| BackgroundBreast cancer is the second most common cancer and the most common female malignant tumor,posing a huge threat to women’s health worldwide.Breast cancer metastasis prevention strategies are less effective,and metastasis is the main cause of death in breast cancer patients.Metastasis is a multi-step phenomenon triggered by epithelial-mesenchymal transition(EMT),and the loss of E-cadherin expression is one of the main markers of EMT.Studies have shown that a large number of long non-coding RNAs(lncRNAs)were abnormal expressed in breast cancer and lncRNA can participate in tumor invasion and metastasis by regulating EMT.LncRNA TCONS＿00068220 as a newly discovered lncRNA has been demonstrated highly expressed in gastric cancer,and sliencing TCONS＿00068220 in gastric cancer cells significantly inhibited cell viability but promoted cell apoptosis.However,the function and underlying mechanism of TCONS＿00068220 in the occurrence and development of breast cancer and whether TCONS＿00068220 can regulate E-cadherin the expression and further participate in the regulation of EMT in breast cancer are still unknown.The investigation of the effect of TCONS＿00068220 on the occurrence and development of breast cancer and the underlying molecular mechanism of E-cadherin could provide potential targets for the diagnosis and treatment of breast cancer.ObjectiveThis study aimed to investigate the function of TCONS＿00068220 and the underlying mechanism of E-cadherin on the occurrence and development of breast cancer.Firstly,experiments were conducted to detect the expression of TCONS＿0006822 and E-cadherin in breast cancer and corresponding adjacent tissues,as well as breast cancer cells and human normal breast epithelial cells.Subsequently,the effect of abnormal expression of TCONS＿00068220 on breast cancer cell viability,migration and invasion was analyzed.Furthermore,a mice breast cancer xenograft tumor model was established to detect the effect of TCONS＿00068220 expression on tumor growth.Taking the regulatory relationship of TCONS＿00068220 on E-cadherin as the starting point,we further detected the targeted regulatory relationship between TCONS＿00068220 and E-cadherin and the function of E-cadherin in regulating the effect of TCONS 00068220 on breast cancer cell viability,migration and invasion.This study was tried to provide experimental support for the application of TCONS＿0006822 and E-cadherin as potential targets for breast cancer diagnosis and treatment.MethodsChapter 1:TCONS＿00068220 was highly expressed in breast cancer tissues and cells and promoted cell proliferation and metastasisBreast cancer tissue samples and adjacent non-cancerous tissues were collected from 60 female patients diagnosed with triple-negative breast infiltrating ductal carcinoma.Human normal breast epithelial cell line MCF-10A and human breast cancer MCF-7,SK-BR-3,MDA-MB-231 and MDA-MB-468 cells were cultured in Dulbecco’s modified Eagle’s medium.qRT-PCR was used to detect the expression level of TCONS＿00068220 in tissues and cells.Subsequently,we constructed the TCONS＿00068220 overexpression vector pLVX-TCONS＿00068220 and the negative control pLVX-Puro and transfected into MCF-7 cells respectively.MDA-MB-231 cells were transfected with mall interfering RNA(siRNA)si-TCONS＿00068220 targeting TCONS＿00068220 or negative control NC-siRNA to silencing TCONS＿00068220.Next,qRT-PCR was used to detect the transfection efficiency of TCONS＿00068220.Cell counting kit-8(CCK-8)and Transwell assays were used to test the effect of TCONS＿0006822 overexpression or silencing on MCF-7 and MDA-MB-231 cell viability,migration and invasion.Chapter 2:TCONS＿00068220 promotes tumor growth in vivoBALB/c-nude male nude mice were used to establish a mice breast cancer xenograft model.To inhibit or overexpressed TCONS＿00068220 in mice,the grouping were as follows:pLVX-TCONS＿00068220,pLVX-Puro,si-TCONS＿00068220 and NC-siRNA group(n=6 for each group).In detailed,human breast cancer MCF-7 cells were firstly transfected with pLVX-TCONS＿00068220 or pLVX-Puro and MDA-MB-231 cells were transfected with si-TCONS＿00068220 or NC-siRNA,and cultured in normal condition.Next,the transfected breast cancer cells were mixed with Matrigel were injected subcutaneously into the left armpit.One week after the injection was completed,the nodules at the injection site were observed,after the successful transplantation of the tumor in the mouse was confirmed,tumor dimensions were measured at day 6,day 9,day 12,day 15,day 18 and day 21 using a caliper to calculate tumor volume.Mice were euthanized after day 21 with 1%sodium pentobarbital was injected intraperitoneally for anesthesia and tumor were excised and weighed.Chapter 3:TCONS＿00068220 regulated breast cancer cell proliferation and metastasis via E-cadherinqRT-PCR was conducted to detect the expression level of E-cadherin in breast cancer tissues and cells.Next,linear regression analysis was performed to test the correlation between TCONS＿00068220 and E-cadherin expression levels in breast cancer tissues.Furthermore,the protein expression level of E-cadherin in TCONS＿00068220-overexpressed MCF-7 cells and TCONS＿00068220-silenced MDA-MB-231 cells were determine via Western blot.Furthermore,the targeting relationship between TCONS＿00068220 and E-cadherin were confirmed through nucleus and cytoplasm isolation assay and RNA-RNA pull-down.Subsequently,E-cadherin overexpression vector(pcE-cad)and E-cadherin siRNA(si-E-cad)were conducted and respectively transfected into MCF-7 and MDA-MB-231 cells.qRT-PCR was used to confirm the transfection efficiency.Finally,after pLVX-TCONS＿00068220 and pcE-cad were co-transfected into MCF-7 cells,while MDA-MB-231 cells were co-transfected with si-TCONS＿00068220 and si-E-cad.The expression level of E-cadherin was detected by qRT-PCR.And CCK-8 and Transwell experiments were performed to detect the effect of the regulatory axis of TCONS＿00068220/E-cadherin on the viability,migration and invasion in MCF-7 and MDA-MB-231 cells.ResultsChapter 1:TCONS＿00068220 was highly expressed in breast cancer tissues and cells and promoted cell proliferation and metastasisResults from qRT-PCR indicated that the expression of TCONS＿00068220 in breast cancer tissues was compared with adjacent tissues(P<0.01).Similarly,compared with normal breast epithelial MCF10A cells,TCONS＿00068220 was highly expressed in breast cancer MCF-7,SK-BR-3,MDA-MB-231 and MDA-MB-468 cells(P<0.05 or P<0.01).pLVX-TCONS＿00068220 transfection significantly increased the expression of TCONS＿00068220 in MCF-7 cells(P<0.01);while the expression of TCONS＿00068220 in si-TCONS＿00068220-transfected MDA-MB-231 cells was significantly suppressed(P<0.01).CCK-8 assay results represented that MCF-7 cell viability was significantly increased after TCONS＿0006822 was overexpressed(P<0.05)while TCONS＿0006822 silencing significantly inhibited MDA-MB-231 cell viability(P<0.05).Results from Transwell assay indicated that the migration and invasion ability of MCF-7 cells were up-regulated after transfected with pLVX-TCONS＿0006822(P<0.05),while the MDA-MB-231 cells that transfected with si-TCONS＿00068220 significantly suppressed the number of migration and invasion of cells(P<0.05).Chapter 2:TCONS＿00068220 promotes tumor growth in vivoThe tumors derived from TCONS＿00068220-overexpressed MCF-7 cells exhibited increased volumes,weights and growth rate than those grown from control cells(P<0.05 or P<0.01).In TCONS＿00068220-silenced mice,the tumors size,weight and growth rate were significantly inhibited(P<0.05 or P<0.01).Chapter 3:TCONS＿00068220 regulated breast cancer cell proliferation and metastasis via E-cadherinqRT-PCR indicated that the expression of E-cadherin in breast cancer tissues was significantly reduced compared with adjacent tissues(P<0.01)and the expression of E-cadherin was down-regulated in breast cancer MCF-7,SK-BR-3,MDA-MB-231 and MDA-MB-468 cells(P<0.05 or P<0.01).Linear regression analysis showed a negatively correlation of TCONS＿00068220 and E-cadherin expression in breast cancer tissues.Western blot demonstrated that TCONS＿00068220 overexpression significantly reduced the E-cadherin protein level in MCF-7 cells,while si-TCONS＿00068220 transfection increased the E-cadherin protein expression in MDA-MB-231 cells.The results of nucleus and cytoplasm isolation experiment proved that TCONS＿00068220 was distributed in both the nucleus and cytoplasm but was expressed in relative abundance in the cytoplasm.The results of RNA-RNA pull-down further indicated that TCONS＿00068220 directly bound to E-cadherin mRNA in MCF-7 and MDA-MB-231 cells(P<0.05).qRT-PCR further performed that pcE-cad transfection significantly increased the E-cadherin expression level(P<0.05),while TCONS＿00068220 overexpression significantly inhibited E-cadherin expression in MCF-7 cells(P<0.05).And the expression level of E-cadherin was up-regulated in pcE-cad and pLVX-TCONS＿00068220 co-transfected group compared with pLVX-TCONS＿00068220 and pcNC co-transfected group in MCF-7 cells(P<0.05).In MDA-MB-231 cells,si-E-cad transfection suppressed E-cadherin the expression(P<0.05)and silencing TCONS＿00068220 expression increased E-cadherin expression level(P<0.05).When si-TCONS＿00068220-transfected MDA-MB-231 cells were further transfected with si-E-cad,the expression of E-cadherin was down-regulated(P<0.05).Results from CCK-8 assay confirmed that compared with the TCONS＿00068220 overexpression group,further transfection of pcE-cad significantly inhibited the MCF-7 cell viability(P<0.05).While si-E-cad transfection significantly alleviated the down-regulation of cell viability which caused by TCONS＿00068220 silencing in MDA-MB-231 cells(P<0.05).Transwell represented that pcE-cad transfection attenuated the increase in MCF-7 cell migration and invasion ability which caused by TCONS＿00068220 overexpression.And si-E-cad transfection reversed CONS＿00068220 silencing induced cell migration and invasion down-regulation in MDA-MB-231 cells.ConclusionsThis study focused on the function of TCONS＿00068220 and the underlying mechanism of E-cadherin in the occurrence and development of breast cancer.Relevant experiments were conducted and the main conclusions were as follows:1.TCONS＿00068220 expression was up-regulated while E-cadherin was down-regulated in breast cancer tissues and cells;TCONS＿00068220 was negatively correlated with E-cadherin expression in breast cancer tissues and cells.2.TCONS＿00068220 overexpression promoted cell viability,migration and invasion ability via down-regulating E-cadherin in MCF-7 cells.3.Silencing TCONS＿00068220 inhibited MDA-MB-231 cell viability,migration and invasion ability through up-regulating E-cadherin.4.TCONS＿00068220 overexpression promoted mouse tumor volume,weight and growth rate,while silencing TCONS＿00068220 significantly inhibited mouse tumor growth.5.TCONS＿00068220 directly targeted E-cadherin in MCF-7 and MDA-MB-231 cells. |
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