The Regulation Role And Molecular Mechanism Of Long Non-coding RNA In Liver Cancer Stem Cells

Part I The Regulation Role and Molecular Mechanism of Long Non-coding RNA in Liver Cancer Stem CellsBackground & AimsHepatocellular carcinoma(HCC) is the sixth most prevalent human malignancy with rising morbidity worldwide. Annually, over 700,000 new patients are diagnosed with HCC. Surgical resection or liver transplantation may provide an opportunity for a cure only when patients are diagnosed at an early stage. For patients with advanced HCC, the introduction of sorafenib may offer but only limited survival benefits. Despite recent progress in HCC prevention, diagnosis and intervention, treatment for this refractory disease remains unsatisfactory due to dismal prognosis and frequent recurrence. Thus, clarifying the underlying mechanism of HCC progression to identify novel targets for effective intervention is urgently needed.Most malignancies are composed of heterogeneous populations that include a distinct subset called cancer stem cells(CSCs). CSCs are believed to be responsible for the initiation, propagation, chemo-resistance, metastasis and relapse of cancers. Previous studies have also verified the existence of liver cancer stem cells(LCSCs), which can be distinguished by various biomarkers, such as CD133, CD90, Ep CAM, OV6, CD24 and CD13 etc. Numerous stemness-associated transcription factors and signaling pathways have been reported to be implicated in LCSC maintenance and propagation. Moreover, recent evidence has demonstrated that epigenetic modifications also contribute to the characteristics of CSCs. Nevertheless, findings reporting the involvement of long non-coding RNAs(lnc RNAs) in LCSCs regulation are rare.lnc RNAs are transcripts longer than 200 bp that do not have any apparent protein-coding ability. Thus far, accumulating evidence has indicated that lnc RNAs can affect disparate cellular functions and participate in diverse physiological and pathological processes. The aberrant expression of lnc RNAs has been demonstrated in multiple malignancies, including HCC, providing new insights into the pathogenesis of cancer. However, the potential role of lnc RNAs in LCSCs propagation remains largely unknown.In present study, we intend to identify lnc RNA especially expressed in LCSCs through high-throughput screening, and elucidate its biological function and molecular mechanism in LCSC expansion. Furthermore, we intend to verify the results in clinical samples and clarify the significant correlation between lnc RNA and HCC clinicopathological features. The results will provide new evidences for understanding mechnisiams of LCSC regulation and provide new targets for the development of effective therapeutic strategies.Methods lnc-DILC(lnc RNA down-regulated in liver cancer stem cells) was identified by microarray and validated by real-time PCR. The role of lnc-DILC in LCSCs was assessed both in vitro and in vivo. Pull down assay and oligoribonucleotides or oligodeoxynucleotides treatment were conducted to evaluate the interaction between lnc-DILC and interleukine-6(IL-6) promoter.Results Depletion of lnc-DILC markedly enhanced LCSC expansion and facilitated HCC initiation and progression, whereas ectopic expression of lnc-DILC dramatically inhibited LCSC expansion. Mechanistically, lnc-DILC inhibited the autocrine IL-6/STAT3 signaling. The putative binding locus of lnc-DILC within IL-6 promoter was confirmed by pull down assay. Consistently, the oligoribonucleotide mimics and an oligodeoxynucleotide decoy of lnc-DILC abrogated the effects on IL-6 transcription, STAT3 activation and LCSC expansion triggered by lnc-DILC depletion and lnc-DILC overexpression. Moreover, our data suggested that lnc-DILC mediated the crosstalk between TNF-α/NF-κB signaling and IL-6/STAT3 cascade. Clinical investigation demonstrated the reduction of lnc-DILC in patient HCCs, and suggested the correlation between lnc-DILC levels and IL-6, Ep CAM or CD24 expression. Decreased lnc-DILC expression in HCCs predicts early recurrence and short survival of patients, highlighting its prognostic value.Conclusions lnc-DILC mediates the crosstalk between TNF-α/NF-κB signaling and autocrine IL-6/STAT3 cascade and connects hepatic inflammation with LCSC expansion, suggesting that lnc-DILC could be not only a potential prognostic biomarker, but also a possible therapeutic target against LCSCs.Part II Shp2 Promotes Liver Cancer Stem Cell Expansion via Augmenting β-catenin SignalingBackground & AimsHepatocellular carcinoma(HCC) is the sixth most common cancer in the world and the second leading cause of cancer death in men. Despite the recent advances in hepatic resection and transplantation, the long-term survival of HCC patients remains unsatisfactory due to the frequent recurrence after surgical resection and the poor response of patients to conventional chemotherapy including TACE(transcatheter arterial chemoembolization).Accumulating evidence has demonstrated that chemo-resistance and recurrence of HCC are closely associated with the existence of liver cancer stem cells(LCSCs). Therefore, more effective therapeutic strategy could be developed by targeting LCSCs if the molecular mechanism underlying CSC regulation is dissected. Nevertheless, the detailed regulatory mechanism of CSC generation and expansion remains far from fully understood.Shp2, encoded by PTPN11, is a non-receptor protein tyrosine phosphatase that contains two Src-homology 2 domains. Intriguingly, distinct from other tyrosine phosphatases that play a negative role in proliferative signaling activation, Shp2 functions as a positive regulator of cell survival and growth. Shp2 acts as a transducer of extracellular pro-survival and proliferative signals, and is required for the full activation of ERK signaling.We previously generated the hepatocyte-specific Shp2 knockout mice and observed that conditional Shp2 ablation facilitated DEN(diethylnitrosamine)-triggered HCC development in mice. However, our most current data demonstrated the overexpression of Shp2 in the majority of human HCCs and its association with advanced stage, poor differentiation and metastasis of liver cancer, which implies the positive role of Shp2 in HCC progression. In further study, we found that Shp2 promotes HCC growth and metastasis by coordinately activating the Ras/Raf/Erk pathway and the PI3-K/Akt/m TOR cascade21. Actually, all of these pro-oncogenic molecules including Shp2, JNK, STAT3, b-catenin exert a pro-survival role in hepatocyte. Therefore, hepatocyte-specific deletion of either of them in mice resulted in the increased cell death and secondary inflammation in liver, which eventually led to enhanced HCC development in DEN-treated mice. Nevertheless, the regulatory role of Shp2 in LCSCs remains unknown.Methods The expression of Shp2 was examined by immunohistochemistry and real-time PCR. The role of Shp2 in LCSCs was assessed both in vitro and in vivo. Western blot and Co-Immunoprecipitation assay were conducted to elucidate the molecular mechanism of Shp2 in LCSCs expansion.Results Herein we observed the high expression of Shp2 in the chemo-resistant HCCs and in the recurrent HCCs from patients. A remarkable increase of Shp2 was detected in the sorted Ep CAM+ or CD133+ LCSCs and in the CSC-enriched hepatoma spheroids of patients. Upregulated Shp2 facilitated LCSC expansion by promoting the dedifferentiation of hepatoma cells and enhancing the self-renewal of LCSCs. Mechanistically, dephosphorylated CDC73 by Shp2 bound β-catenin in cytosol of hepatoma cells and facilitated the nuclear translocation and activation of β-catenin, which promoted the dedifferentiation of hepatoma cells. Shp2 increased β-catenin accumulation by inhibiting GSK3β-mediated β-catenin degradation in LCSCs, thereby enhancing the self-renewal of LCSCs. Blockage of β-catenin abolished the discrepancy in LCSC proportion and the self-renewal capacity between Shp2 depleted hepatoma cells and control cells, which further confirmed that β-catenin is required in Shp2-promoted LCSC expansion. More importantly, HCC patients with low Shp2 levels benefited from TACE treatment, but the patients with high Shp2 expression did not, indicating the significance of Shp2 in personalized HCC treatment.Conclusions Shp2 promotes HCC cell dedifferentiation and LCSC expansion via amplifying β-catenin signaling, and may predict the patient response to chemotherapeutics.