NRDE2 negatively regulates exosome functions by inhibiting MTR4 recruitment and exosome interaction.

The exosome functions in the degradation of diverse RNA species, yet how it is negatively regulated remains largely unknown. Here, we show that NRDE2 forms a 1:1 complex with MTR4, a nuclear exosome cofactor critical for exosome recruitment, via a conserved MTR4-interacting domain (MID). Unexpectedly, NRDE2 mainly localizes in nuclear speckles, where it inhibits MTR4 recruitment and RNA degradation, and thereby ensures efficient mRNA nuclear export. Structural and biochemical data revealed that NRDE2 interacts with MTR4's key residues, locks MTR4 in a closed conformation, and inhibits MTR4 interaction with the exosome as well as proteins important for MTR4 recruitment, such as the cap-binding complex (CBC) and ZFC3H1. Functionally, MID deletion results in the loss of self-renewal of mouse embryonic stem cells. Together, our data pinpoint NRDE2 as a nuclear exosome negative regulator that ensures mRNA stability and nuclear export.

Lysosome-Targeting Bacterial Outer Membrane Vesicles for Tumor Specific Degradation of PD-L1.

Increased expression of immune check point genes, such as PD-L1, is one of the main reasons for immunosuppression, especially for colon cancer. Development of novel therapeutic strategies is of great importance to improve the prognosis. In this study, outer membrane vesicles (OMV) derived from Gram-negative bacteria are engineered to immune checkpoint blockade nanosystem for efficient elicitation of anti-tumor immunity. Briefly, the OMVs are engineered with Lyp1-Traptavidin (S52G, R53D mutant of streptavidin) fusion protein displayed on the surface. The Lyp-1 endows the OMV with the capacity to target tumor tissues, while the Traptavidin ensures easy decoration of biotinylated anti-PD-L1 and biotinylated M6P (mannose 6-phosphate). The simultaneously anchored anti-PD-L1 and M6P (ligand for cation-independent mannose 6-phosphate receptor) on the engineered OMVs coordinately direct the membrane PD-L1 to lysosome for degradation, and thus unleash the anti-tumor immunity. With syngeneic tumor model, the engineered OMVs are confirmed to boost immunity, inhibit cancer growth, and thus prolong survival. Together, A proposed OMV-based modular nanosystem that enables assembly of biotinylated anti-PD-L1 and M6P on the surface for tumor-targeted immune checkpoint blockade.

Spatial transcriptome and single-cell reveal the role of nucleotide metabolism in colorectal cancer progression and tumor microenvironment.

BACKGROUND: The intricacies of nucleotide metabolism within tumor cells specific to colorectal cancer (CRC) remain insufficiently characterized. A nuanced examination of particular tumor clusters and their dynamic interplay with the tumor microenvironment (TME) may yield profound insights into these therapeutically auspicious communicative networks. METHODS: By integrating ten types of single-cell enrichment scoring methods, we carried out enrichment analysis on CRC cell types, which was validated through four additional single-cell cohorts. Groups of tumor cells were determined using the average values of the scores. Using cellphonedb, monocle, inferCNV, SCENIC, and Cytotrace, functional analyses were performed. Utilizing the RCTD approach, single-cell groupings were mapped onto spatial transcriptomics, analyzing cell dependency and pathway activity to distinguish between tumor cell subtypes. Differential expression analysis identified core genes in nucleotide metabolism, with single-cell and spatial transcriptomics analyses elucidating the function of these genes in tumor cells and the immune microenvironment. Prognostic models were developed from bulk transcriptome cohorts to forecast responses to immune therapy. Laboratory experiments were conducted to verify the biological function of the core gene. RESULTS: Nucleotide metabolism is significantly elevated in tumor cells, dividing them into two groups: NUhighepi and NUlowepi. The phenotype NUhighepi was discerned to exhibit pronounced malignant attributes. Utilizing the analytical tool stlearn for cell-to-cell communication assessment, it was ascertained that NUhighepi engages in intimate interactions with fibroblasts. Corroborating this observation, spatial transcriptome cell interaction assessment through MISTy unveiled a particular reliance of NUhighepi on fibroblasts. Subsequently, we pinpointed NME1, a key gene in nucleotide metabolism, affirming its role in thwarting metastasis via in vitro examination. Utilizing multiple machine learning algorithms, a stable prognostic model (NRS) has been developed, capable of predicting survival and responses to immune therapy. In addition, targeted drugs have been identified for both high and low scoring groups. Laboratory experiments have revealed that NME1 can inhibit the proliferation and invasion of CRC tumor cells. CONCLUSION: Our study elucidates the potential pro-tumor mechanism of NUhighepi and the role of NME1 in inhibiting metastasis, further deepening the understanding of the role of nucleotide metabolism in colorectal cancer, and providing valuable targets for disrupting its properties.

ALYREF links 3'-end processing to nuclear export of non-polyadenylated mRNAs.

The RNA-binding protein ALYREF plays key roles in nuclear export and also 3'-end processing of polyadenylated mRNAs, but whether such regulation also extends to non-polyadenylated RNAs is unknown. Replication-dependent (RD)-histone mRNAs are not polyadenylated, but instead end in a stem-loop (SL) structure. Here, we demonstrate that ALYREF prevalently binds a region next to the SL on RD-histone mRNAs. SL-binding protein (SLBP) directly interacts with ALYREF and promotes its recruitment. ALYREF promotes histone pre-mRNA 3'-end processing by facilitating U7-snRNP recruitment through physical interaction with the U7-snRNP-specific component Lsm11. Furthermore, ALYREF, together with other components of the TREX complex, enhances histone mRNA export. Moreover, we show that 3'-end processing promotes ALYREF recruitment and histone mRNA export. Together, our results point to an important role of ALYREF in coordinating 3'-end processing and nuclear export of non-polyadenylated mRNAs.

Microwave ablation versus laparoscopic resection as first-line therapy for solitary 3-5-cm HCC.

BACKGROUND AND AIMS: The study objective was to compare the effectiveness of microwave ablation (MWA) and laparoscopic liver resection (LLR) on solitary 3-5-cm HCC over time. APPROACH AND RESULTS: From 2008 to 2019, 1289 patients from 12 hospitals were enrolled in this retrospective study. Diagnosis of all lesions were based on histopathology. Propensity score matching was used to balance all baseline variables between the two groups in 2008-2019 (n = 335 in each group) and 2014-2019 (n = 257 in each group) cohorts, respectively. For cohort 2008-2019, during a median follow-up of 35.8 months, there were no differences in overall survival (OS) between MWA and LLR (HR: 0.88, 95% CI 0.65-1.19, p = 0.420), and MWA was inferior to LLR regarding disease-free survival (DFS) (HR 1.36, 95% CI 1.05-1.75, p = 0.017). For cohort 2014-2019, there was comparable OS (HR 0.85, 95% CI 0.56-1.30, p = 0.460) and approached statistical significance for DFS (HR 1.33, 95% CI 0.98-1.82, p = 0.071) between MWA and LLR. Subgroup analyses showed comparable OS in 3.1-4.0-cm HCCs (HR 0.88, 95% CI 0.53-1.47, p = 0.630) and 4.1-5.0-cm HCCs (HR 0.77, 95% CI 0.37-1.60, p = 0.483) between two modalities. For both cohorts, MWA shared comparable major complications (both p > 0.05), shorter hospitalization, and lower cost to LLR (all p < 0.001). CONCLUSIONS: MWA might be a first-line alternative to LLR for solitary 3-5-cm HCC in selected patients with technical advances, especially for patients unsuitable for LLR.

Eutrophication decreases ecological resilience by reducing species diversity and altering functional traits of submerged macrophytes.

Positive feedback is key to producing alternative stable states and largely determines ecological resilience in response to external perturbations. Understanding the positive feedback mechanisms in macrophyte-dominated lakes is crucial for resilience-based management and restoration. Based on the field investigation of submerged macrophyte communities in 35 lakes in China, we found that morphological complexity (MC) and morphological plasticity (MP) are correlated with the stoichiometric homeostasis of phosphorus (HP ) and are related to ecosystem structure, functioning, and stability. We also found that the positive feedback strength of lakes dominated by macrophytes is biomass- and diversity-dependent. Eutrophication can decrease the community biomass by decreasing community MC, MP, and HP and the species diversity through low-light availability, ultimately decreasing the positive feedback strength and resilience of clear water states. We argue that functional traits and species diversity should be considered to build more resilient ecosystems in future changing environment scenarios.

A U2-snRNP-independent role of SF3b in promoting mRNA export.

To ensure efficient and accurate gene expression, pre-mRNA processing and mRNA export need to be balanced. However, how this balance is ensured remains largely unclear. Here, we found that SF3b, a component of U2 snRNP that participates in splicing and 3' processing of pre-mRNAs, interacts with the key mRNA export adaptor THO in vivo and in vitro. Depletion of SF3b reduces THO binding with the mRNA and causes nuclear mRNA retention. Consistently, introducing SF3b binding sites into the mRNA enhances THO recruitment and nuclear export in a dose-dependent manner. These data demonstrate a role of SF3b in promoting mRNA export. In support of this role, SF3b binds with mature mRNAs in the cells. Intriguingly, disruption of U2 snRNP by using a U2 antisense morpholino oligonucleotide does not inhibit, but promotes, the role of SF3b in mRNA export as a result of enhanced SF3b-THO interaction and THO recruitment to the mRNA. Together, our study uncovers a U2-snRNP-independent role of SF3b in mRNA export and suggests that SF3b contributes to balancing pre-mRNA processing and mRNA export.

Exosome cofactor hMTR4 competes with export adaptor ALYREF to ensure balanced nuclear RNA pools for degradation and export.

The exosome is a key RNA machine that functions in the degradation of unwanted RNAs. Here, we found that significant fractions of precursors and mature forms of mRNAs and long noncoding RNAs are degraded by the nuclear exosome in normal human cells. Exosome-mediated degradation of these RNAs requires its cofactor hMTR4. Significantly, hMTR4 plays a key role in specifically recruiting the exosome to its targets. Furthermore, we provide several lines of evidence indicating that hMTR4 executes this role by directly competing with the mRNA export adaptor ALYREF for associating with ARS2, a component of the cap-binding complex (CBC), and this competition is critical for determining whether an RNA is degraded or exported to the cytoplasm. Together, our results indicate that the competition between hMTR4 and ALYREF determines exosome recruitment and functions in creating balanced nuclear RNA pools for degradation and export.

mRNAs are sorted for export or degradation before passing through nuclear speckles.

A significant fraction of mRNAs are degraded by the nuclear exosome in normal cells. Here, we studied where and when these exosome target mRNAs are sorted away from properly exported ones in the cells. We show that upon exosome inactivation, polyA RNAs are apparently accumulated in nuclear foci that are distinct from nuclear speckles (NSs), and provide several lines of evidence supporting that these polyA RNAs mainly correspond to accumulating exosome target mRNAs. These results suggest that exosomal mRNA degradation mostly occurs outside of NSs. In support of this possibility, targeting exosome target mRNAs to NSs stabilizes them by preventing exosomal degradation. Furthermore, inhibiting mRNA release from NSs does not attenuate exosomal degradation in normal cells, and results in polyA RNA accumulation both inside and outside of NSs in exosome inactivated cells, suggesting that passage through NSs is not required for sorting mRNAs for degradation or export. Indeed, exosome target mRNAs that normally do not enter NSs are exported upon exosome inactivation. Together, our data suggest that exosome target mRNAs are mainly degraded in the nucleoplasm before entering NSs and rapid removal of these mRNAs is important for preventing their nuclear export.

ALYREF mainly binds to the 5' and the 3' regions of the mRNA in vivo.

The TREX complex (TREX) plays key roles in nuclear export of mRNAs. However, little is known about its transcriptome-wide binding targets. We used individual cross-linking and immunoprecipitation (iCLIP) to identify the binding sites of ALYREF, an mRNA export adaptor in TREX, in human cells. Consistent with previous in vitro studies, ALYREF binds to a region near the 5' end of the mRNA in a CBP80-dependent manner. Unexpectedly, we identified PABPN1-dependent ALYREF binding near the 3' end of the mRNA. Furthermore, the 3' processing factor CstF64 directly interacts with ALYREF and is required for the overall binding of ALYREF on the mRNA. In addition, we found that numerous middle exons harbor ALYREF binding sites and identified ALYREF-binding motifs that promote nuclear export of intronless mRNAs. Together, our study defines enrichment of ALYREF binding sites at the 5' and the 3' regions of the mRNA in vivo, identifies export-promoting ALYREF-binding motifs, and reveals CstF64- and PABPN1-mediated coupling of mRNA nuclear export to 3' processing.

Colorectal cancer-derived small extracellular vesicles establish an inflammatory premetastatic niche in liver metastasis.

Liver metastases develop in more than half of the patients with colorectal cancer (CRC) and are associated with a poor prognosis. The factors influencing liver metastasis of CRC are poorly characterized, but this information is urgently needed. We have now discovered that small extracellular vesicles (sEVs; exosomes) derived from CRC can be specifically targeted to liver tissue and induce liver macrophage polarization toward an interleukin-6 (IL-6)-secreting proinflammatory phenotype. More importantly, we found that microRNA-21-5p (miR-21) was highly enriched in CRC-derived sEVs and was essential for creating a liver proinflammatory phenotype and liver metastasis of CRC. Silencing either miR-21 in CRC-sEVs or Toll-like receptor 7 (TLR7) in macrophages, to which miR-21 binds, abolished CRC-sEVs' induction of proinflammatory macrophages. Furthermore, miR-21 expression in plasma-derived sEVs was positively correlated with liver metastasis in CRC patients. Collectively, our data demonstrate a pivotal role of CRC-sEVs in promoting liver metastasis by inducing an inflammatory premetastatic niche through the miR-21-TLR7-IL-6 axis. Thus, sEVs-miR-21 represents a potential prognostic marker and therapeutic target for CRC patients with liver metastasis.

Effective mRNA Inhibition in PANC-1 Cells in Vitro Mediated via an mPEG-SeSe-PEI Delivery System.

RNA interference (RNAi)-mediated gene therapy is a promising approach to cure various diseases. However, developing an effective, safe, specific RNAi delivery system remains a major challenge. In this study, a novel redox-responsive polyetherimide (PEI)-based nanovector, mPEG-SeSe-PEI, was developed and its efficacy evaluated. We prepared three mPEG-SeSe-PEI vector candidates for small interfering glyceraldehyde-3-phosphate dehydrogenase (siGADPH) and determined their physiochemical properties and transfection efficiency using flow cytometry and PEG11.6-SeSe-PEI polymer. We investigated the silencing efficacy of GADPH mRNA expression in PANC-1 cells and observed that PEG11.6-SeSe-PEI/siGADPH (N/P ratio=10) polyplexes possessed the appropriate size and zeta-potential and exhibited excellent in vitro gene silencing effects with the least cytotoxicity in PANC-1 cells. In conclusion, we present PEG11.6-SeSe-PEI as a potential therapeutic gene delivery system for small interfering RNA (siRNA).

A Sub-Element in PRE enhances nuclear export of intronless mRNAs by recruiting the TREX complex via ZC3H18.

Viral RNA elements that facilitate mRNA export are useful tools for identifying cellular RNA export factors. Here we show that hepatitis B virus post-transcriptional element (PRE) is one such element, and using PRE several new cellular mRNA export factors were identified. We found that PRE drastically enhances the cytoplasmic accumulation of cDNA transcripts independent of any viral protein. Systematic deletion analysis revealed the existence of a 116 nt functional Sub-Element of PRE (SEP1). The RNP that forms on the SEP1 RNA was affinity purified, in which TREX components as well as several other proteins were identified. TREX components and the SEP1-associating protein ZC3H18 are required for SEP1-mediated mRNA export. Significantly, ZC3H18 directly binds to the SEP1 RNA, interacts with TREX and is required for stable association of TREX with the SEP1-containing mRNA. Requirements for SEP1-mediated mRNA export are similar to those for splicing-dependent mRNA export. Consistent with these similarities, several SEP1-interacting proteins, including ZC3H18, ARS2, Acinus and Brr2, are required for efficient nuclear export of polyA RNAs. Together, our data indicate that SEP1 enhances mRNA export by recruiting TREX via ZC3H18. The new mRNA export factors that we identified might be involved in cap- and splicing-dependent TREX recruitment to cellular mRNAs.

Aly and THO are required for assembly of the human TREX complex and association of TREX components with the spliced mRNA.

The mRNA export complex TREX (TREX) is known to contain Aly, UAP56, Tex1 and the THO complex, among which UAP56 is required for TREX assembly. Here, we systematically investigated the role of each human TREX component in TREX assembly and its association with the mRNA. We found that Tex1 is essentially a subunit of the THO complex. Aly, THO and UAP56 are all required for assembly of TREX, in which Aly directly interacts with THO subunits Thoc2 and Thoc5. Both Aly and THO function in linking UAP56 to the cap-binding protein CBP80. Interestingly, association of UAP56 with the spliced mRNA, but not with the pre-mRNA, requires Aly and THO. Unexpectedly, we found that Aly and THO require each other to associate with the spliced mRNA. Consistent with these biochemical results, similar to Aly and UAP56, THO plays critical roles in mRNA export. Together, we propose that Aly, THO and UAP56 form a highly integrated unit to associate with the spliced mRNA and function in mRNA export.

Dual-Functional Nanodroplet for Tumor Vasculature Ultrasound Imaging and Tumor Immunosuppressive Microenvironment Remodeling.

Accurately evaluating tumor neoangiogenesis and conducting precise interventions toward an immune-favorable microenvironment are of significant clinical importance. In this study, a novel nanodroplet termed as the nanodroplet-based ultrasound contrast agent and therapeutic (NDsUCA/Tx) is designed for ultrasound imaging and precise interventions of tumor neoangiogenesis. Briefly, the NDsUCA/Tx shell is constructed from an engineered CMs containing the tumor antigen, vascular endothelial growth factor receptor 1 (VEGFR1) extracellular domain 2-3, and CD93 ligand multimerin 2. The core is composed of perfluorohexane and the immune adjuvant R848. After injection, NDsUCA/Tx is found to be enriched in the tumor vasculature with high expression of CD93. When triggered by ultrasound, the perfluorohexane in NDsUCA/Tx underwent acoustic droplet vaporization and generated an enhanced ultrasound signal. Some microbubbles exploded and the resultant debris (with tumor antigen and R848) together with the adsorbed VEGF are taken up by nearby cells. This cleared the local VEGF for vascular normalization, and also served as a vaccine to activate the immune response. Using a syngeneic mouse model, the satisfactory performance of NDsUCA/Tx in tumor vasculature imaging and immune activation is confirmed. Thus, a multifunctional NDsUCA/Tx is successfully developed for molecular imaging of tumor neoangiogenesis and precise remodeling of the tumor microenvironment.

Sequential therapy for pancreatic cancer patients with synchronous oligo-hepatic metastatic lesions.

BACKGROUND: Treatments for patients suffering from pancreatic cancer with oligo-hepatic metastasis have always been a cause of certain controversy. Herein, we reported 15 pancreatic cancer patients with oligo-hepatic metastasis who accepted sequential therapy of chemotherapy, radiofrequency ablation (RFA), and radical resection of the primary tumor. METHODS: A total of 87 pancreatic cancer patients with synchronous oligo-metastatic hepatic lesions who received treatments in the 2nd Affiliated Hospital of Zhejiang University between January 2017 and July 2020 were enrolled. The chemotherapy regimens included modified folfirinox (54/87) and gemcitabine plus nab-paclitaxel (33/87). Test of blood tumor markers and contrast-enhanced computed tomography (CT) or magnetic resonance (MR) scan was performed at diagnosis and after eight weeks of chemotherapy. RESULTS: Thirty-five patients received just chemotherapy because of poor reaction to the first round of chemotherapy(Overall survival (OS), 6.47±1.80 months); 15 patients reassessed as stable disease (SD)/partial response (PR) continued chemotherapy (OS, 10.35±3.15); nine patients reassessed as progressive disease (PD) after RFA and continued chemotherapy (OS, 10.90±2.60). The primary tumors in 13 patients were unresectable after chemotherapy and RFA (OS, 12.92±2.47), while 15 patients completed the sequential therapy of chemotherapy, radio-frequency ablation, and radical resection (OS, 16.76±6.55). CONCLUSIONS: Sequential chemotherapy and RFA is a good treatment strategy to select the best candidates for surgical treatment among patients with pancreatic cancer with oligo-hepatic metastasis.

Anti-PD-1-based immunotherapy plus lenvatinib to treat advanced gallbladder cancer in the elderly: a case series and review of current literature.

BACKGROUND: Gallbladder cancer (GBC) is a rare malignant tumour of the bile duct. Due to the lack of typical clinical manifestations in the early stage, it is basically at an advanced stage when discovered. Radical resection remains the only curative therapy for patients with GBC. The resection rate is relatively low due to tumour invasion and metastasis, and the overall prognosis is poor. For most patients with unresectable lesions, chemotherapy has been the only recommended treatment for decades. Immunotherapy combined with TKIs (tyrosine kinase inhibitors) was proven to be effective in patients with hepatocellular carcinoma and cholangiocarcinoma. Some physicians have attempted to apply immunotherapy and TKIs combined with traditional chemotherapy in patients with advanced GBC. However, the outcomes were not clear because limited cases were reported. CASE PRESENTATION: We present a case series of four elderly patients with advanced GBC who received tislelizumab and lenvatinib combined with chemotherapy. All four patients responded to this treatment approach. Tumour responses were better in Patient 1 (TMB-H, MSS), Patient 2 (low TMB, MSS), and Patient 3 (low TMB, MSI-H) than in Patient 4 (low TMB, MSS), in whom metastasis occurred during the later stage of treatment. CONCLUSION: The combination of tislelizumab and lenvatinib may be a promising treatment for patients with advanced GBC. The efficacy and safety need further confirmation.

Pancreatic Cancer-Derived Exosomal miR-Let-7b-5p Stimulates Insulin Resistance in Skeletal Muscle Cells Through RNF20/STAT3/FOXO1 Axis Regulation.

Background: Cancers trigger systemic metabolic disorders usually associated with glucose intolerance, which is an initially apparent phenomenon. One of the features of pancreatic cancer (PC) metabolic reprogramming is the crosstalk between PC and peripheral tissues (skeletal muscle and adipose tissues), emphasized by insulin resistance (IR). Our previous study reported that mice pancreatic cancer-derived exosomes could induce skeletal muscle cells (C2C12) IR, and exosomal microRNAs (miRNAs) may exert an important effect. However, the underlying mechanism remains to be further elucidated. Methods: qPCR was used to determine the expression of let-7b-5p in normal pancreatic islet cells and PC cells. Exosomes were purified from PC cell culture medium by ultracentrifugation. The role let-7b-5p on IR-mediated by PC cells-derived exosomes was asses by Oil Red O staining using miRNA inhibitor. Western blot assay was performed to examine the expression of IR-related genes and the activation of signaling pathways. A Luciferase experiment was applied to confirm how let-7b-5p regulated the expression of RNF20. IP/WB analysis further determined whether RNF20 promoted STAT3 ubiquitination. Rescue experiment using RNF20 overexpression plasmid was performed to confirm the role of RNF20 on IR-mediated using PC cell-derived exosomes in C2C12 myotube cells. Results: miRNA-let-7b-5p was identified as the key exosomal miRNA, which could promote the IR in C2C12 myotube cells supported the lipid accumulation, the activation of STAT3/FOXO1 axis, and the decreased expression of IRS-1 and GLUT4. RNF20, an E3 ubiquitin ligase, was confirmed as the target gene of let-7b-5p and was found to improve IR by downregulating STAT3 protein expression via ubiquitination-mediated protein degradation. The ectopic expression of RNF20 could effectively attenuate the IR mediated by the pancreatic cancer-derived exosomes in C2C12 myotube cells. Conclusion: Our data suggest that exosomal miRNA-let-7b-5p may promote IR in C2C12 myotube cells by targeting RNF20 to activate the STAT3/FOXO1 axis.

LncRNA SNHG20 silencing inhibits hepatocellular carcinoma progression by sponging miR-5095 from MBD1.

OBJECTIVE: Long non-coding RNAs (lncRNAs) may have a significant regulatory effect on the progression of hepatocellular carcinoma (HCC), according to recent data. This study aims to investigate how SNHG20, a small nucleolar RNA host gene, contributes to the development of HCC. METHODS: LncRNA SNHG20, miR-5095, and MBD1 gene levels were determined using reverse transcription qPCR (RT-qPCR). Huh-7 and HepG2 cell bioactivities were evaluated using the CCK-8 kit, EdU, flow cytometry, and wound-healing migration tests. To assess the metastasis of Huh-7 and HepG2 cells, a transwell assay was used. The amounts of invasion- and proliferation-associated proteins were determined using western blot. Using the miRDB (www.mirdb.org) software, the possible target genes of lncRNA and miRNA were predicted, and this prediction was then verified by a twofold luciferase reporter test. To determine the pathologic alteration and Ki67 level in tumor tissues, H&E staining and IHC were employed. TUNEL was conducted to assess the presence of apoptotic bodies in the tumor tissues. RESULTS: lncRNA SNHG20 exhibited a high expression in HCC cells (P<0.01). LncRNA SNHG20 knockdown inhibited HCC cell metastasis (P<0.01) and accelerated apoptosis (P<0.01). LncRNA SNHG20 acted as a sponge of miR-5095 in HCC. In addition, miR-5095 overexpression inhibited HCC cell metastasis (P<0.01) and accelerated apoptosis (P<0.01); and miR-5095 negatively targeted MBD1. Furthermore, LncRNA SNHG20 regulated HCC progression through the miR-5095/MBD1 axis, and LncRNA SNHG20 knockdown inhibited HCC growth. CONCLUSION: LncRNA SNHG20 accelerates HCC progression by the miR-5095/MBD1 axis, indicating lncRNA SNHG20 can be used as a biomarker for patients with HCC.

[Identification of HBx-related integration sites in HBsAg-positive hepatocellular carcinoma biopsy].

To identify the integration sites in the host genome for the hepatitis B virus (HBV)-encoded X protein (HBx) in hepatocellular carcinoma (HCC) biopsies that are positive for hepatitis B surface antigen (HBsAg). HCC biopsies were obtained from six patients that were HBV carriers, as demonstrated by the presence of HBsAg in their serum and sero-negativity for antibody to HBsAg. DNA was extracted from the tissue, fractionated, and circularized. Primers were designed according to the HBx sequence and used to amplify the circularized DNA templates by inverse polymerase chain reaction (IPCR). The amplified DNA fragments were checked by electrophoresis, cloned into the PMD18-T expression vector, and sequenced. Sequence alignment was performed by the Blast algorithms. Seven electrophoresis bands yielded 22 sequencing results, which represented a total of three HBx integration sites in the host genome: 19q12, 2q32.2, 22q12. The 19q12 integration site encompasses the CCNE1 gene, which encodes a G1/S-specific cyclin-E1. HBx-related integration sites exist in HBsAg-positive HCC biopsies. The CCNE1 gene may play a role in the development of HBx-related HCC.