| Objective Breast cancer has become a global health problem as the malignant tumor with the highest incidence and the second highest mortality rate among women at home and abroad.As the most common type of tumor in women,some breast cancers are highly invasive and endanger patients’ lives.There are still some breast cancer patients who have distant metastasis at the time of initial diagnosis.The overall survival rate of these patients is far lower than that of patients with early diagnosis and poor prognosis.Breast cancer metastasis is still the major challenges that difficult to overcome,although various treatment strategies including surgery,radiotherapy,chemotherapy and endocrine therapy have been gradually improved with the rapid development of scientific and technological methods,such as genetic testing and drug sensitivity screening.Therefore,searching for molecular markers related to metastasis and clarifying the mechanism of metastasis occurrence and development have become urgent needs to be resolved in clinical and basic research of breast cancer.Long non-coding RNAs(lncRNAs)are long-chain transcripts consisting of more than200 nucleotides.Because there are many different ways to generate long-chain non-coding RNA,it has become a widespread and non-nonsense sequence in the genome.Although it does not have the ability to encode proteins,lncRNAs have gradually been found to participate in and affect the occurrence and progress of many diseases,and become a hot topic for researchers in recent years.Studies have confirmed that lncRNAs can affect protein-coding genes by interfering with transcription,affecting alternative splicing patterns,and regulating protein activity,playing a role in cardiovascular diseases,neurodegenerative diseases,metabolic-related disorders,bacterial infections,and a variety of tumors.The abnormal expression of lncRNAs in a variety of malignant tumors has attracted the attention,and the role and specific mechanism of many lncRNAs in malignant tumors have not yet been elucidated.In this study,we explored the expression level of lncRNA CTD-2108O9.1 in breast cancer tissues and cells,and analyzed its correlation with clinicopathologicalcharacteristics.In order to clarify the role of lncRNA CTD-2108O9.1 in the development of breast cancer,we further studied the effect of its change on the biological function of breast cancer cells.At the same time,this study explored the mechanism of lncRNA CTD-2108O9.1 affecting breast cancer metastasis,and provided new evidence for finding potential biological targets for the diagnosis and treatment of breast cancer metastasis.Methods I.Verify the expression of lncRNA CTD-2108O9.1 in breast cancer:1.Collect the tissues of 97 breast cancer patients from the Department of Breast Surgery,the First Affiliated Hospital of China Medical University.Then use the TRIzol reagent to extract total RNA of tissues,breast normal epithelial cell line MCF-10 A,and breast cancer cell lines MDA-MB-231,MCF7 and ZR-75-1,and perform reverse transcription PCR for reverse transcription;2.Real-time PCR was used to quantitatively detect the expression level of lncRNA CTD-2108O9.1 in tissues and cells.Ⅱ.Analysis of the correlation between lncRNA CTD-2108O9.1 expression level and clinicopathological characteristics and prognosis:1.Use IBM SPSS Statistics V19.0 for statistical analysis,and use chi-square test to analyze the correlation between the expression level of lncRNA CTD-2108O9.1 and the clinicopathological data in breast cancer patients’ tissues;Ⅲ.To verify the effect of lncRNA CTD-2108O9.1 on the biological phenotype of breast cancer cells:1.According to the lncRNA CTD-2108O9.1 sequence,construct a plasmid and knockdown shRNA that overexpress lncRNA CTD-2108O9.1,respectively,and infected the breast cancer cell lines MDA-MB-231 and MCF7 after screening with virus,and puromycin selection stably transfected cells;Qualitative and quantitative detection of lentiviral transfection efficiency using fluorescence microscopy and real-time PCR,respectively,to successfully construct stable expression or knock-down lncRNA CTD-2108O9.1 MDA-MB-231 and MCF7 breast cancer;2.The effect of lncRNA CTD-2108O9.1 on the migration and invasion ability of breast cancer cells MDA-MB-231 and MCF7 was tested using Transwell test and scratchhealing test;3.The effect of lncRNA CTD-2108O9.1 on the proliferation levels of MDA-MB-231 and MCF7 in breast cancer cells was detected by CCK8 experiment;Ⅳ.Explore the effect of lncRNA CTD-2108O9.1 on epithelial-mesenchymal transition(EMT)of breast cancer cells:1.Observe the change of cell morphology of stable cell line after overexpression and knockdown of lncRNA CTD-2108O9.1 by phase contrast microscope;2.Extract total RNA and reverse transcription from stable cell lines,and use real-time PCR to detect the effects of changes in lncRNA CTD-2108O9.1 on the levels of EMT-related markers and transcription of the MMP family;3.Extract the whole protein from the stable cell line and use western blotting to detect the effect of the changes in lncRNA CTD-2108O9.1 on the levels of EMT-related markers and MMP family proteins;4.Use immunofluorescence staining test to detect the effect of the change of lncRNA CTD-2108O9.1 on EMT-related markers and MMP family;5.Use real-time PCR to detect the expression levels of EMT-related markers and MMP families in tissue samples from breast cancer patients;6.Isolate molecules that directly bind to lncRNA CTD-2108O9.1 using TRAP experiments,and use real-time PCR to detect the binding of MMP family and EMT-related markers to lncRNA CTD-2108O9.17.Use Pearson correlation coefficient to analyze the correlation between the expression level of EMT-related markers and MMP family and the expression level of lncRNA CTD-2108O9.1 in breast cancer patients’ tissues;V.Explore the correlation between lncRNA CTD-2108O9.1 and the potential target gene leukemia inhibitory factor receptor(LIFR):1.Use real-time PCR to detect the expression of LIFR in the tissue samples of breast cancer patients;2.Use Pearson correlation coefficient to analyze the correlation between LIFR and lncRNA CTD-2108O9.1 transcription level expression in breast cancer tissues;3.Extract total protein from breast cancer tissue samples,and use western blotting to detect the correlation between LIFR and lncRNA CTD-2108O9.1 protein levels intissues;4.Use real-time PCR and western blotting to detect the effects of lncRNA CTD-2108O9.1 on LIFR transcription level and protein level,respectively;5.Use western blotting and RNA pulldown to detect whether lncRNA CTD-2108O9.1affects LIFR protein translation by binding to EIF4G1;Ⅵ.explore whether lncRNA CTD-2108O9.1 affect breast cancer metastasis by acting on LIFR:1.Knockdown of LIFR in stable breast cancer cell lines that overexpress lncRNA CTD-2108O9.1;2.Use Transwell test and scratch healing test to test whether LIFR can restore the effect of lncRNA CTD-2108O9.1 on the migration and invasion ability of breast cancer cells MDA-MB-231 and MCF7;Ⅶ.in vivo experiments show that lncRNA CTD-2108O9.1 affects breast cancer metastasis by acting on LIFR:1.Nude mouse orthotopic xenograft model and nude mice with over-expressed and knocked down lncRNA CTD-2108O9.1 and over-expressed lncRNA CTD-2108O9.1were constructed using nude mouse fat pad implantation and nude mouse tail vein injection,respectively.Mouse lung colonization and metastasis model;2.After 8 weeks,PET scans were used to detect tumor metastasis in the lungs in the lung colonization metastasis model;3.Record the tumor growth status.Nude mice were sacrificed at 8 weeks,tumor size and weight were measured,and the correlation between tumor growth and metastasis and lncRNA CTD-2108O9.1 and LIFR was analyzed.Results I.Expression of LncRNA CTD-2108O9.1 in breast cancer:1.In the tissues of 97 breast cancer patients,lncRNA CTD-2108O9.1 was generally under-expressed in cancer tissues compared with non-cancerous tissues adjacent to the cancer(86/97,P <0.001);2.Relative to human normal breast epithelial cell MCF-10 A,the relative expression of lncRNA CTD-2108O9.1 in breast cancer cell line MDA-MB-231 is 0.40 ± 0.03 times of MCF-10A(P <0.001),and MCF7 relative expression level was 0.33 ± 0.04 times(P<0.001)and ZR-75-1 was 0.22 ± 0.04 times(P <0.001),both of which were significantly low expression;Ⅱ.Correlation of LncRNA CTD-2108O9.1 expression level with clinicopathological features and prognosis:1.Low expression of LncRNA CTD-2108O9.1 in breast cancer tissues was positively correlated with lymph node metastasis(P = 0.023),and was related to age(P = 0.087),tumor size(P = 0.584),ER(P = 0.387),PR(P = 0.089),HER2(P = 0.272),Ki67(P =0.383)and molecular subtype(P = 0.177)No significant statistical correlation;Ⅲ.Effects of LncRNA CTD-2108O9.1 on the biological phenotype of breast cancer cells:1.After stable transfection,real-time PCR detected that lncRNA CTD-2108O9.1 was up-regulated 142.2 ± 16.4 times(P <0.001)in MDA-MB-231 cells that overexpress lncRNA CTD-2108O9.1(P <0.001).Cells were upregulated by 99.4 ± 22.7 times(P =0.002);the expression of lncRNA CTD-2108O9.1 was down-regulated to 0.15 ± 0.21 times in MDA-MB-231 cells that knocked down lncRNA CTD-2108O9.1(P = 0.002),Lnc RNA CTD-2108O9.1 in MCF-7 cells was down-regulated to 0.59 ± 0.10 times(P =0.002);2.Overexpression of lncRNA CTD-2108O9.1 inhibits the migration and invasion of breast cancer cell MDA-MB-231 and MCF7: In the Transwell migration experiment,compared with OE-NC group,cell migration ability of MDA-MB-231 in OE-2108O9.1group was significantly reduced(303 ± 35 vs.201 ± 38,P = 0.027),and migration ability of MCF-7 cell was significantly reduced in the OE-2108O9.1 group(133 ± 24 vs.64 ± 8,P = 0.009);In the Transwell invasion experiment,compared with the OE-NC group,the invasive ability of MDA-MB-231 cells in the OE-2108O9.1 group was significantly reduced(109 ± 11 vs.71 ± 6,P = 0.006),and the MCF in the OE-2108O9.1group-7 cell invasion ability was significantly reduced(83 ± 13 vs.44 ± 17,P = 0.034);In the scratch healing experiment,compared with the control group OE-NC,the OE-2108O9.1 group MDA-MB-231 and MCF-7 cell scratch healing speed decreased.3.Knockdown of lncRNA CTD-2108O9.1 promotes the migration and invasion of breast cancer cells MDA-MB-231 and MCF7: In the Transwell migration experiment,compared with the KD-NC group,the KD-2108O9.1 group was MDA-MB-231 The cellmigration ability was significantly improved(171 ± 19 vs.251 ± 38,P = 0.032),and the cell migration ability of MCF-7 cells in the KD-2108O9.1 group was significantly enhanced(92 ± 10 vs.165 ± 31,P = 0.009);Transwell In the invasion experiment,compared with the KD-NC group,the invasion ability of MDA-MB-231 cells in the KD-2108O9.1 group was significantly enhanced(130 ± 16 vs.214 ± 15,P = 0.003),and the MCD in the KD-2108O9.1 group was MCF-7 cell invasion ability was significantly improved(73 ± 3 vs.101 ± 14,P = 0.028);in the scratch healing experiment,compared with the control group KD-NC,the KD-2108O9.1 group had MDA-MB-231 and MCF-7 cells heal faster.Ⅳ.Effect of LncRNA CTD-2108O9.1 on epithelial-mesenchymal transition(EMT)of breast cancer cells:1.Phase-contrast microscope photographs showed that breast cancer cells overexpressing lncRNA CTD-2108O9.1 shortened and became blunt,while knockdown lncRNA CTD-2108O9.1 elongated into long spindles;2.After over-expression of lncRNA CTD-2108O9.1 in breast cancer cells,MMP2(0.58± 0.29,P <0.01),MMP9(0.73 ± 0.22,P <0.01),and α-SMA(0.63 ± 0.11,P <0.01))RNA level expression was significantly down-regulated,and laminin RNA expression was significantly increased(1.69 ± 0.20,P <0.01);laminin,ZO-1 and cytokeratin protein levels were increased,MMP2,MMP9,N-cadherin,vimentin and β-catenin were increased.Protein expression was down-regulated and β-catenin nuclear accumulation was reduced;3.The expression level of lncRNA CTD-2108O9.1 in breast cancer tissues was moderately correlated with laminin(r = 0.488,P <0.01),with β-catenin(r = 0.334,P =0.001),N-cadherin(r = 0.261,P = 0.024),Twist1(r = 0.244,P = 0.017)and SNAI1(r =0.282,P = 0.011)are weakly correlated;4.The results of TRAP-qPCR indicated that MMP-2,MMP-9,laminin,N-cadherin andβ-catenin could directly bind to lncRNA CTD-2108O9.1;V.Correlation between LncRNA CTD-2108O9.1 and potential target gene leukemia inhibitory factor receptor(LIFR):1.LIFR was significantly lower in breast cancer tissues compared with non-cancerous non-cancerous tissues(84/97,P <0.001);2.In breast tissue,the expression level of LIFR was only weakly correlated with the expression level of lncRNA CTD-2108O9.1(r = 0.393,P <0.01),but its protein expression level was related to the expression of lncRNA CTD-2108O9.1.3.When lncRNA CTD-2108O9.1 was overexpressed or knocked down,the expression of LIFR protein in breast cancer cells was positively correlated with the change of lncRNA CTD-2108O9.1,but the expression of LIFR RNA level was not significantly changed(P> 0.05).4.After overexpression of lncRNA CTD-2108O9.1,the protein level of EIF4G1 was up-regulated;RNA pulldown results showed that EIF4G1 could directly bind to lncRNA CTD-2108O9.1;Ⅵ.Does LncRNA CTD-2108O9.1 affect breast cancer metastasis by acting on LIFR:1.In the Transwell migration experiment,overexpression of lncRNA CTD-2108O9.1reduced the migration capacity of MDA-MB-231 cells(220 ± 23 vs.168 ± 16,P =0.033),and the LIFR was knocked down to restore the migration capacity(192 ± 6 vs.271 ± 13,P = 0.001),MCF-7 four groups of cells also had the same changes(129 ± 32 vs.69 ± 4 vs.62 ± 11 vs.87 ± 9),and over-expressed lncRNA CTD-2108O9.1 After reducing cell migration ability(P = 0.031),knocking down LIFR again can restore migration ability(P = 0.038);2.In the Transwell invasion experiment,in OE-2108O9.1 compared with OE-NC groups,MDA-MB-231(185 ± 14 vs.156 ± 7,P = 0.032)and MCF-7(85 ± 11 vs.53 ± 8,P =0.020)cell invasion decreased.Compared with the OE-2108O9.1 + sh-NC group,OE-2108O9.1 + sh-LIFR group in MDA-MB-231(153 ± 12 vs.220 ± 20,P = 0.008)and MCF-7(31 ± 19 vs.82 ± 7,P = 0.012)cells increased,the cell invasion ability was improved;3.In the scratch healing experiment,the scratch healing speeds of MDA-MB-231 and MCF-7 cells due to the inhibition of lncRNA CTD-2108O9.1 were restored after knocking down LIFR.Ⅶ.LncRNA CTD-2108O9.1 affects metastasis of breast cancer in vivo by acting on LIFR:1.Compared with the LV-NC group,the SUVmax(0.70 ± 0.10)of lung colonizationmetastases,the OE-2108O9.1 group’s SUVmax(0.49 ± 0.07)decreased(P = 0.039),and OE-2108O9 after LIFR knockdown.1 + sh-LIFR group increased SUVmax(0.59 ± 0.04)(P = 0.081);but there were no significant differences in orthotopic tumor growth and tumor size among the three groups of orthotopic xenograft nude mice models(P> 0.05);2.The SUVmax(1.20 ± 0.19)of the KD-2108O9.1 group that knocked down lncRNA CTD-2108O9.1 was higher than that of the LV-NC group(0.70 ± 0.10,P = 0.016).The SUVmax(0.85 ± 0.02)of the LIFR group decreased(P = 0.034);however,it had no significant effect on tumor growth in situ(P> 0.05).Conclusion1.The lncRNA CTD-2108O9.1 is generally under-expressed in breast cancer tissues and cells;2.The low expression of lncRNA CTD-2108O9.1 in breast cancer tissues is associated with lymph node metastasis;3.Overexpression of lncRNA CTD-2108O9.1 can inhibit the migration and invasion of breast cancer cells MDA-MB-231 and MCF7,and has no significant effect on proliferation and cycle;4.Knocking down lncRNA CTD-2108O9.1 promotes the migration and invasion of breast cancer cells MDA-MB-231 and MCF7,and has no significant effect on proliferation and cycle;5.Overexpression of lncRNA CTD-2108O9.1 inhibits MMP family expression and EMT in breast cancer cells MDA-MB-231;6.Among the EMT-related markers,laminin was moderately correlated with the expression of lncRNA CTD-2108O9.1 in breast cancer tissues,and β-catenin,N-cadherin,Twist1,and Snail were weakly related to it;7.LIFR is generally low expression in breast cancer tissues and cells;8.The lncRNA CTD-2108O9.1 can regulate the expression of LIFR protein level by directly binding EIF4G1;9.Lnc RNA CTD-2108O9.1 affects breast cancer metastasis by regulating LIFR;... |
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