The Study On Function And Mechanism Of NONHSAT029258 In Thyroid Papillary Carcinoma

Background:Thyroid cancer is one of the fastest-growing solid tumors in the last 20 years,and is the most common malignancy among endocrine tumors.Over the past few decades,the incidence of thyroid cancer has increased dramatically.Palillary thyroid carcinoma(PTC),as the most common histological type,accounts for 85% to 90% of all thyroid cancers.Although the malignant degree of PTC is relatively low,which can be cured by traditional treatment,some patients with early cancer metastasis to local lymph nodes may result in higherrate of tumor recurrence and cancer-related mortality.In recent years,long noncoding RNA(lnc RNA)plays an important role in tumor development.lnc RNA usually transcripted by RNA polymerase ?(RNA pol ?)is longer than200 nucleotide,and it was used to be regarded as the "noise" in the process of transcription and did not code proteins.But recent studies have found that lnc RNA plays an important role in regulating gene expression?biology and pathology,which possess a wide range of expression spectrum,high tissue and cell specificity.It performs cis-or trans-regulation at epigenetic,transcriptional and post-transcriptional levels as well as participates in various life processes such as X chromosome silencing,genomic imprinting,chromosome modification,transcriptional activation,transcriptionalinterference,and intra-nuclear transport,which plays a crucial role in the occurrence and development of tumors.In addition,many studies have found abnormal expression of lnc RNA in thyroid cancer.However,the role and functional mechanism of related lnc RNAs in thyroid cancer tissues and cells are not well studied.Therefore,it is of great significance to explore the biological function and molecular mechanism of thyroid cancer-related lnc RNA in the process of tumorigenesis and development.Objective:Basing the second generation of high-throughput sequencing for 12 pairs of PTC and matching tissue adjacent to carcinoma,we got PTC difference lnc RNA expression spectrum.Here,the influence of NONHSAT029258 on PTC cell biology function and possible mechanism were analyzed to explore the diagnosis,prognosis,and target therapyvalueof thyroid cancer-related lnc RNAin PTC Methods:(1)Updating the sequencing results according to thelatest databases,some obviously up-regulated goal lnc RNAs were selected and verified,we validated the main part of intergenic lnc RNAs,their relative expression levels in thyroid cancer cell lines were detected by using q RT – PCR method.then tested the relative expression of cytoplasm and nucleus.Finally a target lnc RNA-NONHSAT029258 was screened and it's chromosome location and tissue expression in PTC spectrum were analyzed.(2)Verifyingthe expression level of thetarget lnc RNA—NONHSAT029258 in thyroid cancer and adjacent tissues in the Cancer Genome Atlas(TCGA)public database.(3)Transfecting thyroid papillary cancer cell line(TPC-1)with specific lnc RNA si RNA to down regulate the expression of target lnc RNA for further cell functional experiments(cck-8 assay,flow cytometry analysisand transwell assay)(4)Constructing an overexpressed lentiviral stable cell line(BCPAP)of the target lnc RNA,which was verified by q RT-PCR,for further cell function experiments(cck-8,flow,transwell).(5)Using mass spectrometry to analyze lnc RNA-related protein molecules for further molecular mechanism study.Results:(1)The expression of NONHSAT029258 was relatively high in the cytoplasm of four thyroid cancer cell lines,especially in the cytoplasm of TPC-1.However,the expression of BCPAP is low,which is suitable for overexpression construction.(2)The expression of NONHSAT029258 in thyroid cancer tissues was higher than that in paracancer tissues(P < 0.001),which was consistent with the second-generation sequencing results.(3)After transfected by si-345,we performed cellular functional assays.The cck-8 proliferation experiment indicated that the proliferation capacity of thyroid cancer cell line TPC-1 decreased significantly after down regulating NONHSAT029258,and with significant cell apoptosis at the later stage.Cell cycle test showed that PTC cells were obviously blocked in G1 phase,however no difference in G2 phase,and the proportion of cells in S phase decreased significantly(P < 0.001).Transwell migration and invasion assays suggested that the invasion and migration of PTC cells were significantly reduced after transfection by si-345.(4)After constructing the overexpressed lentiviral stable cell line BCPAP-NONHSAT029258,the lnc RNA expression levelof the stable cell line was verified by using q RT-PCR,and the level was significantly higher than that of the control group(P<0.001).The results of cck-8 proliferation assay indicated that the proliferation capacity of thyroid cancer cell line was enhanced(P<0.05).The results of flow cytometry cycle showed that cell proportion was decreased in S phase(P<0.05)and increased in G1 phase.Transwell migrationand invasionassays showed that PTC cell motility was obviously inhibited after transfection.(5)To further study the molecular mechanism,the protein molecules that may be related to the lnc RNA were obtained by using mass spectrometry.After GO function clustering analysis,RNA pull-down assay and mass spectrometryindicated that NONHSAT029258 may affect PTC by binding to multiple tumor-associated proteins,such as cyclin dependent kinase 12(CDK12)?cyclin-H(CCNH)?cdc25C-associated protein kinase 1(MARK3)?P21-and CDK-associated protein 1(BCCIP)or cell division cycle protein16 homolog(CDC16).Conclusion: 1.NONHSAT029258 is highly expressed in papillary thyroid carcinoma.2.NONHSAT029258 may promote the progress of PTC cells by regulating cycle,migration and invasion.3.NONHSAT029258 affect PTC progression possibly by binding to multiple tumor-associated proteins,such as CDK12?CCNH?MARK3?BCCIP or CDC16.