Current Biotechnology

The Research Progress on Plant Functional Centromere DNAs
Hui ZHANG, Kai WANG
2022, 12(1): 1-9. DOI: 10.19586/j.2095-2341.2021.0109
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The centromere is an important functional region in eukaryotic chromosomes. During cell mitosis and meiosis in eukaryotes， centromere plays a key role in faithful segregation of chromosomes by recruiting the kinetochore. In eukaryotes， the CenH3 region containing centromere specific histones is defined as the functional centromere region， which is the true centromere. In recent years， based on the application of chromatin immunoprecipitation， centromeric DNAs can be readily isolated and characterized. The composition， construction and evolution of centromeres were revealed， and some genes were found with important biological functions. However， because of the high level of diversity of centromeric repetitive DNAs， the evolution of centromeric repetitive DNAs is still elusive. In this paper， the research progress of functional centromere DNA sequences in plants was summarized， and the new methods and progress of centromere repetitive DNAs research were emphasized， which aimed to provide reference for further research.

Research Progress on the Regulation of Anthocyanin Synthesis in Horticultural Crops
Xin WANG, Tianzhu ZHANG
2022, 12(1): 10-16. DOI: 10.19586/j.2095-2341.2021.0125
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Anthocyanin is an important secondary metabolite that protects plants from biotic and abiotic stress. It has received widespread attention because of its rich colors to plants and its benefit to human health. The related research on the regulation mechanism of anthocyanin synthesis is the current frontier subject of molecular biology of horticultural crops， which has important significance for the improvement of anthocyanin content and germplasm quality of horticultural crops. This article combined the latest research progress in the regulation of anthocyanin biosynthesis in horticultural crops at home and abroad， and introduced the effects of environmental factors， enzymes and hormones， DNA methylation and ubiquitination， and regulatory genes on anthocyanin biosynthesis， and the functional mechanism of anthocyanins against external stresses. The research results of anthocyanin biosynthesis regulation in horticultural crops in recent years were reviewed， in order to using genetic engineering to provide a theoretical reference for enhancing the color richness of horticultural crops.

Research Progress on Plant Endophytic Nitrogen⁃fixing Bacteria and Their Nitrogen Fixation Mechanism
Yuhu WANG, Mingmin ZHAO, Hongli ZHENG
2022, 12(1): 17-26. DOI: 10.19586/j.2095-2341.2021.0126
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Nitrogen is an essential element for plant growth. Plant endophytic nitrogen-fixing bacteria can not only produce nitrogen in the plant body for plant utilization， but also play an active role in the natural nitrogen cycle， which has important significance for the sustainable development of agriculture. The research on endogenous nitrogen-fixing bacteria has gradually become research hotspot in recent years. This article systematically reviewed the research progress of plant endophytic nitrogen-fixing bacteria from many aspects， such as the discovery of plant endophytic nitrogen-fixing bacteria， crop symbiosis， invasion pathways， nitrogen-fixing mechanism， and growth-promoting mechanism. In addition， new research ideas of plant endophytic nitrogen-fixing bacteria and some unresolved problems were discussed. This paper was expected to provide reference for research on plant endophytic nitrogen-fixing bacteria and biological nitrogen fixation.

Research Progress of Histone Methyltransferases ASH2
Wanjie WANG, Nanzhu CHEN, Haisheng HAO, Xueming ZHAO, Huabin ZHU, Weihua DU
2022, 12(1): 27-35. DOI: 10.19586/j.2095-2341.2021.0132
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Epigenetic modification regulates embryo development， cell differentiation， organogenesis and cancer formation via altering chromatin spatial conformation and gene expression. This regulation involves histone methylation， histone acetylation， DNA methylation， gene imprinting， and X chromosome inactivation， etc. Absent， small， homologous 2 （ASH2） is the core component of histone lysine methyltransferase and belongs to the trithorax group. In mammals， ASH2 methylates specifically H3K4 and activates gene transcription. Based on the brief introduction of histone methylation and the trithorax group proteins， this paper reviewed the functions of ASH2 in regulating gene transcription， HOX gene expression， cancer formation and cell differentiation， in order to provide ideas for its application in animal breeding and human disease treatment.

The Status Progress in the Research of Recombinant Vaccine Based on Canarypox Virus
Wengui LI, Yatang CHEN
2022, 12(1): 36-43. DOI: 10.19586/j.2095-2341.2021.0046
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Poxvirus caused by canarypox virus （CNPV） is an infectious disease of poultry， CNPV infection can induce cellular， humoral and mucosal immune responses in the host. It can be remade as a candidate vaccine vector after modification， which can express multiple proteins of viruses and parasites. Therefore， it is of great economic importance to develop recombinant CNPV vector vaccine for immune prevention. The foreign scholars have constructed a variety of similar vaccines， such as equine influenza virus （EIV）， canine distemper virus （CDV）， measles virus （MV）， Nipah virus （NiV）， human immunodeficiency virus type 1（HIV?1）， African horse sickness virus type 4 （AHSV?4）， bluetongue virus serotype 17 （BTV?17）， hepatitis C virus （HCV）， cytomelovirus （CMV）， rabies virus and Leishmania infantum and so on. This article summerized the status in the research of construction and immune mechanism of CNPV?based vaccines， aiming to provide a theoretical basis for the immune prevention of viruses and parasitic diseases.

Research Progress of Raman Spectroscopy in the Field of Dermatology
Boyuan GUO, Pengxiang ZHAO, Hui WANG, Zhen WANG, Tingting YAO, Mengyu LIU, Yao Mawulikplimi ADZAVON, Fei XIE
2022, 12(1): 44-49. DOI: 10.19586/j.2095-2341.2021.0056
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Raman spectroscopy is a new optical detection technology， which is often used in material identification. In recent years， with the increasing demand for noninvasive detection， Raman spectroscopy has been gradually applied to biological fields such as disease diagnosis and substance identification. This paper reviewed the research progress of Raman spectroscopy in the field of skin and its value in the identification of skin tissue components and the diagnosis of skin diseases， in order to promote the wide application of Raman spectroscopy in the mechanism research and clinical diagnosis of dermatology.

Research Progress of Amino Acid Transporters in Tumor Metabolism
Linlin WANG, Zhenliang SUN
2022, 12(1): 50-56. DOI: 10.19586/j.2095-2341.2021.0016
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Metabolic reprogramming is an important feature of tumors. It refers to the reprogramming of metabolic pathways such as carbohydrate metabolism， lipid metabolism and amino acid metabolism by tumor cells to meet their rapid proliferation biosynthesis and energy requirements to maintain their growth rate and compensate for the redox stress caused by its energy metabolism. Although different cancers have different metabolic changes， some features are common to all cancers， and amino acid metabolism reprogramming is one of the important features. The transfer of amino acids into and out of cells require the assistance of amino acid transporters， so many specific amino acid transporters are overexpressed in tumor cells. Targeted amino acid transporter achieves the purpose of anti?tumor by affecting the amino acid metabolism of tumor cells， which is also one of the current research hotspots of anti?tumor drugs. This paper mainly introduced several amino acid transporter that play important roles in tumor metabolism and the research progress of anti?tumor therapy and related mechanism， so as to understand the function of amino acid transporters in anti?tumor research， and promote the development of targeted amino acid transporter antitumor drugs.

Research Progress on the Relationship Between RET Protooncogene and Tumor
Jinping XIAO, Cheng LI, Yundi CAO, Zhijian SUN, Ping KANG, Xiaomei LAN
2022, 12(1): 57-62. DOI: 10.19586/j.2095-2341.2021.0048
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The RET proto-oncogene is an important cancer driver gene， which is closely related to the occurrence and development of a variety of human tumors. The pathogenesis of RET related tumors includes RET gene activation point mutation and RET gene fusion mutation. In recent years， breakthrough progress has been made in the development of precision targeted drugs for carcinogenic RET gene fusion mutation. In this paper， the research progress of RET related pathogenesis， diagnosis and drug development in recent years were reviewed to provide reference for the accurate diagnosis and treatment of cancer patients with RET gene mutation， and improve the disease remission rate and disease control rate by accurately and efficiently inhibiting RET gene mutation.

Correlation Between Magnesiumion and Tumor
Zheng LI, Yamin ZHENG, Xiaonong ZHANG
2022, 12(1): 63-67. DOI: 10.19586/j.2095-2341.2021.0100
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Magnesium ion is an important cation of the human body， and participates in a variety of physiological activities. Imbalance of magnesium intake can not only lead to the corresponding clinical symptoms of hypomagnesemia and hypermagnesemia， but also have a close relationship with the occurrence and development of tumors. At the same time， the latest studies also showed that high concentration of magnesium ion has an anti?tumor effect. In this paper， the relationship between magnesium ions and the occurrence， development， treatment and other aspects of tumor were reviewed， so as to provide further reference for the epidemiological investigation and anti?tumor research of magnesium ions and tumors.

Research Progress on the Correlation Between Ferroptosis and Kidney Disease
Tong JIN, Cheng CHEN
2022, 12(1): 68-74. DOI: 10.19586/j.2095-2341.2021.0133
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Ferroptosis is a newly discovered form of non?apoptotic cell death in 2012. Its essence is the morphological change of mitochondria and damage of phospholipid peroxidation caused by the accumulation of iron?dependent reactive oxygen species （ROS） and lipid hydroperoxides. Ferroptosis is related to the pathophysiological process of many renal diseases. However， the molecular mechanism of ferroptosis in renal disease injury is still lacking of systematic and in?depth understanding. The regulation mechanism of ferroptosis， research progress and its role in renal related diseases were reviewed， aiming to provide new ideas and targets for the treatment of kidney disease.

Photosynthetic Characteristics Studies of Three Maize Yellow Leaf Mutants
Yuhan YUAN, Jiali FAN, Wenzhu YANG, Rumei CHEN
2022, 12(1): 75-82. DOI: 10.19586/j.2095-2341.2021.0097
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Photosynthesis serves as the main way for crops to accumulate biomass in agricultural production， which primarily depends on a variety of photosynthetic pigments as well as complete chloroplast structure and function. Maize leaf color mutants are crucial to study the development of chloroplasts and improve photosynthesis capacity and maize yield. In this study， two maize inbred lines Z58 and B73 were used as control in order to compare the difference between morphological characteristics， photosynthetic pigment content and chlorophyll synthesis precursor content among three maize yellow leaf mutants yl?1 （yellow leaf?1， Z58 background）， yl?2 （yellow leaf?2， B73 background） mutated by ethyl methanesulphonate （EMS） and a natural yellow?leaf mutant yl?3 screened from Z58. The results showed that the three mutants exhibited different levels of yellowing phenotype throughout their growth cycle and no greening， dwarf plants， and delayed growth compared to the control. Total chlorophyll， chlorophyll a and chlorophyll b were significantly decreased （P<0.05）， while the ratio of chlorophyll a/chlorophyll b was significantly increased （P<0.05）. The content of various precursors for chlorophyll synthesis among the different mutants all decreased. The yellow leaf phenotypes of the three mutants may be caused by the abnormal synthesis of related tetrapyrrole compounds due to mutations in different genes. This study laid a theoretical foundation for locating the mutant genes that cause the yellow leaf phenotype of the three mutants and further exploring their utilization potential.

Exploring of Authenticity Identification Method of Transgenic Cotton Plant by Shoot Tip
Rong XIAO, Yunxiao WEI, Yuan WANG, Zhigang MENG, Chengzhen LIANG, Quanjia CHEN, Rui ZHANG
2022, 12(1): 83-89. DOI: 10.19586/j.2095-2341.2021.0159
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The cotton shoot tip transformation method has the advantages of not being restricted by genotype and short transformation cycle， and is an ideal cotton transformation system. However， it is reported that the high?generation plants are generally genetically unstable and the genes are lost in the transgenic plants. In this experiment， the upland cotton TM?1 variety was used as the recipient material， the DsRed2 vector was transferred into cotton by the shoot tip transformation， and 16 resistant plants were obtained by kanamycin screening. The target gene was further amplified by PCR and 6 plants with DsRed2 gene fragment PCR test positive were obtained. The 6 plants were preliminarily judged to be transgenic plants obtained by the shoot tip transformation， but after UV irradiation， no red fluorescence was detected in the 6 transgenic plants. The target gene RT?PCR was performed and found that the DsRed2 gene had only a very low expression or no expression in the 6 transgenic plants. The non?T?DNA fragments of the DsRed2 vector， that is， the backbone of the vector， were subjected to PCR and plant endophyte culture testing. The results showed that the 6 transgenic plants all contained the complete DsRed2 vector and the plant could cultivate endophytes containing the complete vector. The endophyte was positive by PCR of the 16S rRNA fragment of Agrobacterium. It is speculated that the symbiotic relationship between Agrobacterium and plants due to shoop tip infection， which in turn leading to the phenomenon of false positive plants， and the loss of genes and genetic instability of high?generation transgenic plants. The purpose of this research is to establish a complete set of methods for identifying the authenticity of transgenic cotton plants by the shoot tip method， and to provide reference for further in?depth research.

The Non⁃coding RNA Regulated Nitrogen Metabolism in Sinorhizobium meliloti
Ka ZHANG, Yonghao WANG, Yunfeng WU, Lei XU, Haisheng WANG
2022, 12(1): 90-98. DOI: 10.19586/j.2095-2341.2021.0040
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To explore the function of non-coding RNA （ncRNA） in the progress of nitrogen metabolism， we used Sinorhizobium meliloti as starting strain， and identified and obtained ncRNA related to ntrC gene expression. We used lncPRO software to analyze the possibility of interaction between ncRNA and regulatory protein NtrC in Sinorhizobium meliloti， the four genes with higher scores were finally determined， namely SM2011_c06191， SM2011_c06248， SM2011_c07102 and SM2011_c07132， and the SM2011_c06248 gene with the highest score was studied. Northern blot was used to verify the expression of ncRNA， and it was found that the expression level of ncRNA increased first and then decreased after nitrogen-enrichment treatment. The SM2011_c06248 gene knockout strain was constructed in the experiment， and the expression of SM2011_c06248 under natural growth conditions was found to have no obvious regularity by qRT-PCR. After nitrogen enrichment treatment， the ncRNA expression level of wild-type strains first decreased， then increased and then decreased. Compared with natural growth， the ncRNA expression level was significantly increased after 20 min. The expression levels of ntrC and nar genes in wild-type strains first decreased， then increased and then decreased. Compared with the wild type， the expression levels of ntrC and nar genes in SM2011_c06248 knockout strain SM∷248 were significantly down-regulated. The SM2011_c06248， NtrC double mutant strain SM∷248-NtrC and SM2011_c06248 overexpression strain SM：dld-248 were constructed in the experiment. qRT-PCR analysis showed that， compared with the wild type， after nitrogen-enriched treatment， there was no significant change for nar gene expression in the SM∷248-NtrC strain， and the expression levels of ntrC and nar genes in SM：dld-248 strain were significantly up-regulated. ncRNA SM2011_c06248 can respond to the change of nitrogen signal in the environment， and has a positive regulatory effect on the expression of ntrC and nar genes， and ntrC has epistatic effect on nar.

Comparison Study of DNA Extraction Methods for Corn Gluten Meal
Zhen ZHEN, Yinggang DOU, Xiaolan LIU
2022, 12(1): 99-104. DOI: 10.19586/j.2095-2341.2021.0047
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The complexity of the feed sample brings great pressure and challenges to the detection of genetically modified (GM） components of imported and exported products. Corn gluten meal mainly contains protein， starch and lipid， etc.， it is difficult to extract high?quality DNA from them. High?quality DNA is the key to GM testing research， which can reduce "false negative" results rates in imported and exported inspection. Seven commercially available kits（Promega Corporation， Biotecon Corporation， Tiangen Biochemical Technology Co.， Invitrogen Corporation， Qiagen Corporation， TaKaRa Corporation）， CTAB method and modified SDS?CTAB method were used to extract DNA from corn gluten meal in this study. The quality of DNA extracted from maize protein flour by the modified SDS?CTAB method was found to be significantly better than the other two methods by real?time fluorescence PCR， and the C_tvalue of the endogenous gene zSSIIb by the modified SDS?CTAB method was 28.14， which was 27% higher than that of the CTAB method. It is the first application of modified SDS?CTAB method in GM feed production at home and abroad. Seven batches of laboratory samples sent by this extraction method were tested for GM components， the results showed that two batches of positive samples were successfully detected.

The Effect of Deleting the slr1556 Gene Encoding Lactate Dehydrogenase on Ethanol Biosynthesize in Synechocystis sp. PCC 6803
Huilu XU, Min XU, Wei ZHANG
2022, 12(1): 105-111. DOI: 10.19586/j.2095-2341.2021.0058
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To explore the effect of ethanol synthesize in Synechocystis sp. PCC 6803， the exogenous ethanol-producing genes were introduced and the competitive metabolic pathway of pyruvate to lactate was deleted. The pyruvate decarboxylase gene （pdc） from Synechocystis sp. PCC 6803 and the NADPH-dependent aldehyde reductase gene （yqhD） from Escherichia coli in this study were introduced and combined for expression under the drove of the light intensity promoter PrbcL to synthesize ethanol in Synechocystis sp. PCC 6803. Based of this， the slr1556 gene encoding lactate dehydrogenase in Synechocystis sp. PCC6803 was further knocked out to increase the content of pyruvate， the precursor for ethanol synthesis and promote the ethanol production. The results showed that by knocking out the slr1556 gene， the content of pyruvate was improved and the synthesized ethanol output was significantly increased. The block of the competitive metabolic pathway of the conversion of pyruvate to lactic acid can effectively promote the accumulation of pyruvate and then promote the production of ethanol.

Screening and Optimization of Highyield Neutral Proteaseproducing Strains and Pilot Scaleup
Wenjing ZHANG, Ye TONG, Xiwen YANG, Yanjin CAO, Jidong WEI
2022, 12(1): 112-119. DOI: 10.19586/j.2095-2341.2021.0166
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In order to screen and isolate a strain with high-yield neutral protease ability and study the fermentation conditions of the strain， we collected samples during the composting period of cow manure and pig manure， and 19 protease producing strains were screened and isolated on solid medium with casein as the sole carbon source. One of the strains PC2 with the best enzyme-producing effect was selected. Its hydrolysis circle D/d value is 4.25， and the enzyme activity is 10.74 U·mL^-1. Combined with the results of morphology， physiology and biochemistry， and 16S rDNA molecular biology identification， we identified it as Bacillus subtilis， and it is a Gram-positive bacterium. In the follow-up， we optimized the conditions of shake flask and pilot scale-up， which were activated with LB medium at 37 ℃ for 24 h， and fermented with casein fermentation medium at 30 ℃ for 48 h at a speed of 180 r·min^-1. In the specific parameters of the pilot scale-up， the speed of the 50 L seed tank was 180 to 240 r·min^-1， and the aeration rate was 20 to 30 L·min^-1. Add 1% wheat protein powder to the medium of the 500 L fermenter， the rotation speed was 100 to 180 r·min^-1， and the aeration rate was 10 to 25 L·min^-1. After fermentation， the viable bacteria content of PC2 was 4.458 billion·mL^-1， and the enzyme activity was 29.48 U·mL^-1， which was 2.745 times of the initial enzyme activity. The results of this study can lay a theoretical foundation for exploring microbial agents containing neutral protease strains and guide the industrial production of microbial agents.

Improvement Effect of Recombinant Lactoferrin and Recombinant Lysozyme on Ulcerative Colitis in Mice
Ning DING, Ye XU, Weisi ZENG, Yanzhou HU, Lingyu HONG, Kunlun HUANG, Xiaoyun HE
2022, 12(1): 120-128. DOI: 10.19586/j.2095-2341.2021.0094
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Ulcerative colitis （UC） is a chronic， nonspecific inflammatory disease occurring in the rectum and colon， and its incidence has increased significantly in recent years. This study aimed to explore the improvement effect of recombinant human lactoferrin and recombinant human lysozyme derived from genetically modified cow milk on UC， mice UC models were established by dextran sodium sulfate （DSS）. 30 male C57BL/6N mice were randomly divided into blank control group （CON group）， model control group （DSS group）， low-concentration lactoferrin group （L-rLF group， 50 mg·kg^-1·BW^-1）， high-concentration lactoferrin group （H-rLF， 100 mg·kg^-1·BW^-1）， low-concentration lysozyme group （L-rLZM group， 50 mg·kg^-1·BW^-1） and high-concentration lysozyme group （H-rLZM group， 100 mg·kg^-1·BW^-1）. After model building， the mice were given lactoferrin and lysozyme by gavage for one week. The serum and colon of mice were collected to observe the pathological changes of organs， and inflammatory factors and intestinal flora were determined. The results showed that， compared with the model group， disease activity index （DAI）， colon length reduction and histopathological score of mice in the low-concentration lactoferrin group and low-concentration lysozyme group were significantly reduced after lactoferrin and lysozyme gavage. The expression of prionflammatory factors （IL-6， LL-1β， TNF-α） in colon tissue and the concentration of lipopolysaccharide （LPS） in serum and liver were significantly decreased， and the structure of intestinal microflora was also significantly improved in the high-concentration lactoferrin group. The results showed that both recombinant lactoferrin and recombinant lysozyme could improve the ulcerative colitis in mice to varying degrees. This study provided a new idea and theoretical support for the future application of recombinant human lactoferrin and recombinant human lysozyme.

Protective Effect of Echinacoside on Myocardium in db/db Diabetic Mice
Min LIAO, Luo YANG, Zhen WANG, Yarong HAO
2022, 12(1): 129-134. DOI: 10.19586/j.2095-2341.2021.0061
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Diabetic cardiomyopathy （DCM） is a kind of myocardial disease occurring in diabetic patients that can't be explained by coronary heart disease， hypertensive heart disease and other cardiac diseases. At present， the etiology and pathogenesis of DCM have not been fully elucidated and specific treatment is lacking. It was found that echinacoside （ECH）， an extract from Cistanche tubulosa， had a protective effect on cardiomyocytes. In this study， db/m mice were used as normal control group （db/m group）， and db/db mice were divided into model group （db/db group） and ECH intervention group （db/db+ECH group）， to investigate the effect and mechanism of ECH on myocardium of diabetic db/db mice. Mice in db/db +ECH group were intragastrically with ECH， and mice in db/m group and db/db group were intragastrically with 0.9% sodium chloride solution. The cardiac function was observed by echocardiography. Masson staining was used to observe the content of collagen fiber in myocardial tissue. Collagen Ⅰand collagen Ⅲ mRNA expression levels were detected by reverse transcription-polymerase chain reaction， and the protein expression levels of transforming growth factor-β1 （TGF-β1）， phospho-Smad2（p-Smad2） and phospho-Smad3 （p-Smad3） were detected by Western blotting. The results showed that ECH relieved left ventricular hypertrophy and dysfunction， and decreased collagen deposition in db/db mice （P<0.05）. ECH decreased the mRNA expression of collagen Ⅰand collagen Ⅲ （P<0.01）， down-regulated the protein expression of TGF-β1， p-Smad2 and p-Smad3（P<0.05）. The protective effect of ECH on diabetic myocardium may be related to the negative feedback regulation of TGF-β1/Smads signaling pathway， which provides a new idea for the early intervention of DCM.

Effects of Dexmedetomidine on Offspring Development, Spatial Learning and Memory Ability of Pregnancy Rats with Chronic Stress Depression
Xiangyang WANG, Anjun LIU, Jing ZHAO
2022, 12(1): 135-141. DOI: 10.19586/j.2095-2341.2021.0032
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Pregnancy depression can affect the normal growth and development of newborns by affecting the physiological and psychological changes of pregnant women. Dexmedetomidine （Dex） can effectively relieve the depression and reduce the occurrence of postpartum depression. To explore the effects of Dex on offspring development， spatial learning and memory ability of pregnancy rats with chronic stress depression， sixty female pregnant rats were randomly divided into control group， model group， low?dose Dex group and high?dose Dex group. The inoculation rate， average farrowing rate and average survival rate of offspring rats in each group were statistically analyzed. The body mass change and learning and memory ability of offspring rats were recorded after birth. The levels of acetylcholine （Ach）， 5?HT and TChE in brain tissues， and expression of CREB， p?CREB and BDNF in hippocampus of offspring rats were detected. The study showed that Dex could increase the average survival rate by a dose?dependent manner， promoted the levels of Ach， 5?HT， CREB， p?CREB and BDNF levels in the brain tissue， and improved the learning and memory ability of the offspring of chronic stress depression pregnant rats. The results showed that Dex may improve the learning and memory ability of offspring of pregnant rats with chronic stress depression by regulating neurotransmitters and promoting CREB signal transduction. This study aims to explore the effects of Dex on the growth and development and spatial learning and memory abilities of the offspring of rats with depression during pregnancy， so as to guide clinical treatment.

The Role of TWIST1 in the Proliferation, Migration and Angiogenesis of Human Umbilical Vein Endothelial Cells
Yueqiao XIE, Dan GUO, Nan WANG, Na YOU, Wanling HUANG, Xiaotong MA
2022, 12(1): 142-148. DOI: 10.19586/j.2095-2341.2021.0163
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To explore the function of TWIST1 in the proliferation， migration and angiogenesis of primary human umbilical vein endothelial cells （HUVECs）， the cells in the experimental group were infected with lentiviral solution targeting human TWIST1 gene shRNA （pLL3.7-shTwist1-GFP）， and the cells in the control group were infected with lentiviral solution carrying scramble shRNA （pLL3.7-shCtrl-GFP）， the transfection efficiency was accessed by flow cytometry. TWIST1 mRNA expression level was quantified by real-time fluorescent quantitative PCR （qRT-PCR）. The effects of decreased TWIST1 expression on the proliferation， apoptosis， migration， angiogenesis and vascular growth factor receptor 2 （VEGFR2） gene expression of HUVECs were detected by cell growth curves， flow cytometric analysis of Annexin V/7AAD staining， cell scratch assay， tube formation assay， and qRT-PCR， respectively. TWIST1 gene expression in the experimental group decreased to 30% of that in the control group， indicating that shTWIST1 can effectively reduce TWIST1 gene expression. Compared with the control group， knockdown of TWIST1 could significantly inhibit the proliferation of HUVECs （P<0.01） and induce apoptosis （P<0.05）. The wound healing rate， the number of vascular-like structures and the length of total tubular branches of HUVECs were significantly lower than those of the control group （P<0.01）. Compared with the control group， the expression of VEGFR2 in HUVECs in the experimental group significant decreased（P<0.01）. This study explored the role of TWIST1 expressed by HUVECs in endothelial cell proliferation， survival， migration and the formation of capillary-like structure， and may provide a theoretical basis for TWIST1 as a new target for tumor angiogenesis.

Expression Profile Screening of lncRNA and ceRNA Network Construction in Gastric Cancer Based on TCGA and GEO Databases
Jiaxin YONG, Quanfeng WEI, Shanghui LIU
2022, 12(1): 149-157. DOI: 10.19586/j.2095-2341.2021.0042
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Gastric cancer （GC） is one of the most malignant tumors in China， which seriously endangers human health. The tumorigenesis of GC is complicated and lacks specific prognostic biomarkers. Long non?coding RNA （lncRNA） can be used as competitive endogenous RNA （ceRNA） to affect the interaction of microRNA （miRNA） and mRNA， thereby affecting the carcinogenesis and development of GC. Based on the transcriptome data of the TCGA and GEO databases， this study screened the differentially expressed lncRNAs in GC. A lncRNA?miRNA?mRNA network based on six lncRNAs （HAGLROS， TMEM92?AS1， LINC01745， HOXC?AS3， SEMA3B?AS1， FEZF1?AS1） was constructed. The KEGG/GO enrichment and protein interaction analysis showed that lncRNA might regulate the occurrence， development， and metastasis of GC through the mechanism of microRNA sponge. AC011352.1， AC087636.1， AC093627.1， GAS1RR were significant related to the prognosis of GC patients （P<0.05）， which may become potential prognostic biomarkers for GC patients.

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