The Human Host Defense Peptide LL-37 Overexpressed in Lung Cell Lines by Methanolic Extract of Valeriana officinalis

Abstract The present study investigated the effects of valerian methanolic extract and valerenic acid on the expression of LL-37 gene and protein in A549 and MRC5 line cells. After preparing Valerian seeds, sowing them in March 2020, and harvesting the rhizome in October 2020, the extract was prepared from the valerian rhizome by maceration method. Valerian acid content was determined using high performance liquid chromatography (HPLC). Two cell lines (A549 and MRC-5) were used to study the effects of valerian extract, and the MTT test was used to evaluate cell viability. The expression of LL-37 mRNA and protein was assessed by Real-Time PCR and western blot, respectively. In vivo safety assessments and histopathological analysis were also conducted. Data was analyzed by Graphpad Prism 8 software. Valerian methanolic extract and valerenic acid upregulated the LL-37 mRNA and protein expression in both treated cell lines. Valerenic acid showed a greater effect on upregulating LL-37 expression than valerian methanolic extract. A549 cells were more sensitive to valerian methanolic extract compared to MRC5 cells, and its cell viability was reduced. Furthermore, liver and kidney-related safety assessments showed that valerian methanolic extract had no toxic effects. In general, it was concluded that the methanolic extract of valerian as well as the resulting valerenic acid as the most important component of the extract has the ability to upregulate LL-37expression. Therefore, methanolic extract of valerian and valerenic acid can be considered for improving the immune system.

Basic biological characteristics of lncRNA B230352I09 and its role in the process of myocardial injury / 中华急诊医学杂志

Objective:To explore the basic biological characteristics of lncRNA B230352I09 and its role in the process of myocardial injury.Methods:We analyzed the biological characteristics of lncRNA B230352I09 on the UCSC website and predicted the possible binding protein of lncRNA B230352I09 by the catRAPID. Real-time fluorescence quantitative (RT) PCR method was applied to detect the expression of lncRNA B230352I09 in heart tissues at different time points (0, 1, 3, 7d) within 7 days after birth, the organs distribution and expression of lncRNA B230352I09 in neonatal mouse and the expression pattern of lncRNA B230352I09 in the heart of mice with myocardial injury. In addition, we constructed hypoxia model by culturing primary cardiomyocytes to detect the effect of lncRNA 230352I09 overexpression on hypoxic cardiomyocyte apoptosis by Hoechst staining kit, the effect of lncRNA B230352I09 overexpression on ROS content of hypoxic cardiomyocyte by DCFDA probe and changes in mitochondrial membrane potential of hypoxic cardiomyocytes by JC-1 Fluorescent probes.Results:Full-length of mouse B230352I09 was 663bp, located in the chr7:123031415-123066439 forward strand. RBBP6 gene was adjacent to B230352I09, which may be the target of lncRNA B230352I09 by catrapid prediction analysis. With the development of the heart, the expression level of lncRNA B230352I09 showed a gradual downward trend. The main expression organs of lncRNA B230352I09 in 1-day-old mice were heart, brain, kidney and liver. In heart tissue, lncRNA B230352I09 expression in non-cardiomyocytes was significantly less than in cardiomyocytes [ (1.0± 0.03) vs. (9.2± 3.29), P=0.013]. After myocardial injury, the expression level of lncRNA B230352I09 showed an increasing trend compared with the normal developing mice, but there was no statistical significance. Hoechst staining showed that lncRNA B230352I09 could inhibit the apoptosis of hypoxic cardiomyocytes. Detecting the content of ROS in cardiomyocytes showed that compared with the hypoxia group, the generation of ROS was significantly reduced in the lncRNA B230352I09 overexpression group ([(3.8±0.71) vs. (1.65±0.56), P=0.015]). JC-1 fluorescent probe was used to detect the mitochondrial membrane potential, and the results showed that the mitochondrial membrane potential of cardiomyocytes in the lncRNA B230352I09 overexpression group was significantly higher than that in the hypoxia group. Conclusions:In heart tissue, lncRNA B230352I09 was mainly expressed in cardiomyocytes. LncRNA B230352I09 has a protective effect in the process of myocardial injury in mice, mainly by inhibiting apoptosis of cardiomyocytes, reducing ROS production, and protecting mitochondrial membrane potential of cardiomyocytes.

Gene Mutation and Overexpression of Newly Diagnosed Multiple Myeloma Patients / 中国实验血液学杂志

OBJECTIVE@#To analyze the characteristics of gene mutation and overexpression in newly diagnosed multiple myeloma (NDMM) patients.@*METHODS@#Bone marrow cells from 208 NDMM patients were collected and analyzed. The gene mutation of 28 genes and overexpression of 6 genes was detected by DNA sequencing. Chromosome structure abnormalities were detected by fluorescence in situ hybridization (FISH).@*RESULTS@#Gene mutations were detected in 61 (29.33%) NDMM patients. Some mutations occurred in 5 or more cases, such as NRAS, PRDM1, FAM46C, MYC, CCND1, LTB, DIS3, KRAS, and CRBN. Overexpression of six genes (CCND1, CCND3, BCL-2, CCND2, FGFR3, and MYC) were detected in 83 (39.9%) patients, and cell cycle regulation gene was the most common. Single nucleotide polymorphisms (SNP) changes were detected in 169 (81.25%) patients, the TP53 P72R gene SNP (70.17%) was the most common. Abnormality in chromosome structure was correlated to gene overexpression. Compared to the patients with normal chromosome structure, patients with 14q32 deletion showed higher proportion of CCND1 overexpression. Similarly, patients with 13q14 deletion showed higher proportion of FGFR3 overexpression, whereas patients with 1q21 amplification showed higher proportion of CCND2, BCL-2 and FGFR3 overexpression.@*CONCLUSION@#There are multiple gene mutations and overexpression in NDMM. However, there is no dominated single mutation or overexpression of genes. The most common gene mutations are those in the RAS/MAPK pathway and the genes of cyclin family CCND are overexpression.

Construction and identification of a HEK293 cell line with stable TrxR1 overexpression / 南方医科大学学报

OBJECTIVE@#To construct a HEK293 cell line stably overexpressing TrxR1 as a cell model for functional study of TrxR1 and screening of TrxR1-targeting drugs.@*METHODS@#TrxR1 gene was amplified by PCR and ligated with the lentivirus expression vector pLVX-Puro, which was transformed into Escherichia coli and identified by Sanger dideoxy sequencing. HEK293 cells were infected with the recombinant lentivirus vector (pLVX-Puro-TXNRD1) and screened with Puromycin for cell clones with stable TrxR1 overexpression (HEK293-TrxR1-OE cells). HEK293-TrxR1-OE cells, along with HEK293 cells infected with pLVX-Puro vector (HEK293-NC) and normal HEK293 cells, were tested for mRNA and protein expression levels of TrxR1 using RT-qPCR and Western blotting. TrxR1 enzyme activity in the cells was evaluated with insulin endpoint assay and TRFS-green probe imaging. The sensitivity of the cells to auranofin, a specific TrxR1 inhibitor, was determined with CCK8 assay.@*RESULTS@#TrxR1 gene was successfully inserted into the lentiviral vector pLVX-Puro as confirmed by DNA sequencing. The enzyme activity and mRNA and protein expression levels of TrxR1 were significantly higher in HEK293-TrxR1-OE cells than in HEK293 and HEK293-NC cells (P < 0.005). The inhibitory effects of auranofin on proliferation and cellular TrxR1 enzyme activity were significantly attenuated in HEK293-TrxR1-OE cells as compared with HEK293 and HEK293-NC cells (P < 0.005).@*CONCLUSION@#We successfully obtained a HEK293 cell line with stable TrxR1 overexpression, which shows resistance to auranofin and can be used for screening TrxR1 targeting drugs.

The effect and mechanism of epigallocatechol gallate combined with trastuzumab on the proliferation of HER2 overexpressing breast cancer cells / 药学实践杂志

Objective To study the effect and mechanism of epigallocatechol gallate (EGCG) combined with trastuzu-mab on the proliferation of human epidermal growth factor receptor 2 (HER2) overexpressing breast cancer cells. Methods Trastuzumab was expressed and purified. The cell proliferation of HER2 overexpressing breast cancer cells BT474 and SK-BR-3 treated with trastuzumab, EGCG, or trastuzumab plus EGCG was evaluated by CCK8 assay. The effects of EGCG and trastuzumab on the expression of HER2, epidermal growth factor receptor (EGFR), mitogen-activated protein kinase (MAPK), protein kinase B (Akt), and their phosphorylated proteins in BT474 breast cancer cells were detected by Western blot. Results The results of cell proliferation assay indicated that EGCG and trastuzumab, alone or in combination, effectively inhibited the proliferation of BT-474 and SK-BR-3 cells. And within a certain concentration range, EGCG and trastuzumab showed a synergistic proliferation inhibitory effect on HER2 overexpressing breast cancer cells. Consistent with these results, Western blot results showed that trastuzumab and EGCG, alone or in combination significantly reduced the phosphorylation levels of Akt, MAPK, EGFR, and HER2 in BT474 cells. Moreover, the inhibition effect of EGCG plus trastuzumab was significantly more potent than either EGCG or trastuzumab. Conclusion EGCG and trastuzumab could synergistically inhibit the proliferation of HER2 overexpressing breast cancer cells, which may be related to the regulation of Akt and MAPK signaling pathways.

Surfactant Protein (SP) induces preterm birth by promoting oxidative stress via upregulating Storkhead-Box Protein 1

Abstract Preterm birth is the leading cause of infant mortality. The mechanisms that instigate preterm birth remain elusive and this makes it difficult to predict or prevent preterm birth. In this study, the authors found that SP-A induced pathological damage to the placenta and promoted preterm birth. Through mechanism, SP-A promoted the expression of STOX1 which further promoted the oxidative stress in the placenta by inhibiting the activities of a series of antioxidant enzymes including SOD, CAT and GSH-Px. SP-A also induced dysregulation of arginine metabolism by inhibiting NOS2 and ARG2. Overexpression of STOX1 aggravated SP-A induced oxidative stress, pathological damage, and preterm birth, whereas knockdown of STOX1 alleviated SP-A induced oxidative stress, pathological damage and preterm birth. The present study uncovers that SP-A induces preterm birth by promoting oxidative stress via upregulating STOX1, which provides new targets for the prediction and prevention of preterm birth.

Overexpression of (3S)-linalool synthase gene (LIS) regulates the glycyrrhizic acid biosynthesis in Glycyrrhiza uralensis hairy roots / 药学学报

(3S)-Linalool synthase (LIS) is a key enzyme involved in the monoterpene biosynthetic pathway. Based on our previous transcriptome study, the expression level of LIS gene was exceedingly related to glycyrrhizic acid (GA) biosynthesis. Therefore, we used hairy root culturing to further investigate the effect of LIS on the GA biosynthesis. A LIS gene (GenBank accession number: MZ169552) was cloned from Glycyrrhiza uralensis. The plant binary overexpression vector pCA-LIS was constructed by gene fusion. G. uralensis hairy roots overexpressing LIS were induced by the Agrobacterium rhizogenes ATCC15834. The expression levels of LIS were analyzed by real-time quantitative PCR (RT-qPCR) and the contents of GA in hairy root lines were determined by UPLC. It was found that in the hairy root lines overexpressing LIS, the expression levels of LIS were significantly higher than that in the wild type, while the contents of GA were remarkably lower than those in the wild type and negative control. These findings indicate that the expression level of LIS is negatively correlated with the accumulation of GA. In this study, LIS was cloned from G. uralensis for the first time and the negative regulatory effect of LIS on GA biosynthesis was confirmed by reverse genetics. This work provides support for further improvement of the molecular regulatory network of GA biosynthesis in G. uralensis.

The role of licorice DXS knockout and overexpression in glycyrrhizic acid biosynthesis / 药学学报

1-Deoxy-D-xylulose-5-phosphate synthase (DXS) is a rate-limiting enzyme involved in the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway for terpenoid precursor biosynthesis. DXS plays an essential role in glycyrrhizic acid (GA) biosynthesis. Based on our previous transcriptome study, there was a negative correlation between DXS expression and GA content. Therefore, we explored the regulatory role of DXS in GA biosynthesis using both gene overexpression and gene knockout in a hairy root culture system. DXS was cloned from Glycyrrhiza glabra L. (GenBank Accession No. MN158121). A plant binary expression vector pCA-DXS was constructed by a gene fusion method. The sgRNA sequence was designed based on the first exon of DXS to construct the gene editing vector pHSE-DXS. Hairy roots overexpressing or knocking out DXS were generated through an Agrobacterium-mediated method with licorice hypocotyls as explants. Wild-type hairy roots and negative control hairy roots containing empty plasmids were also evaluated. UPLC was used to determine the GA content in each licorice hairy root line. Results showed that the content of GA in the hairy root group knocking out DXS was significantly higher than that in the wild-type and negative control groups, while in the hairy root group overexpressing DXS was significantly lower, suggesting that DXS plays a negative role in GA biosynthesis. This study provides a foundation for determining the function of DXS in terpenoid metabolism and for further establishment of a molecular regulatory network of GA biosynthesis.

Ferulate 5-hydroxylase gene (F5H) regulation of glycyrrhizic acid biosynthesis determined by gene overexpression and knockout / 药学学报

Ferulate 5-hydroxylase (F5H) is a key enzyme involved in the phenylpropane metabolism pathway. Based on our previous transcriptome sequencing study, F5H played a negative regulatory role in glycyrrhizic acid (GA) biosynthesis. Therefore, in this study we cloned the F5H gene and investigated its regulatory effect on GA accumulation through gene overexpression and knockout. F5H was cloned from Glycyrrhiza glabra L. (GenBank Accession No. MK882511). A plant binary expression vector pCA-F5H was constructed by inserting F5H into pCAMBIA1305.1 at Spe I and Bgl II sites. The sgRNA sequences were designed based on the first exon of F5H. The CRISPR/Cas9 gene editing vector pHSE-F5H was constructed by inserting F5H sgRNA into pHSE401 at two Bsa Ⅰ sites. PCA-F5H and pHSE-F5H were transfected into Agrobacterium tumefaciens ATCC15834, which was used to induce hairy root overexpressing or knocking out F5H with licorice hypocotyl as explants. At the same time, wild type and negative control hairy roots were also generated. UPLC was used to assay the GA content in different hairy root lines, and results showed that the GA content in hairy root lines knocking out F5H was significantly higher, whereas in hairy root lines overexpressing F5H GA content was lower than that in the wild-type and negative control. In this work, through a reverse genetics strategy, the negative regulatory effect of F5H on GA biosynthesis was confirmed through gene overexpression and knockout. This work will lay a foundation for further elucidation of the molecular regulatory network of GA biosynthesis.

Overexpressing of chalcone isomerase (CHI) gene enhances flavonoid accumulation in Glycyrrhiza uralensis hairy roots / 药学学报

Chalcone isomerase (CHI) is the second rate-limiting enzyme involved in the biosynthetic pathway of flavonoids in Glycyrrhiza uralensis. Based on our previous studies, we selected the specific CHI haplotype (GenBank Accession No. KY115232) to maximize flavonoid accumulation. We constructed a plant binary expression vector for overexpression of this CHI gene by the gene fusion method and transfected the plasmid into Agrobacterium tumefaciens ACCC10060 by electroporation. The recombinant A. tumefaciens ACCC10060 subsequently was used to infect cotyledons and hypocotyls of G. uralensis to obtain transgenic hairy roots. A qRT-PCR method was used to determine the copy number of CHI and a UPLC method was used to assay the content of four flavonoids in different hairy root lines. The qRT-PCR results showed that the copy number of CHI in hairy roots was 1 or 5. UPLC results showed that the content of total flavonoids, liquiritin, liquiritigenin, and isoliquiritigenin in transgenic hairy root samples was significantly higher than that in wild-type samples. This study demonstrates that overexpression of CHI significantly increases the content of flavonoids in hairy roots of G. uralensis. This work provides a theoretical basis for clarifying the function of CHI. Three transgenic hairy root lines of G. uralensis were isolated which can be used to increase the accumulation of licorice flavonoids in vitro.

Forced expression of FoxGl in specific brain region of App/ps1 mice / 中国药理学通报

Aim To generate the mouse FoxGl overexpression in the specific brain region based on App/psl mice. Methods A FoxGl transgenic mouse line CAG-loxp-stop-loxp-FoxG1-IRES-EGFP was introduced from Southeast University. By crossing B6; 129-5-HT1Btml(CreERT)Cre with this line, we generated a transgenic mouse line Cre; FoxGl. By crossing App/ps1 with this line, combined with tamoxifen induction, FoxG1 was ectopically expressed in the cortex, striatum and hippocampus in app/ ps1 mice. Results The offspring of the mice over expression of FoxGl in the specific brain region of App/ps1 mice were successful in generation. Conclusions FoxG1 overexpression mice in Alzheimer's Disease brain region is obtained by using reasonable gene recombination, providing new model into the mechanism underlying the Alzheimer's disease related with FoxG1.

Construction of rat diacylglycerol kinase gamma lentivirus overexpression vector by homologous recombination / 中国组织工程研究

BACKGROUND: Lentiviral vectors have been widely used as exogenous transgenic vectors. However, a recombinant lentiviral vector containing rat diacylglycerol kinase y (DGKy) gene has not been reported. OBJECTIVE: To construct lentiviral overexpression vector of rat DGKy by homologous recombination. METHODS: Total RNA was extracted from the brain tissue of adult Sprague-Dawley rats, and the cDNA obtained by PCR was used as a template to amplify the 5'-end 1 029 bp and the 3'-end 1 362 bp of the rat DGKy gene CDS. Then, the two homologous recombination fragments were ligated into the plasmid vector. The positive clones were confirmed by PCR and DNA sequencing. The CMV-rat DGKy-GFP lentiviral vector and the lentiviral packaging system were co-transfected into 293T cells for virus packaging and lentivirus was collected to infect 293T cells. The expression of GFP in infected 293T cells was observed under fluorescence microscope. Real-time PCR and western blot assay were used to detect the relative expression of DGKy mRNA in infected 293T cells. RESULTS AND CONCLUSION: The results of PCR simplification and sequencing indicated that the CMV-rat DGKy-GFP lentiviral vector was successfully constructed. In 293T cells infected with CMV-rat DGKy-GFP lentivirus, the expression of GFP was observed under fluorescence microscope and the DGKy mRNA expression was increased significantly than that of the vector group by real-time PCR (P < 0.01). Western blot assay results showed that the DGKy protein expression of the selected GFP-positive 293T cells was increased very significantly (P < 0.001). To conclude, the rat DGKy lentiviral overexpression vector has been successfully constructed and maintains high expression in 293T cells.

Screening Quorum-sensing-associated sRNA and construction of its overexpression and deletion mutant in Pseudomonas aeruginosa / 医学研究生学报

ObjectiveQuorum-sensing (QS) and small regulatory RNA (sRNA) play key regulatory roles in many signaling cascades of Pseudomonas aeruginosa. To investigate whether sRNA is involved in P. aeruginosa QS system, screening QS system-related sRNA, and to construct sRNA overexpression and deletion strains of Pseudomonas aeruginosa for further study of sRNA function.MethodsSRNA associated with the QS system was screened by qPCR and RNA-sequencing (RNA-seq). The target gene were amplified by PCR and inserted into the overexpression vector pROp200 or the homologous recombination vector pGSM-MR, respectively. The connection reaction solution of pROp200-sRNA and pGSM-ΔsRNA was transformed into Escherichia coli DH5a and SM10lp, respectively. The recombinant vectors were identified by PCR. The pROp200-sRNA was transformed into PAO1 by heat shock method, and the pGSM-ΔsRNA was transferred from SM10lp to PAO1 by conjugation. SRNA overexpression and deletion strains were identified by PCR, DNA sequencing and qPCR, the determination of the growth curves and the pyocyanin levels of strains.ResultsFive QS -associated sRNA P26, P5316.1, P30, P34 and AmiL were successfully screened by RNA-seq and qPCR. PCR, DNA sequencing and qPCR showed that sRNA of AmiL, P30 and P34 overexpression and knockout were successful. Compared with wild-type strain, sRNA overexpression and knockout had no significant effect on bacterial growth curve. It were notably that overexpression of AmiL and P30 inhibited and increase the production of pyocyanin, respectively (P0.05).ConclusionThe sRNA overexpression and deletion strains have been successfully constructed and can be used to study the regulatory relationship between sRNA and QS systems, and to further functional study.

Construction of an oral squamous cell carcinoma cell line for stable PA28γ overexpression / 华西口腔医学杂志

OBJECTIVE@#To construct a PA28γ overexpression cell line and determine its effects after infecting an oral squa-mous cell carcinoma (OSCC) cell line.@*METHODS@#The PA28γ gene was cloned into the pLOV.CMV.cherry.2A.EF1a.PuroR lentiviral vector by polymerase chain reaction (PCR), and PCR and DNA sequencing alignment analysis were used for identification. Then, 293T cells were used to package viral diseases. Infected OSCC cells were used to construct a cell line with stable PA28γ overexpression. Finally, the level of PA28γ expression in the OSCC cell line was detected through Western blot.@*RESULTS@#The successful construction of PA28γ recombinant lentiviral vectors was confirmed by DNA sequencing. The results of immunofluorescence showed that the PA28γ overexpression lentivirus successfully infected the OSCC cells and showed cherry red fluorescence. The results of Western blot demonstrated that the constructed cells with stable PA28γ overexpression significantly increased the expression of PA28γ.@*CONCLUSIONS@#The PA28γ overexpression lentiviral vector can significantly increase its protein expression in OSCC cells. We provide a stable OSCC cell line for further study on the effect of PA28γ in OSCC.

c-Met overexpression in breast cancer with positive axillary lymph node

Background: Breast cancer is the second most common cancer in the world and is the most epidemic cancer in women, with approximately 1.67 million cases. Metastasis of tumor cells to other organs is a major cause of the increasing trend of mortality in breast cancer. This study aims to analyze the expression of c-Met associated with metastasis to axillary lymph nodes in invasive breast cancer.Method: The research was conducted at the Laboratory of Anatomical Pathology of Hasanuddin University Hospital. Stratified sampling was performed from January 2014 - January 2019. Immunohistochemical staining technique was applied upon 66 collected samples, followed by evaluating the c-Met expression score in invasive breast cancer group with positive and negative lymph node status.Result: c-Met overexpression was found among the invasive breast cancer incidence with lymph node metastasis. Among 50 cases with c-Met overexpression (c-Met positive), 40 cases (80%) of invasive breast cancer with lymph node metastasis were identified, while 10 cases (20%) were found in invasive breast cancer without metastasis to lymph nodes. On 16 cases with negative c-Met, 3 cases (18.8%) were found in invasive breast cancer with lymph node metastasis, and 13 cases (81.3%) in invasive breast cancer without metastasis to the lymph nodes. The statistical test results indicated a significant correlation between c-Met expression scores and metastasis to axillary lymph nodes in invasive breast cancer (p <0.001).Conclusion: As one of biomarkers, c-Met overexpression plays a vital role in the treatment of patients with invasive breast cancer to predict patient outcomes and to determine modalities. It is possible to apply c-Met overexpression to investigate aggressiveness of metastatic tumor cells in the future.

Analysis of poliovirus receptor, CD155 expression in different human colorectal cancer cell lines: Implications for poliovirus virotherapy

Context: Poliovirus (PV) receptor (CD155) is expressed on several kinds of cells and exerts diverse functions. Various investigations have confirmed that changes in CD155 expression in cancer cell lines affect metastasis, proliferation, and migration. Aims: The purpose of the present study was to investigate the CD155 transcript and protein expression in human colon adenocarcinoma cell lines in comparison to normal fetal human colon (FHC) cells. Materials and Methods: The CD155 expression level in four human adenocarcinoma cell lines and normal colon cell line were assessed using the SYBR green quantitative real-time polymerase chain reaction (PCR) and flowcytometry. Results: The results of real-time PCR indicated that CD155 was significantly overexpressed in all human adenocarcinoma cell lines (P = 0.000). The highest and the lowest expression level of CD155 messenger RNA was observed in SW480 and HT29 cell lines by 491.14, and 12.04 fold changes, respectively, in comparison with the human normal cell line (FHC). Results of flowcytometry indicate that protein was strongly expressed in cancer cell lines. SW480 cells showed the highest CD155 protein expression level of 98.1%, whereas this protein expression was 1.3% in human normal colon cell line (FHC). Totally, these data indicate that CD155 expression is significantly elevated in cancer cell lines. Conclusions: The preferential expression of CD155 on cancer cell lines rather than on normal cell line suggests that CD155 could be targeted for future PV virotherapy

Differential expression of the TwHMGS gene and its effect on triptolide biosynthesis in Tripterygium wilfordii / 中国天然药物

3-Hydroxy-3-methylglutaryl-CoA synthase (HMGS) is the first committed enzyme in the MVA pathway and involved in the biosynthesis of terpenes in Tripterygium wilfordii. The full-length cDNA and a 515 bp RNAi target fragment of TwHMGS were ligated into the pH7WG2D and pK7GWIWG2D vectors to respectively overexpress and silence, TwHMGS was overexpressed and silenced in T. wilfordii suspension cells using biolistic-gun mediated transformation, which resulted in 2-fold increase and a drop to 70% in the expression level compared to cells with empty vector controls. During TwHMGS overexpression, the expression of TwHMGR, TwDXR and TwTPS7v2 was significantly upregulated to the control. In the RNAi group, the expression of TwHMGR, TwDXS, TwDXR and TwMCT visibly displayed downregulation to the control. The cells with TwHMGS overexpressed produced twice higher than the control value. These results proved that differential expression of TwHMGS determined the production of triptolide in T. wilfordii and laterally caused different trends of relative gene expression in the terpene biosynthetic pathway. Finally, the substrate acetyl-CoA was docked into the active site of TwHMGS, suggesting the key residues including His247, Lys256 and Arg296 undergo electrostatic or H-bond interactions with acetyl-CoA.

Potential Oncogenic Role of the Papillary Renal Cell Carcinoma Gene in Non-Small Cell Lung Cancers

PURPOSE: Papillary renal cell carcinoma (PRCC) gene, which located in 1q23.1, is recurrently amplified in non-small cell lung cancer (NSCLC). However, it is unknown whether PRCC is overexpressed in primary NSCLCs and whether PRCC overexpression contributes to lung tumorigenesis. In this study, we aimed to identify the profiles of PRCC expression in Korean NSCLC patients and to elucidate the role of PRCC overexpression on lung tumorigenesis. MATERIALS AND METHODS: We performed immunohistochemistry analysis with a tissue array containing 161 primary NSCLCs. Small interfering RNA targeting PRCC (siPRCC) was transfected into two lung cancer cell lines (NCI-H358 and A549), after which tumor growth, migration, and invasion were observed. Expressions of cell proliferation-, cell cycle-, and metastasis-related molecules were examined by Western blot analysis. We also explored the in vivo effect of PRCC silencing. RESULTS: PRCC overexpression was recurrently observed in NSCLCs (95/161, 59%). After siPRCC treatment, tumor cell proliferation, colony formation, and anchorage independent growth were significantly reduced (p < 0.001 for all three effects). Migration and invasiveness were also significantly repressed (p < 0.001 for both effects). Reflecting cell proliferation, cell cycle, and metastasis, the expressions of Ki67, cyclin D1, AKT-1, pAKT, NF-kB p65, vimentin and CXCL-12 were found to be downregulated. Through mouse xenograft analysis, we confirmed that PRCC silencing significantly repressed a xenograft tumor mass in vivo (p < 0.001). CONCLUSION: The present data provide evidence that PRCC overexpression is involved in the tumorigenesis and progression of lung cancer.

Sub-cellular localization and overexpressing analysis of hydroxylase gene TcCYP725A22 of Taxus chinensis / 生物工程学报

The discovery of hydroxylases in the anticancer drug taxol biosynthesis pathway is a hotspot and difficulty in current research. In this study, a new hydroxylase gene TcCYP725A22 (GenBank accession number: MF448646.1) was used to construct a sub-cellular localization vector pCAMIBA1303-TcCYP725A22-EGFP to get the transient expression in onion epidermal cells. Laser confocal microscopy revealed that the protein encoded by this gene was localized in the cell membrane. Furthermore, the recombinant plant expression plasmid pBI121-TcCYP725A22 was constructed. After transient transformation to the Taxus chinensis mediated by Agrobacterium tumefaciens LBA4404, qRT-PCR and LC-MS were utilized to analyze the effects of TcCYP725A22 overexpression on the synthesis of taxol. The results showed that, in the TcCYP725A22 overexpressed cell line, expression levels of most defined hydroxylase genes for taxol biosynthesis were increased, and the yield of taxanes were also increased. It was concluded that the hydroxylase gene TcCYP725A22 is likely involved in the biosynthetic pathway of taxol.

Construction of miR-331-3p overexpression vector and its effect on cell proliferation / 生物工程学报

To investigate the effect of miR-331-3p on the proliferation of porcine renal epithelial cells (PK15) and its mechanism, the pcDNA 3.1(+) overexpression vector of miRNA-331-3p (pcDNA 3.1(+)-miR-331-3p) was constructed. PK15 cells were divided into four groups, including experimental group, experimental control group, inhibitor group and inhibitor control group. Experimental group and experimental control group were transfected with pcDNA 3.1(+)-miR-331-3p and pcDNA 3.1(+), respectively. Inhibitor group and inhibitor control group were transfected with miR-331-3p inhibitor and miR-331-3p negative control (miR-331-3p NC), respectively. Above all, CCK-8 reagent was used to plot the cell proliferation curve and Propidium (PI) staining was used to detect the proportion of cell stages. Secondly, its expression change were detected by quantitative real-time PCR that included the growth inhibitory protein family member 5 (ING5), cyclin-dependent kinase 2 (CDK2), cyclin-dependent kinase 3 (CDK3), cyclin-dependent kinase 4 (CDK4), Cyclin B and cyclin-dependent kinase inhibitor 1A (CDKN1A). The results showed that the expression of miRNA-331-3p was significantly increased in the experimental group. The cell proliferation curve showed that the number of cells in experimental group was significantly higher than that in experimental control group or inhibitor control group at 48 h and 72 h (P<0.05). Simultaneously, Inhibitor group was significantly lower than experimental control group or inhibitor control group in the number of cells at 48 h and 72 h (P<0.05), but there was no significant difference between the experimental group and the control group. Compared with the experimental control group, the proportion of cells of experimental group in G0/G1 phase decreased, the proportion of S phase and G2/M phase increased, and the inhibitor control group showed the opposite trend. Simultaneously, the expression levels of CDK2, CDK3, CDK4 and Cyclin B genes in the experimental group were significantly increased, while ING5 and CDKN1A genes inhibiting proliferation showed a significant downward trend. These results demonstrate that the miR-331-3p overexpression vector was successfully constructed, and miR-331-3p has the ability to promote the proliferation of PK15 cells. The study lays a solid foundation for further research for its role in pig growth and development.