| Breast cancer is the most common malignancy in women worldwide.With the development of early screening of breast cancer and the continuous improvement of standardized diagnosis and treatment,the mortality rate of breast cancer is gradually decreasing.However,there are still a considerable number of patients with advanced breast cancer,because the tumor has metastasized and invaded other important tissues and organs,resulting in a poor prognosis for patients.Up to now,the specific mechanism of the occurrence and development of breast cancer,especially the molecular metastasis mechanism,is still unclear.Therefore,actively searching for the key factors of breast cancer and exploring its important role and mechanism in the invasion and metastasis of breast cancer has a very important guiding significance for timely finding the occurrence of breast cancer,improving the prognosis of patients and reducing the risk of death.Recent studies have shown that long non-coding RNAs(LncRNAs)can act as oncogenes or tumor suppressors and play a crucial role in the formation and development of human malignant tumors.LncRNAs are widely involved in important regulatory processes such as chromatin modification,chromosome inactivation,genomic imprinting,transcriptional interference,transcriptional activation,and intranuclear trafficking.LncRNA H19 is one of the earliest identified imprinted genes,about 2.3 kb in length,located on chromosome 11p 15.5,and is only from the maternal allele.Our previous study found that the expression level of serum exosomal lncRNA H19 in breast cancer patients was up-regulated,and the diagnostic performance of lncRNA H19 in breast cancer was better than that of traditional diagnostic markers,and it is expected to become a new marker for the diagnosis of breast cancer.However,the role of lncRNA H19 in breast cancer and its molecular mechanism remain unclear.In this study,lncRNA H19 was selected as the research object.First,the expression level of lncRNA H19 in breast cancer tissues was verified,and the relationship between the expression level of lncRNA H19 and the clinicopathological characteristics of patients was explored.Next,breast cancer cell lines with stable knockdown of lncRNA H19 was constructed to detect the effect of lncRNA H19 on the malignant biological characteristics of breast cancer cells.Then,the targeting relationship of lncRNA H19 was predicted by bioinformatics,and it was further verified that lncRNA H19 was involved in the invasion and metastasis of breast cancer cells by targeting and binding to miR-130a-3p to regulate the expression of SATB1.Finally,we established a nude mouse model of subcutaneous xenografts,and verified again that lncRNA H19 affects the occurrence and development of breast cancer cells through in vivo experiments.This study preliminarily explored the clinical significance of lncRNA H19 in breast cancer,further enriched the molecular mechanism of lncRNA H19 involved in breast cancer invasion and metastasis,and was expected to provide a theoretical basis for the targeted therapy of breast cancer.Part Ⅰ Expression of lncRNA H19 in breast cancer tissues and its relationship with clinicopathological features of patientsObjective To investigate the expression level of lncRNA H19 in breast cancer tissues and its relationship with clinicopathological characteristics of patients.Methods The expression levels of lncRNA H19 in 50 breast cancer tissue samples(Tumor)and their corresponding adjacent normal breast tissues(Adjacent)were detected by qRT-PCR.The clinical medical records of the above 50 breast cancer patients were collected,and the relationship between the expression level of lncRNA H19 and the clinicopathological characteristics of breast cancer patients was analyzed.Results The relative expression level of lncRNA H19 in breast cancer tissues was significantly higher than that in adjacent normal breast tissues(2.810±0.599)vs(0.997±0.360),p<0.01.The expression level of lncRNA H19 is associated with lymph node metastasis(x2=6.411),TNM stage(x2=6.762),HR(χ2=9.624),Her-2(χ2=4.482),distant metastasis(χ2=4.614)and Ki67(χ2=5.966)in breast cancer patients,p<0.05,but not with age(χ2=0.410)and tumor size(χ2=0.005).Conclusion LncRNA H19 is highly expressed in breast cancer.The expression level of lncRNA H19 is related to lymph node metastasis,TNM stage,HR,HER-2,distant metastasis and Ki67 in patients with breast cancer.Part Ⅱ Effects of lncRNA H19 on proliferation,migration,invasion and apoptosis of breast cancer cellsObjective To investigate the effect of lncRNA H19 on the proliferation,migration,invasion and apoptosis of breast cancer cells.Methods Five human breast cancer cell lines(MDA-MB-415,T47D,BT474,MDA-MB-231,MCF-7)and human normal breast epithelial cell lines(MCF-10A)were selected,and relative expression levels of lncRNA H19 was detected by qRT-PCR.Three segment H19-RNAi lentivirus vectors were stably transfected into MDA-MB-231 and MCF-7 breast cancer cell lines respectively.qRT-PCR was used to verify the knockdown effect of lncRNA H19.CCK-8 test and plate cloning test were used to evaluate the effect of knockdown of lncRNA H19 on the proliferation of breast cancer cells.Scratch test and Transwell invasion test were used to evaluate the effect of knockdown of lncRNA H19 on the migration and invasion of breast cancer cells.The effect of knockdown of lncRNA H19 on apoptosis of breast cancer cells was observed by flow cytometry.Results LncRNA H19 was detected in human breast cancer cell lines MDA-MB-415(1.411±0.181),T47D(1.912±0.165),BT474(1.632±0.140),MDA-MB-231(2.550±0.156),MCF-7(2.935±0.151)was significantly higher than that in human normal breast epithelial cell line(1.014±0.081),p<0.05.Among them,the relative expression levels of MDA-MB-231 and MCF-7 cell lines were higher,p<0.01.After the three segment H19-RNAi lentiviral vector was transfected into MDA-MB-231,the relative expression levels of lncRNA H19(0.521±0.049),(0.583±0.070),(0.737±0.060)were significantly lower than those in the control group(1.009±0.066),p<0.05;After transfection into MCF-7,the relative expression levels of lncRNA H19(0.784±0.061),(0.633±0.063),(0.743±0.076)were significantly lower than those in the control group(1.012±0.073),p<0.05.The results of the CCK-8 experiment showed that the OD value of MDA-MB-231/MCF-7(0.722±0.069)/(0.622±0.070)was significantly lower than that of the control group(1.253±0.072)/(1.123±0.069)after knockdown of lncRNA H19,P<0.05.The results of the plate clone formation experiment showed that the clone formation ability of MDA-MB-231/MCF-7(52.667±3.786)/(52.333±3.215)was significantly lower than that of the control group(126.667±4.163)/(121.667±3.215)after knockdown of lncRNA H19,p<0.01.The results of the scratch test showed that the scratch repair ability of MDA-MB-231/MCF-7(20.000±1.000)/(19.333±1.155)was significantly lower than that of the control group(44.667±2.082)/(43.667±2.082)after knockdown of lncRNA H19,p<0.01.The results of Transwell invasion assay showed that the number of MDA-MB-231/MCF-7(73.667±3.215)/(72.000±4.000)cells passing through Matrigel-coated was significantly lower than that of the control group(131.000±6.557)/(126.000±6.557)after knockdown of lncRNA H19,p<0.01.The results of flow cytometry showed that the number of apoptotic cells in MDA-MB-231/MCF-7(22.126±1.816)/(23.430±2.223)was significantly higher than that in the control group(8.813±0.621)/(9.014±0.803)after knockdown of lncRNA H19,p<0.01.Conclusion LncRNA H19 is highly expressed in breast cancer cells,and lncRNA H19 can promote the proliferation,migration and invasion of breast cancer cells,and inhibit cell apoptosis.Part Ⅲ Molecular mechanism of lncRNA H19/miR-130a-3p/SATB1 regulating breast cancer cell proliferation,migration,invasion and apoptosisObjective To investigate the molecular mechanism of lncRNA H19 on the proliferation,migration,invasion and apoptosis of breast cancer cells.Methods The Starbase database and DIANA tools database were used to predict the downstream targets of lncRNA H19,and the Targetscan database and miRbase database were used to predict the target genes of miR-130a-3p.The expression levels of miR-130a-3p,miR-29c-3p,miR-29b-3p and miR-454-3p in breast cancer cell lines MDA-MB-231,MCF-7 and human normal breast epithelial cell line MCF-10A were detected by qRT-PCR.The dual-luciferase reporter gene assay verified the targeting binding relationship between lncRNA H19 and miR-130a-3p,miR-130a-3p and SATB1,and the RNA-binding protein immunoprecipitation experiment verified the relationship between lncRNA H19 and miR-130a-3p.At the same time,the relative expression levels of miR-130a-3p in the above 50 breast cancer tissue samples and their corresponding adjacent normal breast tissues were detected by qRT-PCR,and the correlation between the expression levels of lncRNA H19 and miR-130a-3p in breast cancer patients was analyzed.MDA-MB-231 and MCF-7 breast cancer cell lines were transfected with lncRNA H19 siRNA,miR-130a-3p mimic,and miR-130a-3p inhibitor,respectively,and set as NC group,Mimics group,Inhibitor group,and si-H19+inhibitor group.The expression level of miR-130a-3p in the cells of the above groups was detected by qRT-PCR.The changes of cell proliferation,migration,invasion and apoptosis were evaluated by CCK8 assay,plate cloning assay,scratch assay,Transwell invasion assay and flow cytometry.Western blot was used to detect the expression of SATB1 protein in the cells of the above groups.Results The database screened out miR-130a-3p,miR-29c-3p,miR-29b-3p and miR-454-3p as possible downstream targets of lncRNA H19.The relative expression levels of miR-130a-3p in human breast cancer cell lines MDA-MB-231(0.453±0.040)and MCF-7(0.430±0.030)were significantly lower than those in human normal breast epithelial cell lines(1.023±0.051),p<0.01.Next,the results of double luciferase reporter gene experiment showed that in MDA-MB-231/MCF-7 cells,the relative luciferase activity of H19-wt+miR-130a-3p mimic(0.400±0.051)/(0.502±0.072)was significantly lower than that of H19-wt+miR-NC group(1.000±0.102)/(1.000±0.101),p<0.01;while the relative luciferase activity of H19-mut+miR-130a-3p mimic group was not significantly different from that of H19-mut+miR-NC group.The results of the RNA-binding protein immunoprecipitation experiment showed that,compared with the IgG group(0.976±0.153)/(1.002±0.082),both lncRNA H19(6.661±0.307)and miR-130a-3p(6.802±0.195)in the Ago2 antibody enriched RNA Significantly increased,p<0.01.The relative expression level of miR-130a-3p in breast cancer tissue(0.501±0.153)was significantly lower than that in adjacent normal breast tissue(1.090±0.232),p<0.01.The expression levels of lncRNA H19 and miR-130a-3p in breast cancer tissues were negatively correlated,r2=0.6241,p<0.01.In the transfected MDA-MB-231/MCF-7 cell line,compared with NC group(1.050±0.087)/(1.047±0.081),mimics group(3.583±0.112)/(3.553±0.146)significantly increased the expression of miR-130a-3p,p<0.01;Inhibitor group(0.267±0.029)/(0.277±0.038)significantly decreased the expression of miR-130a-3p,P<0.01;The expression level of miR-130a-3p in si-H19+inhibitor group(1.070±0.095)/(1.060±0.095)changed little.The results of CCK-8 showed that compared with NC group(1.272±0.072)/(1.123±0.074),the OD value of MDA-MB-231/MCF-7 cells(0.721±0.073)/(0.612±0.062)decreased significantly after up regulating miR-130a-3p,p<0.05;the OD value of MDA-MB-231/MCF-7 cells(1.650±0.081)/(1.515±0.079)increased significantly after inhibiting miR-130a-3p,P<0.05;the co-transfection of miR-130a-3p inhibitor and si-H19(1.312±0.074)/(1.213±0.072)had no significant change.The results of plate colony formation experiments showed that compared with NC group(126.333±8.145)/(125.000±8.888),the clone forming ability of MDA-MB-231/MCF-7 cells(46.333±6.807)/(44.000±5.292)was significantly decreased after up regulating miR-130a-3p,p<0.01;the clone forming ability of MDA-MB-231/MCF-7 cells(281.667±8.505)/(284.000±14.422)was significantly enhanced after inhibiting miR-130a-3p,P<0.01;the co-transfection of miR-130a-3p inhibitor and si-H19(132.333±6.506)/(131.667±7.638)had no significant change.The results of scratch experiment showed that compared with NC group(46.000±2.646)/(44.667±2.887),the scratch repair ability of MDA-MB-231/MCF-7 cells(22.333±1.528)/(22.667±2.082)was significantly decreased after up regulating miR-130a-3p,p<0.01;the scratch repair ability of MDA-MB-231/MCF-7 cells(80.000±3)/(79.000±2.646)was significantly improved after inhibiting miR-130a-3p,p<0.01;the co-transfection of miR-130a-3p inhibitor and si-H19(42.333±2.082)/(40.667±1.528)had no significant change.The results of Transwell invasion assay showed that compared with NC group(120.000±10.000)/(118.000±8.000),the number of MDA-MB-231/MCF-7 cells(74.667±8.083)/(72.000±8.660)passing through Matrigel-coated matrigel was significantly decreased after up regulating miR-130a-3p,p<0.01;the number of MDA-MB-231/MCF-7 cells(255.333士16.197)/(244.000±13.892)passing through Matrigel-coated matrigel was significantly increased after inhibiting miR-130a-3p,p<0.01;the co-transfection of miR-130a-3p inhibitor and si-H19(124.667±7.371)/(124.333±11.150)had no significant change.The results of flow cytometry experiments showed that compared with the NC group(8.530±0.501)/(8.331±0.735),the apoptosis rate of MDA-MB-231/MCF-7 cells(25.492±1.683)/(24.922±0.804)was significantly increased after up regulating miR-130a-3p,p<0.01;The apoptosis rate of MDA-MB-231/MCF-7 cells(4.491 士0.342)/(4.211±0.350)was significantly decreased after inhibiting miR-130a-3p,P<0.01;the co-transfection of miR-130a-3p inhibitor and si-H19(8.163±0.721)/(8.921±0.686)had no significant change.The database screened out SATB1 as the target gene of miR-130a-3p.The results of double luciferase reporter gene experiment showed that in MDA-MB-231/MCF-7 cells,the relative luciferase activity of SATB1-wt+miR-130a-3p mimic(0.462±0.063)/(0.625±0.065)was significantly lower than that of SATB1-wt+miR-NC group(1.000±0.091)/(1.000±0.093),p<0.01;while the relative luciferase activity of SATB1-mut+miR-130a-3p mimic group was not significantly different from that of SATB1-mut+miR-NC group.In the transfected MDA-MB-231/MCF-7 cell line,WB experiment showed that compared with NC group(1.023±0.051)/(1.013±0.052),the expression of SATB1 protein was significantly inhibited by knocking down lncRNA H19(0.560±0.078)/(0.600±0.075)or up regulating miR-130a-3p(0.527±0.038)/(0.617±0.091),p<0.01,and the expression level of SATB1 protein was increased after inhibiting miR-130a-3p(1.753±0.067)/(1.350±0.087),p<0.01,the co-transfection of miR-130a-3p inhibitor and si-H19(0.970±0.035)/(0.973±0.043)had no significant change.Conclusion The expression of miR-130a-3p is down-regulated in breast cancer cells and tissues.LncRNA H19 regulates the expression of SATB1 in breast cancer cells by competitively binding miR-130a-3p to promote breast cancer cell proliferation,migration and invasion,and inhibit apoptosis.Part Ⅳ Effects of lncRNA H19 on the growth of breast cancer cells in nude mice xenograftsObjective To observe the effect of lncRNA H19 on the proliferation of breast cancer cells in nude mice xenografts.Methods A nude mouse model of subcutaneously transplanted tumor was established,and the changes of tumor volume were measured regularly.Five weeks after inoculation,all nude mice were euthanized and subcutaneous xenografts were collected and weighed.The expression of Ki-67 index in nude mice tumor was detected by immunohistochemistry.Results After knockdown of lncRNA H19,the growth rate of subcutaneous tumors in nude mice in si-H19 group(503.000±47.733)was significantly lower than that in control group(1287.333±125.961),p<0.05.The weight of subcutaneous xenografts in the si-H19 group(0.492±0.046)was significantly lower than that in the control group(1.431±0.137),p<0.05.The results of immunohistochemistry showed that compared with the NC group(80.000±5.000),the number of Ki67 positive cells in the si-H19 group(36.667±2.887)decreased significantly,p<0.01.Conclusion Knockdown of lncRNA H19 can inhibit the growth of breast cancer xenografts in nude mice. |
PDF Full Text Request