Current application and future perspective of CRISPR/cas9 gene editing system mediated immune checkpoint for liver cancer treatment.

Liver cancer, which is well-known to us as one of human most prevalent malignancies across the globe, poses a significant risk to live condition and life safety of individuals in every region of the planet. It has been shown that immune checkpoint treatment may enhance survival benefits and make a significant contribution to patient prognosis, which makes it a promising and popular therapeutic option for treating liver cancer at the current time. However, there are only a very few numbers of patients who can benefit from the treatment and there also exist adverse events such as toxic effects and so on, which is still required further research and discussion. Fortunately, the clustered regularly interspaced short palindromic repeat/CRISPR-associated nuclease 9 (CRISPR/Cas9) provides a potential strategy for immunotherapy and immune checkpoint therapy of liver cancer. In this review, we focus on elucidating the fundamentals of the recently developed CRISPR/Cas9 technology as well as the present-day landscape of immune checkpoint treatment which pertains to liver cancer. What's more, we aim to explore the molecular mechanism of immune checkpoint treatment in liver cancer based on CRISPR/Cas9 technology. At last, its encouraging and powerful potential in the future application of the clinic is discussed, along with the issues that already exist and the difficulties that must be overcome. To sum up, our ultimate goal is to create a fresh knowledge that we can utilize this new CRISPR/Cas9 technology for the current popular immune checkpoint therapy to overcome the treatment issues of liver cancer.

Identification, isolation and expression analysis of eight stress-related R2R3-MYB genes in tartary buckwheat (Fagopyrum tataricum).

KEY MESSAGE: Eight R2R3 - MYB genes in tartary buckwheat were identified, and their expression patterns were comprehensively analyzed, which reveals role in plant response to abiotic stresses. The proteins of the R2R3-MYB superfamily play key roles in the growth and development processes as well as defense responses in plants. However, their characteristics and functions have not been fully investigated in tartary buckwheat (Fagopyrum tataricum), a strongly abiotic resistant coarse cereal. In this article, eight tartary buckwheat R2R3-MYB genes were isolated with full-length cDNA and DNA sequences. Phylogenetic analysis of the members of the R2R3-MYB superfamily between Arabidopsis and tartary buckwheat revealed that the assumed functions of the eight tartary buckwheat R2R3-MYB proteins are divided into five Arabidopsis functional subgroups that are involved in abiotic stress. Expression analysis during abiotic stress and exogenous phytohormone treatments identified that the eight R2R3-MYB genes responded to one or more treatments. This study is the first comprehensive analysis of the R2R3-MYB gene family in tartary buckwheat under abiotic stress.

Characterization of two tartary buckwheat R2R3-MYB transcription factors and their regulation of proanthocyanidin biosynthesis.

Tartary buckwheat (Fagopyrum tataricum Gaertn.) contains high concentrations of flavonoids. The flavonoids are mainly represented by rutin, anthocyanins and proanthocyanins in tartary buckwheat. R2R3-type MYB transcription factors (TFs) play key roles in the transcriptional regulation of the flavonoid biosynthetic pathway. In this study, two TF genes, FtMYB1 and FtMYB2, were isolated from F. tataricum and characterized. The results of bioinformatic analysis indicated that the putative FtMYB1 and FtMYB2 proteins belonged to the R2R3-MYB family and displayed a high degree of similarity with TaMYB14 and AtMYB123/TT2. In vitro and in vivo evidence both showed the two proteins were located in the nucleus and exhibited transcriptional activation activities. During florescence, both FtMYB1 and FtMYB2 were more highly expressed in the flowers than any other organ. The overexpression of FtMYB1 and FtMYB2 significantly enhanced the accumulation of proanthocyanidins (PAs) and showed a strong effect on the target genes' expression in Nicotiana tabacum. The expression of dihydroflavonol-4-reductase (DFR) was upregulated to 5.6-fold higher than that of control, and the expression level was lower for flavonol synthase (FLS). To our knowledge, this is the first functional characterization of two MYB TFs from F. tataricum that control the PA pathway.

Synergistic anti-tumor effect of dual drug co-assembled nanoparticles based on ursolic acid and sorafenib.

Both ursolic acid (UA) and sorafenib (Sora) have been generally utilized in cancer treatment, and the combination of the two has also shown a good anti-tumor effect. However, single-agent therapy for Hepatocellular carcinoma (HCC) has the disadvantages of multi-drug resistance, poor water solubility and low bioavailability, and the application of traditional nanocarrier materials is limited due to their low drug loading and low carrier-related toxicity. Therefore, we prepared US NPs with different proportions of UA and Sora by solvent exchange method for achieving synergistic HCC therapy. US NPs had suitable particle size, good dispersibility and storage stability, which synergistically inhibited the proliferation of HepG2 cells, SMMC7721 cells and H22 cells. In addition, we also proved that US NPs were able to suppress the migration of HepG2 cells and SMMC7721 cells and reduce the adhesion ability and colony formation ability of these cells. According to the results, US NPs could degrade the membrane potential of mitochondrial, participate in cell apoptosis, and synergistically induce autophagy. Collectively, the carrier-free US NPs provide new strategies for HCC treatment and new ideas for the development of novel nano-drug delivery systems containing UA and Sora.

[Molecular cloning of squalene synthase gene form Paris polyphylla and its expression in Escherichia coli].

OBJECTIVE: To clone the cDNA sequence of squalene synthase gene from Paris polyphylla, and characterize the biological features of the obtained SQS. METHOD: Using homology cloning and RACE technique, a full-length cDNA sequence of PpSQS gene was isolated from P. polyphylla. The obtained sequence was analyzed by bioinformatics softwares. A plasmid [named pET-30b (+)-PpSQS] was constructed for prokaryotic expression the recombinant PpSQS. RESULT: The full-length cDNA of PpSQS gene is 1 498 bp, which contains a 1 212 bp ORF. Sequence analysis indicated that PpSQS encoded 403 amino acids residues with a calculated molecular weight (MW) of 46.36 kDa and an isoelectric point (pI) of 6.83. SDS-PAGE results showed that the recombinant PpSQS was expressed in Escherichia coli BL21 (DE3) by inducing with 1 mmol x L(-1) IPTG. CONCLUSION: The full-length cDNA sequence of PpSQS gene was obtained from P. polyphylla, and its molecular features were consisted with classic SQS in plant. The recombinant PpSQS was successfully expressed in E. coli.

CRISPR/Cas9-based application for cancer therapy: Challenges and solutions for non-viral delivery.

CRISPR/Cas9 genome editing is a promising therapeutic technique, which makes precise and rapid gene editing technology possible on account of its high sensitivity and efficiency. CRISPR/Cas9 system has been proved to able to effectively disrupt and modify genes, which shows great potential for cancer treatment. Current researches proves that virus vectors are capable of effectively delivering the CRISPR/Cas9 system, but immunogenicity and carcinogenicity caused by virus transmission still trigger serious consequences. Therefore, the greatest challenge of CRISPR/Cas9 for cancer therapy lies on how to deliver it to the target tumor site safely and effectively. Non-viral delivery systems with specific targeting, high loading capacity, and low immune toxicity are more suitable than viral vectors, which limited by uncontrollable side effects. Their medical advances and applications have been widely concerned. Herein, we present the molecule mechanism and different construction strategies of CRISPR/Cas9 system for editing genes at the beginning of this research. Subsequently, several common CRISPR/Cas9 non-viral deliveries for cancer treatment are introduced. Lastly, based on the main factors limiting the delivery efficiency of non-viral vectors proposed in the existing researches and literature, we summarize and discuss the main methods to solve these limitations in the existing tumor treatment system, aiming to introduce further optimization and innovation of the CRISPR/Cas9 non-viral delivery system suitable for cancer treatment.

FtNAC31, a Tartary buckwheat NAC transcription factor, enhances salt and drought tolerance in transgenic Arabidopsis.

Tartary buckwheat [Fagopyrum tataricum (L.) Gaertn.] is a pseudocereal with strongly abiotic resistance. NACs, one of the largest plant-specific transcription factors (TFs), are involved in various stress responses. However, the characteristics and regulatory mechanisms of NAC TFs remain unclarified clearly in Tartary buckwheat (TB). In this study, it validated that salt, drought, and abscisic acid (ABA) stress significantly up-regulated the expression of NAC TF gene FtNAC31. Its coding protein has a C-terminal transactivated domain and localized in the nucleus, suggesting that FtNAC31 might play a transcriptional activation role in TB. Notably, overexpression of FtNAC31 lowered the seed germination rate upon ABA treatment and enhanced the tolerance to salt and drought stress in transgenetic Arabidopsis. Furthermore, under various stresses, the activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) in FtNAC31 overexpressed lines exhibited a sharp increase trend. Meanwhile, the expression levels of several stress-associated genes including RD29A, RD29B, RD22, DREB2B, NCED3, and POD1, were dramatically upregulated in lines overexpressing FtNAC31. Altogether, overproduction of FtNAC31 could enhance the resistance to salt and drought stresses in transgenic Arabidopsis, which most likely functioned in an ABA-dependent way.

The jasmonate-responsive transcription factor CbWRKY24 regulates terpenoid biosynthetic genes to promote saponin biosynthesis in Conyza blinii H. Lév.

Conyza blinii H. Lév., the most effective component is saponin, is a biennial medicinal material that needs to be overwintered. WRKY transcription factors family is a large protein superfamily that plays a predominant role in plant secondary metabolism, but their characteristics and functions have not been identified in C. blinii. The CbWRKY24 sequence was selectedfrom the transcriptome database of the C. blinii leaves constructed in our laboratory. Phylogenetic tree analysis revealed that it was associated with AaWRKY1 which can regulate artemisinin synthesis in Artemisia annua. Expression analysis in C. blinii revealed that CbWRKY24 was mainly induced by methyl jasmonate (MeJA) and cold treatments. Transcriptional activity assay showed that it had an independent biological activity. Overexpression of CbWRKY24 in transient transformed C. blinii resulted in improved totalsaponins content, which was attributed to upregulate the expression level of keys genes from mevalonate (MVA) pathway in transient transformed plants compared to wild type (WT) plants. Meanwhile, overexpression the CbWRKY24 in transient transformed tomato fruits showed that the transcript level of related genes in lycopene pathway decreased significantly when compared to WT tomatofruits. Additionally, the MeJA-response-element was found in the promoter regions of CbWRKY24 and the histochemical staining experiments showed that promoter had GUS activity in transiently transformed tobacco leaves. In summary, our results indicated that we may have found a transcription factor that can regulate the biosynthesis of terpenoids in C. blinii.

Overexpression of a Tartary Buckwheat Gene, FtbHLH3, Enhances Drought/Oxidative Stress Tolerance in Transgenic Arabidopsis.

bHLH (basic helix-loop-helix) transcription factors play important roles in the abiotic stress response in plants, but their characteristics and functions in tartary buckwheat (Fagopyrum tataricum), a flavonoid-rich cereal crop with a strong stress tolerance, have not been fully investigated. Here, a novel bHLH gene, designated FtbHLH3, was isolated and characterized. Expression analysis in tartary buckwheat revealed that FtbHLH3 was mainly induced by polyethylene glycol 6000 (PEG6000) and abscisic acid (ABA) treatments. Subcellular localization and a yeast one-hybrid assay indicated that FtbHLH3 has transcriptional activation activities. Overexpression of FtbHLH3 in Arabidopsis resulted in increased drought/oxidative tolerance, which was attributed to not only lower malondialdehyde (MDA), ion leakage (IL), and reactive oxygen species (ROS) but also higher proline (Pro) content, activities of antioxidant enzymes, and photosynthetic efficiency in transgenic lines compared to wild type (WT). Moreover, qRT-PCR analysis indicated that the expression of multiple stress-responsive genes in the transgenic lines was significantly higher than in WT under drought stress. In particular, the expression of AtNCED, a rate-limiting enzyme gene in ABA biosynthesis, was increased significantly under both normal and stress conditions. Additionally, an ABA-response-element (ABRE) was also found in the promoter regions. Furthermore, the transgenic Arabidopsis lines of the FtbHLH3 promoter had higher GUS activity after drought stress. In summary, our results indicated that FtbHLH3 may function as a positive regulator of drought/oxidative stress tolerance in transgenic Arabidopsis through an ABA-dependent pathway.

ß-Amyrin synthase from Conyza blinii expressed in Saccharomyces cerevisiae.

Conyza blinii H.Lév. is a widely used medicinal herb in southwestern China. The main pharmacological components of C. blinii are a class of oleanane-type pentacyclic triterpene glycosides known as conyzasaponins, which are thought to be synthesized from ß-amyrin. However, no genes involved in the conyzasaponin pathway have previously been identified. Here, we identify an oxidosqualene cyclase (OSC), a ß-amyrin synthase, which mediates cyclization of 2,3-oxidosqualene to yield ß-amyrin. Ten OSC sequences were isolated from C. blinii transcript tags. Phylogenetic analysis was used to select the tag Cb18076 as the putative ß-amyrin synthase, named CbßAS. The open reading frame of CbßAS is 2286 bp and encodes 761 amino acids. Its mature protein contains the highly conserved motifs (QXXXGXW/DCTAE) of OSCs and (MWCYCR) of ß-amyrin synthases. Transcription of CbßAS was upregulated 4-24 h after treatment of the seedlings of the C. blinii cultivar with methyl jasmonate. Furthermore, expression of CbßAS in Saccharomyces cerevisiae successfully yielded ß-amyrin. The chemical structures and concentrations of ß-amyrin were confirmed by GC-MS/MS. The target yeast ultimately produced 4.432 mg·L-1 ß-amyrin. Thus, CbßAS is an OSC involved in conyzasaponin biosynthesis.

Overexpression of a tartary buckwheat R2R3-MYB transcription factor gene, FtMYB9, enhances tolerance to drought and salt stresses in transgenic Arabidopsis.

Tartary buckwheat (Fagopyrum tataricum) is a traditional coarse cereal that exhibits strong plasticity in its adaptation to harsh and complicated environmental stresses. In an attempt to study the strong tolerance of tartary buckwheat, the FtMYB9 gene, which encodes an R2R3-MYB transcription factor protein, was functionally investigated. FtMYB9 expression was rapidly and strongly induced by ABA, cold, salt, and drought treatments in the seedling stage. A yeast one-hybrid system assay indicated that FtMYB9 is an activator of transcriptional activity, consistent with its roles as a transcription factor. Its overexpression in plants resulted in increased sensitivity to ABA at the germination and seedling stages compared to wild type. The overexpression of FtMYB9 increased tolerance to drought and salt stresses by the activation of some stress-related genes from both ABA-independent and ABA-dependent pathways in transgenic Arabidopsis. Furthermore, enhanced proline content and the activation of the P5CS1 gene implied that FtMYB9 may be involved in proline synthesis in plants. Collectively, these results suggest that FtMYB9 functions as a novel R2R3-MYB TF which plays positive roles in salt and drought tolerance by regulating different stress-responsive signaling pathways.

Characterization of Three Glucosyltransferase Genes in Tartary Buckwheat and Their Expression after Cold Stress.

Anthocyanins confer the red color in the hypocotyl of tartary buckwheat sprouts. Uridine diphosphate (UDP)-glucose:flavonoid 3-O-glycosyltransferase (UFGT) stabilizes anthocyanin by attaching the glucosyl moiety from UDP-glucose to the C3 hydroxyl of anthocyanin. In this study, we characterized three UFGT-like genes, designated FtUFGT1, 2, and 3 from tartary buckwheat. The results revealed that FtUFGT1, FtUFGT2, and FtUFGT3 can convert cyanidin to cyanidin 3-O-glucoside, with specific activities of 20.01 × 10(-3), 8.93 × 10(-3), and 20.24 × 10(-3) IU/mg, respectively. The active-site residues of the C-terminal domains and the N-terminal domains are important for the donor and acceptor recognition of these proteins. The expression of the three FtUFGTs paralleled the tissue-specific anthocyanin accumulation. After cold treatment, the increased content of anthocyanin was accompanied by the up-regulated expression of the three FtUFGTs. Among these three UGFT gene members, FtUFGT3 showed the highest expression level and the highest specific activity, suggesting that FtUFGT3 might be the major gene involved in anthocyanin biosynthesis. These results suggested that the FtUFGT genes, FtUFGT3 in particular, might be important candidates for anthocyanin formation in tartary buckwheat sprouts.