| Objective: Ovarian cancer(OC)has the highest mortality in gynecologic malignancies,accounting for 3% of the gynecological cancer total incidence.Due to insidious onset and the lack of effective methods for early diagnosis,most patients are at the refractory advanced stage when it is clearly diagnosed.This leads to poor prognosis and lower 5-year survival rate.Therefore,an in-depth study of the pathogenesis of ovarian cancer and looking for effective early diagnostic markers are of great significance for patients with ovarian cancer.Recent years,long non-coding RNA(lncRNA)has been researched as one of the new hotspots in oncology field,which involve in multiple biological processes,including tumor cells proliferation,migration,apoptosis,invasion,chemotherapy resistance,angiogenesis,immunity and tumorigenesis.Bladder cancer associated transcript 1(BLACAT1)is a new reported lncRNA,which has been identified as a poor prognosis factor in the previous researches,it promoted the proliferation and invasion of breast cancer,cervical cancer,lung cancer,colorectal cancer,gastric cancer,osteosarcoma and glioma.However,to our knowledge,whether BLACAT1 participated in the biological processes of ovarian cancer has not been reported.We predict the bind site of miR-519d-3p on lncRNA BLACAT1 through online bioinformatic tool.It has been reported that mi R-519d-3p could inhibit the proliferation of ovarian cancer cells and improve their sensitivity to cisplatin by targeting XIAP.miR-519d-3p could regulate its downstream target genes to inhibit the proliferation and invasion of prostate cancer,gastric cancer,trophoblast cells,and pancreatic cancer.However,its role in the invasion and migration of ovarian cancer cells is unknown.Multimodal ultrasound mainly refers to the combination of 2-Dimentional Ultrasound(2DUS),Color Doppler Flow Imagining(CDFI),Power Doppler Imagining(PDI),superb microvascular imaging(SMI),contrast-enhanced ultrasonography(CEUS),and ultrasound elastography(UE),which can acquire the acoustic parameters of the tissues or lesions.Then,a comprehensive evaluation of the morphology,blood flow perfusion and tissue stiffness of the lesions,which can be used for the diagnosis,treatment and therapy evaluation.Therefore,noprevious study has reported the utility of multiple ultrasound in evaluating the efficacy of targeted gene therapy for subcutaneous xenograft of ovarian cancer in nude mice.Method: 1.Thirty ovarian cancer tissues and twenty-nine normal ovarian tissues were collected from patients who had been taken surgical resection at the gynecology department of Shenjing Hospital of China Medical University(Shenyang,China),all the tissue samples were pathologically confirmed,they were immediately frozen by liquid nitrogen and then stored at-80°C.The patients defined complicating with any malignant tumors of other system,and none of them has received any preoperative radiotherapy,chemotherapy or hormone therapy.The clinicopathological data of all the patients were collected.The expression level of BLACAT1 in the tissue samples and four types of ovarian cell lines(SK-OV-3、ES-2、NIH-OVCAR-3 and Caov-3)were quantified by Quantitative Real-Time Polymerase Chain Reaction(qRT-PCR),and the relationship between the expression level and clinicopathologic features was analyzed,while two types of ovarian cell lines with higher expression level were screened out.2.The SK-OV-3 and NIH-OVCAR-3 cells were divided into control group,NC shRNA group,and BLACAT1 shRNA group.The expression level of BLACAT1 was knocked down by gene silencing,and BLACAT1 shRNA or NC shRNA was transiently transfected into the two cell lines.Then,qRT-PCR was used to identify the knocking down effect.The effect of BLACAT1 shRNA on the proliferation of SK-OV-3 and NIH-OVCAR-3 cells was assessed by methyl thiazolyl tetrazolium(MTT)assay and EdU staining.Flow cytometric analysis was carried out to determine the effect of BLACAT1 on ovarian cancer cell cycles.The effect on the migration,invasion and metastasis of ovarian cancer cells were assessed by wound healing assay and Transwell invasion test.3.HEK-293 T cells were divided into four groups: wt-BLACAT1+NC mimic group,mut-BLACAT1+NC mimic group,wt-BLACAT1+miR-519d-3p mimic group,and mut-BLACAT1+miR-519d-3p mimic group.Luciferase reporter assay was used to detect the binding relationship between BLACAT1 and mi R-519d-3p.qRT-PCR was used to detect the effect of BLACAT1 on the expression of mi R-519d-3p in SK-OV-3 and NIH-OVCAR-3 cells.The effect of BLACAT1 on the expression levels of Ribosomal protein S15A(RPS15A),nuclearβ-catenin,cytoplasmic β-catenin and vascular endothelial growth factor(VEGF)in the two cell lines were investigated by Western blot.4.SK-OV-3 cells were divided into control group,NC mimic group and miR-519d-3p mimic group.SK-OV-3 cells were transiently transfected by NC mimic or miR-519d-3p mimic.MTT assay was used to evaluate the effect of the up-regulation of miR-519d-3p on the proliferation of SK-OV-3 cells.The effect of miR-519d-3p on SK-OV-3 cell migration,invasion and metastasis was assessed by wound healing assay and Transwell invasion test.5.The SK-OV-3 cells that stably transfected with BLACAT1 shRNA or NC shRNA were subcutaneously injected into the right armpit of nude mice.The tumor volumes were measured and calculated every 3-day as the tumor being visible.Multimodal ultrasound was performed on the 25 th day after the injection,including 2DUS,CDFI,SMI and UE.Then,qRT-PCR was used to detect the expression levels of BLACAT1 and miR-519d-3p of the xenografts,while Western blot was used to identify their expression levels of RPS15 A,nuclear β-catenin,cytoplasmic β-catenin and VEGF.Ki67 and CD31 were investigated by immunohistochemistry(IHC)staining,while microvessel density(MVD)was measured according to the result of CD31 staining.Results: 1.BLACAT1 was significantly overexpressed in ovarian cancer tissue than in normal ovarian tissue(P<0.05),and the level of expression was correlated with the pathological type of ovarian cancer tissue(P<0.05).BLACAT1 expression levels were positively correlated with FIGO stage and lymph node metastasis(P<0.05),but not correlate with age,histological grade,level of serum CA125,existence of ascites and tumor size(P>0.05).BLACAT1 was positively expressed in the four ovarian cancer cell lines,the expression level of the cells from high to low was SK-OV-3,NIH-OVCAR-3,ES-2 and Caov-3,the expression levels of SK-OV-3 and NIH-OVCAR-3 cells were significantly higher than the other cell lines(P<0.05).2.qRT-PCR confirmed the down-regulation of BLACAT1 expression in SK-OV-3 and NIH-OVCAR-3 cells by transfected with BLACAT1 shRNA(P<0.05).The MTT assay and EdU staining showed that BLACAT1 knockdown suppressed the cell viability of both of these two cells(P<0.05),and flow cytometric analysis showed that BLACAT1 knockdown induced cell cycle arrest of both of these two cells.Wound healing assay and Transwell test showed that inhibition of BLACAT1 exp,,essioncould significantly suppress the migration and invasion of the ovarian cancer cells compared with the cells which was transfected with NC shRNA(P<0.05).3.The binding sites of BLACAT1 to miR-519d-3p was predicted through the online bioinformatic tool,http://starbase.sysu.edu.cn/.Luciferase reporter assay showed that after 293 T cell being transfected with pmiR-GLO-wt-BLACAT1,miR-519d-3p mimic could cause a significant reduction in the luciferase activity of the cells transfected with pmiR-GLO-wt-BLACAT1(P<0.05),while it had no effect on the luciferase activity of the cells with pmiR-GLO-mut-BLACAT1 transfection(P>0.05).The expression levels of miR-519d-3p in the two cell lines assessed by qRT-PCR were obviously up-regulated according to the knockdown of BLACAT1expression(P<0.05).Western blot showed the BLACAT1 inhibition could significantly reduce the level of RPS15 A,nuclear β-catenin and VEGF(P<0.05),but could not affect cytoplasmic β-catenin accumulation in ovarian cancer cells(P>0.05).4.MTT assay displayed that miR-519d-3p mimic significantly inhibited the proliferation of ovarian cancer cells compared with the cells transfected with NC mimic.The migrated and invasive capacity of SK-OV-3 cells were remarkably inhibited by transfecting miR-519d-3p mimic compared with their corresponding controls according to the wound healing assay and Transwell invasion test(P<0.05).5.The xenograft assay showed that BLACAT1 knockdown significantly decreased the tumor volume and weight of the subcutaneous tumor of the nude mice(P<0.05).2DUS features of the tumors of the two groups were similar without obvious difference.The tumors transfected with NC shRNA showed more blood flow on CDFI and SMI compared to the tumors transfected with BLACAT1 shRNA,but the difference was not statistically significant(P>0.05).Real-time tissue elastography(RTE)scores of the tumors of the two groups were mainly 3 and 4,while there was no significantly difference of the RTE results between the two groups(P>0.05).Shear wave elastography(SWE)showed that the Emax、Emean、Eratio of the tumors of NC shRNA group were higher than those of tumors of BLACAT1 shRNA group,and the difference between the two groups was statistically significant(P<0.05).qRT-PCR showed significantly lower BLACAT1 expression level and higher miR-519d-3p expression level in the tumors of BLACAT1 shRNA group compared with the tumors of NC shRNA group(P<0.05).The expression RPS15 A,nuclear β-catenin and VEGF were significantly decreased by BLACAT1 knockdown in the tumors of ovarian cancer(P<0.05),while the level of cytoplasmic β-catenin was not affected(P>0.05).IHC staining assay showed that the ovarian cancer cells that transfected with BLACAT1 shRNA had a lower expression of ki67 and MVD.The semiquantitative analysis SMI showed positive correlation between MVD and VEGF.Conclusions: 1.The high expression of BLACAT1 in ovarian cancer tissues and cell lines suggests that BLACAT1 may play an oncogenic role in tumorigenesis of ovarian cancer.2.BLACAT1 could promote the proliferation,migration,as well as invasion of ovarian cancer cells in vitro.3.BLACAT1 could promote the ovarian cancer proliferation,migration,invasion,as well as angiogenesis through Wnt/β-catenin signaling pathway via regulating miR-519d-3p in vitro.4.MiR-519d-3p could suppress the proliferation,migration,as well as invasion of ovarian cancer cells in vitro.5.BLACAT1 could promote the growth and angiogenesis of xenograft through activating Wnt/β-catenin signaling pathway via regulating miR-519d-3p and its target gene.Multimode ultrasound(2DUS,CDFI,SMI,RTE and SWE)can be used to verify the effect of BLACAT1 shRNA on subcutaneous xenograft of ovarian cancer in nude mice in terms of tumor morphology,neovascularization,and tissue stiffness. |
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