龙眼LEAFY同源基因片段的克隆和表达

研究克隆的龙眼(Dimdcarpus Longan Lour.)LFY同源基因(LLFY)保守区片段在不同组织器官以及“冲梢”逆转成的营养芽中的表达情况,为进一步鉴定龙眼LFY同源基因的功能及分析其在龙眼花序的“冲梢”过程中的作用机理奠定基础。根据不同物种LEAFY同源基因保守区序列设计简并引物,从“红核子”龙眼(D.longan Lour.cv.Red Seed)中克隆了425bp的cDNA片段。同源性比较结果表明,获得的序列为龙眼LEAFY同源基因(LLFY);同时还获得了1816bp的LLFY基因组序列片段并进行了序列分析。通过RT-PCR研究LLFY在6种龙眼不同组织器官的表达发现LLFY在花序芽和花芽中的表达量最高,叶芽中有微量表达,叶片和茎段中没有表达;在“冲梢”逆转成的营养芽中,LLFY的表达量明显高于叶芽。

猪TGFβ1基因及其受体组织表达特性的研究

以妊娠母猪为研究材料,采用RT-PCR半定量方法对猪转化生长因子β1(TGFβ1)及其3种受体组织表达特点进行研究。结果表明:TGFβ1在11种组织中的表达量,由高到低依次为肾上腺、下丘脑、肌肉、卵巢、脾、大脑、肾、子宫、肺、肝和心脏。3种受体的表达,总体上来说在子宫中相对较高。TGFβ1及其受体在肾上腺和子宫等组织中有相对较高的表达量,提示其在繁殖过程中可能发挥作用。

平菇漆酶基因在毕赤酵母中的分泌表达及酶学性质研究

采用RTPCR技术克隆到一个平菇(Pleurotusostreatus)漆酶基因的全长cDNA,命名为lccPo1,其序列提交GenBank,登录号为AY450404。将其ORF克隆到毕赤酵母表达载体pHBM906,转化3株毕赤酵母GS115、KM71和SMD1168,该漆酶基因在3种毕赤酵母菌株中均实现了分泌表达。3种摇瓶培养条件①25℃,1.0%(VV)甲醇;②20℃,1.0%(VV)甲醇;③20℃,0.5%(VV)甲醇,进行比较研究后发现适当提高甲醇浓度有利于漆酶在低温条件下表达,而降低培养温度到20℃则可以提高漆酶的产量2～6倍。3株重组毕赤酵母在其最适反应条件下测得三者粗酶液最高漆酶酶活分别为3.19UmL[GS115(pHBM565)]、2.56UmL[KM71(pHBM565)]和2.49UmL[SMD1168(pHBM565)]。对重组酶进行相关的酶学性质分析表明,三者的最适反应pH值约为4.2,最适反应温度约为60℃。重组毕赤酵母GS115(pHBM565)所产酶的热稳定性稍好,在pH稳定性方面三者没有太大差异。

快速老化小鼠海马差异表达cDNA芯片的制作

老年性痴呆(Alzheimer’s disease,AD)是老年人群中最普遍的痴呆类型,是一种神经退行性紊乱疾病,目前临床上还没有有效的治疗方法。快速老化小鼠亚系P8(senescence-accelerated mouse prone8,SAMP8)是研究增龄相关性认知缺陷机制以及研究脑老化机制的良好动物,同时也是研究AD较为理想的实验动物模型之一。cDNA芯片技术可以同时规模研究成千上万个基因的表达,尤其适于AD这种多机制、多靶标、多途径的复杂疾病的研究,为了揭示AD的发病机制,发现用于治疗AD的药物靶标,以SAMP8和SAMR1海马抑制消减cDNA文库中的cDNA片段为材料,以β-actin和G3PDH为内参,设计了16×(1×14)点阵方案,并点制了含有3136个点的SAM海马差异表达cDNA芯片。芯片背景均匀一致,点的大小均一,排列规则整齐。在靶分子与探针杂交过程中,进行了杂交条件和洗涤芯片的优化。将杂交结果进行统计分析,选择差异表达的cDNA进行测序并进行生物信息学分析,用实时定量RT-PCR对部分基因的表达进行了验证,检测了芯片筛选结果的可靠性。该芯片的成功制备为进一步进行差异表达基因的筛选和研究提供了良好的手段,并将成为揭示SAMP8脑老化和AD发病机制的有力手段。

重组人B淋巴细胞刺激因子基因原核表达载体的构建及鉴定

目的构建人BLyS基因原核表达载体。方法采用RT-PCR方法,从人外周血淋巴细胞的总cDNA中扩增得到474bp的人BLySDNA片段,再用BamHI和EcoRI双酶切后,克隆到原核表达载体pGEX-5X-3中,用限制性内切酶酶切分析和DNA序列分析鉴定重组质粒。结果BLyScDNA已经正确克隆到原核表达载体pGEX-5X-3中。结论本实验为研制抗人BLyS单克隆抗体来研究人BLyS与自身免疫性疾病的关系奠定了基础。

Detection of Survivin mRNA in nasopharyngeal carcinoma by real-time fluorescence quantitative RT-PCR

Objective： To establish the method of real time fluorescence quantitative RT-PCR for detecting the expression of Survivin mRNA in nasopharyngeat carcinoma （NPC） tissues. Methods： The total RNA was extracted from NPC cell line CNE-2 and tissues with Trizol and then been transcribed reversely to cDNA, a method of real time fluorescence quantitative RT-PCR for detecting the expression of Survivin mRNA in NPC tissues had been established, in which chronic nasopharyn-gitis patients＇ nasopharynx tissues treated as control group. Results： The expression of Survivin mRNA all could be detected either in CNE-2 cells, NPC tissues or in chronic nasopharyngitis patients＇ nasopharynx tissues, and there was higher the expression level of Survivin mRNA in NPC tissues than which in chronic nasopharyngitis patients＇ nasopharynx tissues, the difference was significant （P 〈 0.01）. The expression of Survivin mRNA could be detected both in stage Ⅰ ＋ Ⅱ and stage Ⅲ ＋ Ⅳ NPC, and there was no significant difference in relative quantifications of gene expression between these two groups （P 〉 0.05）. There was no relationship between Survivin mRNA expression and age and sex of NPC patients （P 〉 0.05）. Conclusion： Real time fluorescence quantitative RT-PCR is a rapid, effective and high sensitive method for detecting the expression of Survivin mRNA in NPC tissues. The overexpression of Survivin mRNA may play some roles in pathogenesis of NPC.

Selection of reference genes for qRT-PCR analysis of gene expression in sea cucumber Apostichopus japonicus during aestivation

Quantitative real-time reverse transcription-polymerase chain reaction （qRT-PCR） is a technique that is widely used for gene expression analysis, and its accuracy depends on the expression stability of the internal reference genes used as normalization factors. However, many applications of qRT-PCR used housekeeping genes as internal controls without validation. In this study, the expression stability of eight candidate reference genes in three tissues （intestine, respiratory tree, and muscle） of the sea cucumber Apostichopus japonicus was assessed during normal growth and aestivation using the geNorm, NormFinder, delta CT, and RefFinder algorithms. The results indicate that the reference genes exhibited significantly different expression patterns among the three tissues during aestivation. In general, the β-tubulin （TUBB） gene was relatively stable in the intestine and respiratory tree tissues. The optimal reference gene combination for intestine was 40S ribosomal protein S18 （RPSI 8）, TUBB, and NADH dehydrogenase （NADH）; for respiratory tree, it was β-actin （ACTB）, TUBB, and succinate dehydrogenase cytochrome B small subunit （SDHC）; and for muscle it was α-tubulin （TUBA） and NADH dehydrogenase [ubiquinone] 1α subcomplex subunit 13 （NDUFA13）. These combinations of internal control genes should be considered for use in further studies of gene expression in A.japonicus during aestivation.

Detection of Syndecan-1 and Heparanase-1 Genes in Esophageal Carcinoma by Quantitative RT-PCR

OBJECTIVE To quantitatively explore the expression of Syndecan-1 and heparanase-1 in esophageal cancer tissue as well as their relationship with the clinicopathological factors, in order to evaluate their roles in tumor invasion and metastasis.METHODS Real-time fluorescence quantitative PCR （Q-PCR） was used to analyze the expression levels of Syndecan-1 and heparanase-1 genes￡？participants included 67 cases with esophageal cancers and 32 healthy volunteers.RESULTS The expression of Heparanase-1 gene in esophageal cancers was higher than that in normal esophageal tissue （P 〈 0.001）, and the expression of Syndecan-1 gene in the normal esophageal tissue was higher compared with esophageal cancers （P 〈 0.001）. The positive rates of Syndecan-1 and Heparanase-1 gene in esophageal cancer were 13.4% （9/67） and 85.1% （57/67）.The expression of Syndecan-1 and Heparanase-1 genes was signifi cantly related to di. erentiation, depth of infi ltration, lymph node metastasis, vessel metastasis, and TNM stages of disease （P 〈 0.05）. In an attempt to measure the association between the 2 agents, this study found that the expression of Syndecan-1 mRNA had a significantly negative correlation with the expression of Heparanase-1 mRNA by using Spearman rank correlation test （OR = -0.572, P 〈 0.001）.CONCLUSION Syndecan-1 and Heparanase-1 play important roles in the invasion and metastasis of esophageal cancer. The reduction of Syndecan-1 and/or the increase of Heparanase-1 may influence the invasion and metastasis of malignant tumors.Thus the combination assay of Syndecan-1 and Heparanase-1 may contribute to the diagnosis and treatment of malignant tumors.

Identification of normalization factors for quantitative realtime RT-PCR analysis of gene expression in Pacific abalone Haliotis discus hannai

Quantitative real-time reverse transcription-polymerase chain reaction （qRT-PCR） is widely used in studies of gene expression. In most of these studies, housekeeping genes are used as internal references without validation. To identify appropriate reference genes for qRT-PCR in Pacific abalone Haliotis discus hannai, we examined the transcription stability of six housekeeping genes in abalone tissues in the presence and absence of bacterial infection. For this purpose, abalone were infected with the bacterial pathogen Fibrio anguillarum for 12 h and 48 h. The mRNA levels of the housekeeping genes in five tissues （digestive glands, foot muscle, gill, hemocyte, and mantle） were determined by qRT-PCR. The PCR data was subsequently analyzed with the geNorm and NormFinder algorithms. The results show that in the absence of bacterial infection, elongation factor-l-alpha and beta-actin were the most stably expressed genes in all tissues, and thus are suitable as cross-tissue type normalization factors. However, we did not identify any universal reference genes post infection because the most stable genes varied between tissue types. Furthermore, for most tissues, the optimal reference genes identified by both algorithms at 12 h and 48 h post-infection differed. These results indicate that bacterial infection induced significant changes in the expression of abalone housekeeping genes in a manner that is dependent on tissue type and duration of infection. As a result, different normalization factors must be used for different tissues at different infection points.

抗草甘膦基因mEPSPS转化油菜研究

mEPSPS基因是编码5-烯醇式丙酮酸莽草酸-3-磷酸合酶的抗草甘膦基因。本研究通过根癌农杆菌介导的遗传转化,将人工合成改造的草甘膦抗性基因mEPSPS导入甘蓝型油菜品系甲572,获得了4株转基因植株。分子检测证明,外源mEPSPS基因已整合到转基因油菜基因组中并能稳定遗传到下一代。各转基因植株中mEPSPS基因能正确表达,但不同转化株的基因表达量之间有差异。转mEPSPS油菜自交一代在稀释100倍的41%农达异丙胺盐制剂(含草甘膦3 039mg/L)喷洒条件下仍能正常生长,而不含转基因的对照植株在稀释200倍农达(含草甘膦1 519mg/L)之后全部死亡。

Cloning of cDNA Encoding COMT from Chinese White Poplar ( Populus tomentosa ), Sequence Analysis and Specific Expression

The cDNA fragment encoding caffeic acid 3_O_methyltransferase (COMT) in Chinese white poplar ( Populus tomentosa Carr.) was isolated and cloned by RT_PCR technique. The size of the cDNA fragment is 1 080 bp, which almost covers the whole cDNA_encoding region. Authors’ cDNA fragment in P. tomentosa shares 98.7% homology with the reported corresponding cDNA in the P. tremuloids at nucleotide level, 99.4% homology at amino acid level, respectively. The analysis of Northern dot hybridization showed that COMT is expressed specifically in the developing secondary xylem of stem during the season of xylem differentiation, which means the linkage between the gene expression for a monolignol biosynthetic enzyme and seasonal regulation of xylem development in woody plant.

Cloning and Expression of a Profilin Gene from Rapeseed

Profilin has recently been identified as an actin-binding protein in higher plants. A cDNA clone (designated Repro) encoding profilin gene was isolated from rapeseed ( Brassica napus L. cv. canadian Tween) using RT-PCR technique. Sequence analysis showed 82% similarity to Zea mays L. ZmPro3, 85% to Arabidopsis AthPRF1, 82% to Nicotiana tabacum L. NTPRO, 81% to Oryza sativa L. profilin A. A new full-length cDNA was obtained by 5'-RACE and 3'-RACE techniques. Sequence analysis showed that the size of full-length cDNA is 672 bp which contains a major open reading frame of 134 amino, acids, 5' and 3' untranslated regions and a long Poly (A) tail. Northern blot analysis showed that the profilin gene is a pollen and anther specific gene.

Construction and Preliminary Identification of Eukaryotic Expression Vector of Cryptosporidium parvum miR-2980

[Objective] This study aimed to construct and preliminarily identify the eu- karyotic expression vector of Cryptosporidium parvum miR-2980. [Method] The cp-miR- 2980 precursor was amplified from C. parvum genomic DNA and cloned into pMD18- T vector. The amplified precursor was then subcloned into pVAX I vector and identi- fied with restriction endonuclease digestion and sequencing. The recombinant plasmid pVAX-miR2980 was transfected into HCT-8 cells. Total RNA was extracted and the expression of cp-miR-2980 was evaluated by RT-PCR detection. [Result] The results showed that the recombinant eukaryotic expression vector pVAX-miR2980 was suc- cessfully constructed, which can express cp-miR-2980 in HCT-8 cell. [Conclusion] This study laid the foundation for further exploring the biological function of cp-miR-2980.

Clinical implication of 14-3-3 epsilon expression in gastric cancer

AIM:To evaluate for the first time the protein and mRNA expression of 14-3-3εin gastric carcinogenesis.METHODS:14-3-3εprotein expression was determined by western blotting,and mRNA expression was examined by real-time quantitative RT-PCR in gastric tumors and their matched non-neoplastic gastric tissue samples.RESULTS:Authors observed a significant reduction of 14-3-3εprotein expression in gastric cancer(GC)samples compared to their matched non-neoplastic tissue.Reduced levels of 14-3-3εwere also associated with diffuse-type GC and early-onset of this pathology.Our data suggest that reduced 14-3-3εmay have a role in gastric carcinogenesis process.CONCLUSION:Our results reveal that the reduced 14-3-3εexpression in GC and investigation of 14-3-3ε interaction partners may help to elucidate the carcino-genesis process.

Identification of differential gene expressions in colorectal cancer and polyp by cDNA microarray

AIM： TO screen the differential expressed genes in colorectal cancer and polyp tissue samples. METHODS： Tissue specimens containing 16 cases of colorectal adenocarcinoma and colorectal polyp vs nor- mal mucosae were collected and subjected to cDNA microarray and bioinformatical analyses. Quantitative reverse transcription-polymerase chain reaction （qRT- PCR） was used to confirm some of the cDNA microarray data.RESULTS： The experimental data showed that eight genes were differentially expressed, most of which were upregulated in adenomatous polyp lesions. Forty-six genes expressions were altered in colorectal cancers, of which 29 were upregulated and 17 downregulated, as compared to the normal mucosae. In addition, 18 genes were similarly altered in both adenomatous polyps and colorectal cancer, qRT-PCR analyses confirmed the cDNA microarray data for four of those 18 genes： MTA1, PDCD4, TSC1 and PDGFRA. CONCLUSION： These differentially expressed genes likely represent biomarkers for early detection of co- Iorectal cancer and may be potential therapeutic targets after confirmed by further studies.

Identification and characterization of a Dna J gene from red alga Pyropia yezoensis(Bangiales, Rhodophyta)

Members of the Dna J family are proteins that play a pivotal role in various cellular processes, such as protein folding, protein transport and cellular responses to stress. In the present study, we identified and characterized the full-length Dna J c DNA sequence from expressed sequence tags of Pyropia yezoensis( Py Dna J) via rapid identification of c DNA ends. This c DNA encoded a protein of 429 amino acids, which shared high sequence similarity with other identified Dna J proteins, such as a heat shock protein 40/Dna J from Pyropia haitanensis. The relative m RNA expression level of Py Dna J was investigated using real-time PCR to determine its specific expression during the algal life cycle and during desiccation. The relative m RNA expression level in sporophytes was higher than that in gametophytes and significantly increased during the whole desiccation process. These results indicate that Py Dna J is an authentic member of the Dna J family in plants and red algae and might play a pivotal role in mitigating damage to P. yezoensis during desiccation.

Protease-activated receptor(PAR)1, PAR2 and PAR4 expressions in esophageal squamous cell carcinoma

Here,we used reverse transcription-PCR(RT-PCR) and western blot to detect protease-activated receptor(PAR) 1,PAR 2 and PAR 4 expression in cancer tissues and cell lines of esophageal squamous cell carcinoma,and investigated the co-relationship between PAR expression and clinic-pathological data for esophageal cancer.The methylation of PAR4 gene promoter involved in esophageal carcinoma was also analyzed.By comparing the mRNA expressions of normal esophageal tissue and human esophageal epithelial cells(HEEpiC),we found that among the 28 cases of esophageal squamous cell carcinoma,PAR1(60%) and PAR2(71%) were elevated in 17 and 20 cases,respectively,and PAR4(68%) expression was lowered in 19 cases.Whereas,in human esophageal squamous cells(TE-1 and TE-10),PAR1 and PAR2 expression was increased but PAR4 was decreased.Combined with clinical data,the expression of PAR1 in poorly differentiated(P=0.016) and middle and lower parts of the esophagus(P=0.016) was higher; expression of PAR4 in poorly differentiated carcinoma was lower(P=0.049).Regarding TE-1 and TE-10 protein expression,we found that in randomized esophageal carcinoma,PAR1(P=0.027) and PAR2(P=0.039) expressions were increased,but lowered for PAR4(P=0.0001).In HEEpiC,TE-1,TE-10,esophageal and normal esophagus tissue samples(case No.7),the frequency of methylation at the 19 CpG loci of PAR4 was 35.4%,95.2%,83.8%,62.6% and 48.2%,respectively.Our results indicate that the expression of PAR1 and PAR2 in esophageal squamous cell carcinoma is increased but PAR4 is decreased.Hypermethylation of the promoter of the PAR4 gene may contribute to reduced expression of PAR4 in esophageal squamous cell carcinoma.

Epigenetic inactivation of secreted frizzled-related protein 2 in esophageal squamous cell carcinoma

AIM： To investigate the expression and methylation status of the secreted frizzled-related protein 2 （SFRP2） in esophageal squamous cell carcinoma （ESCC） and ex- plore its role in ESCC carcinogenesis.METHODS： Seven ESCC cell lines （KYSE 30, KYSE150, KYSE410, KYSE510, EC109, EC9706 and TE-1） and one immortalized human esophageal epithelial cell line （Het- 1A）, 20 ESCC tissue samples and 20 paired adjacent non-tumor esophageal epithelial tissues were analyzed in this study. Reverse-transcription polymerase chain reaction （RT-PCR） was employed to investigate the expression of SFRP2 in cell lines, primary ESCC tumor tissue, and paired adjacent normal tissue. Methylation status was evaluated by methylation-specific PCR and bisulfite sequencing. The correlation between expres- sion and promoter methylation of the SFRP2 gene was confirmed with treatment of 5-aza-2＇-deoxycytidine. To assess the potential role of SFRP2 in ESCC, we es-tablished stable SFRP2-transfected cells and examined them with regard to cell proliferation, colony formation, apoptosis and cell cycle in vivo and in vitro.RESULTS： SFRP2 mRNA was expressed in the im- mortalized normal esophageal epithelial cell line but not in seven ESCC cell lines. By methylation-specific PCR, complete methylation was detected in three cell lines with silenced SFRP2 expression, and extensive methylation was observed in the other four ESCC cell lines. 5-aza-2＇-deoxycytidine could restore the expres- sion of SFRP2 mRNA in the three ESCC cell lines lack- ing SFRP2 expression. SFRP2 mRNA expression was obviously lower in primary ESCC tissue than in adjacent normal tissue （0.939 ± 0.398 vs 1.51 ± 0.399, P 〈 0.01）. SFRP2 methylation was higher in tumor tissue than in paired normal tissue （95% vs 65%, P 〈 0.05）. The DNA methylation status of the SFRP2 correlated inversely with the SFRP2 expression. To assess the potential role of SFRP2 in ESCC, we established stable SFRP2 transfectants and control counterparts by in- troducing pcDNA3.1/v5 hisA -SFRP2 or pcDNA3.1/v5 hisA -empty vector into KYSE30 cells lacking SFRP2 expression. After transfection, the forced-expression of SFRP2 was confirmed by the RT-PCR. In comparison with the control groups, stably-expressed SFRP2 in KYSE 30 cells significantly reduced colony formation in vitro （47.17% 4± 15.61% vs 17% ：1： 3.6%, P = 0.031） and tumor growth in nude mice （917.86：1：249.35 mm3 vs 337.23 ± 124.43 mm3, P 〈 0.05）. Using flow cytom- etry analysis, we found a significantly higher number of early apoptotic cells in SFRP2-transfected cells than in the control cells （P = 0.025）. The mean cell number in the S and G2-M phases of the cell cycle was also significantly lower in SFRP2-transfected KYSE30 cells compared with mock transfected counterparts. CONCLUSION： Silencing of SFRP2 expression through promoter hypermethylation may be a factor in ESCC carcinogenesis through loss of its tumor-suppressive activity.

Hexahydrocurcumin enhances inhibitory effect of 5-fluorouracil on HT-29 human colon cancer cells

AIM： To investigate the ability of hexahydrocurcumin （HHC） to enhance 5-fluorouracil （5-FU） in inhibiting the growth of HT-29 cells by focusing on cyclooxygenase （COX）-2 expression.METHODS： Antiproliferative effects of HHC and 5-FU, alone and in combination, on growth of HT-29 human colon cancer cells were assessed using 5-diphenyltetrazolium bromide （MTT） reduction assay. In combinationtreatment, low doses of 5-FU were used combined with various concentrations of HHC to minimize the toxic- ity and side effects of 5-FU. The therapeutic effects of these drugs on down-regulation of COX-2 mRNA and protein expression were examined using semi-quantitative reverse transcription-polymerase chain reaction （RT-PCR） and Western blotting analysis.RESULTS： Ml-I- reduction assay indicated that HHC alone markedly decreased the viability of HT-29 human colon cancer cells compared to control. Semi-quantitative RT-PCR analysis indicated that HHC is a selective COX-2 inhibitor. This finding was supported by the ob- servation that HHC significantly down-regulates COX-2 mRNA expression compared to the control （control： 100.05% ± 0.03% vs HHC： 61.01% ± 0.35%, P 〈 0.05） but does not alter COX-1 mRNA. In combined treatment, addition of HHC to a low dose of 5-FU exerts a synergistic effect against the growth of HT-29 cells by markedly reducing cell viability to a greater degree than monotherapy. Semi-quantitative RT-PCR indicated that 5-FU at the concentration of 5 pmol/L in combina- tion with HHC at the concentration of 25 pmol/L signifi- cantly down-regulates COX-2 mRNA expression when compared with values in cells treated with 5-FU or HHC alone （HHC ＋ 5-FU： 31.93% ± 5.69%, 5-FU： 100.66% ± 4.52% vs HHC： 61.01% ±0.35%, P 〈 0.05）.CONCLUSION： HHC together with 5-FU exerts a synergistic effect and may prove chemotherapeutically useful in treating human colon cancer.

High dose glargine alters the expression profiles of microRNAs in pancreatic cancer cells

AIM: To investigate the effect of high dose glargine on the expression profiles of microRNAs in human pancreatic cancer cells. METHODS: Real-time polymerase chain reaction array (RT-PCR) was applied to investigate miRNAs differentially expressed in Sw1990 cells treated with or without 100 IU/L glargine. Stem-loop RT-PCR was used to confirm the results of the array assay in Sw1990 and Panc-1 cells. The effects of miR-95 on cell growth, apoptosis, invasion and migration abilities were respectively examined by CCK8 assay, apoptosis assay, Matrigel invasion and migration assay in Sw1990 and Panc-1 cells. Nude mice xenograft models with Sw1990 cells were built to investigate pancreatic cancer growth in vivo after transfection by the lentivirus pGLV3-GFP-miR-95. RESULTS: Ten miRNAs were significantly up-regulated and 2 miRNAs down-regulated in glargine treated Sw1990 cells when compared with non-treated cells (2.48-fold changes on average, P < 0.01). miR-95, miR-134 and miR-34c-3p are the top three miRNAs regulated by glargine (3.65-fold, 2.67-fold and 2.60-fold changes respectively, P < 0.01) in Sw1990 cells. Stem-loop RT-PCR confirmed that high dose glargine up-regulated the expression of miR-95 and miR-134 in both Sw1990 and Panc-1 cells. The most obvious change is the apparent increase of miR-95. Forced expression of miR-95 significantly increased cell proliferation (Sw1990: 2.510 ± 0.129 vs 2.305 ± 0.187, P < 0.05; Panc-1: 2.439 ± 0.211 vs 2.264 ± 0.117, P < 0.05), invasion (Sw1990: 67.90 ± 12.33 vs 47.30 ± 5.89, P < 0.01; Panc-1: 37.80 ± 8.93 vs 30.20 ± 5.14, P < 0.01), migration (Sw1990: 101 ± 6.00 vs 51.20 ± 8.34, P < 0.01; Panc-1: 91.80 ± 9.22 vs 81.50 ± 7.47, P < 0.01) and inhibited cell apoptosis (Sw1990: 22.05% ± 1.92% vs 40.32% ± 1.93%, P < 0.05; Panc-1: 20.17% ± 0.85% vs 45.60% ± 1.43%, P < 0.05) when compared with paired negative controls, whereas knockdown of miR-95 obtained the opposite effect. Nude mice xenograft models confirmed that miR-95 promoted the growth of pancreatic cancer in vivo when compared with negative control (tumor volume: 373.82 ± 23.67 mLvs 219.69 ± 17.82 mL,P < 0.05). CONCLUSION: These observations suggested that mod- ulation of miRNA expression may be an important mechanism underlying the biological effects of glargine.