| Objective:ERp57 has been found to be closely associated with the chemoresistance of human ovarian cancer.After a period of chemotherapy,chemotherapy resistance is inevitable.Multidrug resistance(MDR)phenotype has developed eventually after chemotherapy treatment for a certain period,leading to a high recurrence rate(>50%)and poor prognosis for these patients.Therefore,it is very important to find molecular markers and molecular mechanism of chemotherapy resistance in ovarian cancer,which is useful for guiding clinical medication and improving the survival rate of cancer patients.ERp57,also referred to as PDIA3,is a widely expressed protein of multiple biological functions.ERp57 was reported first time to be relevant with chemotherapy resistance of ovarian cancers by Bernardini et al in 2005.ERp57 previously showed to form a nuclear complex associated with resistance to DNA conformation–altering chemotherapeutic drugs in in vitro systems,represented a novel class of genes associated with in vivo drug response in ovarian cancer patients.Later,Cicchillitti et al.used a comparative proteomic approach to analyze the paclitaxel sensitivity of A2780 epithelial ovarian cancer cell and identified that ERp57 is one of the proteins as being altered in paclitaxel-resistant cell lines in comparison with the paclitaxel-sensitive cell lines.ERp57 interacted with class III β-tubulin(TUBB3)in paclitaxel-resistance ovarian cells and this ERp57/TUBB3 interaction occurs in a novel location of cytoskeleton rather than the nuclear compartment.These results indicate that ERp57 plays an important role in chemoresistance in ovarian cancer by modulating the attachment of microtubules to chromosomes following paclitaxel treatment through its interaction with TUBB3.The relationship between ERp57 and tumor resistance was further verified in 2015.Choe et al.found that inhibition of ERp57-STAT3 complex can activate Mcl-1 protein and restore the sensitivity of esophageal cancer cells to radiotherapy.Hussmann et al.found that inhibition of ERp57 in GCT116 colon cancer cells has been shown to bolster ER-stress induced apoptosis with concomitant chemotherapy and irradiation treatment through p53-mediated PERK UPR(unfolded protein reaction)activation.However,the biological roles of ERp57 in the chemoresistance of ovarian cancer still remain unknown and there is no study was reported regarding the effects of down-regulation of ERp57 on the improvement of the paclitaxel sensitivity of the chemoresistant ovarian cancer.In summary,in order to understand the relationships between ERp57 and paclitaxel resistance,and explore the role of ERp57 in ovarian cancer cells to paclitaxel resistance,we construct a taxol-resistance ovarian cancer cell SKOV3/tax.In this study,we compared the expression levels of ERp57 in ovarian cancer cell SKOV3 and its paclitaxel resistance cell SKOV3/tax.Then,we utilized small interfering RNA approach to repress the ERp57 expression and reexamined the biological effects of ERp57-si RNA silencing on the possible MDR reversal of SKOV3/tax cells.Furthermore,we conducted a bioinformatics analysis to identify the biological processes and pathways closely related with ERp57 and chemoresistant ovarian cancer.In order to solve the problem of drug resistance and metastasis,various strategies were used to reverse the drug resistance,such as therapeutic antibodies and small molecule inhibitors on the key factors of drugresistant and metastasis signaling pathways.Some synthetic oligonucleotide can fold into G-quadruplex and have the ability to anti-tumor.,AS1411 is a typical one of the G-quadruplex.AS1411 was uptake by cancer cells through binding with the nucleolin on cell membrane,and AS1411 induce tumor cell apoptosis by regulating genes expression.The effects of G-quardruplex on ovarian cancer drugresistant and metastasis are unclear.Based on the results of our group,we research on the biological effects of G-quardruplex on drugreistant and metastasis for providing experimental evidence and theoretical basis for ovarian cancer patients to choose the following treatments.Methods:1.The cell viability was measured by MTT assay,the IC50 of SKOV3 and SKOV3/tax cells in ovarian cancer cell lines was calculated,and the expression level of drugresistant-related molecules in SKOV3 and SKOV3/tax cells was analyzed by Western Blot.2.After transfection of ERp57-si RNA,the clone formation test detected the cell colony formation ability and the Transwell cell was used to detect the cell migration ability.3.After transfection of ERp57-si RNA,MTT assay was used to detect the survival rate of ovarian cancer cells under paclitaxel,the apoptosis level was detected by flow cytometry,and colony forming ability for single cell was detected by colony forming assay.4.Ovarian cancer Cells were pretreated by ERp57-si RNA and Paclitaxel.Drugresistance-related proteins P-gp and TUBB3,proliferation related proteins PCNA and nucleolin,apoptosis related factors Bcl-2,Bax and Bcl-xl,and invasion and metastasis related molecules such as molecules,such as Bax,TUBB3,and expression were detected by Western blot.5.ERp57 and protein protein interaction network were constrvted by STRING,ERp57 gene/protein gene/protein network were constrcted by Gene MANIA,Annotation of biological process and gene co-occurrence analysis were performed using COREMINE online database/tool.The pathway enrichment analysis was performed using DAVID online tool6.MTT assay was used to screen the G-quadruplex with antitumor activity to the ovarian cancer sensitive and drug resistant cells.7.CD spectroscopy was used to analyze the structure of the G-quadruplex in the medium and the simulated cell physiological ion condition solution.8.The effect of G-quadruplex on the colony formation of ovarian cancer cell was detected by cloning and formation.9.The morphological effects of G-quadruplex body on ovarian sensitive and drug-resistant cells were observed under the optical microscope.10.Ovarian cancer cells were treated by long term and high dose G-quadruplex,Western Blot were used to detected the protein expression level.11.Ovarian cancer cells were treated by short term and low dose G-quadruplex,cell migration and invasion ability were analyzed by Transwell cell experiment,the protein level was detected by Western Blot.Results:1.IC50 of SKOV3 is 3.24 + 0.03 n M and IC50 of SKOV3/tax is 101.06 + 0.99 n M,RI of SKOV3/tax cell resistance index is 31.19 + 0.59.Western Blot detected the protein expression level of SKOV3 and SKOV3/tax.Compared with the parental cell protein SKOV3,the expression level of ERp57 in resistant cells increased significantly.At the same time,the expression of p-STAT3 was significantly increased.The expression of cell proliferation related molecules PCNA decreased in paclitaxel resistant cells.The expression of paclitaxel resistant molecule marker TUBB3 and transporter P-gp were increased.The expression of anti apoptotic protein Bcl-2 and Bcl-xl increased,and the expression of apoptotic protein Bax decreased in drug resistant cell SKOV3/tax.The expressions of MMP2 and MMP9 related to invasion and metastasis were significantly decreased.The level of EMT related molecules marker vimentin are not obvious different.2.ERp57-si RNA can obviously reduce the clonogenic ability and migration ability of SKOV3 and SKOV3/tax by reducing the PCNA and nucleolin.3.ERp57-si RNA increased the apopotosis level by reducing the Bcl-2,Bcl-xl,increasing Bax,p53/p47 expression level.4.The bioinformatics software were used to predict the interaction proteins and signaling pathways of ERp57 involved in the mechanism of ovarian cancer resistance.5.AS1411 had therapeutic effect on the ovarian cancer resistant and sensitive cells.AS1411 formed a mixed G-quadruplex structure under the condition of cell culture medium and simulated physiological ions.6.AS1411 inhibited the proliferation of ovarian cancer cells,and AS1411 changed the cell morphology of ovarian cancer cells to round.7.Long time and high dose of AS1411 can induce the expression of p53 or p53/p47 subtypes,reduce the expression of Bcl-2,increase the expression of Bax,and caspase 3,AS1411 induce the apoptosis of ovarian cancer cells.AS1411 can inhibit the expression of P-gp,nucleolin and STAT3 to increase the sensitivity of taxol.8.Short term and small doses of AS1411 inhibit the migration and invasion of ovarian cancer cells through ERp57/STAT3/MMP9.Conclusion:1.ERp57 is closely related to the sensitivity of paclitaxel in ovarian cancer.ERp57 can be used as a molecular marker for ovarian cancer chemotherapy resistance phenomenon.2.AS1411 can restore the sensitivity of ovarian cancer resistant cells to taxol and inhibited the migration and invasion of ovarian cancer cells. |
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