Functions Of Long Non-coding RNAs In Lung Cancer Initiation And Progression

Lung cancer is the leading cause of cancer-related deaths worldwide.The mechanisms of its initiation and progression have not been fully elucidated.Long non-coding RNAs(lnc RNAs)are the non-coding RNAs with a length of more than 200 nucleotides.The functions and mechanisms of lnc RNAs in lung cancer as well as the upstream regulatory mechanisms need to be further investigated.In this study,we uncovered the roles of lnc-LINP1 and lnc-CCTT in lung cancer initiation and progression.In lung cancer,transforming growth factor-?(TGF-?)signaling pathway promotes cancer progression by inducing epithelial-mesenchymal transition(EMT).The function of lnc-LINP1(lnc RNA in nonhomologous end joining pathway 1)in lung cancer are not defined.We found that TGF-?1 repressed lnc-LINP1 transcription in a SMAD4-dependent manner in lung cancer cells.Lnc-LINP1 inhibited EMT process of lung cancer cells,as shown by the upregulation of epithelial cell marker E-Cadherin and the downregulation of mesenchymal cell markers vimentin,N-Cadherin,and SNAIL,a key EMT transcriptional factor.Functionally,lnc-LINP1 suppressed lung cancer cell migration,invasion,and stemness acquisition.Furthermore,lnc-LINP1 partially inhibited TGF-?-induced EMT and cell invasion in lung cancer cells.Additionally,lncLINP1 expression was downregulated in some human lung cancer samples.The results showed that TGF-? promotes lung cancer progression by repressing lnc-LINP1.Moreover,we identified a new lnc RNA CCTT(CENP-C targeting transcript),which promotes the initiation and progression of lung cancer.Lnc-CCTT was amplified in 72% of human lung adenocarcinoma samples.The gain and loss-offunction assays demonstrated that lnc-CCTT promoted lung cancer cell proliferation,clone formation,and xenograft tumor growth.Overexpression of lnc-CCTT induced the transformation of a normal lung bronchial epithelial cell line BEAS-2B,indicating its oncogenic role in lung cancer.Mechanistically,lnc-CCTT upregulated the expression of its nearby oncogene ERBB2,and led to the activation of its downstream pathways such as AKT and STAT3.We also uncovered an important role of lnc-CCTT in regulating centromere functions.CENP-C is the core component of constitutive centromere associated network(CCAN)complex for kinetochore assembly.Previous studies have established that CENP-A is required for recruiting CENP-C to centromeres.However,it is largely unknown whether CENP-A independent mechanisms involve in the loading of CENPC to centromere.It has not been reported that any lnc RNA transcribed from noncentromeric DNA could localize to centromeres and regulate centromere functions in trans.In this study,we found that lnc-CCTT knockdown led to abnormal centromere functions,as shown by alignment defects,chromosome bridge,multipolar,binuclear and micronuclear cells,and aneuploid cells.We further showed that lnc-CCTT interacted with CENP-C and recruited it to centromeres.Using chromatin isolation by RNA purification(Ch IRP)-seq,we found that lnc-CCTT bound to centromeric DNA of all chromosomes.The 44-79 nucleotides of lnc-CCTT were predicted to form an RNAds DNA triplex with centromeric DNA by bioinformatics analysis,which was further confirmed by RNase H treatment and EMSA analysis.The results of UV-crosslinked immunoprecipitation(CLIP)-seq,selective 2'-hydroxyl acylation analyzed by primer extension(SHAPE)-seq and CENP-C truncation assay indicated that the 127-177 nucleotides of lnc-CCTT bound to the C-terminal domain of CENP-C.Finally,we found that lnc-CCTT might recruit CENP-C to a non-centromere locus.In summary,we revealed the functions of lnc RNA LINP1 and CCTT in lung cancer initiation and progression,providing new knowledge for better understanding the roles of lnc RNAs in lung cancer.In addition,we uncovered an important role of lnc-CCTT,a lnc RNA transcribed from a non-centromere locus,in recruiting CENP-C to centromeres by forming RNA-ds DNA triplex with centromeric DNA.