miR-92a/DUSP10/JNK signalling axis promotes human pancreatic cancer cells proliferation

Pancreatic cancer is one of the most common types of cancers in the whole world with a poor prognosis. Finding out how the cancer form and develop is the most important way to cure this cancer. miRNAs, 21–22 nucleotides regulatory small non-coding RNAs, have been found to be critical involved in the growth of pancreatic cancer. In this study, we found that miR-92a was up regulated in three kinds of human pancreatic cancer cell lines. There is a correlation between miR-92a and malignant degree of human pancreatic cancer cell lines. Then we found that miR-92a was essential for promoting cell proliferation in human pancreatic cancer. Inhibition of the function of miR-92a repressed the proliferation of pancreatic cancer cells. Further, we found that miR-92a enhanced the activation of JNK signalling pathway by directly targeting the JNK signalling inhibitor DUSP10. DUSP10 is responsible for miR-92a induced JNK signalling and cell proliferation. Altogether, our study showed a miR-92a/DUSP10/JNK signalling pathway that plays an important role in regulating the proliferation of pancreatic cancer cells.     

MiR-125a regulates ovarian cancer proliferation and invasion by repressing GALNT14 expression

Increasing evidence has suggested that dysregulation of microRNAs (miRNAs) could contribute to tumor progression. The miR-125a was downregulated in several types of cancer, however, the molecular mechanism of miR-125a in the ovarian cancer remains unclear. The aim of the paper was to reveal the mechanism of miR-125a regulating cell proliferation and metastasis in ovarian cancer. In this study, western blotting, immunohistochemistry and serum-ELISA assay revealed that polypeptide N-acetylgalactosaminyl transferase 14 (GALNT14) expression was upregulated and correlated with the cancer stage in ovarian cancer. The expression levels of miR-125a were downregulated and negatively related to GALNT14 expression in clinical ovarian cancer tissues. Moreover, luciferase reporter assay identified polypeptide N-acetylgalactosaminyl transferase 14 (GALNT14) as a direct target of miR-125a, and overexpression of miR-125a markedly reduced the expression of GALNT14 in ovarian cancer. Functional characterization of miR-125a was accomplished by reconstitution of miR-125a and silencing GALNT14 expression in ovarian cancer cells to determine changes in proliferation and invasion. The MTT assay and transwell assay revealed that miR-125a transfectant significantly inhibits cell proliferation and invasion, by repressing GALNT14 expression. Furthermore, the gelatin zymography assay miR-125a mimics and GALNT14 siRNA suppressed the activity of MMP2 and MMP9. Taken together, our findings show that miR-125a functions as tumor suppressor in ovarian cancer by targeting GALNT14, and miR-125a may therefore serve as a biomarker for diagnosis and therapeutics in ovarian cancer.     

miR-935 promotes gastric cancer cell proliferation by targeting SOX7

Gastric cancer is the most common cancer in the world, miRNAs have been demonstrated to play critical role in the development and progression of gastric cancer, such as miR-7, miR-217 and miR-335. Here, we found miR-935 was upregulated in gastric cancer tissues and cells. Overexpression of miR-935 promoted cell proliferation and tumorigenesis in vitro determined by MTT analysis, colony formation analysis, BrdU cell proliferation analysis and soft agar growth analysis, knockdown of miR-935 reduced these effects. Tumor suppressor sex-determining region Y-box 7 (SOX7) was the direct target of miR-935, overexpression of miR-935 inhibited SOX7 expression, but promoted the levels CCND1 and C-MYC which promotes cell proliferation and tumorigenesis, knockdown of miR-935 increased SOX7 level, and inhibited CCND1 and C-MYC expression. Synchronous knockdown of miR-935 and SOX7 promoted cell proliferation and tumorigenesis in vitro, confirming miR-935 regulated gastric cancer cell proliferation by inhibiting SOX7. In summary, we found miR-935 contributed to cell proliferation of gastric cancer through targeting SOX7.     

miR-421靶向PDCD4促进前列腺癌DU145细胞增殖的实验研究

目的研究MicroRNA-421(miR-421)促进前列腺癌DU145细胞增殖的作用及潜在的分子机制。方法培养前列腺癌DU145细胞,分为对照组、miR-阴性对照(NC)组、miR-421组、miR-421+pcDNA组、miR-421+pcDNA-细胞程序性死亡基因4(PDCD4),miR-NC组转染miR-NC、miR-421组转染miR-421、miR-421+pcDNA组转染miR-421及pcDNA质粒、miR-421+pcDNA-PDCD4组转染miR-421及pcDNA-PDCD4质粒,MTS法测定细胞增殖活力,荧光定量PCR测定PDCD4的mRNA表达水平,western blot测定PDCD4的蛋白表达水平,双荧光素酶报告基因实验验证miR-421与PDCD4的靶向结合。结果与对照组及miR-NC组比较,miR-421组的增殖活力增加,PDCD4的表达水平及野生型PDCD4双荧光素酶报告基因的荧光活力均降低(P<0.05);与miR-421+pcDNA组比较,miR-421+pcDNAPDCD4组的增殖活力降低,PDCD4的表达水平增加(P<0.05)。结论 miR-421对前列腺癌DU145细胞的增殖具有促进作用,靶向抑制PDCD4是miR-421发挥这一作用的潜在分子机制。     

Selective monitoring of vitamin D2 and D3 supplementation with a highly specific 25-hydroxyvitamin D3 immunoassay with negligible cross-reactivity to 25-hydroxyvitamin D2

Background

The effects of vitamin D₂ and D₃ supplementation on circulating concentrations of 25(OH)D₃ require reliable analytical tools for specific determination of 25(OH)D₃ and 25(OH)D₂. We have developed a highly specific 25-OH Vitamin D3 ELISA with negligible cross-reactivity towards 25(OH)D₂.

Methods

25(OH)D₃ concentrations were measured in several study participants; 1) 641 healthy men and women; 2) 39 postmenopausal women receiving 400-800 IU vitamin D₃ daily for 4 months; 3) 45 men and women with hip fracture receiving 1000 IU vitamin D₂ daily for 3 months.

Results

This 25-OH Vitamin D3 ELISA had minimal cross-reactivity to 25(OH)D₂, (0.7%), and demonstrated a high correlation (r² = 0.93) with 25(OH)D₃ determined by HPLC. 25(OH)D₃ increased by 14% in subjects receiving vitamin D₃ for 4 months (p < 0.01), whereas there was no significant change in 25(OH)D₃ levels in those receiving vitamin D₂.

Conclusions

We report that 25(OH)D₃ ELISA was used for evaluation of 25(OH)D₃ concentrations in subjects receiving vitamin D₂ and D₃ supplementation. The increase of 25(OH)D₃ in circulation with vitamin D3 supplementation and lack of increase with vitamin D₂ supplementation suggest that this assay has sufficient sensitivity and specificity to be used as a reliable measurement of nutritional vitamin D₃ status in humans.     

miR-1184 regulates the proliferation and apoptosis of colon cancer cells via targeting CSNK2A1

MicroRNA (miRNA) exerts an important part in colon cancer cell proliferation and apoptosis. Meanwhile, the dysregulation of some miRNAs is detected in colon cancer cells. However, it remains unclear about the underlying mechanism of their effects on tumor pathogenesis. The current work aimed to examine the miR-1184 effect on colon cancer cells. The differentially expressed miRNAs (DEMs), including miR-9-3p, miR-1184, miR-492, miR-92a-1-5p and miR-20a-3p, were obtained from the GSE115108 and GSE132619 data sets using the ‘GEO2R’ online tool. Based on the findings, miR-1184 was significantly down-regulated within colon cancer cells and tissues. Moreover, the experimental results of CCK8, flow cytometry, colony formation and Western blotting assays showed that, miR-1184 over-expression suppressed colon cancer cell proliferation through inhibiting Ki67 expression and promoted their apoptosis through up-regulating cleaved caspase-3 and down-regulating Bcl-2 expression. By contrast, miR-1184 inhibition exerted the opposite effects. A total of 110 target genes of miR-1184 were predicted using the TargetScan and miRTarBase databases, which were then used to construct the protein-protein interaction (PPI) network based on the DAVID and STRING websites and to perform GO and KEGG pathway enrichment analyses. The MCODE plug-in of cytoscape was utilized to verify that CSNK2A1 was the target gene and key gene in significant modules. MiR-1184 directly targets CSNK2A1 via using RNA immunoprecipitation assay and luciferase reporter gene assay. According to the results, CSNK2A1 over-expression reversed the functions of miR‐1184 over-expression in suppressing colon cancer cell proliferation and enhancing their apoptosis. In conclusion, over-expression of miR-1184 inhibits colon cancer cell proliferation but promotes their apoptosis through down-regulating CSNK2A1 expression.     

Tumor apoptosis in prostate cancer by PGD2 and its metabolite 15d-PGJ2 in murine model

Fifteen-deoxy-Δ^12,14-PGJ₂ (15d-PGJ₂) is one of non-enzymatically converted metabolite from prostaglandin D₂ (PGD₂). Anti-tumor effects of 15d-PGJ₂ in various tumors are partially known, but the detail of in vivo mechanisms of action is still unclear. In this study, we investigated the effects of 15d-PGJ₂ and PGD₂ on murine prostate cancer in vitro and in vivo. Murine prostate cancer cells RM9 were transfected with murine prostaglandin D₂ synthase (mPGDS) gene by using defective retrovirus vector, designated as RM9-mPGDS. In addition, RM9 was also transfected with only defective retrovirus vector, designated as RM9-EV and used as control in this study. The expression and production of the gene were confirmed by RT-PCR and ELISA, respectively. For in vivo study, RM9-mPGDS was injected into the back of C57BL/6 mice, then resulted tumor was used for pathological analysis 14 days after the inoculation. Tumor cell apoptosis in the tissue was detected by TUNEL staining. Retrovirally transfected mPGDS in RM9 significantly induced apoptosis in vivo but not in vitro, by TUNEL staining and cell death ELISA, respectively. Our results strongly suggested that the apoptosis induced in RM9-mPGDS in vivo was probably achieved in tumor environment such as hypoxic condition. The introduction of PGDS gene into cancer cells might be a novel therapy against cancer.     

miR-892a regulated PPP2R2A expression and promoted cell proliferation of human colorectal cancer cells

Colorectal cancer (CRC) is one of the most common malignancy cancers in the world. Aberrant microRNA expression is involved in human diseases including cancer. In the present study, we investigated the miR-892a's role in CRC cell proliferation. We found that miR-892a was frequently upregulated in human colorectal cancer tissues and cell lines compared with the matched tumor adjacent tissues and normal colonic cell line FHC. Overexpression of miR-892a promoted cell proliferation and colony formation of CRC. Bioinformatics analysis further revealed PPP2R2A was identified as a potential miR-892a. Overexpression of miR-892a-in SW480 cells reduced PPP2R2A protein expression. Subsequently, data from luciferase reporter assays showed that PPP2R2A 3′-untranslated region (3′-UTR) carried the directly binding site of miR-892a. Furthermore, siRNA-mediated silencing of PPP2R2A blocked the inhibitory effect of miR-892a-in on CRC cell growth. In sum, our data provided compelling evidence that overexpression of miR-892a may provide a selective growth promotion for CRC cells by direct suppression of PPP2R2A expression.     

MiR-572 prompted cell proliferation of human ovarian cancer cells by suppressing PPP2R2C expression

Ovarian cancer (OC) remains one of the most common types of malignant cancer, and the molecular mechanism underlying its proliferation is still largely unclear. It is reported that microRNAs acted as important regulators of cell proliferation by regulating its targeted gene. In this study, our result showed that miR-572 was markedly upregulated in OC cell lines and clinical tissues. Results of both gain-of-function and loss-of-function experiments revealed that upregulation of miR-572 expression dramatically promoted OC cell proliferation, whereas decreased miR-572 expression significantly reduced cell proliferation. Bioinformatics analysis and luciferase reporter assays further revealed PPP2R2C, a putative tumor suppressor as a potential target of miR-572. Moreover, silencing of PPP2R2C using small interfering RNA (siRNA) counteracted the proliferation arrest by miR-572-in in OC cells. In sum, our data provide that miR-572 promoted cell proliferation in OC by targeting PPP2R2C and might serve as a therapeutic target of OC.     

MiR-200a在上皮性卵巢癌中的表达及临床意义

目的探讨miR-200a上皮性卵巢癌组织中的表达及与临床病理特征关系。方法采用Taqman茎环探针实时荧光定量PCR方法检测40例上皮性卵巢癌和30例良性上皮性卵巢肿瘤组织标本中miR-200a的表达,分析miR-200a的表达量与临床病理特征关系。结果 miR-200a在卵巢癌组织中的相对表达量显著高于良性卵巢肿瘤组织(P<0.01),在晚期卵巢癌组织中表达低于早期卵巢癌组织(P<0.05),在低分化卵巢癌中表达有下降趋势。miR-200a表达与病理类型及淋巴结转移无相关性。结论 miR-200a在上皮性卵巢癌组织中表达升高,在晚期卵巢癌中表达下降,提示其可能在卵巢癌的发生发展中起原癌基因的作用,并参与晚期卵巢癌的侵袭转移。     

超顺磁性Fe2O3用作磁共振胃肠对比剂

目的 制备一种由Fe2O3为主要原料的口服磁共振胃肠造影混悬剂,研究Fe2O3在磁共振磁场中保持超顺磁性和稳定悬浮的临界尺寸.方法 根据超顺磁性理论,推导出Fe2O3微粒显示超顺磁现象的临界尺寸理论值和在1.5T磁共振磁场中的临界值;根据磁学理论,分析磁共振对比剂稳定的机制,推导在磁共振磁场中,Fe2O3微粒在对比剂中稳定悬浮的极限尺寸.以小于该尺寸的Fe2O3微粒作主料制备对比剂,并进行磁共振成像对比增强实验研究.结果 在1.5T磁共振磁场中,Fe2O3微粒在对比剂中既能稳定悬浮又具有超顺磁性的极限尺寸为11.1 nm,Fe3O4为7.1 nm.用尺寸为10 nm的Fe2O3微粒制备的对比剂,具有稳定、超顺磁性、安全、不沉降、不团聚的特性,且在磁共振成像上呈现明显的阴性对比作用.结论 从微粒尺寸角度考虑,用Fe2O3微粒作对比剂中的磁性粒子比Fe3O4更有优势.     

miR-125b inhibits keratinocyte proliferation and promotes keratinocyte apoptosis in oral lichen planus by targeting MMP-2 expression through PI3 K/Akt/mTOR pathway

Oral lichen planus (OLP) is a chronic inflammatory mucosal disease that involves the degeneration of keratinocytes. However, the etiology and mechanisms of OLP pathogenesis have not been fully elucidated. In this study, we used keratinocytes HaCaT stimulated with lipopolysaccharide (LPS) to mimic a local OLP immune environment, and investigated the regulatory role of miR-125b in keratinocyte proliferation and apoptosis under OLP conditions. Immunohistochemical analysis and quantitative real-time PCR (qRT-PCR) assay showed that MMP-2 expression was up-regulated and miR-125b expression was down-regulated in both OLP mucosa tissues and LPS-incubated HaCaT cells. Western blot analysis indicated that miR-125b overexpression suppressed LPS-induced MMP-2 expression in HaCaT cells. Molecularly, our results confirmed that MMP-2 is a target gene of miR-125b in HaCaT cells. The effect of miR-125b on cell proliferation was revealed by CCK-8 assay, BrdU assay and cell cycle analysis, which illustrated that miR-125b overexpression impeded LPS-induced HaCaT cell proliferation. Flow cytometry analysis further demonstrated that miR-125b overexpression promoted HaCaT cell apoptosis. Moreover, these effects were involved in PI3 K/Akt/mTOR activation, as miR-125b overexpression inhibited LPS-enhanced expression of p-Akt and p-mTOR proteins. Taken together, these data confirm that miR-125b might inhibit keratinocyte proliferation and promote keratinocyte apoptosis in OLP pathogenesis by targeting MMP-2 through PI3 K/Akt/mTOR pathway.     

miR-29a在乳腺癌组织中的表达及其对乳腺癌细胞增殖和迁移的影响

摘要：目的：检测miR-29a在乳腺癌组织中的表达水平并探讨其对人乳腺癌细胞增殖和迁移的影响。 方法：用荧光定量RT-PCR检测乳腺癌组织及癌旁组织中miR-29a的表达水平;用脂质体法将miR-29a mimic及miR-29a inhibitor分别转染乳腺癌细胞系MDA-MB-231,通过MTT实验和 Transwell实验观察miR-29a对MDA-MB-231细胞增殖和迁移能力的影响;miRanda软件预测miR-29a的靶基因,western blot验证其表达结果。 结果：荧光定量RT-PCR检测结果表明,与癌旁组织（2.17±0.89）比较,miR-29a在乳腺癌组织（5.65±1.45）中的表达水平上调（t=3.94,P＜0.01）;MTT实验和Transwell实验结果显示,与mimics阴性对照组［（106.36±5.15)%,(216.70±7.20）个］相比,miR-29a mimics组［（133.32±6.31）%,(294.30±8.60)个］乳腺癌细胞的增殖和迁移能力增加（t分别为3.36和6.85,P均＜0.01）;与inhibitor阴性对照组［（105.35±3.42）%,(240.30±9.50）个］相比,miR-29a inhibitor组［（66.63±3.82）%,(156.30±7.80）个］乳腺癌细胞的增殖和迁移能力降低（t分别为8.18和6.81,P均＜0.01）。miRanda软件预测人第10号染色体缺失的同源性磷酸酶-张力蛋白基因（PTEN）可能为miR-29a的靶基因。western blot结果显示,与mimics阴性对照组（0.55±0.01）相比,乳腺癌细胞转染miR-29a mimic后,其PTEN蛋白质的表达水平（0.22±0.01）下调（t=14.29,P＜0.01）;与inhibitor阴性对照组（0.49±0.02）相比,转染miR-29a inhibitor后,PTEN蛋白质的表达水平（1.25±0.02）上调（t=19.39,P＜0.01）。 结论：miR-29a在乳腺癌组织中的表达水平增高,并可能通过下调PTEN蛋白的表达促进乳腺癌细胞的增殖和转移。     

MiR-520a对胃癌SGC7901细胞增殖、侵袭、迁移以及顺铂药物敏感性的影响

目的:探讨miR-520a在胃癌中的表达以及对胃癌细胞生物学功能的影响。方法:通过real-time PCR检测miR-520a在胃癌组织和胃癌细胞中的表达。采用慢病毒在胃癌细胞SGC7901中过表达miR-520a,在体外观察miR-520a对胃癌细胞增殖、凋亡、侵袭和迁移以及对顺铂(DDP)敏感性的影响。并通过异种移植瘤实验观察过表达miR-520a对胃癌细胞在裸鼠体内生长的影响。结果:与癌旁正常组织相比,miR-520a在胃癌组织中的表达明显降低。与正常胃黏膜上皮细胞GES-1相比,miR-520a在胃癌SGC7901细胞中的表达明显降低,且在顺铂耐药SGC7901/DDP细胞中的表达更低。miR-520a过表达能够促进SGC7901细胞凋亡,抑制细胞增殖、侵袭和迁移。miR-520a过表达促进SGC7901细胞对DDP的药物敏感性增加。异种移植瘤实验发现miR-520a过表达的细胞在裸鼠体内形成的肿瘤生长速度减慢。结论:miR-520a可抑制胃癌发生与发展,提高胃癌细胞对顺铂的敏感性。     

FoxP3 demethylation is increased in human colorectal cancer and rat cholangiocarcinoma tissue

Objectives

FoxP3 expression is a marker for Tregs which are known to be involved in tumor immunity. We aimed to evaluate FoxP3 promoter demethylation in human colorectal cancer (CRC) and rat intrahepatic cholangiocarcinoma (ICC).

Design and methods

Bisulfite-treated genomic DNA templates of shock frozen paired samples were studied from 13 anonymous CRC patients and from 10 male rats (n = 6 ICC induced by thioacetamide and n = 4 age-matched controls). Real-time PCR was carried out using a LightCycler 480 system. Human FoxP3 and CD3 promoter demethylations were estimated using previously described assays; and rat FoxP3 promoter demethylation using a newly developed assay.

Results

A significant 3.5-fold increase of the demethylation in FoxP3 promoter region was found in human CRC and rat ICC (P < 0.05). The average frequency of cells with FoxP3 demethylation in patients suffering from CRC was 0.26% in normal tissue and 0.92% in tumor tissue (n = 11 paired samples). Although, no significant difference was found between the mean frequency of CD3 demethylation in normal tissue (4.80%, n = 6) and in tumor tissue (4.14%, n = 6) from CRC patients, the ratio of demethylated CD3/FoxP3 promoter areas was significantly lower in tumor specimens (P < 0.05). Using our novel assay, we found a significant increase in mean frequencies of cells with FoxP3 demethylation in rats with ICC (7.42%, n = 6) in comparison to controls (2.14%, n = 4).

Conclusion

FoxP3 seems to be an interesting biomarker for immune response to epithelial tumors. Functional consequences from the increase of Tregs remain to be demonstrated. Further studies with outcome data are necessary.     

miR-152通过调控FOXR2表达抑制人前列腺癌细胞增殖的研究

目的 探讨人前列腺癌PC-3细胞中miR-152调控叉头框蛋白R2(FOXR2)对细胞增殖的影响.方法 采用实时荧光定量PCR检测人正常前列腺上皮细胞RWPE-1和前列腺癌PC-3细胞中miR-152、FOXR2 mRNA的相对表达水平,Western blot检测FOXR2蛋白水平.miR-152 mimics、mi...     

MiR-551b-3p靶向USP9X对人肺癌A549细胞凋亡和放射敏感性的影响

目的:探讨miR-551b-3p对肺癌A549细胞凋亡和放射敏感性的影响及分子机制。方法:将miRNC,miR-551b-3p,anti-miR-NC,anti-miR-551b-3p,si-NC,si-USP9X分别转染至A549中,记为miR-NC组、miR-551b-3p组、anti-miR-NC组、anti-miR-551b-3p组、si-NC组、si-USP9X组;将miR-551b-3p分别与pc DNA和pc DNA-USP9X共转染至A549中,记为miR-551b-3p+pc DNA组、miR-551b-3p+pc DNA-USP9X组。采用实时荧光定量PCR(RT-q PCR)检测miR-551b-3p和USP9X的表达水平;蛋白质印迹法检测X染色体连锁的泛素特定蛋白水解酶9(ubiquitin-specific peptidase 9 X-linked,USP9X)、 B细胞淋巴瘤/白血病-2 (B-cell lymphoma/leukemia-2, Bcl-2)、 Bcl-2相关X蛋白(Bcl-2 related X protein,Bax)的表达;荧光素酶报告实验检测miR-551b-3p和USP9X的靶向关系;流式细胞术检测细胞凋亡;细胞克隆集落形成实验检测放射敏感性。结果:肺癌组织和肺癌细胞A549中miR-551b-3p表达水平显著降低,USP9X mRNA和蛋白质表达水平显著升高(均P<0.001)。miR-551b-3p靶向调控USP9X,过表达miR-551b-3p和抑制USP9X表达,细胞凋亡率显著升高,Bcl-2表达水平显著降低,Bax表达水平显著升高,细胞的存活曲线明显下移(均P<0.001)。USP9X过表达逆转了miR-551b-3p过表达对肺癌A549细胞凋亡和放射敏感性的作用。结论:过表达miR-551b-3p促进肺癌A549细胞凋亡,增强肺癌细胞放射敏感性,其机制可能与USP9X有关。     

MiR-95 induces proliferation and chemo- or radioresistance through directly targeting sorting nexin1 (SNX1) in non-small cell lung cancer

MicroRNAs are emerging as a class of small regulatory RNAs whose specific roles and significant functions in the majority of carcinomas have yet to be entirely illustrated. The aim of this study is to explore the effect of miR-95 and determine whether miR-95 could be a potential therapeutic target for human non-small cell lung cancer. First of all, our study showed that miR-95 was highly expressed in both NSCLC cell lines (compared with normal cells) and tumor tissues (compared with corresponding normal tissues), whereas the protein level of SNX1 was downregulated in NSCLC cell lines. Next, we found that ectopic overexpression of miR-95 in A549 or H226 contributed to tumor growth in xenograft mouse models. In addition, the results also indicated that upregulation of miR-95 could significantly enhance the susceptibilities of NSCLC cells to chemo- or radiotherapy. Furthermore, using the luciferase reporter, we demonstrated that SNX1 is a direct target of miR-95. Meanwhile, overexpression of SNX1 could abrogate the growth of NSCLC cells induced by miR-95. Taken together, these results suggest that miR-95 functions as an oncogene role in NSCLC cells by directly targeting SNX1.     

miR-99b-5p,miR-380-3p,and miR-485-3p are novel chemosensitizing miRNAs in high-risk neuroblastoma

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