| Background: Lung cancer remains the most common cancer diagnosed worldwide and has one of the lowest survival rates of all cancers. The high mortality rate is related to the low cure rate, which in turn is associated with the lack of adequate screening and early detection measures. The understanding of lung cancer on molecular level is obviously insufficient, it is urgent need for improving diagnosis by novel molecular signatures and novel targets and strategies for personalized treatment.Numerous effective anticancer drugs have been developed from natural sources.Most anticancer drugs are secondary metabolites of plants and animals and other small molecule compounds. However, the activity of biological macromolecules has attracted great attention of researchers in recent years. The earthworm fibrinolytic enzyme(EFE)has shown significant antitumor activity in hepatoma cells both in vitro and in vivo. In addition, protein-bound polysaccharide(PSK) derived from the CM-101 strain of the fungus Coriolus versicolor has shown anticancer activity in vitro, in in vivo experimental models, and in human cancers. Moreover, trichosanthin, a type I ribosome-inactivating protein, induces cell death in various cell types, including several tumor cell lines, and induces apoptosis in K562 cells. Lentinula edodes is the second most popular edible mushroom in the world market, is also a specialty in China. In the folk is known as the king of the mountain. Lentinula edodes belongs to basidiomycotina,Agaricales, Tricholomataceae, cartinellin. Lentinula edodes contains high protein, low fat, polysaccharides, amino acids and vitamins. It is rich in nutritional and medicinalvalue. Lentinan edodes contains lentinan can increase the body’s immunity. The extract of Lentinus edodes can eliminate the hydrogen peroxide in vivo, also delay aging. In addition, Lentinan edodes have effect on diabetes, tuberculosis, infectious hepatitis,neuritis, constipation, reduce blood pressure, cholesterol, and blood lipid, but also the prevention of arteriosclerosis, cirrhosis and other diseases.Since 1990, according to theinternational study of pharmaceutical research for Lentinula edodes, six strains of mycels of Lentinula edodes have been fermented with the fermentation technology of bioengineering. One of the strain’sfermentation broths was found to have a direct anti-tumor effect. Because the strain was studied beginning in March 1991, it was named as “C91-3”. “C” represents “China”. The in vivo and in vitro experiments confirmed that some of the protein components have significant effects on inducing cell apoptosis.As the protein has the anti-tumor effect, our group conducted high-throughput sequencing of the transcriptome, access to a large number of Unigene and its functional annotation information. Unigene was 28923, with the coding frame for Unigene was18120. According to the preliminary results of gene function annotation, domain analysis using online software tool Sanger Pfam, screening of apoptosis related gene Latcripin-13 from Unigene sequences. We designed primer according to the transcriptome sequences of Latcripin-13, with 3 ’-Full RACE(Rapid Amplificatioon of c DNA Ends, RACE) and 5’-Full RACE method to obtain the coding sequence(CDS)of Latcripin-13 gene. At present, we have completed the CDS of Latcripin-13 on Gen Bank registered(Accession Number:KF682439).Objective: Construction of the prokaryotic expression vector to apoptosis related proteins Latcripin-13 domain of Lentinula edodes C91-3 in Ecoli. Rosetta-gami(DE3)induced expression, while the expression products were identified. Induced expression of Lentinula edodes C91-3 apoptosis related protein of Latcripin-13 domain, to study the influence on tumor cell biological function, to explore the mechanism of inducing apoptosis of tumor cells and provide the basis for the new anti-tumor drugs.Methods:(1)Total RNA was extracted from the mycelium of Lentinula edodes C91–3。The CDS of Latcripin-13 was isolated with 3’-Full Rapid Amplification of c DNA Ends(RACE) and 5’-Full RACE methods according to the Lentinula edodes transcriptome sequencing results. Through the software of bioinformatics analysis of Latcripin-13 protein, we get the amino acid composition and physicochemical properties of secondary structure, and predict its functional domain.(2)The specific primers were designed, acquired Latcripin-13 domain sequences and the gene was cloned into the prokaryotic expression vector p ET32a(+) by using the technology of RCR, construct Prokaryotic Recombinant Expression Plasmid p ET32a(+)-truncated-latcripin-13, the plasmid was transformed into Ecoli. Rosetta-gami(DE3) and inducible expression, the expression products were identified by Western blot method. Separation and purification of the target protein by affinity chromatography method and determination of protein concentration by Bradford method. For the purpose of protein refolding method using urea gradient dialysis, and the refolded protein was identified by the method of circular dichroism spectra.(3)Identification of antitumor activity of functional domain of Latcripin-13: MTT(Methyl Thiazoly Ltetra Zolium, MTT) method for the determination of the proliferation inhibition rate on human lung cancer cells of A549; With the FITC-labeled Latcripin-13 domain, the localization of the FITC-Latcripin-13 domain in A549 cells using fluorescence microscopy; Latcripin-13 domain effects on A549 cells morphology detection by electron microscopy;Latcripin-13 domain effects on apoptosis of A549 cells by 33258 methods; The influence of apoptosis and cell cycle with Latcripin-13 domain on A549 cells were detected by flow cytometry; JC-1 method to detect the A549 cell mitochondrial membrane potential; Spectrophotometry to detect the Caspase-3,-8,-9 enzyme activity;Western blot detect changes in the expression levels of Bcl-2, Bax, CCD1, CDK4, NF-,NF-κ B, GSK3β, p-GSK3β.Results:(1)Latcripin-13 was obtained by RACE method. Through the analysis of Latcripin-13 amino acid sequence, we found that the three level structure of the protein containing the chromosomes condense regulator(RCC1) and plant homeodomain(PHD).(2)Application of PCR technology, we has obtained the latcripin-13 genefragment, by Bam HⅠand Xho I double enzyme digestion to verify insertion of p ET32a(+) is the fragment of latcripin-13 gene fragment. SDS-PAGE and Western results show that Latcripin-13 functional domain in Ecoli. Rosetta-gami(DE3) was successfully expressed. Latcripin-13 domain was successfully purified by affinity chromatography.CD spectra results showed the target protein refolded successfully.(3)The A549 cells were treated with various concentrations of the Latcripin-13 domain, growth was significantly inhibited in a dose- and time-dependent manner(P < 0.05); Latcripin-13 domain was distributed in the cytoplasm of A549 cells and electron microscopy showed that the apoptosis of A549 cells induced by Latcripin-13 domain, and presents the basic phenomenon of early apoptosis; Hoechst 33258 staining showed that the protein can induce A549 cell apoptosis, and showed nuclear fragmentation and apoptotic chromosomal crimple phenomenon; Flow cytometry results showed, 100μg/ml Latcripin-13 domain induced the death of A549 cells through apoptosis and 100μg/ml BSA-treated group showed almost no early apoptotic or late apoptotic cells and only some mechanical damage of necrotic cells; Cell cycle results showed that, with the increasing concentration of Latcripin-13 domain, the percentage of G0/G1 phase increased, the percentage of S phase decreased significantly, Latcripin-13 domain arrested A549 cells in the cell cycle at G0/G1 phase; A549 cells treated with Latcripin-13 domain demonstrated significant dissipation of the MMP in a concentration-dependent manner, indicating that treatment with Latcripin-13 domain led to loss of mitochondrial membrane potential of A549 cells; Caspase Colorimetric Assay showed 100 μg/ml of Latcripin-13 domain significantly induced the activation of caspase-3, caspase-8 and caspase-9; Western blot results showed that, with the increase concentration of Latcripin-13 domain, the Bax/Bcl-2 ratio was increased(p<0.05), the GSK3β /p-GSK3β ratio was increased(p<0.05), NF- κB, CCD1, CDK4 expression was decreased.Conclusion:(1)According to the transcriptome of Lentinula edodes C91-3, apoptosis related gene Latcripin-13 was successfully screened, and successfully obtained the CDS of Latcripin-13 gene.(2)Latcripin-13 fragment sequences was obtained successfully.Recombinant plasmid p ET32a(+)-truncated-latcripin-13 has been constructed successfully. The apoptosis-related protein Latcripin-13 domain has been successfully expressed in E.coli Rosetta-gami(DE3). Latcripin-13 domain was successfully purified by affinity chromatography. Inclusion body protein Latcripin-13 domain was refolded by urea gradient dialysis.(3) Latcripin-13 domain inhibits cell proliferation of A549 cells via G1 cell cycle arrest and the mitochondria-mediated pathway and stimulates apoptosis through caspase-8 and NF-κB signaling. |
PDF Full Text Request