Differential expression of miR‐21, miR‐125b and miR‐191 in breast cancer tissue

Aims: To develop new biomarkers for early detection and to inform effective clinical management of breast cancer. Methods: Real‐time polymerase chain reaction was used to profile microRNA (miRNA) in tumor tissue from 50 breast cancer patients using non‐tumor breast tissue from each patient as a control. We have focussed on three miRNA; miR‐21, miR‐125b and miR‐191, all of which have been implicated in breast cancer with either proven or predicted target genes involved in critical cancer‐associated cellular pathways. Results: Upregulation of miR‐21 and miR‐191 and downregulation of miR‐125b, was found in breast cancer tissue. Combined expression analysis of miR‐125b/miR‐191 increased sensitivity to 100% and specificity to 94% while miR‐21/miR‐191 increased to 92% and 100%, respectively. Therefore, combination of two miRNA gives a better prediction than individual miRNA. Conclusions: We could differentiate between breast cancer and adjacent non‐tumor breast tissue as a control with a high degree of sensitivity and specificity in the Mexican population using a combined expression analysis of only two miRNA. These observations, although a proof of principle finding at this time, show that a combined expression profile of two miRNA (miR‐125b/miR‐191 and miR‐21/miR‐191) can discriminate between breast cancer and non‐tumor tissue with high specificity and sensitivity.     

MicroRNA‐143/Musashi‐2/KRAS cascade contributes positively to carcinogenesis in human bladder cancer

It has been well established that microRNA (miR)‐143 is downregulated in human bladder cancer (BC). Recent precision medicine has shown that mutations in BC are frequently observed in FGFR3, RAS and PIK3CA genes, all of which correlate with RAS signaling networks. We have previously shown that miR‐143 suppresses cell growth by inhibiting RAS signaling networks in several cancers including BC. In the present study, we showed that synthetic miR‐143 negatively regulated the RNA‐binding protein Musashi‐2 (MSI2) in BC cell lines. MSI2 is an RNA‐binding protein that regulates the stability of certain mRNAs and their translation by binding to the target sequences of the mRNAs. Of note, the present study clarified that MSI2 positively regulated KRAS expression through directly binding to the target sequence of KRAS mRNA and promoting its translation, thus contributing to the maintenance of KRAS expression. Thus, miR‐143 silenced KRAS and MSI2, which further downregulated KRAS expression through perturbation of the MSI2/KRAS cascade.     

Suppression of AKT expression by miR‐153 produced anti‐tumor activity in lung cancer

Lung cancer is one of the leading causes of cancer death worldwide. microRNAs have been shown to be a novel class of regulators in lung cancer. Here, we explored the role of miR‐153 in the pathogenesis of lung cancer and its therapeutic potential. miR‐153 was significantly decreased in lung cancer tissues than the adjacent tissues. The protein and mRNA levels of protein kinase B (AKT), which were shown to promote tumor growth, were both increased in lung cancer tissues than adjacent tissues. Overexpression of miR‐153 significantly inhibited AKT protein expression, which were abrogated by co‐transfection of AMO‐153, the specific inhibitor of miR‐153. Luciferase assay showed that transfection of miR‐153 markedly suppressed the fluorescent intensity of chimeric vectors carrying the 3'UTR of AKT1, while produced no effect on the mutant construct, indicating that AKT is regulated by miR‐153. Overexpression of miR‐153 significantly inhibited the proliferation and migration, and promoted apoptosis of cultured lung cancer cells in vitro, and suppressed the growth of xenograft tumors in vivo. Interestingly, lung cancer cells with lower endogenous miR‐153 expression are more sensitive to ectopic overexpressed miR‐153. The IC₅₀ of miR‐153 on lung cancer cells is positive correlated with the endogenous miR‐153 level, while negative correlated with AKT level. Knockdown of AKT expression suppressed lung cancer cell proliferation. In summary, miR‐153 exerted anti‐tumor activity in lung cancer by targeting on AKT. The sensitivity of lung cancer cells to miR‐153 is determined by its endogenous miR‐153 level.     

Circulating microRNA‐590‐5p functions as a liquid biopsy marker in non‐small cell lung cancer

Despite the availability of various diagnostic procedures, a tissue biopsy is still indispensable for the routine diagnosis of lung cancer. However, inaccurate diagnoses can occur, leading to inefficient cancer management. In this context, use of circulating microRNAs (miRNAs) may serve as diagnostic tools as liquid biopsies, and as biomarkers to better understand the molecular mechanisms involved in the progression of cancer. We identified miR‐590‐5p as a potential prognostic marker in the progression of non‐small cell lung cancer (NSCLC). We were able to detect this miRNA in blood plasma samples of NSCLC patients through quantitative real‐time PCR. Our data showed an ~7.5‐fold downregulation of miR‐590‐5p in NSCLC patients compared to healthy controls, which correlated with several clinicopathological features. Further, overexpression of miR‐590‐5p led to decreased cell viability, proliferation, colony formation, migration, and invasion potential of lung cancer cells, whereas its knockdown showed the opposite effect. In addition, the levels of several proteins involved in the epithelial‐to‐mesenchymal transition negatively correlated with miR‐590‐5p levels in lung adenocarcinoma cells and tumors of NSCLC patients. Further, dual‐luciferase reporter assays identified STAT3 as a direct target of miR‐590‐5p, which negatively regulated STAT3 activation and its downstream signaling molecules (eg, Cyclin D1, c‐Myc, Vimentin, and β‐catenin) involved in tumorigenesis. Taken together, our study suggests that miR‐590‐5p functions as a tumor suppressor in NSCLC through regulating the STAT3 pathway, and may serve as a useful biomarker for the diagnosis/prognosis of NSCLC, and as a potential therapeutic target for the treatment of NSCLC.     

Clinicopathological significance of the microRNA‐146a/WASP‐family verprolin‐homologous protein‐2 axis in gastric cancer

Gastric cancer (GC) is one of the most common malignancies, and cancer invasion and metastasis are the leading causes of cancer‐induced death in GC patients. WASP‐family verprolin‐homologous protein‐2 (WASF2), with a role controlling actin polymerization which is critical in the formation of membrane protrusions involved in cell migration and invasion, has been reported to possess cancer‐promoting effects in several cancers. However, data of WASF2's role in GC are relatively few and even contradictory. In this study, we analyzed WASF2 expression in GC tissues and their corresponding adjacent normal tissues. We found that WASF2 was upregulated in GC tissues and high level of WASF2 was associated with lymph node metastasis of GC. Through gain‐ and loss‐of‐function studies, WASF2 was shown to significantly increase GC cells migration and invasion, but had no effect on proliferation in vitro. Importantly, WASF2 was also found to enhance GC metastasis in vivo. Our previous research suggested that WASF2 was a direct target of microRNA‐146a (miR‐146a). Furthermore, we analyzed miR‐146a's level in GC tissues and their corresponding adjacent normal tissues. We found that miR‐146a was downregulated in GC tissues and low miR‐146a level was associated with advanced TNM stage and lymph node metastasis. The level of WASF2 in GC tissues was negatively correlated with miR‐146a expression and had inverse clinicopathologic features. The newly identified miR‐146a/WASF2 axis may provide a novel therapeutic target for GC.     

Co‐delivery of microRNA‐21 antisense oligonucleotides and gemcitabine using nanomedicine for pancreatic cancer therapy

Tumor metastasis occurs naturally in pancreatic cancer, and the efficacy of chemotherapy is usually poor. Precision medicine, combining downregulation of target genes with chemotherapy drugs, is expected to improve therapeutic effects. Therefore, we developed a combined therapy of microRNA‐21 antisense oligonucleotides (ASO‐miR‐21) and gemcitabine (Gem) using a targeted co‐delivery nanoparticle (NP) carrier and investigated the synergistic inhibitory effects on pancreatic cancer cells metastasis and growth. Polyethylene glycol–polyethylenimine–magnetic iron oxide NPs were used to co‐deliver ASO‐miR‐21 and Gem. An anti‐CD44v6 single‐chain variable fragment (scFv_CD44v6) was used to coat the particles to obtain active and targeted delivery. Our results showed that the downregulation of the oncogenic miR‐21 by ASO resulted in upregulation of the tumor‐suppressor genes PDCD4 and PTEN and the suppression of epithelial–mesenchymal transition, which inhibited the proliferation and induced the clonal formation, migration, and invasion of pancreatic cancer cells in vitro. The co‐delivery of ASO‐miR‐21 and Gem induced more cell apoptosis and inhibited the growth of pancreatic cancer cells to a greater extent than single ASO‐miR‐21 or Gem treatment in vitro. In animal tests, more scFv_CD44v6‐PEG‐polyethylenimine/ASO‐magnetic iron oxide NP/Gem accumulated at the tumor site than non‐targeted NPs and induced a potent inhibition of tumor proliferation and metastasis. Magnetic resonance imaging was used to observed tumor homing of NPs. These results imply that the combination of miR‐21 gene silencing and Gem therapy using an scFv‐functionalized NP carrier exerted synergistic antitumor effects on pancreatic cancer cells, which is a promising strategy for pancreatic cancer therapy.     

ASC amino acid transporter 2, defined by enzyme‐mediated activation of radical sources,enhances malignancy of GD2‐positive small‐cell lung cancer

Ganglioside GD2 is specifically expressed in small‐cell lung cancer (SCLC) cells, leading to enhancement of malignant phenotypes, such as cell proliferation and migration. However, how GD2 promotes malignant phenotypes in SCLC cells is not well known. In this study, to reveal the mechanisms by which GD2 increases malignant phenotypes in SCLC cells, we used enzyme‐mediated activation of radical sources combined with mass spectrometry in GD2⁺ SCLC cells. Consequently, we identified ASC amino acid transporter 2 (ASCT2), a major glutamine transporter, which coordinately works with GD2. We showed that ASCT2 was highly expressed in glycolipid‐enriched microdomain/rafts in GD2⁺ SCLC cells, and colocalized with GD2 in both proximity ligation assay and immunocytostaining, and bound with GD2 in immunoprecipitation/TLC immunostaining. Malignant phenotypes of GD2⁺ SCLC cells were enhanced by glutamine uptake, and were suppressed by L‐γ‐glutamyl‐p‐nitroanilide, a specific inhibitor of ASCT2, through reduced phosphorylation of p70 S6K1 and S6. These results suggested that ASCT2 enhances glutamine uptake in glycolipid‐enriched microdomain/rafts in GD2⁺ SCLC cells, leading to the enhancement of cell proliferation and migration through increased phosphorylation of the mTOR complex 1 signaling axis.     

VEGF, b-FGF,NOS2 和 NOS3在非小细胞肺癌的表达及其临床意义

目的 :研究血管内皮生长因子 (VEGF)、碱性成纤维细胞生长因子 (b -FGF)、一氧化氮合酶 2 (诱生型 ,NOS2 )、一氧化氮合酶 3(内皮型 ,NOS3)在非小细胞肺癌 (NSCLC)中的表达及其与肿瘤血管形成和淋巴结转移的关系。方法 :用免疫组化 (S -P法 )染色技术对 95例NSCLC石腊组织标本的VEGF、b -FGF、NOS2 和NOS3及肿瘤内微血管密度 (IMVD)进行检测和分析。结果 :VEGF、b FGF表达与TNM分期、IMVD及淋巴结转移有关 (P<0 .0 5) ,两者共表达与IMVD有关 (P <0 .0 1 )。NOS3与组织学分型、IMVD和淋巴结转移有关 (P <0 .0 5) ;NOS2与IMVD有关 (P <0 .0 5)。相关分析显示促血管形成因子 (VEGF、b FGF)与一氧化氮合酶 (NOS2 、NOS3)的表达呈正相关 (r=0 .30 1 8,P <0 .0 5)。结论 :VEGF和b FGF在促进血管形成和促进肿瘤转移方面可能起到协同作用 ,并且有NO的参与 ;VEGF、b FGF、NOS2 和NOS3的检测对于判断NSCLC转移和预后及具有临床应用价值     

Downregulation of microRNA‐100/microRNA‐125b is associated with lymph node metastasis in early colorectal cancer with submucosal invasion

A majority of early colorectal cancers (CRCs) with submucosal invasion undergo surgical operation, despite a very low incidence of lymph node metastasis. Our study aimed to identify microRNAs (miRNAs) specifically responsible for lymph node metastasis in submucosal CRCs. MicroRNA microarray analysis revealed that miR‐100 and miR‐125b expression levels were significantly lower in CRC tissues with lymph node metastases than in those without metastases. These results were validated by quantitative real‐time PCR in a larger set of clinical samples. The transfection of a miR‐100 or miR‐125b inhibitor into colon cancer HCT116 cells significantly increased cell invasion, migration, and MMP activity. Conversely, overexpression of miR‐100 or miR‐125b mimics significantly attenuated all these activities but did not affect cell growth. To identify target mRNAs, we undertook a gene expression array analysis of miR‐100‐silenced HCT116 cells as well as negative control cells. The Ingenuity Pathway Analysis, TargetScan software analyses, and subsequent verification of mRNA expression by real‐time PCR identified mammalian target of rapamycin (mTOR) and insulin‐like growth factor 1 receptor (IGF1R) as direct, and Fas and X‐linked inhibitor‐of‐apoptosis protein (XIAP) as indirect candidate targets for miR‐100 involved in lymph node metastasis. Knockdown of each gene by siRNA significantly reduced the invasiveness of HCT116 cells. These data clearly show that downregulation of miR‐100 and miR‐125b is closely associated with lymph node metastasis in submucosal CRC through enhancement of invasion, motility, and MMP activity. In particular, miR‐100 may promote metastasis by upregulating mTOR, IGF1R, Fas, and XIAP as targets. Thus, miR‐100 and miR‐125b may be novel biomarkers for lymph node metastasis of early CRCs with submucosal invasion.     

miR‐203 inhibits cell proliferation,invasion, and migration of non‐small‐cell lung cancer by downregulating RGS17

Involvement of the RGS17 oncogene in the promotion of non‐small‐cell lung cancer (NSCLC) has been reported, but the regulation mechanism in NSCLC remains unclear. MicroRNAs (miRNAs) negatively regulate gene expression, and their dysregulation has been implicated in tumorigenesis. To understand the role of miRNAs in Regulator of G Protein Signaling 17 (RGS17)‐induced NSCLC, we showed that miR‐203 was downregulated during tumorigenesis, and inhibited the proliferation and invasion of lung cancer cells. We then determined whether miR‐203 regulated NSCLC by targeting RGS17. To characterize the regulatory effect of miR‐203 on RGS17, we used lung cancer cell lines, A549 and Calu‐1, and the constructed miR‐203 and RGS17 overexpression vectors. The CCK8 kit was used to determine cell proliferation, and the Transwell® assay was used to measure cell invasion and migration. RT‐PCR, western blots, and immunofluorescence were used to analyze expression of miR‐203 and RGS17, and the luciferase reporter assay was used to examine the interaction between miR‐203 and RGS17. Nude mice were used to characterize in vivo tumor growth regulation. Expression of miR‐203 inhibited proliferation, invasion, and migration of lung cancer cell lines A549 and Calu‐1 by targeting RGS17. The regulatory effect of miR‐203 was inhibited after overexpression of RGS17. The luciferase reporter assay showed that miR‐203 downregulated RGS17 by direct integration into the 3′‐UTR of RGS17 mRNA. In vivo studies showed that expression of miR‐203 significantly inhibited growth of tumors. Taken together, the results suggested that expression of miR‐203 inhibited tumor growth and metastasis by targeting RGS17.