LINC00936/microRNA-221-3p Regulates Tumor Progression in Ovarian Cancer by Interacting with LAMA3.

BACKGROUND: Ovarian cancer remains a leading cause of mortality in women. It is known that long non-coding RNA (lncRNA) controls various biological processes and pathogenesis of many diseases, including cancers. This study aimed to determine whether LINC00936 and microRNA-221-3p (miR-221-3p) influence the laminin alpha 3 chain gene (LAMA3) in the development of ovarian cancer. METHODS: The expressions of LINC00936, miR-221-3p, and LAMA3 in ovarian cancer and adjacent tissues were assessed. Furthermore, ovarian cancer cells were transfected with vectors with overexpressed LINC00936, miR-221-3p mimic, miR-221-3p inhibitor, and si-LAMA3 to elucidate their functions in ovarian cancer cell proliferation, migration, invasion, angiogenesis, and tumorigenesis. The binding relationship between LINC00936 and miR-221-3p and the relationship between miR-221-3p and LAMA3 were verified to explore the mechanism of action of LINC00936 in ovarian cancer. LINC00936 binds to miR-221-3p as a ceRNA and regulates the expression of LAMA3. RESULTS: LINC00936 and LAMA3 were poorly expressed, while miR-221-3p was highly expressed in ovarian cancer tissues. Over-expression of LINC00936 contributed to decreasing miR- 221-3p expression and increasing LAMA3 expression. LINC00936 overexpression or miR-221- 3p silencing downregulated the levels of PCNA, MMP-2, MMP-9, and VEGF and decreased cell proliferation, migration, invasion, angiogenesis, and ovarian cancer tumorigenesis. CONCLUSION: Collectively, overexpression of LINC00936 suppressed the development of ovarian cancer by competitively binding to miR-221-3p and controlling LAMA3 expression. These results could serve as a novel theoretical base for the treatment of ovarian cancer.

Cervical Cancer Cells-Derived Extracellular Vesicles Containing microRNA-146a-5p Affect Actin Dynamics to Promote Cervical Cancer Metastasis by Activating the Hippo-YAP Signaling Pathway via WWC2.

Application of extracellular vesicles (EVs) for cancer treatment has been well-documented. We probed into the potential role of cervical cancer cells-secreted EVs by transferring miR-146a-5p in cervical cancer. After characterization of miR-146a-5p expression in clinical cervical cancer tissue samples, gain- and loss-of-function experiments were implemented to test the effect of miR-146a-5p on the invasion, epithelial-mesenchymal transition (EMT), and anoikis in cervical cancer cells. EVs were isolated from high-metastatic cervical cancer cells, after which their effects on the malignant behaviors of low-metastatic cervical cancer cells were assessed in a co-culture system. Luciferase assay was implemented to validate the putative binding relationship between miR-146a-5p and WWC2, followed by further investigation of downstream pathway (Hippo-YAP). Finally, nude mouse lung metastasis model was developed for in vivo validation. miR-146a-5p was elevated in cervical cancer tissues and high miR-146a-5p expression promoted the metastatic potential of cervical cancer cells through enhancing their invasiveness and anoikis resistance, and inducing EMT. Furthermore, miR-146a-5p carried by EVs secreted by highly metastatic cervical cancer cells could promote the metastasis of low-metastatic cervical cancer cells. Mechanistically, miR-146a-5p targeted WWC2 to activate YAP, by which it inhibited the phosphorylation of cofilin, and promoted the process of cofilin-mediated depolymerization of F-actin to G-actin. In vivo data demonstrated that EVs-carried miR-146a-5p promoted tumor metastasis through the WWC2/YAP axis. Cancer-derived EVs delivered pro-metastatic miR-146a-5p to regulate the actin dynamics in cervical cancer, thereby leading to cancer metastasis. This experiment highlighted an appealing therapeutic modality for cervical cancer.

Neuropeptide Y in the amygdala contributes to neuropathic pain-like behaviors in rats via the neuropeptide Y receptor type 2/mitogen-activated protein kinase axis.

Neuropeptide Y (NPY) is a highly conserved endogenous peptide in the central and peripheral nervous systems, which has been implicated in nociceptive signaling in neuropathic pain. However, downstream mechanistic actions remain uncharacterized. In this study, we sought to investigate the mechanism of NPY and its receptor NPY2R in the amygdala in rats with neuropathic pain-like behaviors induced by chronic constriction injury (CCI) of the sciatic nerve. The expression of NPY and NPY2R was found to be aberrantly up-regulated in neuropathic pain-related microarray dataset. Further, NPY was found to act on NPY2R in the basolateral amygdala (BLA). As reflected by the decrease in mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) as well as the increase of NPY expression in the amygdala of rats with neuropathic pain-like behaviors, NPY was closely related to the effect of amygdala nerve activity in neuropathic pain. Subsequently, mechanistic investigations indicated that NPY2R activated the MAPK signaling pathway in the amygdala. NPY2R-induced decrease of MWT and TWL were also restored in the presence of MAPK signaling pathway antagonist. Moreover, it was revealed that NPY2R overexpression promoted the viability while inhibiting the apoptosis of microglia. Taken together, NPY in the amygdala interacts with NPY2R to activate the MAPK signaling pathway, thereby promoting the occurrence of neuropathic pain.

Combination therapy with Nab-paclitaxel and the interleukin-15 fused with anti-human serum albumin nanobody as a synergistic treatment for colorectal cancer.

This study determines the effect of Nab-paclitaxel in combination with IL-15 fusion protein, containing IL-15 and an anti-HSA nanobody domain, on colorectal cancer bearing mice. In vitro binding test of IL15 fusion protein to HSA and Nab-paclitaxel, as well as CTLL-2 cell stimulation assay were performed. The tumor inhibitory effects of Nab-paclitaxel in combination with IL-15 fusion protein was evaluated in the HCT116 bearing murine model. Moreover, the population and function of cytotoxic T cells and M1 macrophages, as well as MDSCs and Treg cells, were also further examined. As a result, combination therapy of Nab-paclitaxel and IL-15 fusion protein effectively inhibits the tumor growth and produced a 78% reduction in tumor size for HCT116, as compared to vehicle group. In the TDLN for the combination group, there were 18% of CD8+ IFN-γ + T-cells and 0.47% CD4+CD25+FOXP3+ regulatory T-cells, as opposed to 5.0% and 5.1%, respectively, for the model control group. Combination therapy further exhibited enhanced suppressive effects on the accumulation of CD11b+GR-1+ MDSC in spleen and bone marrow. Furthermore, Nab-paclitaxel and IL-15 fusion protein showed a significant suppression of NF-κB-mediated immune suppressive markers and increased expression of CD8, Granzyme B, CD62L, CD49b, and CD86 without obvious organ toxicity. In conclusion, combination therapy of Nab-paclitaxel and IL-15 fusion protein can effectively stimulate the antitumor activity of immune effector cells, thereby inhibiting immunosuppressive cells within the TME of colorectal cancer, and the overall therapeutic effect has a significant advantage over monotherapy.AbbreviationsInterleukin 15, IL-15; Human serum albumin, HSA; Myeloid-derived suppressor cells, MDSC; Albumin binding domain, ABD; Tumor drainage lymph node, TDLN; Natural killer (NK); Tumor-draining lymph node (TDLN); Tumor infiltrating lymphocyte, TIL; Immunogenic cell death, ICD; Enhanced permeability retention, EPR; Liposomal doxorubicin, Doxil; 5-fluorouracil, 5-FU.

Intravenous Delivery of RNA Encoding Anti-PD-1 Human Monoclonal Antibody for Treating Intestinal Cancer.

Recently, antibody-based therapeutic agents are becoming most leading biologics for treating many diseases, especially for cancer. However, large-scale application of antibody drugs is still hampered by high cost and complex technical process. Endogenous expression of proteins or antibodies can be achieved by applying in vitro transcription (IVT) technique to produce mRNA and then deliver into body, which supplies opportunity to avoid many disadvantages in antibody production as well as clinical applications. Here, we designed the IVT-mRNA encoding the Pembrolizumab, as a commercial anti-PD-1 monoclonal antibody (mAb). The in vitro functional properties and in vivo antitumor activities of the Pembrolizumab expressed from mRNA were both assessed. Maximized expression level of the Pembrolizumab from IVT-mRNA was achieved via optimizing the usage of signal peptide and molar ratio of heavy/light chain. Then the mRNA was further formulated by lipid nanoparticle (LNP), which enable efficient in vivo delivery and protect mRNA from degradation. Intravenously delivered the single dose of mRNA-LNPs in mice resulted in duration of serum Pembrolizumab level over 25 µg/mL more than 35 days. Pharmacokinetic study exhibited significantly enhanced drug exposure of mRNA-encoded mAbs compared with direct injection of Pembrolizumab at same dose. Chronic treatment of the tumor-bearing mice with LNP-encapsulated Pembrolizumab mRNA effectively downregulated the growth of intestinal tumors and improved the animal survival. In brief, our present research demonstrated that the application of LNP-encapsulated IVT-mRNA can change the human body into a protein drug manufacturing site to express full-size mAbs for treating cancer and hold potential to be a novel alternative to protein-based therapies.

Engineered mRNA-expressed bispecific antibody prevent intestinal cancer via lipid nanoparticle delivery.

The potential of antibodies, especially for the bispecific antibodies, are limited by high cost and complex technical process of development and manufacturing. A cost-effective and rapid platform for the endogenous antibodies expression via using the in vitro transcription (IVT) technique to produce nucleoside-modified mRNA and then encapsulated into lipid nanoparticle (LNP) may turn the body to a manufactory. Coinhibitory pathway of programmed death ligand 1 (PD-L1) and programmed cell death protein 1 receptor (PD-1) could suppress the T-cell mediated immunity. We hypothesized that the coblocking of PD-L1 and PD-1 via bispecific antibodies may achieve more potential antitumor efficacies compare with the monospecific ones. Here, we described the application of mRNA to encode a bispecific antibody with ablated Fc immune effector functions that targets both human PD-L1 and PD-1, termed XA-1, which was further assessed the in vitro functional activities and in vivo antitumor efficacies. The in vitro mRNA-encoded XA-1 held comparable abilities to fully block the PD-1/PD-L1 pathway as well as to enhance functional T cell activation compared to XA-1 protein from CHO cell source. Pharmacokinetic tests showed enhanced area under curve (AUC) of mRNA-encoded XA-1 compared with XA-1 at same dose. Chronic treatment of LNP-encapsulated XA-1 mRNA in the mouse tumor models which were reconstituted with human immune cells effectively induced promising antitumor efficacies compared to XA-1 protein. Current results collectively demonstrated that LNP-encapsulated mRNA represents the viable delivery platform for treating cancer and hold potential to be applied in the treatment of many diseases.Abbreviations: IVT: in vitro transcription; LNP: lipid nanoparticle; hPD-1: human PD-1; hPD-L1: human PD-L1; ITS-G: Insulin-Transferrin-Selenium; Pen/Strep: penicillin-streptomycin; FBS: fetal bovine serum; TGI: tumor growth inhibition; IE1: cytomegalovirus immediate early 1; SP: signal peptide; hIgLC: human immunoglobulin kappa light chain; hIgHC: human IgG1 heavy chain; AUC: area under the curve; Cl: serum clearance; Vss: steady-state distributed volume; MLR: mixed lymphocyte reaction.

The Applicability of ADA, AFU, and LAC in the Early Diagnosis and Disease Risk Assessment of Hepatitis B-Associated Liver Cirrhosis and Hepatocellular Carcinoma.

Objective: This study aimed to evaluate the applicability of adenosine deaminase (ADA), α-l-fucosidase (AFU), lactic acid (LAC), and their combined detection in the early diagnosis of chronic hepatitis B (CHB), liver cirrhosis (LC), and hepatocellular carcinoma (HCC). Methods: A retrospective analysis of hepatitis B-positive liver disease patients admitted between 2015 and 2020 was conducted. The receiver operating characteristic (ROC) curve was used to determine the diagnostic value of each indicator in LC and HCC, and binary logistic regression analysis was performed to determine the factors and risks related to the occurrence of the two conditions. Results: The levels of ADA, AFU, and LAC were significantly increased in patients with CHB, LC, and HCC (p < 0.05). The ROC curve showed that the sensitivity and specificity of ADA, AFU, LAC, and their combined detection in the CHB and LC groups as well as in the LC and HCC groups reflected different degrees of clinical value. In the CHB and LC groups, the adjusted odds ratio (OR) values of ADA, AFU, and LAC among patients in the high-level group were 3.218, 1.859, and 11.474, respectively, when the median was considered the cutoff point. When quartiles were considered the cutoff point, the OR risk values of the adjusted levels of ADA, AFU, and LAC were higher than those of the lowest-level group (Q1) (p < 0.05). In the LC and HCC groups, the adjusted OR values of ADA, AFU, and LAC among patients in the high-level group were 0.967, 2.365, and 38.368, respectively. When quartiles were considered the cutoff point, the OR risk values of AFU and LAC levels were higher than those of the lowest-level group (Q1) (p < 0.05). Conclusion: ADA, AFU, and LAC demonstrated good value in the early diagnosis of LC and HCC. The combined detection of ADA+AFU+LAC is more effective than single detection for the early diagnosis of the two conditions. ADA, AFU, and LAC can serve as risk predictors of LC, while AFU and LAC can be considered early risk predictors of HCC.

Lipopolysaccharides increase the risk of colorectal cancer recurrence and metastasis due to the induction of neutrophil extracellular traps after curative resection.

BACKGROUND: Intra-abdominal infection after curative surgery for colorectal cancer is a serious complication associated with an increased risk of recurrence. Lipopolysaccharides (LPS)-an essential component of the cell wall of Gram-negative bacteria-were found to exert a protumorigenic effect by stimulating the inflammatory pathology and formation of neutrophil extracellular traps (NETs). This study was conducted to test whether LPS-induced formation of NETs promotes the development of cancer and metastasis. METHODS: The clinical characteristics, incidence of relapse, and serum myeloperoxidase-DNA complexes of 40 patients with infection and 40 patients without infection after curative surgery were analyzed. The effects of LPS on the induction of NETs were evaluated in a mouse model of colorectal cancer and liver metastasis. The toll-like receptor 9 (TLR9)-a DNA receptor-was knocked down to assess its effect on the mitogen-activated protein kinase pathway and activities implicated in the formation of NETs. RESULTS: Analysis of the clinical data obtained from these patients showed the significant relation of the formation of NETs and incidence of metastasis and survival rates. Subsequent in vitro experiments revealed an increased level of citrullinated-histone H3 and myeloperoxidase-DNA in LPS-injected mice with colorectal cancer. In the mimic metastatic model, injection of LPS enhanced the metastatic capacity, which was then attenuated by DNase I. This suggested that the formation of NETs was activated by LPS. Injection of TLR9-knockdown HCT116 cells in mice, followed by induction through LPS, mitigated the level of citrullinated-histone H3 and myeloperoxidase-DNA. This finding implied that the formation of NETs was suppressed. CONCLUSION: These findings shed light on the mechanism underlying the relationship between the elevated rate of colorectal cancer recurrence in patients who underwent surgery and the incidence of infection. This mechanism involves the protumorigenic activities of LPS-induced formation of NETs. The NETs which could be mediated by the TLR9 and the mitogen-activated protein kinase signaling pathway.

Toxoplasma Gondii Rhoptry Protein 16 (ROP16) Modifies Apoptosis in Human 293T Cells.

Toxoplasma Gondii is an obligate intracellular protozoan. T. gondii tachyzoites can invade nucleated host cells and inhibit their apoptosis. Therefore, the goal of the present study was to determine whether rhoptry protein 16 (ROP16) secreted by the invading T. gondii can reduce the apoptotic response of the host cell. For this purpose, a vector for in vitro overexpression of T. gondii ROP16 was constructed and used to transfect human 293T cells. Cells transfected with the vector robustly expressed ROP16, as evidenced by Western blotting. Apoptosis of 293T cells was induced by incubation with 0.5 µg/mL actinomycin D (ActD) for 24 h, and its magnitude was measured using Annexin V-FITC/PI and TUNEL assays. Cells transfected by ROP16-expressing vector were characterized by a significantly lower level of apoptosis measured by both techniques.Moreover, caspase-3 activity was also reduced. Thus, ROP16 inhibited ActD-induced apoptosis of human 293T cells, documenting the ability of this rhoptry protein to modulate apoptosis of host cells infected by T. gondii.

Induction of FAS II Metabolic Disorders to Cause Delayed Death of Toxoplasma gondii.

The exact mechanism of delayed death of Toxoplasma gondii is not known. FAS II synthesis in the apicoplast of T. gondii is essential for the survival of Toxoplasma gondii, while ß-hydroxyacylacyl carrier protein dehydratase (FabZ) is indispensable for fatty acid synthesis. The present study investigated the relationship between the delayed death of T. gondii by inducing metabolic disorders due to suppression the expression of FabZ. A tetracycline-induced knockout vector inserted with T. gondii fabZ gene was constructed, and transfected into T. gondii TATi strain by electroporation. The stable mutants with tetracycline-induced knockout were selected, expression of FabZ was suppressed by using anhydrotetracycline (ATc), and FAS II deficient tachyzoites were prepared. The Western blot and qPCR results revealed that proliferation of FAS II deficient tachyzoites was not significantly different compared to the normal tachyzoites at 24 h and 48 h; however, after 72 h, the number of T. gondii tachyzoites in the ATc treated group was significantly (p < 0.05) less than that of non-treated group, indicating the delayed death of T. gondii caused by the loss of apicoplast and decrease in the expression of FabZ, which inhibited the FAS II metabolism. The results of this study can be used for prevention of toxoplasmosis by inducing delayed death of T. gondii.

miR-124 inhibits progression of hepatocarcinoma by targeting KLF4 and promises a novel diagnostic marker.

Hepatocarcinoma is one of the most lethal malignancy haunting the Chinese population, which is partially due to the difficulties in diagnosis at an early stage. The search for a biomarker that could signify the presence and progress of hepatocarcinoma is never ended. MicroRNAs are 22-nt RNAs that could bind to 3' UTR of target mRNAs, mediating degradation of mRNAs or inhibiting the translation. Although much has been investigated, the role of miR-124 in hepatocarcinoma remained elusive. We first detected aberrant expression level of miR-124 in HCC tissues of 112 patients. By exploring the clinical parameters, we found a significantly inverse correlation between miR-124 level and TNM stages. Consistent with this, the survival analysis indicated the association of low miR-124 with longer survival time. Subsequent forced expression miR-124 resulted in reduced cell viability of Hep3B and SMMC-7221, which cell lines have high and low background expression of miR-124, respectively. TargetScan prediction rendered a subset of target candidates, which were selected for experimental validation, KLF4 was subject to luciferase assay. Ectopic expression of KLF4 increased the sphere formation ability and CD44/133-positive cell numbers, which can be reversed by abundant expression of miR-124, suggesting that KLF4 is a functional target of miR-124 in tumourigenesis and cancer progression of HCC.

Detection of clarithromycin-resistant Helicobacter pylori in clinical specimens by molecular methods: A review.

Various molecular methods have been developed to rapidly detect clarithromycin (CLR) resistance in Helicobacter pylori isolates in clinical specimens. All of these assays for detecting CLR resistance in H. pylori are based on detection of mutations in the 23S rRNA gene. In this article, we summarise current knowledge regarding the detection of H. pylori CLR resistance in clinical specimens by molecular tests. The available data showed that restriction fragment length polymorphism (RFLP), 3'-mismatch PCR, DNA sequencing, the PCR line probe assay (PCR-LiPA) and fluorescence in situ hybridisation assay (FISH) are able to detect CLR-resistant H. pylori in clinical specimens with excellent specificity and sensitivity. However, several factors limit their clinical application, including fastidious, time-consuming preparation and low-throughput as well as carrying a risk of contamination. Furthermore, as an invasive method, FISH is not suitable for children or the elderly. Among the molecular methods, one that is most promising for the future is real-time PCR probe hybridisation technology using fluorescence resonance energy transfer (FRET) probes, which can rapidly detect CLR resistance with high sensitivity and specificity in biopsies and stool specimens, even though mixed infections are present in clinical specimens. Moreover, due to the advantages that this method is simple, rapid and economical, real-time PCR is technically feasible for clinical application in small- and medium-sized hospitals in developing countries. Second, with high sensitivity, specificity and throughput, DNA chips will also be a valuable tool for detecting resistant H. pylori isolates from cultures and clinical specimens.