by TPX2-siRNA5TPX2is expected to be a candidate target for gene therapy of cervical cancer. |
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