Search

SummaryThe COVID-19 pandemic and the SARS-CoV-2 with its variants have posed unprecedented challenges worldwide. Existing vaccines have limited effectiveness against the SARS-CoV-2 variants. Therefore, novel vaccines to match current mutated viral lineages with long-term protective immunity are urgently in demand. In the current study, we for the first time designed a recombinant Adeno-Associated Virus 5 (rAAV5)-based vaccine named as rAAV-COVID-19 vaccine (Covacinplus) by using RBD-plus of spike protein with both the single-stranded and the self-complementary AAV5 delivering vectors (ssAAV5 and scAAAV5), which provides excellent protection from SARS-CoV-2 infection. A single dose vaccination induced the strong immune response against SARS-CoV-2. The induced neutralizing antibodies (NAs) titers were maintained at a high peak level of over 1:1024 even after more than one year of injection and accompanied with functional T-cells responses in mice. Importantly, both ssAAV- and scAAV-based RBD-plus vaccines exhibited high levels of serum NAs against current circulating variants including variants Alpha, Beta, Gamma and Delta. SARS-CoV-2 virus challenge test showed that ssAAV5-RBD-plus vaccine protected both young and old age mice from SARS-CoV-2 infection in the upper and the lower respiratory tracts. Moreover, whole genome sequencing demonstrated that AAV vector DNA sequences were not found in the genome of the vaccinated mice after one year vaccination, demonstrating excellent safety of the vaccine. Taken together, this study suggests that rAAV5-based vaccine is powerful against SARS-CoV-2 and its variants with long-term protective immunity and excellent safety, which has great potential for development into prophylactic vaccination in human to end this global pandemic.

Background: Overexpression of epidermal growth factor receptor (EGFR) has been reported to be implicated in the pathogenesis of non-small cell lung cancer (NSCLC). Several EGFR inhibitors have been used in clinical treatment of NSCLC, but the emergence of EGFRL858R/T790M resistant mutation has reduced the efficacy of the clinical used EGFR inhibitors. There is an urgent need to develop novel EGFRL858R/T790M inhibitors for better NSCLC treatment. Methods: By screening a natural product library, we have identified gossypol as a novel potent inhibitor targeting EGFRL858R/T790M. The activity of gossypol on NSCLC cells was evaluated by cell proliferation, cell apoptosis and cell migration assays. Kinase activity inhibition assay and molecular docking were used to study the inhibition mechanism of gossypol to EGFRL858R/T790M. Western blotting was performed to study the molecular mechanism of gossypol inhibiting the downstream pathways of EGFR. Results: Gossypol inhibited the cell proliferation and cell migration of NSCLC cells, and induced caspase-dependent cell apoptosis of NSCLC cells by upregulating the expression of pro-apoptotic protein BAD. Molecular docking revealed that gossypol could bind to the kinase domain of EGFRL858R/T790M with good binding affinity through hydrogen bonds and hydrophobic interactions. Gossypol inhibited the kinase activity of EGFRL858R/T790M with EC50 of 150.1 nM. Western blotting analysis demonstrated that gossypol inhibited the phosphorylation of EGFR and its downstream signal pathways in a dose-dependent manner. Conclusion: Gossypol inhibited cell proliferation and induced apoptosis of NSCLC cells by targeting EGFRL858R/T790M. Our findings provided a basis for developing novel EGFRL858R/T790M inhibitors for treatment of NSCLC.

The Regorafenib is a broad-spectrum kinase inhibitor that has been approved to treat colorectal cancer (CRC). However, evidences have shown that the agent is also implicated in drug interaction with microRNA-21 (miR-21), an oncogenic miRNA which plays a key role in resisting programmed cell death in CRC cells. Here, we supposed that, instead of kinase inhibition, Regorafenib can directly bind to and then stabilize miR-21 pre-element, thus preventing RNase Dicer-meditated cleavage of the pre-element to mature miR-21. In order to verify the notion, an in silico-in vitro integrated investigation of the direct intermolecular interaction between Regorafenib and miR-21 pre-element was performed by using active pocket identification, RNA-ligand docking, molecular dynamics (MD) simulation, binding energetic analysis, and fluorescence-based assay. It was revealed that the Regorafenib can bind at the major groove-like stem region of miR-21 pre-element through three geometrically satisfactory hydrogen bonds (H-bonds) as well as a number of hydrophobic forces and π-π stacking, conferring strong specificity and high stability to the RNA-ligand complex system (Kd=0.73μM). Separate inversion mutation of two base pairs (G6C, C12G) and (A13U, U4A) that are involved in the H-bonding can considerably impair the affinity of Regorafenib to miR-21 pre-element, with Kd increase to 27 and 96μM, respectively. All these supported that Regorafenib can directly bind to miR-21 pre-element at molecular level and the binding mode can be properly modeled by using the proposed integrated strategy. This study would provide a potential, alternative mechanism for anti-colorectal cancer chemotherapy with Regorafenib.

MicroRNAs are a class of small noncoding RNAs that function as key regulators of gene expression at the post-transcriptional level. Recently, microRNA-373 (miR-373) has been found to function as an oncogene in testicular germ cell tumors. In our study, we found that miR-373 is upregulated in human hepatocellular carcinoma (HCC) tissues as compared with adjacent normal tissues, and promotes the proliferation of the HCC cell lines HepG2 and QGY-7703 by regulating the transition between G1-phaseand S-phase. The gene encoding the protein phosphatase 6 catalytic subunit (PPP6C ), a negative cell cycle regulator, was identified as a direct target gene of miR-373 by use of a fluorescent reporter assay. The mRNA and protein levels of PPP6C were both inversely correlated with the miR-373 expression level. Overexpression of PPP6C abolished the regulation of cell cycle and cell growth exercised by miR-373 in HepG2 cells. These results indicate that miR-373 plays an important role in the pathogenesis of HCC, and may be a new biomarker in HCC. Our results demonstrate that miR-373 can regulate cell cycle progression by targeting PPP6C transcripts and promotes the growth activity of HCC cells in vitro. The downregulation of PPP6C by miR-373 may explain why the expression of miR-373 can promote HCC cell proliferation.