LncRNA SEMA3B-AS1 Inhibits Breast Cancer Progression By Targeting MiR-3940-3p/KLLN Axis

Background The most common cancer type,in females worldwide,is breast cancer(BC).The molecular mechanisms of BC pathogenesis have been extensively studied,resulting in the classification of BC into three main subtypes: Luminal with positive status for estrogen receptor(ER+)and progesterone receptors(PR+),human epidermal growth factor receptor 2(HER2+),and basal-like tumors,which lacks hormone receptor and HER2 expression,called triple-negative breast cancer(TNBC).TNBC accounts for 10%-15% of all BC in the United States and is more common in women of African descent and women<50 years of age.Increasing published studies illustrated that LncRNAs act as competing endogenous RNAs(ce RNAs)involved in tumorigenesis by acting as sponges for micro RNA(miRNA).Long noncoding RNAs(lnc RNAs)play a crucial regulatory role in BC progression.In our study,we found that,by examining the lnc RNA data from The Cancer Genome Atlas(TCGA)database,the level of lnc RNA SEMA3B-AS1(SEAS1)in TNBC samples was significantly lower than that in adjacent tissues.ObjectiveTo explore the effect of SEAS1 on the proliferation,migration,invasion,and apoptosis of TNBC,and to further study the mechanism of SEAS1 in the carcinomas.MethodsThe research molecules(SEAS1)were screened out by the TCGA database,and the expression differences between cancer and adjacent cancer were determined by an online database and clinical samples.RT-q PCR was used to analyze the expression difference of SEAS1 in cell lines of BC.The localization of this molecule in cell localization was determined by FISH.The clinicopathological characteristics of patients corresponding to the samples were collected,and the correlation between SEAS1 and clinicopathological characteristics was analyzed by chi-square test.The expression of SEAS1 was knocked down by si RNA in TNBC cells.The effects of SEAS1 on the proliferation,migration,invasion,and apoptosis of TNBC were verified by CCK8 assay,colony formation assay,Ed U assay,flow cytometry,and Transwell assay.The BC cell line stably overexpressing SEAS1 was constructed by lentivirus transfection.The subcutaneous xenotransplantation model was carried out to verify the biological function of SEAS1 in vivo.Using online database DIANA Tools to predict the miRNA downstream of SEAS1,RT-q PCR confirmed the downstream molecule miR-3940-3p.The expression of pri-miR-3940-3p and pre-miR-3940-3p were assessed after SEAS1 knockdown.RNA immunoprecipitation(RIP)assay and luciferase assay verified the interaction between SEAS1 and miR-3940-3p.The effects of miR-3940-3p overexpression and knockdown on the proliferation,migration,invasion,and apoptosis of TNBC were evaluated.The rescue assay was conducted to illustrate that miR-3940-3p was involved in the development of TNBC as the downstream of SEAS1.miR-3940-3p target genes were screened by online databases miRDB,miRPath DB,miRBase,and Target Scan.The expression levels of miR-3940-3p target genes in MDA-MB-231,BT-549,and MCF10 A cells were analyzed by RT-q PCR.The binding site of miR-3940-3p to its target gene was predicted by miRWalk database,and its binding was verified by RIP assay and luciferase assay.Knockdown of miR-3940-3p and knockdown of its target gene expression verified the effects on proliferation,migration,invasion,and apoptosis of TNBC.The products of RNA pulldown assay were analyzed by mass spectrometry,and a protein was obtained by crossing the mass spectrometry results with RNA binding proteins and transcription factors.The expression level of SEAS1 was observed by knocking down the protein.RIP assay and luciferase assay verified their binding.The binding of SMAD3 to SEAS1 promoter was predicted by the PROMO website and verified by chromatin immunoprecipitation(Ch IP)experiment.ResultsIn the present study,through transcriptomic profiling analysis of the TCGA database and further validated in a cohort of BC tissues,we identify a dysregulated LncRNA SEAS1 in TNBC.SEAS1 was downregulated in TNBC tissues compared with para-carcinoma tissue,which was associated with poor prognosis of TNBC patients.We further demonstrated that knockdown or overexpression of SEAS1 significantly enhanced or suppressed proliferation migration and invasion of TNBC cells in vivo and in vitro experiments.Mechanistically,RIP and luciferase reporter assay was performed to reveal that lnc RNA SEAS1 could act as a miRNA sponge to competitively bind with miR-3940-3p,preventing the degradation of its target gene KLLN,which acts as a tumor-inhibiter in BC.Moreover,through RNA pulldown assay and mass spectrometry analysis,we found a transcription factor SMAD3 that negatively regulated SEAS1,thus aggravating the progression of TNBC.Ch IP assay confirmed that SMAD3 binds to the SEAS1 promoter.The clinical samples of TNBC further confirmed SEAS1 was correlated with lymphatic metastasis and distant metastasis of TNBC.ConclusionsOur study revealed a novel pathway for breast cancer progression via SMAD3/lnc RNA SEAS1/miR-3940-3p/KLLN and suggest that SEAS1 may be a potential biomarker and therapeutic target for TNBC patients.