iRGD-modified exosomes-delivered BCL6 siRNA inhibit the progression of diffuse large B-cell lymphoma.

Clinical applications of siRNA therapeutics have been limited by the immunogenicity of the siRNA and low efficiency of siRNA delivery to target cells. Recently, evidence have shown that exosomes, endogenous nano-vesicles, can deliver siRNA to the tumor tissues in mice. Here, to reduce immunogenicity, we selected immature dendritic cells (DCs) to produce exosomes. In addition, tumor targeting was achieved by engineering the DCs to express exosomal membrane protein (Lamp2b), fused to av integrin-specific iRGD peptide (CRGDKGPDC). Next, iRGD targeted exosomes (iRGD-Exo) were isolated from the transfected DCs, and then the isolated exosomes were loaded with BCL6 siRNA by electroporation. Our results found that integrin (αvß3) receptors were highly expressed on OCI-Ly8 cells. In addition, iRGD-Exo showed high targeting ability with avß3 integrins positive OCI-Ly8 cells. Significantly, iRGD-Exo loaded with BCL6 siRNA suppressed DLBCL cell proliferation in vitro. Furthermore, intravenously injected iRGD-Exo delivered BCL6 siRNA to tumor tissues, resulting in inhibition of tumor growth in DLBCL. Meanwhile, exosomes mediated BCL6 siRNA delivery did not exhibit appreciable toxicity in mice. Collectively, our study demonstrates a therapeutic potential of exosomes as a promising vehicle for RNAi delivery to treat DLBCL.

LncRNA FIRRE stimulates PTBP1-induced Smurf2 decay, stabilizes B-cell receptor, and promotes the development of diffuse large B-cell lymphoma.

Sustained expression of B-cell receptor (BCR) critically contributes to the development of diffuse large B-cell lymphoma (DLBCL). However, little is known on the mechanism regulating BCR expression. In the present study, we explored the biological significance of functional intergenic repeating RNA element (FIRRE) in DLBCL and its regulation on BCR. Functional impacts of FIRRE on cell viability, transformation, and apoptosis were examined by MTT, colony formation, and flow cytometry, respectively. The interaction between FIRRE and polypyrimidine tract binding protein 1 (PTBP1) was identified by RNA pull-down and verified using RNA immunoprecipitation (RIP) assays. The effects of FIRRE and PTBP1 on Smurf2 mRNA were examined by RIP, RNA pull-down, and mRNA stability assays. Smurf2-mediated BCR ubiquitination was investigated using co-immunoprecipitation, ubiquitination, and protein stability assays. In vivo, xenograft models were used to assess the impacts of targeting FIRRE on DLBCL growth. FIRRE was specifically up-regulated in and essentially maintained multiple malignant behaviors of BCR-dependent DLBCL cells. Through the interaction with PTBP1, FIRRE promoted the mRNA decay of Smurf2, a ubiquitin ligase for the degradation BCR protein. Targeting FIRRE was sufficient to regulat Smurf2 and BCR expressions and inhibit DLBCL malignancy both in vivo and in vitro. FIRRE-PTBP1 interaction, by simulating Smurf2 mRNA decay and stabilizing BCR, promotes the development of DLBCL. Consequently, targeting this signaling mechanism may provide therapeutic benefits for DLBCL.

CDK12 activates MYC to repress miR-28-5p/EZH2 and amplifies tonic BCR signaling to promote the development of diffuse large B-cell lymphoma.

Cyclin-dependent kinase 12 (CDK12) is a transcription-associated kinase that participates in various cellular processes. However, its regulatory role in the progression of diffuse large B-cell lymphoma (DLBCL), which is the most prevalent subtype of non-Hodgkin lymphoma (NHL), is still elusive and controversial.The expression of CDK12 was detected by immunohistochemistry (IHC), RT-qPCR was performed to detect miR-28-5p expression of OCI-LY3 and SU-DHL-4 cells. MTT and soft agarose colony formation assays were used to detect cell proliferation. The cell apoptosis was determined by flow cytometry. The protein expressions changes of MYC, EZH2 and the biomarkers of BCR signaling were also detected. A subcutaneous transplantation tumor model of OCI-LY3 cells in nude mice was established to evaluate anticarcinogenic activities of CDK12 knockdown. Elevated expression of CDK12 was observed while miR-28-5p was downregulated in DLBCL tissues. CDK12 knockdown or miR-28-5p overexpression could inhibit proliferation and promote apoptosis of DLBCL cells. miR-28-5p inhibition could reverse the effect of CDK12 knockdown on proliferation and apoptosis of DLBCL cells. In addition, CDK12 knockdown could inhibit DLBCL tumor growth in the mice model. CDK12 activated MYC to repress miR-28-5p/EZH2 and amplified tonic BCR signaling to promote the development of DLBCL, which might provide potential therapeutic targets for future therapeutic intervention in DLBCL.

LncRNA HCP5 acts as a miR-128-3p sponge to promote the progression of multiple myeloma through activating Wnt/ß-catenin/cyclin D1 signaling via PLAGL2.

BACKGROUND: Although long non-coding RNA (lncRNA) HCP plays essential roles in human cancers, its function and mechanism in multiple myeloma (MM) have not crystallized. METHODS: HCP5 level in MM was assessed through qRT-PCR. A series of functional investigations were conducted to evaluate the influences of HCP5 on proliferation and apoptosis. Bioinformatics analysis and RIP/RNA pull-down assays were carried out to determine the relationships among HCP5, miR-128-3p, and PLAGL2. Relative protein level was determined through Western blot. A xenograft tumor model was applied for validating the roles of HCP5/miR-128-3p/PLAGL2 axis in vivo. RESULTS: HCP5 was significantly increased in MM. HCP5 knockdown effectively thwarted the proliferative rate and cell cycle of MM cell lines and suppressed tumor growth. HCP5 regulated PLAGL2 expression by sponging miR-128-3p. PLAGL2 overexpression effectively rescued cells from influences by sh-HCP5 on cell proliferative and apoptotic rates. Additionally, HCP5 knockdown significantly inhibited Wnt/ß-catenin/cyclin D1 signaling, and these effects were eliminated by PLAGL2 overexpression. CONCLUSION: Our study revealed that HCP5/miR-128-3p/PLAGL2 is closely correlated to MM development by modulating Wnt/ß-catenin/cyclin D1 signaling. HCP5 promoted cell proliferation and tumor formation of MM cells by activating the Wnt/ß-catenin/CCND1 signaling pathway by sponging miR-128-3p to increase PLAGL2 expression.

[Preliminary experiences of application of sentinel lymph node navigation technique in early gastric cancer].

OBJECTIVE: To investigate the feasibility and clinical significance of sentinel lymph node (SLN) navigation limited surgery in early gastric cancer (EGC). METHODS: Thirty-nine patients confirmed with EGC between January 2002 and December 2006 were randomly divided into tailored surgery group (20 cases) and conventional surgery group (19 cases). By combining the mapping agents of (99m)Tc labeled sulfur colloid solution and blue violet, SLN biopsy was conducted in tailored surgery group, in which a limited gastric resection with D0-D1 lymphadenectomy was performed in 17 cases with negative SLN examined by routine HE staining during operation; standard radical gastrectomy with lymphadenectomy (D2) was conducted in the other 3 cases with positive SLN and in all the cases of conventional surgery group. The diagnostic accuracy and false-negative rate of SLN status were calculated respectively. The operation outcome and postoperative complication and survival rate were compared between the two groups. RESULTS: SLNs were detected in all 20 patients with a successful detection rate of 100% in tailored surgery group. The number of detected SLNs ranged from 1 to 3, with a mean of 2.2 per case. The diagnostic accuracy and false-negative rate was 95% and 5%, respectively. The hospital stay and recovery time of gastrointestinal functions in patients undergoing limited surgery were significantly shorter than in conventional surgery group and with similar postoperative survival and less complications. CONCLUSIONS: SLN biopsy may provide an accurate diagnostic procedure for detecting lymph node metastasis in EGC. Patients with node-negative EGC receiving limited surgery are likely to benefit from minimally invasive approach with the similar survival as standard radical surgery.

Killing effects of cytosine deaminase gene mediated by adenovirus vector on human pancreatic cancer cell lines in vitro.

OBJECTIVE: To evaluate the killing effects of the cytosine deaminase (CD) gene mediated by adenovirus vector on human pancreatic cancer cell lines in vitro. METHODS: The CD gene was cloned into pAdTrack-CMV-CD, and pAdTrack-CMV-CD and pAdEasy-1 were recombinated in bacteria. The newly recombinated Ad-CD containing green fluorescent protein (GFP) was propagated in 293 cells and purified by cesium chloride gradient centrifugation. Human pancreatic cancer cell lines Patu8988 and SW1990 were infected with this virus, then 5-FC was added. XTT assay was used to estimate relative numbers of viable cells. RESULTS: The positive clones were selected by using endonuclease to digest the combinatants and the concentration of viral liquids containing the CD gene was 2X10(11) pfu/ml. It was found that significant cytotoxic activities were possessed by 5-FC for the CD gene transduced pancreatic cell lines, but little effects exerted on the nontransduced pancreatic carcinoma cells. CONCLUSIONS: The CD gene mediated by adenovirus with a high infectivity is efficient for gene therapy of pancreatic carcinoma cell lines. These data demonstrate the therapeutic efficacy of an enzyme prodrug strategy in experimental pancreatic cancer.