Silencing LncRNA-ATB Targeting MiR-141-3p Inhibits Metastasis And Invasion Of Breast Cancer

Background: Breast cancer(BC)is a kind of malignant tumor derived from breast tissue.It is one of the most common malignant tumors among women in the world and it is the second killer of female death.Many factors have a potential link with the occurrence of breast cancer,including age,weight,use of hormones,and smoking.In our country,with the continuous improvement of people’s living standards,the number of patients suffering from breast cancer has also shown an upward trend year by year.Therefore,in order to reduce the mortality rate of breast cancer patients,to understand the pathogenesis of breast cancer,and actively explore the treatment methods to inhibit the occurrence and development of breast cancer,so as to improve the treatment efficiency of breast cancer patients has a profound theoretical and practical significance.Epithelial-mesenchymal transition(EMT)refers to the process by which epithelial cells lose polarity and acquire the phenotype of mesenchymal cells.It affects embryonic development,wound healing and tumor metastasis by transforming differentiated epithelial cells into undifferentiated mesenchymal cells capable of migrating.It is an important step in the ability of tumor cells to acquire invasion and migration.When tumor cells invade and metastasize,EMT can induce tumor cells to acquire stem cell characteristics such as anti-apoptosis,immune exemption,and so on,thereby promoting tumor invasion and metastasis,adding difficulty for treatment.Therefore,exploring new targets for the inhibition of EMT and its underlying molecular mechanisms contribute to the treatment of breast cancer.As more and more long non coding RNAs(LncRNAs)have been discovered,the research on LncRNAs and tumors has received increasing attention from experts and scholars.LncRNAs are a subclass of non-coding RNAs,generally more than 200 nt in length,with a relatively conserved secondary structure but lacking protein coding ability.The data shows that LncRNA has stronger tissue-specificity and cell-specificity during life activities and can influence biological behaviors by regulating mRNA and protein expression levels.Studies have shown that in colorectal cancer cells,upregulation of LncRNA-PVT1 can significantly inhibit the proliferation and invasion of colorectal cancer cells.In addition,in pituitary adenoma cells,through upregulation of E2F1,LncRNA-CCAT2 can be activated and interact with PTTG1,thereby playing an important regulatory role in the development of pituitary adenomas.In breast cancer,LncRNA also has an important regulatory role.In addition,in pituitary adenoma cells,through upregulation of E2F1,LncRNA-CCAT2 can be activated and interact with PTTG1,thereby playing an important regulatory role in the development of pituitary adenomas.In breast cancer,LncRNA also has an important regulatory role.Transforming growth factor β(TGF-β)-activated LncRNA(LncR-ATB),as a member of the LncRNA family,plays an important regulatory role in the development of tumors.LncRNA-ATB has been shown to be abnormally expressed in a variety of tumor cells.Studies have shown that there is a high expression of LncRNA-ATB in colorectal cancer patients.LncRNA-ATB is high expressed in clinical samples of cervical cancer tissues and cervical cancer cells.In gastric cancer cells,LncRNA-ATB can promote the proliferation,migration and invasion of gastric cancer cells.In addition,some of studies have found that LncRNA-ATB also plays an important regulatory role in breast cancer cells.According to related literature reports,LncRNA-ATB promotes the metastasis of breast cancer cells through the regulation of epithelial-mesenchymal transition(EMT)-related factors,but whether this regulation is related to miRNAs is rarely reported.MiRNA is a type of endogenous non-coding RNA that is about 18-25 nucleotides in length.It is well known that miRNAs can regulate the expression of many target mRNAs at the same time,thus exerting regulatory effects on cell proliferation,differentiation,tumor metastasis,and tumor angiogenesis.Nowadays,the application potential of miRNA as a new antitumor therapeutic target has been recognized by more and more scholars.The study found that the presence of miR-150-5p,miR-222-3p,and miR-423-5p increased in patients with oral cancer.In many cancer patients,miR-448 can target and regulate ROCK1,and then influence the occurrence and development of tumors through PI3K/AKT.In breast cancer,miRNA also has an important regulatory role.The study found that in breast cancer patients,there are a variety of abnormal expression of miRNAs,including miR-548c-5p,miR-7-5p,miR-210-3p,and miR-128-3p.MiR-664 inhibits proliferation and invasion of breast cancer cells through targeted regulation of IRS1.In addition,studies have shown that miR-577 targets inhibition of Rab25,which in turn inhibits the expression of EMT-related factors,and plays an important regulatory role in the development of breast cancer.MiR-141-3p is a member of the miRNA family and closely related to the occurrence and development of tumors.According to related literature,miR-141-3p can promote prostate cancer cell proliferation by inhibiting KLF9 expression.MiR-141-3p also plays an important role in the chemotherapy resistance of esophageal cancer.In addition,in breast cancer cells,miR-141-3p plays an important regulatory role in the migration and invasion of breast cancer cells through targeted regulation of ANP32 E.In vivo experiments,the regulation of miR-141-3p on the development of breast cancer has also been confirmed.However,little is known about whether miR-141-3p plays an important regulatory role in the development of breast cancer through EMT.Recent studies have found that,in addition to being directly involved in the regulation of gene expression,LncRNA can also be used as a ceRNA to compete with other RNA transcripts for the same miRNA,thereby achieving mutual exchange and regulation.Studies have shown that LncRNA-SNG1 can act as a molecular sponge of miR-145-5p,inhibit the expression of miR-145-5p,and then up-regulate the target gene MTGH of miR-145-5p,ultimately affecting the development of non-small cell lung cancer.In prostate cancer cells,LncRNA-PVT1 inhibits the expression of miR-186,which in turn regulates the expression of EMT-related factors and promotes the invasion of prostate cancer cells.In breast cancer cells,LncRNA-SNHG15 can promote the proliferation,migration and invasion of breast cancer cells by inhibiting the expression of miR-211-3p.In addition,LncRNA-H19 can play an important regulatory role in the development of breast cancer cells through the process of EMT and MET as the "molecular sponge" of miR-200b/c and let-7b.However,whether LncRNA-ATB acts as a ceRNA has been reported rarely through the competitive inhibition of miR-141-3p,thereby affecting the invasion and metastasis of breast cancer cells.This study examined the expression of LncRNA-ATB and miR-141-3p in normal breast cells and breast cancer cells and analyzed the association of LncRNA ATB and miR-141-3p with breast cancer.Furthermore,the effects of LncRNA-ATB and miR-141-3p on invasion and EMT of breast cancer cells were demonstrated by cell transfection,lentivirus infection,transwell,Real-time PCR and Western blot techniques.Finally,the interaction of miR-141-3p with target genes,the EMT-related factors ZEB1 and ZEB2,was verified by dual luciferase reporter gene technology and Western blot technique.The effects of LncRNA-ATB/miR-141-3p on the invasion and metastasis of breast cancer cells and related molecular mechanisms were described,which provided new targets and new technologies for the clinical treatment of breast cancer.It has important theoretical and practical significance.Method: Real-time PCR was used to detect the relative expression of LncRNA-ATB and miR-141-3p in human breast cells MCF10 A and human breast cancer cells MDA-MB-231 and BT549.LncRNA-ATB low expression human breast cancer stable cell line was constructed by transient transfection technique and on this basis miR-141-3p inhibitor was transfected.Real-time PCR was used to detect the expression of LncRNA-ATB and miR-141-3p in LncRNA-ATB-low expressing human breast cancer cell lines.Transwell technique was used to detect the effect of LncRNA-ATB and miR-141-3p on the invasive ability of breast cancer cells.Changes in expression of EMT-related factors wasdetected by Real-time PCR and Western blot.The targeted regulation of miR-141-3p on EMT-related factors ZEB1 and ZEB2 was detected by dual luciferase assay.The effect of miR-141-3p on the expression of EMT-related proteins ZEB1 and ZEB2 was detected and analyzed by Western blot.Result:Compared with normal breast epithelial cells,the relative expression of LncRNA-ATB was significantly increased in breast cancer cells MDA-MB-231 and BT549,and the relative expression of miR-141-3p was significantly reduced.Compared with the negative control infected cells,the relative expression of LncRNA-ATB in LncRNA-ATB-low expressing human breast cancer transfected cells was significantly decreased,and the relative expression of miR-141-3p was significantly increased.Compared with the negative control infected cells,the LncRNA-ATB low expression of breast cancer cell invasion ability was significantly reduced,and the relative expression levels of the EMT-related factor E-cadherin mRNA and protein levels were significantly increased,EMT-related factor N-cadherin,vimentin,ZEB1,ZEB2 were significantly reduced.Compared with LncRNA-ATB-low expressing human breast cancer transfected cells,the invasive ability of LncRNA-ATB low-expressing human breast cancer transfected cells was significantly increased after transfection of miR-141-3p inhibitor,EMT-related factor E-cadherin mRNA The relative expression levels of protein and protein were significantly decreased,and the relative expression levels of mRNA and protein levels of EMT-related factors N-cadherin,vimentin,ZEB1,and ZEB2 were significantly increased.MiR-141-3p has targeted regulation of EMT related factors ZEB1 and ZEB2,and miR-141-3p inhibits the protein levels of EMT related factors ZEB1 and ZEB2.Conclusion:LncRNA-ATB and miR-141-3p are abnormal expressedin breast cancer cells.LncRNA ATB competitively inhibits the expression of miR-141-3p and promotes the invasion and metastasis of breast cancer cells.LncRNA ATB competitively inhibits the expression of miR-141-3p and regulates the mRNA and protein levels of EMT-related factors E-cadherin,N-cadherin,vimentin,ZEB1 and ZEB2.MiR-141-3p targets the inhibition of mRNA and protein of EMT-related factors ZEB1 and ZEB2.