MiRNA-335 Modulates Hepatoma Cell Lines Apoptosis and Proliferation by Targeting Forkhead Box O3a (FOXO3a)

This study intends to elucidate MiRNA-335’s role in hepatoma cell lines (HCC). Real-time PCR was used to detect MiRNA-335 expression in HCC, flow cytometry and MTT were used to detect apoptosis and proliferation. Luciferase reporting system analyzed the targeting relationship between Foxo3a and MiRNA-335. HCC (SMMC7721 cell) exhibited significantly reduced MiRNA-335 compared to normal hepatocyte cell (HL7702). MiRNA-335 mimic inhibited HCC proliferation and enhanced apoptosis, which were reversed by MiRNA-335 inhibitor. Luciferase reporter gene system showed that MiRNA-335 significantly inhibited the fluorescent activity of Foxo3a 3′-UTR, indicating that MiRNA-335 could target Foxo3a RNA. In conclusion, the decrease of MiRNA-335 can promote the proliferation of hepatoma cells and inhibit apoptosis possibly through regulating Foxo3a, which provides a new direction for the treatment of liver cancer.

Study on the Mechanism of Mirna-21 Affecting the Differentiation of Bone Marrow Mesenchymal Stem Cells into Cardiomyocyte-Like Cells By Targeting Ajuba / Isl1 Axis

Purpose: To study the mechanism of miRNA-21 targeting ajuba/ Isl1 axis to affect BMSC differentiation to cardiac myoid cells. Methods: BMSC was cultured and miRNA-21 was constructed to infect BMSC. The miRNA-21 was directly regulated by luciferase reporter gene system. The expression of cTnI, ajuba and Isl1 was detected by RT-qPCR and WB. The expression of cTnI, ajuba and Isl1 was detected by RT-qPCR and WB, and miR-21 was detected by RT-qPCR; The differentiation ability of BMSC in all groups was detected by RT-qPCR and WB; To evaluate the effect of ajuba and Isl1 on the differentiation ability of BMSC. Results: BMSC was cultured successfully, BMSC was successfully constructed by mir-21-OE, mir-21-KD, ajuba-OE and ajuba KD slow virus; RT-qPCR and WB were used to detect the high expression of cTnI in mir-21-OE and ajuba KD groups, and the expression of miR-21 was increased, the expression of ajuba was inhibited and Isl1 expression was enhanced. Conclusion: The expression of ajuba was inhibited by up regulation of miRNA-21 target, and the expression of Isl1 enhanced to promote BMSC differentiation, that is, miRNA-21 regulated the axis of ajuba/ Isl1 to influence the differentiation of BMSC to cardiomyocyte-like cells.

hsa-miR-9-5p Down-Regulates HK2 and Confers Radiosensitivity to Nasopharyngeal Carcinoma

Background: This study was designed to explore the effects of hsa-miR-9-5p on radiotherapy sensitivity of nasopharyngeal carcinoma (NPC) by targeting hexokinase 2 (HK2). Methods: The levels of hsa-miR-9-5 and HK2 in NPC patients and radiosensitive and resistant cells were determined using qRT-PCR. The dual luciferase reporter gene system was used to determine hsa-miR-9-5p targeting HK2. The level of HK2 expression in NPC were determined using qRT-PCR and western blotting after the administration of hsa-miR-9-5p agomir. The effects of hsa-miR-9-5p on proliferation and apoptosis with or without irradiation (IR) were examined using CCK-8, flow cytometry and colony formation assays. (18F)-Flourodeoxyglucose uptake was used to evaluate the growth of tumor with or without radiation therapy in vivo. Results: hsa-miR-9-5p target to inhibit HK2. Moreover, the cell proliferation was seen in a decreased trend while the cell apoptosis increased in the hsa-miR-9-5p group following radiation therapy hsa-miR-9-5p also showed a significant inhibitory effect on the growth of tumor in vivo with radiation therapy. Conclusions: hsa-miR-9-5p improved the radiosensitivity of NPC by targeting HK2.

Differential Effects of Components in Artemisia annua Extract on the Induction of Drug-Metabolizing Enzyme Expression Mediated by Nuclear Receptors

Artemisia annua tea is a popular dosage form used to treat and prevent malaria in some developing countries. However, repeated drinking leads to an obviously decreased efficacy, which may be related to the induction of metabolizing enzymes by artemisinin. In the present study, the ability of different components in A. annua to activate the pregnane X receptor and constitutive androstane receptor was evaluated by the dual luciferase reporter gene system. The changes in mRNA and protein expression of CYP3A4 and CYP2B6 were determined by quantitative real-time PCR and Western blotting. Results showed that in the pregnane X receptor-mediated CYP3A4 reporter gene system, chrysosplenetin and arteannuin B exhibited a weak induction effect on pregnane X receptor wt, while arteannuin A had a strong induction effect on pregnane X receptor wt and pregnane X receptor 370 and a weak induction effect on pregnane X receptor 163. In the pregnane X receptor-mediated CYP2B6 reporter gene system, arteannuin A had a moderate induction effect on pregnane X receptor wt and pregnane X receptor 379, and a weak induction effect on pregnane X receptor 403, while arteannuin B had a weak induction effect on pregnane X receptor wt and pregnane X receptor 379. Arteannuin A had a strong induction effect on constitutive androstane receptor 3 in constitutive androstane receptor-mediated CYP3A4/2B6 reporter gene systems, while arteannuin B showed a weak induction effect on constitutive androstane receptor 3 in the constitutive androstane receptor-mediated CYP2B6 reporter gene system. The mRNA and protein expressions of CYP3A4 and CYP2B6 were increased when the pregnane X receptor or constitutive androstane receptor was activated. Various components present in A. annua differentially affect the activities of pregnane X receptor isoforms and the constitutive androstane receptor, which indicates the possibility of a drug-drug interaction. This partly explains the decline in efficacy after repeated drinking of A. annua tea.

The effect of miR-23b-3p on growth hormone in pituitary cells of Yanbian yellow cattle

Background There is a relationship between miR-23b-3p and GH in pituitary of Yanbian yellow cattle. However, the specific mechanism of the effect of miR-23b-3p on GH in pituitary of Yanbian yellow cattle is still unclear.This study aimed to evaluate the effect of miR-23b-3p on growth hormone (GH) in pituitary cells of Yanbian yellow cattle. Methods The primary culture of Yanbian yellow cattle pituitary cells was carried out, and mimics (miR-23b-3p-mi group), mimics reference substance (NC group), inhibitor (miR-23b-3p-in group) and inhibitor reference substance (iNC group) of miR-23b-3p were transfected into the established pituitary primary cells. After 48 h, the cells were collected and the total RNA and protein were extracted.The mRNA transcription and protein expression level of GH and miR-23b-3p target genes were detected by real time fluorescence quantitative PCR (qPCR) and Western blot, respectively. The target relationship of miR-23b-3p was validated by double luciferase reporter gene system. Results Compared with the NC control group, GH mRNA transcription and protein expression level in pituitary cells of Yanbian yellow cattle was significantly decreased by adding miR-23b-3p minics ( P <0.01), while compared with the iNC control group, GH mRNA transcription and protein expression level were significantly increased by adding miR-23b-3p inhibitor( P <0.05). The result of bioinformatics analysis and double luciferase reporter gene system validation proved that miR-23b-3p targeted 3'UTR of pituitary specific transcription factor 1 (POU1F1). Compared with the NC control group, POU1F1 mRNA transcription and protein expression level were significantly inhibited by the addition of miR-23b-3p minics ( P <0.01), while compared with the iNC control group, POU1F1 mRNA transcription and protein expression level were significantly increased by the addition of miR-23b-3p inhibitor ( P <0.01). Conclusions miR-23b-3p could regulate GH in pituitary cells by regulating POU1F1 gene.

USP21 modulates Goosecoid function through deubiquitination

The homeobox gene Goosecoid (GSC), which is known to regulate craniofacial development, is activated by mono-ubiquitination; however, the deubiquitylase responsible for GSC deubiquitination and inhibition has yet to be identified. In the present study, we constructed the recombinant plasmid pFlag-CMV-2-GSC and the SRY (sex-determining region Y)-box 6 (Sox6) reporter gene system to identify deubiquitylases that regulate GSC expression. We demonstrate that the ubiquitin carboxyl-terminal hydrolase 21 (USP21) regulates the deubiquitination of GSC negatively, as demonstrated by its inhibition of Sox6 reporter gene transcription. USP21 interacted with GSC to promote GSC deubiquitination while having no effect on GSC protein stability. Cell viability, migration, and function in ATDC5 cells were probably influenced by USP21 through GSC. These findings suggest that USP21 modulates GSC function through deubiquitination.

Monoterpenes induce the heat shock response in Arabidopsis

Monoterpenes are common constituents of essential oils produced by plants. Although it has been reported that monoterpenes enhanced the heat tolerance of plants, the mechanism has not been elucidated. Here, we tested whether 13 monoterpenes promoted the heat shock response (HSR) in Arabidopsis. To assess the HSR-inducing activity of monoterpenes, we produced transgenic Arabidopsis, which has the β-glucuronidase gene driven by the promoter of a small heat shock protein (HSP17.6C-CI) gene. Results indicated that two monocyclic and four bicyclic monoterpenes showed HSR-inducing activities using the reporter gene system. In particular, (−)-perillaldehyde, which is a monocyclic monoterpene, demonstrated the most potent HSR-inducing activity. (−)-Perillaldehyde significantly inhibited the reduction of chlorophyll content by heat shock in Arabidopsis seedlings. Our previous study indicated that chemical HSR inducers such as geldanamycin and sanguinarine inhibited the activity of plant chaperones in vitro. (−)-Perillaldehyde also inhibited chaperone activity, indicating that it might promote the expression of heat shock protein genes by inhibiting chaperones in the plant cell.