An Assay Method for Characterizing Bacteriophage T7 RNA Polymerase Activity by Transcription-Translation (TX-TL) System.

T7 system is a commonly used in protein expression and the highest transcription activity of T7 RNAP usually caused the instability of T7 system. In order to apply T7 system extensively, it is essential to characterize T7 RNAP activity. In the present paper, an assay method for T7 RNAP activity was developed with a transcription-translation (TX-TL) system. After the optimization of TX-TL system, the operating parameters were determined as 34°C, 60 min with 20 ng/µl of plasmid DNA template. The standard curve of TX-TL assay method indicated an excellent correlation (r = 0.998), and the sensitivity was better than that of western blotting method. The precision investigation indicated a mean-relative error of 2.58% and a standard-relative error of 7.01%. Moreover, the cell lysate could be added directly to the optimized TX-TL system without affecting T7 RNAP activity assay. The feasibility of present method was further confirmed by characterizing T7 RNAP activity in cell lysate of five strains of Escherichia coli (E. coli) DH5α with different T7 RNAP activities and seven commercial strains of E. coli (DE3). The present assay method for T7 RNAP activity would have a great application in synthetic biology, metabolic engineering, enzyme engineering and biomedicine.

Exosome Marker Proteins of Tumor-Associated Fibroblasts and Exosome-Derived miR-92a-3p Act as Potential Biomarkers for Liver Cancer.

Many recent studies have shown that microRNAs (miRNAs) in exosomes can be absorbed by nearby or distant cells, and the abnormal expression of these exosomal miRNAs is associated with most pathological progresses. In this study, we explored the diagnostic value of exosome marker proteins and exosome-derived miR-92a-3p in liver cancer. The clinicopathological data of 60 patients with liver cancer admitted to Tanghan Gongren Hospital from October 2017 to October 2019 were collected. Tumor tissue and adjacent tissue were collected during surgery. Quantitative reverse transcription polymerase chain reaction and Western blot were used to detect the expression levels of miR-92a-3p in exosomes of fibroblasts and tumor tissue, and exosome marker proteins. In liver cancer tissue and fibroblast exosomes, the expression of miR-92a-3p was significantly increased. The receiver operator characteristic curve of the expression level of miR-92a-3p in exosomes and tissue showed that the area under the curve was 0.906 and 0.911, respectively. HSP70 and CD63 were highly expressed in the tissue of liver cancer and fibroblast exosomes. miR-92a-3p was positively correlated with HSP70 and CD63 in the exosomes of liver cancer fibroblasts. In addition, miR-92a-3p and exosome marker proteins (HSP70 and CD63) were highly expressed in tumors with a diameter greater than 5 cm, and were higher in liver cancer patients with BCLC stage B/C. Tumor fibroblast-derived exosome marker proteins and miR-92a-3p have good diagnostic value in liver cancer, indicating that they may be new diagnostic markers for liver cancer.

m5C RNA methyltransferase-related gene NSUN4 stimulates malignant progression of hepatocellular carcinoma and can be a prognostic marker.

Hepatocellular carcinoma (HCC) is a cancer with relatively high mortality, yet little attention has been devoted for related prognostic biomarkers. This study analyzed differential expression of m5C RNA methyltransferase-related genes in normal samples and tumors samples in TCGA-LIHC using Wilcoxon test. K-means consensus clustering analysis was implemented to subdivide samples. Independent prognostic factors were screened by univariate and multivariate Cox regression analyses. KEGG pathway enrichment analysis was performed on the screened independent prognostic factor using GSEA tools. qPCR was conducted to test mRNA expression of key m5C RNA methyltransferase-related genes in tissues and cells. There were 7 m5C RNA methyltransferase-related genes (NOP2, NSUN4, etc.) differentially expressed in HCC tumor tissues. HCC samples were classified into 3 subgroups through clustering analysis according to the expression mode of m5C RNA methyltransferase-related genes. It was also discovered that patients in different subgroups presented significant differences in survival rate and distribution of grade. Additionally, NOP2, NSUN4 and NSUN5 expression notable varied in different grades. Through regression analyses combined with various clinical pathological factors, it was displayed that NSUN4 could work as an independent prognostic factor. KEGG analysis showed that NSUN4 mainly enriched in signaling pathways involved in ADHERENS JUNCTION, RNA DEGRADATION, MTOR SIGNALING PATHWAY, COMPLEMENT and COAGULATION CASCADES. As examined by qPCR, NSUN4 was conspicuously upregulated in HCC patient's tissues and cells. Altogether, our study preliminarily developed a novel biomarker that could be independently used in prognosis of HCC, which may provide a new direction for the study of related molecular mechanism or treatment regimen.

MiR-18a-5p Facilitates Progression of Hepatocellular Carcinoma by Targeting CPEB3.

Objective: To explore the function of the miR-18a-5p/CPEB3 axis in regulating the occurrence of hepatocellular carcinoma (HCC). Methods: Differentially expressed miRNAs and mRNAs were acquired by bioinformatics analysis. qRT-PCR was used for miR-18a-5p and CPEB3 mRNA expression detection. Cell functional assays were implemented to examine the biological functions of HCC cells. The binding relationship between miR-18a-5p and CPEB3 was verified by a dual luciferase assay. Results: In HCC, miR-18a-5p was remarkably highly expressed, while CPEB3 was markedly lowly expressed. HCC cell progression was facilitated after cells transfecting miR-18a-5p mimic, whereas silencing miR-18a-5p caused the opposite result. Overexpressing CPEB3 could restore promoting effect of miR-18a-5p on the growth of HCC cells. Conclusion: Oncogene miR-18a-5p accelerates malignant phenotype by suppressing CPEB3. MiR-18a-5p/CPEB3 axis in HCC identified in this study provides a new target for HCC treatment.

MiR-92a-3p Promotes the Malignant Progression of Hepatocellular Carcinoma by Mediating the PI3K/AKT/mTOR Signaling Pathway.

BACKGROUND: As one of the most common cancers globally, hepatocellular carcinoma (HCC) usually has a poor prognosis. Many HCC patients are usually diagnosed at advanced stages. Therefore, new potential biomarkers for the diagnosis and prognosis of HCC are urgently needed. More and more studies have shown that miR-92a-3p can regulate the occurrence and development of various cancers, but its clinical significance and molecular mechanism in HCC are still elusive. Here, we tried to clarify the regulatory mechanism of miR-92a-3p in HCC. METHODS: In this study, we conducted qRT-PCR and revealed that miR-92a-3p was notably upregulated in HCC cells. MTT, flow cytometry, wound healing, Transwell invasion assays and western blot were conducted to uncover that overexpressed miR-92a-3p could boost the proliferation, migration, invasion and epithelial-mesenchymal transition (EMT) of HCC cells while inhibiting cell apoptosis. In addition, the proteins associated with the PI3K/AKT/mTOR pathway were also detected by western blot. RESULTS: It was suggested that miR-92a-3p could activate the PI3K/AKT/mTOR signaling pathway. CONCLUSION: These results suggest that miR-92a-3p plays a tumor-promoting role in HCC and may be a potential biomarker for the diagnosis and prognosis of HCC.

miR-9-5p facilitates hepatocellular carcinoma cell proliferation, migration and invasion by targeting ESR1.

The study aimed to explore the relationship between miR-9-5p and ESR1, and clarify the underlying functional mechanism in the occurrence and development of hepatocellular carcinoma (HCC). Expression data including miRNAs and mRNAs of HCC downloaded from TCGA database were processed for differential analysis, and corresponding clinical data were collected for survival analysis to identify the target miRNA miR-9-5p. Bioinformatics databases were applied for predicting downstream target mRNAs of miR-9-5p. qRT-PCR was used to evaluate expression of miR-9-5p. Western blot was used to detect protein expression of ESR1. MTT, wound healing assay and Transwell assay were used to detect HCC cell proliferation, migration and invasion, respectively. Dual-luciferase reporter gene assay was used to identify the targeting relationship between miR-9-5p and ESR1. Research suggested that miR-9-5p was highly expressed in HCC cells but ESR1 was poorly expressed. Overexpression of miR-9-5p could improve the proliferation, invasion and migration of cells. Dual-luciferase reporter assay showed that ESR1 was the downstream target of miR-9-5p in HCC. Overexpression of miR-9-5p markedly reduced ESR1 mRNA and protein levels in HCC cells, whereas inhibition of miR-9-5p expression produced the contrary results. Silencing ESR1 could noticeably reverse the effect of miR-9-5p knockdown on the proliferation, migration and invasion of HCC cells. As an oncogene, miR-9-5p fostered the proliferation, migration and invasion of HCC cells by targeting and inhibiting ESR1 expression.

Silencing of FoxM1 blocks growth, migration and invasion of papillary thyroid carcinoma cells.

The aim of the present study was to investigate the effect of Forkhead box transcription factor M1 (FoxM1)-silencing on the growth, migration and invasion of K1 human papillary thyroid carcinoma (PTC) cells. The effect of FoxM1-small interfering RNA (siRNA) in K1 cells was detected by western blot analysis. FoxM1-siRNA and control siRNA were transfected into K1 cells using Lipofectamine® 2000 (transfection group, T) and the non-meaning sequence group (NM). K1 cells exposed to PBS solution comprised the blank control group (CON). Cell proliferation ability was detected using an MTT assay. Cell migration and invasion was detected by the single cell scratch test and Transwell invasion assay, respectively. Western blot analysis indicated that FoxM1 siRNA downregulated the expression of FoxM1 protein. Cell proliferation, migration and invasion were significantly lower in the T group compared with the NM and CON groups (P<0.05). These results indicated that silencing of FoxM1 expression could block growth, invasion and migration of K1 cells. This study may provide a novel target gene for targeted therapy of PTC.

Construction of indazolo[3,2-a]isoquinolines via [3 + 2] cycloaddition of benzynes.

A [3 + 2] annulation protocol for the construction of N-substituted indazolo[3,2-a]isoquinolines starting from benzynes and C,N-cyclic azomethine imines was developed. A diverse range of highly functionalized products indazolo[3,2-a]isoquinolines featuring an indazole scaffold can be easily accessed via a one-step reaction under mild conditions, and they show good anti-proliferative activity on cancer cells.

miR-138-5p acts as a tumor suppressor by targeting hTERT in human colorectal cancer.

Colorectal cancer (CRC) is the second most common cancer in the world. The incidence of this cancer is increasing in the developing countries. Mechanism of CRC tumorigenesis has been widely studied at the molecular levels, and has been recently proposed microRNAs as novel players in CRC. It has been reported that microRNA-138-5p (miR-138-5p) play key roles in different kinds of human cancers. However, the roles and underlying molecular mechanisms of miR-138-5p in CRC have not been adequately elucidated. Thus, the aim of the present study was to investigate the roles and possible regulatory mechanisms of miR-138-5p in CRC. In this study, we demonstrated that miR-138-5p was significantly down-regulated in CRC tissue samples and cell lines. Functional analyses indicated that the overexpression of miR-138-5p significantly delayed cell proliferation, reduced colony formation and increased apoptosis in CRC cell lines. Moreover, human telomerase reverse transcriptase (hTERT), an important oncogene in the management of tumors, was confirmed as a direct target of miR-138-5p in CRC cells. We also found that the hTERT expression was increased in CRC tissues and was inversely correlated with miR-138-5p. Further study showed that the restoration of hTERT expression by an overexpressing plasmid could reverse the effects of miR-138-5p on proliferation and apoptosis of CRC cells. Taken together, these data defines a major suppresses proliferation and promotes apoptosis role for miR-138-5p, a microRNA functions as a tumor suppressor in CRC, by directly targeting hTERT, which would be provide a new strategy for future CRC therapies.

Ligustrazine alleviates acute pancreatitis by accelerating acinar cell apoptosis at early phase via the suppression of p38 and Erk MAPK pathways.

OBJECTIVE: This study aimed to investigate the role of ligustrazine on apoptosis and inflammatory reaction in acute pancreatitis. METHODS: Rats and acinar cells were treated with caerulein to induce acute pancreatitis models. Cell models were treated with saline, p38 inhibitor, Erk inhibitor and ligustrazine. Then, the levels of TNF-α, IL-1ß and IL-6 were determined by ELISA assay, the protein levels of p38, Erk1/2, p53 and cleaved caspase3 were determined by western blotting, and apoptosis were measured by flow cytometry. Rat models were treated with saline and ligustrazine. Plasma amylase and pancreatic myeloperoxidase activity and the levels of TNF-α, IL-1ß and IL-6 in rats were determined. The protein levels of p38, Erk1/2, p53 and cleaved caspase3 in pancreas tissues were determined by western blotting, and pancreas tissues were also performed TUNEL staining to observe apoptosis status. RESULTS: Ligustrazine downregulated the levels of TNF-α, IL-1ß, IL-6. The protein levels of p38 and Erk were reduced by p38 inhibitor, Erk inhibitor and ligustrazine, while the levels of p53 and cleaved caspase 3 were upregulated. Apoptosis of AP acinar cells and cells in AP rat models was promoted after treated with ligustrazine. Plasma amylase and pancreatic myeloperoxidase activity in AP rat models were reduced by ligustrazine. CONCLUSION: Ligustrazine alleviates acute pancreatitis by accelerating acinar cell apoptosis at early phase via the suppression on p38 and Erk MAPK pathways. It is capable of attenuating the severity of acute pancreatitis and may have a therapeutic effect on patients with acute pancreatitis.

Construction of a host-independent T7 expression system with small RNA regulation.

It is desirable to build a universal and efficient protein expression system for wild-type prokaryotic strains in biotechnology industry and the outstanding T7 expression system could be a good candidate. However, the current utilization of T7 system depends on the specific DE3 lysogenic hosts, which severely limits its application in wild-type strains. In this study, a host-independent T7 expression system without relying on DE3 lysogenic hosts to provide T7 RNA Polymerase was developed. T7 RNA Polymerase gene (Gene1) and T7 Promoter were successfully integrated into a single plasmid with the regulation of proper antisense RNA to limit T7 RNA Polymerase expression at a non-lethal level. This host-independent T7 expression system realized efficient protein expression in 4 non-DE3 Escherichia coli strains and a wild-type Sinorhizobium strain TH572.