The Study Of The Diagnostic Value And Molecular Mechanism Of Triple Negative Breast Cancer Specific Serum-based Biomarker LncRNA T376626

ObjectiveWe aimed to explore the serum expression,diagnostic value and molecular mechanism of lncRNA T376626 in TNBC.Methods1.LncRNA array was used for 3 cases of Luminal A subtype,3 cases of Luminal B subtype,3 cases of Her-2 overexpression subtype,3 cases of TNBC subtype and 3 cases of healthy serums,to profile serum-based TNBC differentially expressed lncRNAs.2.Screening and validating the differentially expressed lncRNAs with qPCR in 134 serum samples（include 28 cases of Luminal A subtype,27 cases of Luminal B subtype,21 cases of Her-2 overexpression subtype,15 cases of TNBC subtype,43 cases of healthy controls）.3.The diagnostic value of lncRNA T376626 was determined by the receiver operating characteristic（ROC）curve and the stability of serum lncRNA T376626 was investigated.The relationship between lncRNA T376626 and clinicopathological characteristics,as well as,the relationship between lncRNA T376626 and the expression of CEA,CA15-3,and CA-125 were analyzed.4.The intracellular location of lncRNA T376626 was clarified by FISH.The characteristics of lncRNA T376626 were analyzed with RNAfold,UCSC database,PhyloCSF and ORFfinder.5.The effect of hypoxia and.serum deprivation on the expression of serum lncRNA T376626 in three TNBC cell lines was revealed.6.Gain-and loss-of-function experiments were performed with siRNA and overexpressed plasmid in cells.CCK8 assay,colon forming assay,wound healing assay and transwell assay were performed to investigate the biological function of lncRNA T376626.7.The protein level expressions of cell apoptosis and cell cycle-related factors in the overexpressed/knockdown lncRNA T376626 MDA-MB-231 cells were detected by western blot,which may revealing lncRNA T376626’s possible mechanism involved in the promotion of TNBC cell proliferation.8.RNA pulldown and mass spectrometry were used to detect the possible RNA-binding proteins of lncRNA T376626.GO analysis and KEGG pathway analysis were performed to further reveal the potential molecular mechanism of lncRNA T376626 involved in the promotion of TNBC cell proliferation and metastasis.Results1.A total of eight TNBC differentially expressed serum lncRNAs（T376626,T342620,T279792,T096933,T273036,T220927,TCONS₀0015540,T127589）were screened out from five groups.2.In the validation stage,five differentially expressed lncRNAs（T220927,TCONS₀0015540,T127589,T273036,T279792）were excluded because their expression patterns were inconsistent with the results of lncRNA array.The expression of the remaining three lncRNAs（T376626,T342620,T096933）were significantly increased in the serum of patients in TNBC group,decreased in the serum of patients in non-TNBC group,and the lowest in healthy control group（P<0.05）.We ranked the three lncRNA according to the expression foldchange in the original data of lncRNA array.Finally lncRNA T376626 was chosen for further research.3.Stability experiment result showed that different temperature for different period,or repeated freezing thaws had small effect on the expression of lncRNA T376626.Serum lncRNA T376626 expression level was related to medullary carcinoma,ER status negative,PR status negative and Her-2 status negative（P<0.05）.Compared with patients with invasive ductal carcinoma,the expression level of serum lncRNA T376626 in patients with invasive medullary carcinoma was increased.There was no significant correlation between the expression of serum lncRNA T376626 and the expression of serum CEA,CA15-3,and CA-125（P>0.05）.When the three biomarkers were used to detect TNBC separately,lncRNA T376626 had the best sensitivity and specificity,which was 80%and 74.4%,respectively,with an AUC of 0.758（95%CI（0.616～0.900））.The AUC of lncRNA T376626 combined with CEA and CA15-3 to detect TNBC were 0.769（0.633-0.906）and 0.785（0.747-0.986）.When lncRNA T376626 combined with CA15-3 to detect TNBC,the sensitivity was the highest（86.7%）.4.UCSC database,PhyloCSF,ORFfinder analysis results showed that lncRNA T376626 may be not able to encode proteins.FISH results showed that lncRNA T376626 was localized in predominantly in cytoplasm,with some localization in the nuclear.Hypoxia conditions could promote the secretion of extracellular lncRNA T376626.5.CCK8 assay and colon forming assay results showed that overexpressed lncRNA T376626 promoted TNBC cells proliferation,while knockdown of lncRNA T376626 inhibited TNBC cells proliferation.Wound healing assay and transwell migration assay results showed that overexpression of lncRNA T376626 significantly increased the migratory ability of TNBC cells（P<0.05）,while knockdown of lncRNA T376626 decreased the migratory ability of TNBC cells（P<0.05）.Transwell invasion assay results showed that overexpressed lncRNA T376626 significantly promoted TNBC cells invasion（P<0.05）,however,knockdown of lncRNA T376626 inhibited TNBC cells invasion（P<0.05）.6.Western blot result found that overexpressed lncRNA T376626 could promoted the expression of Bcl-2,on the contrary,knockdown lncRNA T376626 inhibited the expression of Bcl-2.Overexpression of lncRNA T376626 increased the expression of three anti-cell cycle factors P18,P21 and P27.Knockdown lncRNA T376626 decreased the expression of them.7.RNA pulldown and mass spectrometry analysis identified 131 possible RNA-binding proteins of lncRNA T376626.GO analysis and KEGG pathway analysis found that lncRNA T376626-binding proteins were mainly enriched in biological processes such as Mrna splicing,cell adhesion and RNA alternative splicing.Conclusion1.LncRNA T376626 is a TNBC serum-based lncRNA,it highly expresses in TNBC patients’serum.2.LncRNA T376626 exists stably in serum and it sets a high diagnostic value on TNBC.3.LncRNA T376626 promotes the TNBC cells proliferation,migration and invasion.Further experiments find that lncRNA T376626 may affect TNBC proliferation via regulating the expression of cell apoptosis and cell cycle-related factors.