| Objective:Ovarian cancer(OC)is one of the three major gynecological malignancies,and its mortality rate is the first among them,ovarian serous cancer is the most common,with high morbidity,high recurrence rate and high mortality.Paclitaxel combined with platinum-based chemotherapy is the preferred chemotherapy regimen for patients with ovarian serous carcinoma after primary tumor cell reduction.In the past 20 years,although the complete remission rate of ovarian cancer has reached more than 80%,most patients have undergone multiple chemotherapy due to repeated recurrence,and the tumor-free survival period after each chemotherapy is gradually shortened from chemotherapy-sensitive to chemotherapy-resistant.Therefore,the5-year survival rate of ovarian cancer patients is still lower than 40%.The drug resistance of chemotherapy for ovarian cancer has always been one of the urgent problems to be solved.Many studies have found that tumor drug resistance is closely related to tumor proliferation,apoptosis,migration,invasion,EMT and other malignant biology.Therefore,in-depth research on the molecular mechanism of the occurrence and development of ovarian cancer,especially drug resistance,plays an important role in the early diagnosis,treatment and prognosis of ovarian cancer.RNA(circular RNA,circ RNA)is a kind of non-coding RNA with special closed circular structure,which is not easy to be degraded and has a high degree of sequence conservation,so it has gradually become a research hotspot.At present,a large number of studies have found that circ RNA participates in malignant biological behaviors such as tumor proliferation,metastasis and drug resistance,and also plays an important regulatory role in the occurrence and development of ovarian cancer.However,the research on paclitaxel resistance in ovarian cancer has not been carried out in depth.Therefore,the differentially expressed circ＿0000231 was screened out by gene chip of ovarian cancer drug-resistant cell line.Circ＿0000231 is formed by connecting the head and tail of exon 2 and exon 3 of its ontology gene ARHGAP12.It is found that circ＿0000231 can promote the development of atherosclerosis in colon cancer and nasopharyngeal carcinoma and regulate the toxic effects of adriamycin on the heart through ce RNA mechanism.Up to now,the research of circ＿0000231 in ovarian cancer has not been reported,so we should study circ＿0000231 in ovarian cancer in order to find new diagnostic or therapeutic targets.Bioinformatics analysis predicted that circ＿0000231 might be combined with mi R-140.Mi R-140 is located on chromosome 16.Studies have found that mi R-140 can inhibit the proliferation,migration,invasion and epithelial-mesenchymal transition(EMT)of ovarian cancer,and promote cell apoptosis and other malignant biological behaviors.However,the study on paclitaxel resistance of mi R-140 to ovarian cancer has not been carried out yet.The bioinformatics database predicts that the member of RAS oncogene family(RAP1B)is the target gene of mi R-140.RAP1 B is a member of RAS small G protein family,which is widely recognized as an oncogene.RAP1 B also plays a role in promoting cancer in many kinds of tumors.Similarly,the study of RAP1B’s resistance to paclitaxel in ovarian cancer has not been reported.At the same time,there is no report about the combination of mi R-140 and RAP1 B.Therefore,this study hopes to explore the effects of circ＿0000231 on proliferation,apoptosis,migration,invasion of ovarian cancer and malignant biological behavior of EMT and paclitaxel resistance through mi R-140/RAP1 B axis through in vitro and in vivo experiments,and explore the mechanism among them,so as to provide a new theoretical basis for the basic research of paclitaxel resistance in ovarian cancer.At the same time,through related research,we can find a new target to reverse paclitaxel resistance of ovarian cancer,and provide scientific and powerful reference for clinical treatment of ovarian cancer.Methods:1.In this study,the differentially expressed circ RNAs were screened by using gene chip technology,taking human ovarian serous cancer cell line SKOV3 and human ovarian serous cancer cell line SKOV3-TR30 as samples.The expression level of circ＿0000231 in chemotherapy-resistant and sensitive tissues and cell lines of ovarian serous carcinoma was detected by q RT-PCR method,and the relationship between the expression level of circ＿0000231 and clinicopathological characteristics of ovarian serous carcinoma was analyzed.The stability of circ＿0000231 was verified by RNase R digestion experiment,and the cell localization of circ＿0000231 was detected by fluorescence in situ hybridization.Circ＿0000231 ovarian cancer cell lines with silent expression and overexpression were constructed,and the transfection efficiency was detected by q RT-PCR.CCK-8 assay was used to detect the effects of circ＿0000231 on the proliferation and taxol drug sensitivity of ovarian cancer cells,and flow cytometry and Transwell assay were used to detect the effects of circ＿0000231 on the apoptosis,migration and invasion of ovarian cancer cells.Meanwhile,Western Blot analysis showed that the silence and overexpression of circ＿0000231 caused the expression changes of EMT marker proteins(E-cadherin,Vimentin,MMP9)and drug resistance-related protein(β-tubulin III).To construct a stable knockdown circ＿0000231 cell line,and to verify the effect of circ＿0000231 on the proliferation and drug resistance of ovarian cancer in vivo by nude mice transplantation experiment.2.Predict mi RNAs(mi R-558,mi R-622,mi R-140,mi R-135b-5p)combined with circ＿0000231 according to bioinformatics analysis,and detect the expression of mi RNAs in silent circ＿0000231 by q RT-PCR.The expression changes of circ＿0000231 before and after interference with mi R-140 were further detected by q RT-PCR.Double luciferase experiment and RIP experiment were used to detect whether circ＿0000231 and mi R-140 can be directly combined.Co-localization of circ＿0000231 and mi R-140 cells was detected by fluorescence in situ hybridization.q RT-PCR was used to detect the expression of chemoresistance and sensitive tissues and cell lines in ovarian serous carcinoma.Spearman correlation coefficient evaluates the correlation between expression of circ＿0000231 and mi R-140.Up-regulated and down-regulated mi R-140 cell lines were constructed,and the effects of cell proliferation,taxol drug sensitivity,apoptosis,migration and invasion were detected by CCK-8,flow cytometry,Transwell and other experiments.Western Blot was used to detect the expression changes of EMT marker protein and drug resistance-related protein in each transfection group.Bioinformatics analysis predicted that mi R-140 might bind to the downstream target gene RAP1 B.Double luciferase assay was used to detect whether mi R-140 could bind to RAP1 B.Further,q RT-PCR and Western Blot were used to detect the expression changes of RAP1 B after the interference of circ＿0000231 and mi R-140 respectively.q RT-PCR and Western Blot were used to detect the expression of RAP1 B in chemotherapy-resistant and sensitive tissues and cell lines of ovarian serous carcinoma.Spearman correlation coefficient evaluates the correlation between expression of mi R-140 and RAP1 B.The overexpression cell line of RAP1 B was constructed,the transfection efficiency was detected by q RT-PCR,and the effects on cell proliferation and taxol drug sensitivity were detected by CCK-8 experiment.3.Construct the RAP1 B cell line that knocks down circ＿0000231 to down-regulate mi R-140 and knocks down circ＿0000231 to over-express Rap1 b,and detect the effects of cell proliferation,taxol drug sensitivity,apoptosis,migration and invasion by CCK-8,flow cytometry,Transwell and other experiments.Western Blot was used to detect the expression changes of EMT marker protein,drug resistance-related protein and RAP1 B in each transfection group.Results:1.The expression level of circ＿0000231 in paclitaxel-resistant ovarian serous cancer cell line SKOV3-TR30 is higher than that of ovarian serous cancer cell line SKOV3,and it is highly expressed in sensitive tissues in chemotherapy-resistant ovarian cancer tissues,which is closely related to FIGO stage,and has nothing to do with age,differentiation degree and lymph node metastasis.Circ＿0000231 can’t be degraded by RNase R,and it is localized in cytoplasm.Silencing circ＿0000231 can inhibit cell proliferation and increase the drug sensitivity of ovarian cells to paclitaxel,while overexpression is the opposite.Flow cytometry showed that silencing circ＿0000231 increased the apoptosis rate,while overexpression decreased the apoptosis rate.Transwell experiment showed that silencing circ＿0000231 reduced the number of cells penetrating the membrane,while overexpression increased the number of cells penetrating the membrane.Western Blot showed that silence of circ＿0000231increased the expression of E-cadherin,and decreased the expression of Vimentin,MMP9 and β-tubulin III,while over-expression was the opposite.The results of nude mouse tumorigenesis experiment showed that silencing circ＿0000231 inhibited the growth of tumor.Immunohistochemical results showed that silencing circ＿0000231 decreased the expression of β-tubulin III in transplanted tumor tissues of nude mice.The differences were statistically significant.2.The mi RNAs(mi R-558,mi R-622,mi R-140,mi R-135b-5p)that may bind to circ＿0000231 were predicted by bioinformatics.q RT-PCR showed that mi R-140 was the highest expression in the silent circ＿0000231 group.The double luciferase experiment and RIP experiment proved that circ＿0000231 and mi R-140 can be directly combined.FISH co-localization indicated that circ＿0000231 and mi R-140 were co-located in cytoplasm.q RT-PCR showed that the expression of mi R-140 in ovarian serous cancer cell line was higher than that in paclitaxel-resistant ovarian serous cancer cell line.At the tissue level,the expression of mi R-140 in sensitive ovarian cancer tissue was higher than that in drug-resistant ovarian cancer tissue,and the expression of mi R-140 was negatively correlated with circ＿0000231.The results of CCK-8 showed that down-regulation of mi R-140 could promote the proliferation of ovarian cancer cells and enhance the drug sensitivity to paclitaxel,while up-regulation of mi R-140 could inhibit it.Flow cytometry showed that down-regulation of mi R-140 decreased the apoptosis rate,while up-regulation increased the apoptosis rate.Transwell experiment showed that down-regulation of mi R-140 increased the number of cell membrane penetration,while over-expression decreased the number of cell membrane penetration.Western Blot showed that down-regulation of mi R-140 decreased the expression of E-cadherin,increased the expression of Vimentin,MMP9 and β-tubulin III,but the over-expression was the opposite.The differences were statistically significant.It was predicted by bioinformatics that RAP1 B might combine with mi R-140.The double luciferase experiment confirmed that they could be combined.q RT-PCR and Western Blot showed that the expression level of RAP1 B had the same trend as that of circ＿0000231,but had the opposite trend to that of mi R-140.q RT-PCR and Western Blot showed that the expression level of RAP1 B in SKOV3-TR30 was significantly higher than that in SKOV3,and it was also highly expressed in chemotherapy-resistant tissues of ovarian serous carcinoma.Spearman correlation coefficient evaluation shows that mi R-140 is negatively correlated with the expression level of RAP1 B.Through CCK-8 experiment,we found that the expression of RAP1 B can promote the proliferation of ovarian cancer cells and the drug resistance to paclitaxel.3.We constructed two co-transfected cell lines in SKOV3-TR30,which were silencing circ＿0000231 and downregulating mi R-140,silencing circ＿0000231 and overexpressing RAP1 B.In the cell lines of silencing circ＿0000231 and downregulating mi R-140,CCK-8 experiment showed that anti-mi R-140 could reverse the inhibition of silencing circ＿0000231 on the proliferation of ovarian cancer cells and paclitaxel resistance.The results of flow cytometry showed that compared with the silent circ＿0000231 group,the apoptosis rate of anti-mi R-140 co-transfection group decreased.Transwell test was used to detect the migration and invasion of cells,and the results showed that compared with the silent circ＿0000231 group,the number of cells passing through the membrane increased in the anti-mi R-140 group.Western Blot results showed that the expression of E-cadherin increased,while the expression of Vimentin,MMP9,β-tubulin III and RAP1 B decreased in the anti-mi R-140co-transfection group compared with the silent circ＿0000231 group,and the differences were statistically significant.In silence circ＿0000231 and overexpression RAP1 B cell lines,CCK-8 experiment was used to detect the effect of co-transfection of silence circ＿0000231 and overexpression RAP1 B on the proliferation of ovarian cancer cells.The results showed that overexpression of RAP1 B could reverse the inhibitory effect of silencing circ＿0000231 on the proliferation of ovarian cancer cells and paclitaxel resistance.The results of flow cytometry showed that compared with the silent circ＿0000231 group,the apoptosis rate of co-transfection group with overexpression RAP1 B decreased.Transwell assay was used to detect the migration and invasion of cells.The results showed that compared with the silent circ＿0000231 group,the number of cells passing through the membrane increased in the co-transfection group with over-expression RAP1 B.Western Blot results showed that compared with the silent circ＿0000231group,the co-transfection group with over-expression RAP1 B increased the expression of E-cadherin,and decreased the expression of Vimentin,MMP9,β-tubulin III and RAP1 B,with statistical significance.Conclusion :1.circ＿0000231 is highly expressed in sensitive tissues and cell lines of ovarian serous carcinoma resistant to chemotherapy,and is related to FIGO stage in clinical pathobiological characteristics of ovarian serous carcinoma,but not related to age,tissue differentiation and lymph node metastasis.2.circ＿0000231 promotes the proliferation,migration and invasion of ovarian cancer cells,inhibits cell apoptosis and promotes the drug resistance of ovarian cancer to paclitaxel.3.This study confirmed that circ＿0000231 promoted the malignant biological behavior of ovarian serous carcinoma and paclitaxel resistance by binding mi R-140 to regulate RAP1 B. |
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