Identification Of Subtype Specific LincRNAs And The Function Study Of EGOT And EPB41L4A-AS2 In Breast Cancer

Background and PurposeBreast cancer is the most common cancer in women worldwide,and it is one of the main causes of death with complex pathogenesis.Generally,therapy regimen was performed according to the molecular subtype of breast cancer,while the molecular subtype was not perfect for clinical practice.Subtype specific long noncoding RNA(lncRNA)can be applied as marker for diagnosis and therapy and it can monitor the disease relapse,drug resistance,and prognosis prediction.The lncRNA studies involved in malignancy disease were accumulated,excepting the solid tumor breast cancer.By whole genome high throughput sequencing,we identified and investigated the subtype specific lncRNA,EGOT was aberrant expressed significantly in breast cancer and it is closely related to worse prognosis.Another purpose of this study was to investigate the expression of the EGOT and EPB41L4A-AS2 in invasive ductal breast carcinomas,to evaluate its relationship with clinicopathological characteristics and the role and function in breast cancer.MethodsWhole genome high throughput sequencing was performed for each subtype breast cancer of 33 samples.Subtype specific lincRNA was identified and investigated and a novel breast cancer classification system,termed as linctype,was established via bioinformatics approaches.Subtype specific lincRNA,EGOT and EPB41L4A-AS2 expression was evaluated by real-time RT-PCR in 250 cancer tissuesand 50 adjacent normal tissues.In addition,the data from TCGA was also applied for further validation and the comparison between PAM50 molecular subtype system and linctype was applied to analyses the capacity of prognosis prediction.Finally,the regulation of EGOT was explored,and the biology function of EGOT and EPB41L4A-AS2 was also investigated via proliferation by CCK-8 assay and clongenetic assay,apoptosis by flow cytometry,migration and invasion by transwell assay.ResultsBy whole genome high throughput sequencing,79340 transcripts were identified and 26477 lincRNAs including 715 subtype specific lincRNAs.Breast cancer in TCGA can be divided into five different subtypes with distinct prognoses via the novel subtype classification system linctype.The expression EGOT was lower in breast cancer tissue than in corresponding normal tissue(P<0.001).The decreased expression level of EGOT was markedly associated with factors such as larger tumor size(P=0.038),lymph node involvement(P=0.02),higher Ki-67(P=0.025).High expression of EGOT was significantly associated with better overall survival in breast cancer patients(P<0.05).EGOT expression was downregulated when estrogen was added to the culture in MCF-7 cell line(P<0.05).Overexpression of EGOT in MDA-MB-231 and UACC812 cell lines indicated a decreased number of clones and the migration and invasion capability(all P<0.05).For EPB41L4A-AS2,the expression EPB41L4A-AS2 was lower in lung cancer tissue and renal cancer tissue(P<0.001)than in corresponding normal tissue(all P<0.05).High expression EPB41L4A-AS2 was significantly associated with better overall survival in breast cancer,lung cancer and renal cancer patients(all P<0.05).Cell proliferation assays showed that overexpression of EPB41L4A-AS2 inhibited tumor cell growth compared with the control in MDA-MB-231,A549 and 786-Ocancer cell lines.Furthermore,all three cell lines exhibited increased apoptosis with EPB41L4A-AS2 overexpression as assessed by flow cytometry.Finally,in a clonogenic assay,overexpression of EPB41L4A-AS2 reduced clone numbers in these three cancer cell lines.ConclusionsThese data suggest that subtype specific lincRNAs may contribute the accuracy for breast cancer diagnosis and therapy.Breast cancer patients in TCGA can be divided into five different subtypes with distinct prognoses via linctype.The expression EGOT and EPB41L4A-AS2 was downregulated in breast cancer and low expression of the two genes are associated with worse prognosis.They may be act as tumor suppressor gene for suppression of tumor cell proliferation and invasion.The exact mechanism was not clear.From the character of gene location and the literature,we hypothesize that EGOT may be involved in autophagy via regulation of ITPR1 expression,and EPB41L4A-AS2 suppresses the proliferation of tumor cell via regulation of the expression of EPB41L4 A.However,little is known about how and why EGOT and EPB41L4A-AS2 behave like a tumor suppressor.Further studies are needed to determine how EGOT and EPB41L4A-AS2 are involved in mediating tumor biology.