| Part 1 The expression,biological function and clinical significance of SNHG10 in epithelial ovarian cancer tissues and cells.Objective: to detect the expression of SNHG10 in epithelial ovarian cancer(EOC)tissues and cells and its biological function,and to analyze its relationship with survival and prognosis of EOC patients.Methods:1.Bioinformatics were used to find differentially expressed lncRNAs in EOC patients.2.A total of 85 pairs of tumor tissue and fallopian tube tissue from EOC patients who underwent ovarian tumor reduction surgery in the Department of Gynecology,The Fourth Hospital of Hebei Medical University from May2015 to May 2016 were collected,and quantitative real-time polymerase chain(qRT-PCR)was used to detect the expression of SNHG10 in EOC tissues.Meanwhile,qRT-PCR was used to detect the SNHG10 expression in EOC cells.3.Kaplan-meier method was used to analyze the correlation between SNHG10 expression level and the survival of EOC patients.4.Cox proportional hazard regression model were used to analyze the potential influencing factors for prognosis in EOC patients.5.EOC cell lines with overexpression of SNHG10 were constructed by liposome transfection6.CCK-8 assay was used to detect the effect of overexpression of SNHG10 on proliferation of EOC cells.Plate colony formation test was used to detect the effects of overexpression of SNHG10 on the clonogenesis of EOC cells.Wound healing assay was used to detect the effect of overexpression of SNHG10 on migration of EOC cells.Transwell assay was used to detect the effect of overexpression of SNHG10 on the invasion of EOC cells.Results:1.The results of bioinformatics showed that SNHG10,TRHDE-AS1,linc00476,linc00893 and NR2F2-AS1 were differentially expressed in EOC patients.2.The results of qRT-PCR showed that the mRNA expression levels of SNHG10,TRHDE-AS1,LINC00476,LINC00893 and NR2F2-AS1 in EOC tissues were significantly downregulated compared with the corresponding fallopian tube tissues,and the expression level of SNHG10 was the lowest(P<0.01);compared with normal ovarian epithelial cells IOSE80,SNHG10 mRNA expression levels in EOC cells A2780,SKOV3,OVCAR3 and OV90 were significantly downregulated(P<0.01).3.Kaplan-meier survival analysis showed that Progress-free survival(PFS)and Overall survival(OS)were significantly reduced in patients with low SNHG10 expression compared with those with high SNHG10 expression(P<0.01).4.Multivariate Cox proportional risk regression analysis showed that peritoneal metastasis and SNHG10 expression were significantly correlated with PFS in EOC patients(P<0.001);lymphnode metastasis,peritoneal metastasis and SNHG10 expression was significantly correlated with OS in EOC patients(P<0.05).SNHG10 expression was an independent risk factor for the prognosis of EOC patients.5.The results of qRT-PCR showed that the SNHG10 expression in SNHG10-ov group was signally higher than that in EV group after overexpression of SNHG10(P<0.01),suggesting that the EOC cell line with overexpression of SNHG10 was successfully constructed.6.The result of CCK-8 and plate colony formation assay revealed that over-expression of SNHG10 could inhibit the proliferation of SKOV3 and A2780 cells(P<0.01).Wound healing and transwell assay revealed that overexpression of SNHG10 could inhibit the migration and invasion of SKOV3 and A2780 cells(P<0.001).Summary:1.The expression of SNHG10 is low in EOC tissues and cells,the low expression level of SNHG10 is closely associated with poor prognosis in EOC patients,and can be an important biomarker affecting the prognosis of EOC patients.2.SNHG10 can inhibit proliferation,migration and invasion of EOC cells,and plays a tumor suppressive role in EOC.Part 2 Mechanism of SNHG10 inhibiting epithelial ovarian cancer.Objective: To explore the possible mechanism that SNHG10 plays an anti-tumor role in EOC by adsorbing miR-200a-3p to regulate its downstream target gene bridging intergrator 1(BIN1)and then inhibit the epithelial-mesenchymal transitions(EMT)process.Method:1.Lnc Base v.2 was used to predict the potential miRNAs binding with SNHG10,and the top ten miRNAs were selected according to Pearson Score.2.qRT-PCR was used to detect the expression changes of 10 candidate miRNAs in epithelial ovarian cancer cell A2780 after overexpression of SNHG10,and the miRNA with the most significant expression differences was selected as the research object.3.qRT-PCR was used to explore the expression of miR-200a-3p in EOC tissues,and the correlation between miR-200a-3p and SNHG10 expression level was analyzed.4.The subcellular localization of SNHG10 in epithelial ovarian cancer cells was determined by fluorescence in situ hybridization(FISH).5.RNA Binding Protein Immunoprecipitation(RIP)assay and double luciferase reporter gene assay were used to confirm the interaction between SNHG10 and miR-200a-3p.6.qRT-PCR was used to detect the miR-200a-3p expression in EOC cells.7.The effects of knock-down miR-200a-3p on the proliferation,migration and invasion of EOC cells were detected by CCK-8 assay,plate colony formation assay,wound healing assay and transwell assay.8.PITA,miRan Da and miRWALKS were used to predict downstream genes of miR-200a-3p.9.Dual luciferase reporter gene was used to confirm whether miR-200a-3p could interact with BIN1.10.qRT-PCR was used to explore the changes of BIN1 mRNA expression level in A2780 and SKOV3 cells after miR-200a-3p knockdown;Western blot method was used to detect the changes of BIN1 protein expression level in A2780 and SKOV3 cells after miR-200a-3p knockdown.11.Immunohistochemical staining(IHC)was used to explore BIN1 expression in EOC tissues.12.Kaplan-meier method was used to analyze the pertinence between the BIN1 expression and the survival of EOC patients,and the pertinence between the co-expression of SNHG10 and BIN1 and the survival of EOC patients.13.Lipofectamine TM2000 was transfected into EOC cells SKOV3 and A2780 by liposome transfection.They were divided into EV,SNHG10-ov,EV+miRNA inhibitor NC,EV+miR-200a-3p inhibitor,SNHG10-ov+miRNA mimic NC and SNHG10-ov+miR-200a-3p mimic.The expression levels of BIN1 and EMT-associated protein markers in each group were detected by Western blot.14.The effects of overexpression of SNHG10 and overexpression of miR-200a-3p on the proliferation,migration and invasion of EOC cells were detected by salvage experiment.Results:1.In the prediction results of Lnc Base V.2,the top 10 miRNAs that may bind to SNHG10 were screened out according to Pearson Score.2.The results of qRT-PCR showed that among the ten candidate miRNAs,miR-544 a,miR-24-3p,miR-361-5p,miR-449 a and miR-200a-3p were downregulated by SNHG10 overexpression in EOC cells(P<0.05),and miR-200a-3p showed the lowest expression(P<0.001),and we finally chose mir200A-3p as our research object.3.qRT-PCR results showed that miR-200a-3p was highly expressed in EOC tissues and negatively correlated with the expression level of SNHG10(P<0.001).4.FISH assay results suggested that SNHG10 and miR-200a-3p were co-located in the cytoplasm of A2780 and SKOV3 cells.5.RNA Binding Protein Immunoprecipitation and dual luciferase reporter assay results revealed that SNHG10 could mutual effect with miR-200a-3p as a ceRNA in A2780 and SKOV3 cells(P<0.001).6.qRT-PCR results showed that compared with normal ovarian epithelial cells IOSE80,miR-200a-3p expression levels in EOC cells A2780,SKOV3,OVCAR3 and OV90 were significantly upregulated(P<0.001).7.The results of CCK-8 and plate colony formation assay revealed that knockdown of miR-200a-3p inhibited proliferation and clonogenesis of EOC cells(P<0.001).Wound healing and Transwell assay revealed that knockdown of miR-200a-3p inhibited migration and invasion of EOC cells(P<0.001).8.PITA,miRan Da and miRWALK databases predicted the target genes that can bind to miR-200a-3p,and BIN1 was identified by overlapping.9.Dual luciferase reporter gene revealed that miR-200a-3p could interact with BIN1(P<0.001).10.The results of qRT-PCR revealed that knockdown of miR-200a-3p promoted the expression of BIN1 mRNA in A2780 and SKOV3 cells(P<0.001);Western blot results showed that knockdown of miR-200a-3p promoted the expression of BIN1 protein in A2780 and SKOV3 cells(P<0.001).11.Immunohistochemical staining indicated that BIN1 was low expression in EOC tissues.12.Kaplan-meier analysis showed that in epithelial ovarian cancer patients with low expression of BIN1,PFS and OS were significantly shortened(P<0.01);SNHG10 and BIN1 co-expressed patients had significantly longest PFS and OS(P<0.05).13.Western blot showed that compared with EV group,BIN1 and E-cadherin expression increased and N-cadherin,Vimentin and SNAIL expression decreased in SNHG10-ov group(P<0.01).Compared with EV+miRNA inhibitor NC group,BIN1 and E-cadherin expression increased and N-cadherin,Vimentin and SNAIL expression decreased in EV+miR-200a-3p inhibitor group(P<0.01).Compared with SNHG10-OV+miRNA mimic NC group,BIN1 and E-cadherin expression decreased and N-cadherin,Vimentin and SNAIL expression increased in the SNHG10-OV +miR-200a-3p mimic group(P<0.01).The above results suggested that lncRNA SNHG10 upregulated BIN1 expression through SNHG10/miR-200a-3p axis and reversed epithelial-mesenchymal transformation of EOC,thus participating in the development of EOC as a tumor suppressor gene.14.The results of rescue experiments showed that overexpression of SNHG10 could inhibit the proliferation,migration and invasion of EOC cells,while overexpression of miR-200a-3p could reverse the decrease of proliferation,migration and invasion of EOC cells induced by overexpression of SNHG10(P<0.001).Summary:1.miR-200-3p is the target miRNA of lncRNA SNHG10 and is highly expressed in EOC tissues.miR-200a-3p can promote the proliferation,migration and invasion ability of EOC cells and play a carcinogenic role in EOC.2.BIN1 is the target gene of miR-200a-3p,and is low expressed in EOC tissues,which is closely related to the poor prognosis of patients with EOC.3.SNHG10 promotes the expression of BIN1 through the adsorption of miR-200a-3p,thereby inhibiting the EMT process,thus playing a tumor suppressive role in EOC.Part 3 SNHG10 inhibits epithelial ovarian cancer in vivo.Objective: To explore the anticancer effect of SNHG10/miR-200a-3p/BIN1 axis in mice,and lay a theoretical foundation for EOC reversal therapy.Methods:1.Through stable transfection of human ovarian cancer cell line A2780,the cells were divided into SNHG10 overexpression control group(EV)and SNHG10 overexpression group(SNHG10-ov).2.A2780 cells were injected into the groin of female BALB/c nude mice with 5 mice in each group to establish the transplanted tumor model.3.The mice were weighed every 3 days,the volume of transplanted tumor was measured every week,and plot tumor growth curve.5 weeks later,all mice were killed by neck dislocation method,and the primary tumor was removed and weighed.4.The expression levels of SNHG10 and miR-200a-3p in tumor tissues of mice were detected by qRT-PCR.5.The expression levels of BIN1,E-cadherin and N-cadherin in transplanted tumor tissues of mice were detected by IHC.Results:1.Tumor growth curve revealed that the growth of SNHG10-ov group was significantly lower than that of EV group(P<0.05).The nude mice were sacrificed 5 weeks later,and the transplanted tumor was stripped and weighed.The results showed that the size and weight of tumor in SNHG10-ov group was significantly smaller than that in EV group(P<0.001),indicating that overexpression of SNHG10 can significantly inhibit tumor growth in mice.2.The results of qRT-PCR showed that SNHG10 expression in SNHG10-ov group was observably higher than that in EV group,while the expression of miR-200a-3p in SNHG10-ov group was observably lower than that in EV group(P<0.01).3.IHC showed that compared with EV group the expression of BIN1 and E-cadherin was higher in SNHG10-ov group,while the expression level of N-cadherin was lower in SNHG10-ov group(all P<0 05).Summary:1.SNHG10 can inhibit the growth of transplanted tumor in mice.2.SNHG10 may reverse the EMT process through miR-200a-3p /BIN1 axis,and thus play a tumor suppressive role in mice.Conclusions:1.lncRNA SNHG10 is underexpressed in EOC tissues,and its low expression is closely related to poor prognosis of patients with EOC.It is a biomarker that affects the prognosis of patients with EOC and plays a tumor suppressive role EOC.2.SNHG10 can directly bind miR-200a-3p as a ceRNA to promote the up-regulation of BIN1 expression,and then inhibit EMT process,thus exerting its tumor suppressive effect in EOC. |
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