The Regulation Of New Gene KIAA1529on The Resistance To Paclitaxel In Ovarian Cancer And The Mechanism

Background and ObjectiveOvarian cancer is one of the three malignant diseases in reproductive system, and is the most malignant disease in the gynecological oncology. According to the statistics, there was about22200women newly diagnosed with ovarian cancer and more than15000women dead because of the disease. The early symptoms of ovarian cancer are not obvious. When diagnosed with this disease, the women always present at an advanced stage of ovarian cancer. Nowadays the standard treatment of ovarian cancer is the cytoreductive surgery followed by the chemotherapy combined platinum with paclitaxel. But most patients will develop the drug resistance to the chemotherapeutics eventually, which limits the treatment outcome. Thus the5-year survival for the patients with ovarian cancer remains40%approximately, which was not improved in the past decades. In the clinic treatment, the prognosis of the patients with ovarian cancer is depended on the reaction to the therapy. The chemotherapy options would be designed according to multi-factors such as the disease condition, the operation and the pathological diagnosis. Implementation of individualized treatment programs is the main research direction for gynecologic oncologist at home and abroad.Paclitaxel is one of the first-line chemotherapeutic agents in the treatment of ovarian cancer. The Pharmacological mechanism of paclitaxel is to accelerate tubulin polymerization and make the stable structure of microtubule. Paclitaxel could prevent the arrangement and separation of the chromosomes in the cancer cells, and then make the cell cycle arrest, which induce the apoptosis of cancer cells ultimately. It is generally recognized that the antineoplastic mechanism of paclitaxel is to promote the polymerization of tubulin and prevent the depolymerization of microtubules, which arrests the cancer cells in the G2/M phase.In our preliminary study, we used the RNAi library and proteomics such as co-immunoprecipitation and mass spectrometric analysis to discover that a new gene named KIAA1529might be involved in the resistance to paclitaxel in ovarian cancer cells in the AKT/GSK-3β pathway. The aim of this research was to detect the expressions of KJAA1529in ovarian cancer cell lines and tissue, and use the RNAi technology to explore the effect of KA on the resistance to paclitaxel in ovarian cancer cells in the GSK-3β pathway and the related mechanism. It was expected to provide a new target for molecular targeting therapy to reverse the drug resistance to paclitaxel in ovarian cancer.MethodsTumor cell lines were collected and TRIzol was used to extract the total RNA in the tumor cell lines. And then real-time PCR was introduced to detect the mRNA expressions of new gene KIAA1529in ovarian cancer cell lines and breast cancer cell lines, while the expressions of protein KA was detected by western blotting.In the treatment of paclitaxel, the expressions of proteins GSK-3β, p-GSK-30, KA in the ovarian cancer SKOV3cell line were detected by western blotting after GSK-3β being inhibited by the specific inhibitor LiCl. Flow cytometry was used to examine the changes in the cell cycle and the mitotic index in SKOV3cells. The immunofluorescence method was used to stain with p-H3and a-tubulin to observe the changes of cell proportions in mitosis and the changes in the morphology of cytoskeleton. Then flow cytometry was selected to examine the apoptosis in each group. RNAi technology was used to transfect KA siRNA into ovarian cancer SKOV3cells. Real-time PCR and western blotting were introduced to detect the inhibitory effect of the KA siRNA in SKOV3cells. After GSK-3β being inhibited by the specific inhibitor LiCl, flow cytometry was used to detect differences in the cell cycle and the mitotic index between the control group and the transfective group in the treatment of paclitaxel. Immunofluorescence method was used to stain with p-H3and a-tubulin to observe the changes of cell proportions in mitosis and the changes in the morphology of cytoskeleton in the control group and the transfective group. The apoptosis in the control group and the transfective group were examed by flow cytometry.After GSK-3β being inhibited by the specific inhibitor LiCl, western blotting was carried out to observe the changes in the expressions of the mitosis checkpoint relative proteins in the SKOV3cells with paclitaxel treatment. RNAi technology was used to transfect KA siRNA into ovarian cancer SKOV3cells, and then western blotting method was taken to detecet the changes in the expressions of the mitosis checkpoint relative proteins in the SKOV3cells after inhibition GSK-3β with LiCl in paclitaxel treatment.The RNAi technology was chosen to transfect Aurora siRNA into ovarian cancer SKOV3cells. Western blotting was taken to exam the inhibitory effect of the Aurora siRNA in SKOV3cells. Flow cytometry was used to detect differences in the cell cycle and the mitotic index between the control group and the transfective group in the treatment of paclitaxel. Immunofluorescence method to stain with p-H3and α-tubulin was made to observe the changes of cell proportion in mitosis and the changes in the morphology of cytoskeleton in the control group and the transfective group. Finally, the apoptosis in the control group and the transfective group were examined by flow cytometry. Results1. Real-time PCR revealed that the new gene KIAA1529could express in the lung cancer cell line, breast cancer cell lines and ovarian cancer cell lines. Western blotting showed that KA protein could express in the ovarian cancer cell lines such as SKOV3, A2780, C13k and OV2008. In the tissues of ovarian lesion, the expression of KA protein in benign lesions was higher than that in the malignant lesions and among the malignant tissues, the expression of KA protein was lower in poorly differentiated carcinoma than the expression in highly and moderately differentiated tissues.2. Western blotting showed that the expression of p-GSK-3β was increased by using the GSK-3β specific inhibitor LiCl. The expression of KA protein was increased when the SKOV3cells were treated SKOV3cells with LiCl and paclitaxel compared to treated with paclitaxel alone. Flow cytometry test showed that the GSK-3β specific inhibitor LiC1would reduce the G2/M arrest induced by paclitaxel in ovarian cancer SKOV3cells and the mitotic index decreased. Immunofluorescence revealed that in the treatment of combining LiCl and paclitaxel, the cell proportion in the mitosis was reduced compared with using paclitaxel alone. Meanwhile the cells with the mitotic distinctive structure which are condensed nuclei and round cytoskeleton were reduced. Flow cytometry test showed that the apoptosis was less in the treatment of combining LiCl and paclitaxel than that in the treatment of paclitaxel alone. There were significant differences between the two groups.3. Real-time PCR and western blotting showed that the expression of KIAA1529mRNA and protein in the SKOV3cells were reduced obviously after transfected with KA siRNA. With the treatment of LiC1and paclitaxel flow cytometry test showed that the SKOV3cells arrested in G2/M phrase in the transfective group were more than the control group. And the mitotic index was increased in the transfective group. With the treatment of LiCl and paclitaxel immunofluorescence revealed that the number of SKOV3cells in the mitosis was higher in the transfective group, while the cells with condensed nuclei and round cytoskeleton were also more in the transfective group. In addition, the apoptosis rate was increased in the transfective group compared to the control group.4. After GSK-3β being inhibited by the specific inhibitor LiCl, the expressions of the mitosis checkpoint relative proteins such as Aurora A, Aurora B, BUBR1, MAD2and CDC20in the SKOV3cells were detected in the treatment with paclitaxel. The results shown that only the expressions of Aurora A, Aurora B and BUBR1were changed compares to the treatment with paclitaxel alone and all expressions of these proteins were decreased. When transfected the ovarian cancer SKOV3cells with KA siRNA, in the combination treatment with LiCl and paclitaxel, the expression of Aurora B was increased in the transfective group after KA inhibited by siRNA, while the expressions of Aurora A and BUBR1were not change in the transfective group compared to the control group.5. Western blotting showed that the expression of Aurora B was decreased in the SKOV3cells when the cells were transfected with Aurora B siRNA. It revealed that Aurora B siRNA could inhibit the expression of Aurora B. Flow cytometry test showed that the mitotic index was decreased with the treatment of paclitaxel after transfected the SKOV3cells with Aurora B siRNA. Immunofluorescence indicated that the number of SKOV3cells in the mitosis was lower in the transfective group, while the cells with condensed nuclei and round cytoskeleton were also less in the transfective group with the treatment of paclitaxel alone compared to the control group. Finally, with the treatment of paclitaxel flow cytometry test showed that the apoptosis rate was less in the transfective group than that of the control group. ConclusionsThe new gene KIAA1529could express in the lung cancer cell line, breast cancer cell lines and ovarian cancer cell lines. And the KA expression in ovarian cancer tissue is associated with the tumor differentiation.In the GSK-3β pathway, the specific inhibitor LiC1can inhibit the function of GSK-3β by phosphorylation. When GSK-3β was inhibited by LiCl, the expression of KA in the SKOV3cells was increased with the treatment of paclitaxel. Meanwhile, the G2/M arrest in SKOV3cells induced by paclitaxel was diminished, which made the cancer cells escape from the mitotic arrest induced by paclitaxel. Thus the apoptosis induced by paclitaxel was decreased and the drug-resistance to paclitaxel can be conducted eventually.However, if the expression of KA was inhibited by siRNA, the drug-resistance to paclitaxel induced by inhibition of GSK-3β would be reversed. It indicates that the drug-resistance to paclitaxel induced by inhibition of GSK-3β is accomplished through increasing the expression of KA.Our research revealed that when GSK-3β was inhibited by phosphorylation, the expression of the chromosome passenger protein Aurora B was inhibited as the expression of KA was increased in treatment of paclitaxel. Moreover, as the the expression of KA was inhibited by KA siRNA, the expression of Aurora B recovered in SKOV3cells treated with paclitaxel. The research illustrates the increased expression of KA inhibits the expression of the chromosome passenger protein Aurora B, thereby impacting the function of mitotic checkpoint and leading to make the tumor cells escape form mitotic arrest induced by paclitaxel. Ultimately, it causes the the drug-resistance to paclitaxel.In conclusion, our resaerch illuminate the function of the new gene KIAA1529and its coding protein in the ovarian cancer for the first time. In the GSK-3β pathway, as a downstream regulatory factor, KA could modulate the expression of the important chromosome passenger protein Aurora B to participate in the drug-resistance to paclitaxel. In addition, the new gene KIAA1529coding protein KA connects the GSK-3P pathway with the function of mitotic checkpoint. Thus, the new gene KIAA1529coding protein KA, which has a large molecular weight, could be a new target for molecular targeting therapy to reverse the drug resistance to paclitaxel.