| Objective：During the clinical cancer treatment, the reduction of chemotherapy sensitivity even leadingto chemotherapy resistance in cancer cells is the important cause of recurrence and metastasis. Inrecent years, though the combination of platinum drugs and paclitaxel have increased thefive-year survival rate of ovarian cancer patients, the inevitable chemotherapy resistance inovarian cancer (especially in platinum-resistant ovarian cancer) becomes the key factor whichaffect the survival of patients. The cause of tumor drug resistance is unclear, however, to increasethe chemotherapeutic sensitivity may reduce the toxic side effects of drugs and improve thecurative effect. On the other hand, it may reduce the incidence of drug resistance. In this study,the expression of α2,3-sialyltransferase (ST3GalⅢ) influenced by different concentrations ofcisplatin (DDP) in ovarian cancer cells, the differences of toxicity tolerance and apoptosis effectof DDP with vary expression of ST3GalⅢ in the ovarian cancer cell lines, and furthercombining used the sialyltransferase inhibitor (ST3GalⅢ-siRNA) with DDP to enhance thechemotherapy sensitivity in ovarian cancer cells were investigated. The results may give us anew method for the clinical treatment of ovarian cancer.Methods:1. To identify the differences of ST3GalⅢ expression in different ovarian cancer cell lines:Use qRT-PCR to detect and compare the expression level of ST3GalⅢ mRNA in four ovariancancer cell lines (OVCAR3, SKOV3, HO8910, HO8910PM), and select the cell line with highestexpression of ST3GalⅢ (HO8910PM) and the lowest expression of ST3GalⅢ (SKOV3).2. To determine the half inhibition rate of cell proliferation induced by DDP in ovarian cancercell lines: Use MTT to detect the inhibition rate of cell proliferation induced by DDP in theovarian cancer cell line with high expression of ST3GalⅢ and the low one (HO8910PM,SKOV3), and calculate their IC50values, to compare the chemosensitivity to DDP of different levels of ST3GalⅢ in ovarian cancer cells preliminarily.3. To determine the concentration and duration of DDP in the ovarian cancer cell line with lowexpression of ST3GalⅢ: Choose the SKOV3cell line which with low expression of ST3GalⅢ,treated with different concentrations (0μmol/L,1μmol/L,20μmol/L,100μmol/L) of DDP fordifferent time（24h,48h,72h）respectively, and use qRT-PCR to detect the expression of ST3GalⅢ mRNA in each group of the cells. Explore whether DDP may regulate the expression ofST3Gal Ⅲ in ovarian cancer cells, and screen out the optimal concentration and the bestduration of DDP.4. To observe the knock-down effect of ST3GalⅢ mRNA by siRNA in HO8910PM cells:Choose the HO8910PM cell line which with high expression of ST3GalⅢ, transfected withST3GalⅢ-siRNA sequence (Ⅰ, Ⅱ, Ⅲ) respectively, after48h, the transfected cells wereharvested and the siRNA-directed inhibition efficiency of ST3GalⅢ was detected by qRT-PCRand Western-blot, to explore the effectiveness of ST3GalⅢ-siRNA and select the most efficientsequence.5. Select the SKOV3cell line which with low expression of ST3GalⅢ, first treated with DDPand then transfected with ST3GalⅢ-siRNA, to detect the expression of CAS-apoptotic proteins(caspase8and caspase3) in different levels of ST3GalⅢ by Western-blot, to analyze theapoptosis by FACS and TUNEL, and investigate the effect of DDP chemotherapy in SKOV3cells with different levels of ST3GalⅢ.6. Choose the HO8910PM cell line which with high expression of ST3GalⅢ, first transfectedwith ST3GalⅢ-siRNA and then treated with DDP, to detect the expression of CAS-apoptoticproteins (caspase8and caspase3) in different levels of ST3GalⅢ by Western-blot, analyze theapoptosis by FACS and TUNEL, and investigate the effect of DDP chemotherapy in HO8910PMcells with different levels of ST3GalⅢ.Results:1. There are obvious differences of ST3GalⅢ mRNA expression in different ovarian cancer celllines: with qRT-PCR analysis, the results confirm that ST3GalⅢ mRNAs are differentiallyexpressed in the four ovarian cancer cell lines (OVCAR3, SKOV3, HO8910, HO8910PM)(P<0.01); Wherein the HO8910PM cells has the highest expression level of ST3GalⅢ mRNA,while the SKOV3cells has the lowest expression level; Hence we choose the HO8910PM and SKOV3cell lines for subsequent experiments.2. Identify the optimal concentrations of DDP for proliferation inhibition in SKOV3andHO8910PM cells: The inhibition rate of cell proliferation induced by different concentrations ofDDP for24h in SKOV3cells by MTT assay was detected. The results confirm that the inhibitionof cell proliferation of SKOV3cells by DDP is significantly higher than that of HO8910PM cells(P <0.05); The IC50value of SKOV3cells is69.29±1.46μmol/L, while that of HO8910PM cellsis425.77±6.58μmol/L; We choose three concentrations of DDP (1μmol/L,20μmol/L,100μmol/L)for subsequent experiments.3. The low concentration of DDP can induce the expression of ST3Gal Ⅲ: With qRT-PCRexamination, ST3GalⅢ expression has certain tendency with dose and time-dependent by DDP;ST3GalⅢ can be upregulated in different extent by1μmol/L DDP for different time, theexpression level of ST3GalⅢ mRNA was more fundamental by1μmol/L DDP for48h, about2.5times higher than the control group; so the optimal concentration of DDP was screened as1μmol/L, and the best duration was48h.4. siRNA can specifically know-down the expression of ST3GalⅢ mRNA in ovarian cancercells: With ST3GalⅢ-siRNA transfection for48h, the expressions of ST3GalⅢ mRNA are14.4%,27.4%and35.2%in the group of siRNAⅠ, Ⅱ, Ⅲ,respectively, all are lower than theControl, Lipo, NC and PC groups; There are statistically significant differences (P <0.01);Wherein siRNAⅠgroup has the lowest expression of ST3GalⅢ. The results also confirm thatsiRNAⅠcan effectively inhibit the expression of ST3GalⅢ protein detected by Western-bolt, sowe select siRNAⅠsequence for subsequent transfection experiments..5. The expression of CAS-apoptotic proteins and cell apoptosis increase in SKOV3cells treatedwith DDP+siRNA: By Western-blot detection, the results confirm that ST3GalⅢ is up-regulatedin cells by1μmol/L DDP for48h (DDP group)；ST3GalⅢ is significantly down-regulated in thecells which are first treated with DDP and then transfected with ST3GalⅢ-siRNA (DDP+siRNAgroup), and their expression of caspase8and caspase3are up-regulated; While the expression ofST3GalⅢ, caspase8and caspase3in the corresponding control groups (DDP+Lipo, DDP+NCand DDP+Control) do not increase. Similarly, the FACS and the TUNEL assay also show that(DDP+siRNA) group has a significant increase of apoptosis compared with the other groups.6. The expression of CAS-apoptotic proteins and cell apoptosis were increased in HO8910PM cells treated with siRNA+DDP: By Western-blot detection, the results confirm that ST3GalⅢ isnot up-regulated in HO8910PM cells by1μmol/L DDP for48h (DDP group), as same as theirexpression of caspase8and caspase3；When ST3GalⅢ is silenced by siRNA, and thencombined with DDP (siRNA+DDP group), in which caspase8and caspase3are significantup-regulated, while the expression of ST3GalⅢ, caspase8, caspase3in the correspondingcontrol groups (siRNA, Taxol+Lipo and Taxol+NC) has no increase; Similarly, the FACS and theTUNEL assay also show that (siRNA+DDP) group has a significant increase of apoptosiscompared with the other groups.Conclusions:1. ST3GalⅢ is differentially expressed in the four ovarian cancer cell lines (OVCAR3, SKOV3,HO8910, HO8910PM); Wherein the HO8910PM cells express the highest level of ST3GalⅢ,while the SKOV3cells express the lowest.2. Overexpression of ST3GalⅢ in ovarian cancer cells enhance the tolerance to DDP, anddecrease the inhibition rate of cell proliferation.3. ST3GalⅢ can be up-regulated in ovarian cancer SKOV3cells by appropriate concentration(1μmol/L) of DDP for a suitable time (24h,48h,72h), the up-regulation of ST3GalⅢ is moresignificant by1μmol/L DDP for48h.4. ST3GalⅢ can be effectively down-regulated in ovarian cancer by ST3GalⅢ-siRNA.5. Down-regulation of ST3GalⅢ may enhance the apoptotic efficacy induced by DDP in ovariancancer cells.6. ST3GalⅢ-siRNA may enhance the effect of apoptosis induced by DDP in ovarian cancer cellsthrough caspase activation.7. ST3GalⅢ-siRNA combined with DDP may enhance chemotherapy sensitivity in ovariancancer. |
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