The Mechanism Of LncRNA SNHG16 Contributing To Breast Cancer Cell Migration

Breast cancer is the most commonly diagnosed cancer and the leading cause of cancer death among women younger than 45 years in China,accounting for 15%of all new cancers among females in 2015.In addition,there is a significant upward trend in incidence and mortality rates of breast cancer.Morbidity and mortality in patients with solid tumors always result from the disruption of normal biological function by disseminating tumor cells,and tumor cell migration has been intensely investigated as the underlying cause of cancer metastasis.However,the molecular mechanisms which mediate breast cancer cell migration remain largely unclear.Thus,it’s important to find key molecules and pathways participating in the development of breast cancer,which will conduct early diagnosis and therapy and will further improve the prognosis.The long noncoding RNAs(IncRNAs)are a category of noncoding RNAs with over 200 nucleotides that do not encode for proteins,and the emerging impact of IncRNAs in cancer research has been discovered in prevalent cancer types.Through diverse mechanisms,lncRNAs play critical roles as drivers of tumor suppressive and oncogenic functions.Modes of direct post-transcriptional interaction among lncRNAs and miRNAs include miRNA-triggered lncRNA decay,lncRNA as miRNA sponge/decoy,and IncRNA generating miRNAs.Small nucleolar RNA host gene 16(SNHG16),also known as non-coding RNA expressed in aggressive neuroblastoma(ncRAN),was newly identified as a potential oncogene in colorectal cancer,neuroblastoma and bladder cancer.However,whether SNHG16 contributes to the progression of breast cancer and the underlying mechanism remains poorly understood.Thus,our current study aims to investigate the function of SNHG16 in breast cancer cell proliferation and migration and the molecular mechanism which includes the four following parts:1.Expression of SNHG16 in breast cancer tissues and its function on breast cancer cell proliferation and migration;2.SNHG16 is predicted to be ceRNA of E2F5 by directly binding miR-98;3.MiR-98 inhibits cell migration by targeting E2F5;4.The regulation of E2F5 and the promotion of breast cancer cell migration by SNHG16 in miR-98 dependent manner.Part I.Expression of SNHG16 in breast cancer tissues and its function on breast cancer cell proliferation and migrationOBJECTIVE:Recent studies showed that SNHG16 is upregulated and acts as an oncogene in neuroblastoma,bladder cancer and colorectal cancer.However,the function of SNHG16 in breast cancer remains largely unknown.The current study is to define the role of SNHG16 in breast cancer by evaluating SNHG16 expression level in breast cancer tissue samples.In vitro assay will be used to assess the impact of SNHG16 on breast cancer cell proliferation and migration.METHODS:We collected 30 pairs of cancerous tissues and adjacent noncancerous tissues and isolated RNA.QRT-PCR was used to detect the aberrant expression of SNHG16 in breast cancer tissues.Simultaneously,we examined the SNHG16 expression levels of four common breast cancer cells and chose the cells in which SNHG16 expressed at higher levels for knockdown assay,cells in which SNHG16 expressed at relatively low levels were selected for overexpression experiment.We then performed MTT assay to test the influence of SNHG16 on breast cancer cell proliferation.Transwell migration assay were used to examine the function of SNHG16 on cell migration.RESULTS:SNHG16 expressed at higher levels in cancerous tissues compared with adjacent noncancerous tissues in 21 out of 30 pairs of breast tissues.SNHG16 expression was measured in MDA-MB-231,MCF-7,MDA-MB-468 and SK-BR-3 cell lines,which was obviously higher in MDA-MB-231 and MCF-7 cells.Then MDA-MB-231 and MCF-7 cells were selected for SNHG16 knockdown by three siRNAs.Moreover,we constructed a plasmid containing SNHG16 sequence,and MDA-MB-468 and SK-BR-3 cells were used for overexpression experiment due to relatively low innate expression of SNHG16.SNHG16 knockdown significantly decreased the migration ability while overexpression of SNHG16 promoted cell migration noticeably.On the other hand,SNHG16 knockdown or overexpression had shown no significant effect on cell proliferation compared with controls.CONCLUSION:SNHG16 acts as an oncogene in breast cancer by promoting breast cancer cell migration,while SNHG16 has no significant influence on cell proliferation.Part Ⅱ.SNHG16 is predicted to be ceRNA of E2F5 by directly binding miR-98OBJECTIVE:Recent studies have suggested that lncRNA could act as a ceRNA to exert its regulatory functions.To further explore the underlying mechanism responsible for SNHG16 functions in breast cancer,we examined a set of mRNAs which were predicted to competitively bind miRNAs with SNHG16 by bioinformatic methods and proved the direct binding of SNHG16 and the downstream miRNA.Then the correlation of SNHG16 and the downstream miRNA in breast cancer tissues was verified.METHODS:We predicted the possible ceRNAs of SNHG16 and the common binding miRNAs by using the starBase,v2.0 program(http://starbase.sysu.edu.cn).Then the expression levels of candidate ceRNAs and miRNAs after knockdown or overexpression of SNHG16 were examined by qRT-PCR to further determine the key downstream molecules.DIANA TOOLS were used to predict the binding site of SNHG16 and the ceRNA with the common binding miRNA.We then constructed tool vectors,wildtype and mutant vectors to perform RNA immunoprecipitation(RIP)assay and dual luciferase reporter assay,validating the direct binding and specific binding site of SNHG16 and the downstream miRNA.Meanwhile,we examined the SNHG16 expression levels after overexpression of the candidate miRNA to further demonstrate the reciprocal repression of them.Then,the expression levels of SNHG16 and the candidate downstream miRNA in breast cancer tissues were tested using qRT-PCR and the correlation between them was analyzed.RESULTS:The expression profiles of possible targets including Rap2B,HMGA2,AMOT,SMAD2,E2F5,ZEB1,were explored.After transfection with siRNAs of SNHG16 or pcDNA3.1-SNHG16 vector,qRT-PCR detection revealed that SNHG16 knockdown or overexpression had no effect on the expression of candidate mRNAs except for E2F5.Subsequently,bioinformatics analysis using DIANA Tools suggested that there was a common binding site of miR-98 between SNHG16 and E2F5.The miR-98 expression was significantly increased after silencing of SNHG16 in MDA-MB-231 and MCF-7 cells,and were decreased after overexpression of SNHG16 in MDA-MB-468 and SK-BR-3 cells.RIP assay showed that SNHG16 was significantly enriched for miR-98 compared to the mutant and empty vector.Overexpression of miR-98 significantly reduced the luciferase activities of the WT reporter vector but not empty vector or mutant reporter vector.A significant suppression of SNHG16 expression was observed after miR-98 mimics were transfected into breast cancer cells.Furthermore,the negative association between miR-98 and SNHG16 expression was confirmed in 12 breast cancer tissues.CONCLUSION:There is a direct binding between SNHG16 and miR-98.SNHG16 may function as an oncogene by competitively binding miR-98 with E2F5.Part Ⅲ.MiR-98 inhibits cell migration by targeting E2F5OBJECTIVE:To further investigate whether SNHG16 promotes breast cancer cell migration through the miR98-E2F5 regulation axis,we examined the function of miR-98 and E2F5 in breast cancer and the targeting effect between them.METHODS:We performed Transwell migration assay after overexpression of miR-98.Based on the predicted binding site,we constructed a luciferase reporter vector containing 3’-UTR of E2F5 and performed dual luciferase reporter assay.The expression of E2F5 after miR-98 overexpression was examined by qRT-PCR and Western Blot.The function of E2F5 on cell migration was investigated by Transwell assay after E2F5 knockdown.Furthermore,we measured the RNA levels of E2F5 in breast cancer tissues and matched noncancerous tissues via qRT-PCR.RESULTS:Transwell migration assay showed that miR-98 overexpression significantly suppressed cell migration.We found that miR-98 inhibited luciferase activity of the E2F5 3’-UTR reporters but not empty vectors.Expectedly,miR-98 significantly suppressed the expression of E2F5 in RNA and protein levels in breast cancer cells.After silencing of E2F5,the number of migrated cells was significantly decreased,which mimicked the effect of miR-98 on breast cancer cells.Furthermore,we revealed that E2F5 expression was up-regulated in breast cancer tissues.CONCLUSION:MiR-98 can inhibit breast cancer cell migration by targeting E2F5.Part IV.The regulation of E2F5 and the promotion of breast cancer cell migration by SNHG16 in miR-98 dependent manner.OBJECTIVE:To demonstrate that SNHG16 promotes breast cancer cell migration by competitively binding miR-98 with E2F5,we further investigated the regulation of E2F5 by SNHG16 and proved whether this regulation and the influence of SNHG16 on breast cancer cell migration depend on miR-98.METHODS:The expression of E2F5 was examined by qRT-PCR and Western Blot after SNHG16 knockdown in MDA-MB-231 and MCF-7 cells or overexpression o.f SNHG16 in MDA-MB-468 and SK-BR-3 cells.We then detected the expression levels of SNHG16 and E2F5 in four breast cancer cell lines and 12 tissue samples and analyzed the correlation between them.Rescue assay were performed to determine whether miR-98 can reverse the promotion of breast cancer cell migration and the regulation of E2F5 expression by SNHG16.RESULTS:Knockdown of SNHG16 decreased E2F5 expression in RNA and protein levels in MDA-MB-231 and MCF-7 cells.Inversely,ectopic expression of SNHG16 resulted in the upregulation of E2F5 in MDA-MB-468 and SK-BR-3 cells.Cells with relatively high endogenous SNHG16 expression levels exhibited higher levels of E2F5 as determined by qRT-PCR,and vice versa.SNHG16 transcript levels were positively correlated with E2F5 mRNA levels in breast cancer tissues.For the rescue experiment,forced expression of miR-98 significantly abrogated SNHG16 overexpression and SNHG16-inducing upregulation of E2F5,strongly indicating that SNHG16 regulated E2F5 expression in a miR-98 dependent manner.We were interested whether restoration of miR-98 could reverse the SNHG16-mediated promotion of cell migration in breast cancer.The results showed that forced expression of miR-98 could significantly attenuate SNHG16-mediated promotion of migration in breast cancer cells.CONCLUSION:SNHG16 can positively regulate the expression of E2F5,in addition,this regulation and the promotion of breast cancer cell migration by SNHG16 is miR-98 dependent.Collectively,the study presented analysis of the role of SNHG16 in breast cancer progression and the underlying mechanism.The bullet points of the study are reflected in the following aspects:1.SNHG16 was first shown to work as a ceRNA by competitively binding miR-98 with E2F5.2.SNHG16 promotes breast cancer cell migration in a miR98-dependent manner.3.Direct combination of SNHG16 with miR-98 was proved using RIP and luciferase assay.4.The positive regulation of E2F5 by SNHG16 in breast cancer was revealed.The defects and the future directions of this study include:1.Our study mainly investigated the function of SNHG16 on breast cancer cell proliferation and migration,however,we didn’t research the effect of SNHG16 on breast cancer cell invasion,apoptosis and chemoresistance,which remain further research.2.The current study used breast cancer cell lines to investigate the influence of SNHG16 on breast cancer in vitro,while the function of SNHG16 in vivo needs to be proved by nude mice tumorigenesis and metastasis assay.3.In this study,we analyzed the expression difference and correlation of SNHG16 and downstream molecules only in breast cancer tissues and adjacent noncancerous tissues.However,we didn’t carry out comparative study with breast cancer classification indexes,which need further expandation of specimen quantity for a specific pathological classification analysis.