Long Non-coding RNA NEAT1 Regulate The Self-renewal Of Liver Cancer Stem Cells Via The Hippo Signal Pathway

Background & Aims Primary hepatocellular carcinoma(HCC)is the sixth most prevalent malignancy in the world,with the second mortality rate only to lung cancer.China has the highest morbidity and mortality of liver cancer,so the diagnosis and treatment of HCC is very important.The main treatment of HCC is surgical resection,but most of patients diagnosed are in advanced stage,which can only take chemoradiotherapy,transcatheter arterial chemoembolization or sorafenib treatment.However,most patients will have recurrence,metastasis and drug resistance after treatment with poor prognosis.Therefore,it is urgent to find new diagnostic markers and therapeutic targets for HCC.It is widely accepted that most tumors are heterogenous in their cellular composition.Tumors of most types can be divided into cellular sub-populations consisting of differentiated cells and cancer stem cells(CSCs),which undergo self-renewal potential and tumor initiating ability.Many chemotherapy methods can significantly inhibit the tumor at the beginning and reduce the tumor load,but fail to eliminate CSCs completely,leading to cancer recurrence eventually.Numerous evidences have proved the existence of liver cancer stem cells(LCSCs)in HCC,which are responsible for the development and recurrence of HCC.Long non-coding RNAs(lnc RNAs)are defined as transcripts longer than 200 nucleotides without protein-coding ability.Accumulating evidences have indicated that lnc RNAs can participate in a variety of biological processes including gene expression,subcellular structure and protein complex stability through different mechanisms.They can also participate in cellular inflammation,tumorigenesis and other pathological processes.Recently,more and more evidences show that lnc RNAs play an important role in regulating the function of CSCs,and many lnc RNAs have been found to be highly expressed in CSCs.Similar to other tumors,various lnc RNAs are significantly up-regulated in HCC,including HEIH,MALAT1,HULC and HOTAIR.These functional lnc RNAs regulate gene expression in a variety of ways,thus participating in the occurrenceand development of liver cancer.The study of lnc RNAs related to HCC has brought great hope for the discovery of new markers for early diagnosis,prognosis and treatment of HCC.In this study,we have identified lnc RNA NEAT1 is up-regulated in LCSCs.Hippo signaling pathway,which is involved in the regulation of various cell and organ homeostasis,has received extensive attention.Recent studies have shown that Hippo signaling pathway plays an important role in the processes of liver development,changing of liver cell fate,liver regeneration and hepatocarcinogenesis in mammals.Yes-associated protein(YAP)is a key transcription factor in Hippo signaling pathway,and its regulation of CSCs can be found in many tumors.However,studies on lnc RNA regulation of LCSCs via Hippo pathway have not yet been reported.In present study,we intend to verify the regulation role of NEAT1 on the self-renewal of LCSCs both in vitro and in vivo.And we will clarify the molecular mechanism of NEAT1 in regulating LCSCs via Hippo pathway.Furthermore,we will analyze the correlation between NEAT1 and the clinical pathology of HCC.This study is expected to provide a new theoretical basis for the regulation mechanism of LCSCs,and provide new targets for the diagnosis and treatment of HCC.Methods 1.The up-regulated lnc RNAs in LCSCs was chose from microarray of lnc RNAs with the intersection of the lnc RNAs upregulated in the TCGA cohort of 50 paired HCC tissues and adjacent non-tumour tissues.2.LCSCs were obtained by either hepatoma spheroid formation or FACS using stemness markers.The expression of NEAT1 in LCSCs was detected by Real-time PCR and FISH.3.The expression of NEAT1 in the liver cancer and the adjacent tissues was detected by using Real-time PCR and in situ hybridization.4.The expression of Ep CAM and CD24 in liver cancer tissues was detected by Real-time PCR,and was further sent to analyzed the correlation with NEAT1.5.According to the expression of NEAT1 in liver cancer patients,the relationship between NEAT1 and HCC patients' prognosis was analyzed.6.The NEAT1 knockdown or knockin cell lines were established by using the lentivirus.7.LCSCs were enriched by spheroid formation assay in established cell lines,and the expressions of LCSC markers and stemness-associated transcription factors weredetected by Real-time PCR.8.The population of Ep CAM+ or CD133+ cells in HCC was detected by flow cytometry analysis.9.The effect of NEAT1 on the self-renewal ability of LCSCs was verified by using spheroid formation assay,soft agar clone formation assay and limited dilution assay.10.The effect of NEAT1 on the Hippo signal pathway in LCSCs was verified by using luciferase reporter assay,Western blot,Real-time PCR,FISH,IF and IHC.11.RNA pulldown and RIP experiments were used to clarify the protein which interacts with NEAT1.12.The direct target of NEAT1 and their relationship with Hippo signal pathway in LCSCs was verified by Western blot and Real-time PCR.13.The direct target of NEAT1 and their relationship with Hippo signal pathway in LCSCs was confirmed by functional assays.Results 1.NEAT1 is highly expressed in LCSCs.2.The overall and disease-free survival of HCC patients with high expression of NEAT1 were worse than that of patients with low expression of NEAT1.3.The NEAT1 inhibited the enlargement and self-renewal ability of LCSCs,while overexpression of NEAT1 promoted the enlargement and self-renewal ability of LCSCs.4.NEAT1 regulates the activation of LCSCs through the Hippo signal pathway.5.NEAT1 physically interacts with AKAP8.6.NEAT1 can regulate the phosphorylation of LATS and YAP in the Hippo signal pathway by interacting with AKAP8 protein to regulate distribution of PKA in nucleus and cytoplasm in LCSCs.Conclusion In this study,we found that NEAT1 is highly expressed in LCSCs,and NEAT1 can regulate the phosphorylation of LATS and YAP in the Hippo signal pathway by interacting with AKAP8 protein to regulate the distribution of PKA in nucleus and cytoplasm,thus affecting the amplification and self-renewal function of LCSCs.Clinical studies have also confirmed that the overall and disease-free survival of patients with high NEAT1expression will be worse than that of patients with low NEAT1 expression.In conclusion,our findings provide a new theoretical basis for the regulation of the NEAT1 and Hippo pathways in LCSCs.