The main approach to treat HIV-1 infection is combination antiretroviral therapy (cART). Although cART is effective in reducing HIV-1 viral load and controlling disease progression, it has many side effects, and is expensive for HIV-1 infected patients who must remain on lifetime treatment. HIV-1 gene therapy has drawn much attention as studies of genome editing tools have progressed. For example, zinc finger nucleases (ZFN), transcription activator like effector nucleases (TALEN) and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 have been utilized to successfully disrupt the HIV-1 co-receptors CCR5 or CXCR4, thereby restricting HIV-1 infection. However, the effects of simultaneous genome editing of CXCR4 and CCR5 by CRISPR-Cas9 in blocking HIV-1 infection in primary CD4+ T cells has been rarely reported. Furthermore, combination of different target sites of CXCR4 and CCR5 for disruption also need investigation. In this report, we designed two different gRNA combinations targeting both CXCR4 and CCR5, in a single vector. The CRISPR-sgRNAs-Cas9 could successfully induce editing of CXCR4 and CCR5 genes in various cell lines and primary CD4+ T cells. Using HIV-1 challenge assays, we demonstrated that CXCR4-tropic or CCR5-tropic HIV-1 infections were significantly reduced in CXCR4- and CCR5-modified cells, and the modified cells exhibited a selective advantage over unmodified cells during HIV-1 infection. The off-target analysis showed that no non-specific editing was identified in all predicted sites. In addition, apoptosis assays indicated that simultaneous disruption of CXCR4 and CCR5 in primary CD4+ T cells by CRISPR-Cas9 had no obvious cytotoxic effects on cell viability. Our results suggest that simultaneous genome editing of CXCR4 and CCR5 by CRISPR-Cas9 can potentially provide an effective and safe strategy towards a functional cure for HIV-1 infection.

The family of potassium channel tetramerizationdomain (KCTD) proteins consists of 26 members with mostly unknown functions. The name of the protein family is due to the sequence similarity between the conserved N-terminal region of KCTD proteins and the tetramerization domain in some voltage-gated potassium channels. Dozens of publications suggest that KCTD proteins have roles in various biological processes and diseases. In this review, we summarize the character of Bric-a-brack,Tram-track, Broad complex(BTB) of KCTD proteins, their roles in the ubiquitination pathway, and the roles of KCTD mutants in diseases. Furthermore, we review potential downstream signaling pathways and discuss future studies that should be performed.

Hepatitis B virus (HBV) is a major risk factor for the development and progression of hepatocellular carcinoma. It has been reported that viral infection can interfere with cellular microRNA (miRNA) expression and participate in the pathogenesis of oncogenicity. Here, we report that decreasing levels of the expression of the miRNA miR‐192‐3p is associated with rising levels of HBV DNA in the serum of HBV patients. We revealed that HBV infection repressed the expression of miR‐192‐3p through hepatitis B x protein interaction with c‐myc. We further showed that miR‐192‐3p was repressed by HBV transfection in vitro and in a mouse model, leading to cellular autophagy. Using an miRNA target prediction database miRBase, we identified X‐linked inhibitor of apoptosis protein (XIAP) as a target gene of miR‐192‐3p and demonstrated that miR‐192‐3p directly targeted the XIAP 3′‐untranslated region of XIAP messenger RNA. Importantly, we discovered that HBV promoted autophagy through miR‐192‐3p‐XIAP axis and that this process was important for HBV replication in vitro and in vivo. We demonstrated that miR‐192‐3p functioned through the nuclear factor kappa B signaling pathway to inhibit autophagy, thereby reducing HBV replication. Conclusions: Our findings indicate that miR‐192‐3p is a regulator of HBV infection and may play a potential role in hepatocellular carcinoma. It may also serve as a biomarker or therapeutic target for HBV patients.

Extracellular vesicles (EVs) play important roles in intercellular communication through the delivery of their cargoes, which include proteins, lipids, and RNAs. Increasingly, multiple studies have reported the association between EV small non-coding RNAs and cancer, due to their regulatory functions in gene expression. Hence, analysis of the features of small non-coding RNA expression and their incorporation into EVs is important for cancer research. We performed deep sequencing to investigate the expression of small RNAs in plasma EVs from lung adenocarcinoma (ADC) patients, lung squamous cell carcinoma (SQCC) patients, and healthy controls. Then, eighteen differently expressed miRNAs in plasma EVs was validated by QRT-PCR. The small RNA expression profiles of plasma EVs were different among lung ADC, SQCC patients, and healthy controls. And many small RNAs, including 5′ YRNA hY4-derived fragments, miR-451a, miR-122-5p, miR-20a-5p, miR-20b-5p, miR-30b-5p, and miR-665, were significantly upregulated in non-small cell lung cancer (NSCLC) EVs. And the cell viability assays indicated that hY4-derived fragments inhibited the proliferation of lung cancer cell A549. By comparing the cellular and EV expression levels of six miRNAs in NSCLC cells, we found that miR-451a and miR-122-5p were significantly downregulated in NSCLC cell lysates, while significantly upregulated in NSCLC EVs. The differently expressed EV small RNAs may serve as potential circulating biomarkers for the diagnosis of NSCLC. Particularly, YRNA hY4-derived fragments can serve as a novel class of biomarkers, which function as tumor suppressors in NSCLC. Additionally, miR-451a and miR-122-5p may be sorted into NSCLC EVs in a selective manner.

Extracellular vesicles (EVs) released by tumor cells play important roles on the remodeling of the tumor–stromal environment and on promoting tumor metastasis. Our earlier studies revealed that miR-122-5p, a type of small non-coding RNA, was dysregulated in non-small cell lung cancer (NSCLC) cell-derived EVs. In this study, we found that miR-122-5p was selectively sorted and secreted into lung cancer EVs through binding to RNA-binding protein hnRNPA2B1. In addition, we found that hnRNPA2B1 interacted with miR-122-5p through the EXO-motif. The delivering of lung cancer EVs-miR-122-5p promoted the migration of liver cells, which may play roles in establishing a pre-metastatic micro-environment and hepatic metastasis of lung cancer. Importantly, our findings revealed the molecular mechanism that RNA-binding protein controls the selective sorting of tumor-derived EV miR-122-5p, which potentially promotes lung cancer progression.

MicroRNA-720 (miR-720), a nonclassical miRNA, is involved in the initiation and progression of several tumors. In our previous studies, miR-720 was shown to be significantly upregulated in cervical cancer tissues compared with normal cervical tissues. However, the precise biological functions of miR-720, and its molecular mechanisms of action, are still unknown. Microarray expression profiles, luciferase reporter assays, and western blot assays were used to validate Rab35 as a target gene of miR-720 in HEK293T and HeLa cells. The regulation of Rab35 expression by miR-720 was assessed using qRT-PCR and western blot assays, and the effects of exogenous miR-720 and Rab35 on cell migration were evaluated in vitro using Transwell® assay, wound healing assay, and real-time analyses in HeLa cells. The influences of exogenous miR-720 on cell proliferation were evaluated in vitro by the MTT assay in HeLa cells. In addition, expression of E-cadherin and vimentin associated with epithelial-mesenchymal transition were also assessed using western blot analyses after transfection of miR-720 mimics and Rab35 expression vectors. The results showed that the small GTPase, Rab35, is a direct functional target of miR-720 in cervical cancer HeLa cells. By targeting Rab35, overexpression of miR-720 resulted in a decrease in E-cadherin expression and an increase in vimentin expression and finally led to promotion of HeLa cell migration. Furthermore, reintroduction of Rab35 3′-UTR(−) markedly reversed the induction of cell migration in miR-720-expressing HeLa cells. The miR-720 promotes cell migration of HeLa cells by downregulating Rab35. The results show that miR-720 is a novel cell migration-associated gene in cervical cancer cells.