Silencing Scamp1-TV2 Inhibited The Malignant Biological Behaviors Of Breast Cancer Cells By Interaction With PUM2 To Facilitate INSM1 MRNA Degradation

Objective:Breast cancer is one of the most common malignancies,and also one of the cancer killers endangered all women in the world.At present,molecular targeted therapy plays an important role in the comprehensive treatment of breast cancer.However,the 5-year survival period of some breast cancer patients has not improved significantly,especially the three negative breast cancer patients.It is very important for the selection of breast cancer treatment strategies to explore the mechanism of breast cancer development.More studies have shown that long-noncoding RNAs play an important role in the evolution of a variety of tumors,including breast cancer.Long noncoding RNAs(lncRNAs),which are a novel class of transcripts with the size than 200 nucleotidesare involved in the occurrence and development of many kinds of tumors,as well as the role of oncogenes or tumor suppressor genes.As a transcript of SCAMP1 gene,lncRNA SCAMP1-TV2(Homo sapiens secretary carrier membraneprotein 1,transcript variant 2,genebank,NR 110885.1)cannot be translated into protein,and the expression and effects of SCAMP 1-TV2 in breast cancer have notbeen reportedRNA binding proteins(RBPs)can bind to hnRNAs so as to playan important role in the process of gene regulation.Most RBPs interact with target RNA molecules in various post-translational stages,including the alternative splicing,mRNA stabilization,localization,translation and degradation.Therefore,the research on the interaction between RNA and protein is the key to explore the function of RNA.PUM2(pumilio RNA binding family member 2)belongs to a PUF family of RBPs.PUM2 can bind with 750 unique mRNA targets in humans,and plays a post-transcriptional regulatory role in myeloid leukemia and bladder cancer.However,the expression and role played by PUM2 in breast cancer have not yet been reported.LncRNAs serve as a“molecular sponge”or“molecular scaffold”for RBP to regulate the expression ofdownstream genes.Based on the prediction with thebioinformatics software in this study,there was a binding site between SCAMP1-TV2and PUM2,which indicates that SCAMP1-TV2 may play its biological role bybinding with PUM2Transcription factors(TFs)are a type of protein molecules that can specifically bind to a specific sequence upstream of the 5 'end of a gene,thereby ensuring that the target gene is expressed at a specific time and space with a specific intensity.The cis-acting element can achieve covalent binding with the DNA binding domain of the transcription factor,thereby inhibiting or enhancing gene expression.Human insulinoma-associated 1(INSM1)gene is located at chromosome20p11.2,and consists of a 2838 base pair sequence containing an open reading frame of 1530 nucleotides,encoding 510 amino acids.Studies have shown thatINSMl is highly expressed in medullary thyroid carcinoma,small celllung cancer,and cervical carcinoma,and can regulate the biological behaviors oftumor cells.At present,the expression and potential regulatory effects of INSM1 in breast cancer currentlyremains unclear.Further,PUM2 can bind with mRNA3'-UTR of downstream molecules in a targeted way to promote their degradation andthus inhibit the gene expression.Based on theprediction with thebioinformatics software in this study,there was a binding site between the 3'-UTRof INSM1 mRNAand PUM2,which indicates that INSM1 may play itsbiological role bybinding with PUM2SASH1 has been regarded as a tumour suppresser in various type of tumors,such as lung cancer,gastric cancer and hepatocellular carcinoma.Some reports suggested that SASH1 blocks the malignantbiological behaviors of breast cancer cells through the inhibition of PI3K/AKTsignaling pathway.SASH1 expression was decreased in 74%of mammary tumors in comparison with normalmammary epithelial tissues.However,the role played by SAAH1 in breast cancer have not yet been reportedIn this study,the endogenous expression of SCAMP1-TV2,PUM2,INSMland SASH1 in breast cancer tissues and cells was determined.Then,further investigation was done on the relationship between these molecules and their effects on the biological behaviors of breast cancer cells,so as to reveal the novel mechanismfor the morbidity and progress of breast cancer,and offer another therapy for curing the breast cancerMethods:1.The endogenous expression levels of SCAMP1-TV2 was detected in breast cancer tissues,MCF-7 and MDA-MB-231 cells byqRT-PCR.2.The endogenousexpression levels of PUM2and INSM1were detected in breast cancer tissues,MCF-7 and MDA-MB-231 cells by IHC,qRT-PCR and Western blot.3.The effects of SCAMP1-TV2,PUM2,INSM1 and SASH1 on the apoptosis of MCF-7 and MDA-MB-231 cells were assessed by flow cytometry.4.The stable transfected MCF-7 and MDA-MB-231 cells of sh-SCAMP1-TV2,sh-PUM2,sh-INSM1,sh-SASH1 and over-expression of PUM2,INSM1 and SASH1 were established,and the changes in the biologicalbehaviors of cells were detected by Transwell and CCK-8 cell proliferation experiment.5.The relationship ofSCAMPl-TV2,PUM2 and INSM1was confirmed by RIP and pull-down experiments.6.The relationship of INSM1 and SASH1 promoter regionwas confirmed by dual-luciferase reporter assay and ChIP.7.The levels of p-PI3K/PI3K andp-AKT/AKT were detected inover-expression of SASH1 or sh-SASH1 cells byWestern blot.8.Tumour growth was verified by the silencing of SCAMP1-TV2 incombination with the over-expression of PUM2by the xenograft tumor experiment in nude mice.Results:1.The expression of SCAMP 1-TV2 was significantly increased in luminal A,triple negative breast cancer tissues by qRT-PCR.The expression of SCAMP1-TV2 was significantly increased in MCF-7 cells and MDA-MB-231 cells by qRT-PCR.The viability,migration and invasion of sh-SCAMP1-TV2 cells weresignificantly decreased,whereas the apoptosis was markedly increased.2.The expression of PUM2was significantly decreased in luminal A,triple negative breast cancer tissues by IHC and qRT-PCR.The expression of PUM2was significantly decreased inMCF-7 cells and MDA-MB-231 cells byqRT-PCR and Western blot.The result of Transwell and CCK-8 cell proliferation experiment showed thatthe viability,migration and invasion of sh-INSM1 cells were markedly increased.Additionally,the results of flow cytometry confirmed that the apoptosis of sh-INSM1 cells was significantly decreased.The opposite results wereobserved in over-expression of INSM1cells.RIP assayandRNA pull-down assayvalidated the interaction between SCAMP1-TV2 and PUM2.3.The expression of INSM1 was significantly increased in luminal A,triple negative breast cancer tissues,by IHC and qRT-PCR.The expression of INSM1 was significantly increased inMCF-7 cells and MDA-MB-231 cells byqRT-PCR and Western blot.The result of Transwell and CCK-8 cell proliferation experiment showed thatthe viability,migration and invasion of sh-INSM1 cells weresignificantly decreased.Additionally,the results of flow cytometry confirmed that the apoptosis of sh-INSM1 cells was markedlyincreased.The opposite results wereobserved in over-expression of INSM1 cells.4.The result of qRT-PCR and Western blot showed that Silencing SCAMP1-TV2 reduced its binding to PUM2,and further increased the binding of PUM2 to INSM1 mRNA.And this decreased the expression of INSM1 mRNA,which down-regulates the expression of INSM1.And the inhibitory effects ofPUM2 over-expression and the promotion of INSMlover-expression on the malignant biological behaviors of breast cancer cells was reversed by PUM2 over-expression combined with INSM1 over-expression by Transwell and CCK-8 cell proliferation experiment.5.Both the mRNA and protein expression levels of SASH1 were significantly down-regulated in over-expression of INSM1 cells by qRT-PCR and Western blot.The opposite results were observed in sh-INSM1 cells CHIP assays and dual-luciferase validated that INSM1 binding site was found inthe promoter region of SASH1.The viability,migration and invasion of over-expression of SASH1 cells were significantly decreased by Transwell and CCK-8 cell proliferation experiment.Additionally,the results of flow cytometry confirmed that the apoptosis of over-expression of SASH1cells was markedly increased.The levels of p-PI3K/PI3K and p-AKT/AKT in over-expression of SASH1 cells were both significantly reduced,thereby inhibiting the activity of PI3K/AKTsignaling pathway by Western blot.6.The xenograft tumor experiment showed that theweight and size of xenograft tumors were both significantly reduced in SCAMP1-TV2 silencing group,over-expression of PUM2 group and SCAMP1-TV2 silencing in combination with PUM2 over-expression group,and SCAMP1-TV2 silencing in combination with PUM2 over-expression grouphad the smallest tumorsConclusion:1.The expression of SCAMP1-TV2 and INSM1 were significantly increased in MCF-7 cells and MDA-MB-231 cells,while PUM2 was decreased.The viability,migration and invasion of sh-SCAMP1-TV2,sh-INSM1 cells or over-expression of PUM2weresignificantly decreased2.SCAMP1-TV2 promoted the malignant biological behaviors of breast cancer cells by inhibiting its binding to PUM2,and further decreasing the binding of PUM2 to INSM1 mRNA.And this increased the expression of INSM1 mRNA,which up-regulates the expression of INSM1.INSM1 bound to the predicted binding site in the SASH 1 promoter,and transcriptionally inhibited SASH1 expression3.Silencing SCAMP1-TV2 inhibited the malignant biological behaviors of breast cancer cells by reducing its binding to PUM2,and further increasing the binding of PUM2 to INSM1 mRNA.And this decreased the expression of INSM1 mRNA,which down-regulates the expression of INSM1.Silencing INSM1 transcriptionally promoted SASH1 expression.And SASH1 over-expression inhibited the malignant biological behaviors of breastcancer cells by inhibiting the activity of PI3K/AKT signaling pathway4.Theweight and size of xenograft tumors were both significantly reduced in SCAMP1-TV2 silencing group,over-expression of PUM2 group and SCAMP1-TV2 silencing in combination with PUM2 over-expression group,and SCAMP1-TV2 silencing in combination with PUM2 over-expression grouphad the smallest tumors.