In recent years, microRNAs (miRNAs) have been found to play an essential role in tumor development. In lung tumorigenesis, targets and pathways of miRNAs are being revealed, and further translational research in this field is warranted. MiR-155 is one of the miRNAs most consistently involved in various neoplastic diseases. We aimed to investigate the prognostic impact of the multifunctional miR-155 in non-small cell lung cancer (NSCLC) patients.
Tumor tissue samples from 335 resected stage I to IIIA NSCLC patients were obtained and tissue microarrays (TMAs) were constructed with four cores from each tumor specimen. In situ hybridization (ISH) was used to evaluate the expression of miR-155.
There were 191 squamous cell carcinomas (SCCs), 95 adenocarcinomas (ACs), 31 large cell carcinomas and 18 bronchioalveolar carcinomas. MiR-155 expression did not have a significant prognostic impact in the total cohort (P = 0.43). In ACs, high miR-155 expression tended to a significant negative prognostic effect on survival in univariate analysis (P = 0.086) and was an independent prognostic factor in multivariate analysis (HR 1.87, CI 95% 1.01 - 3.48, P = 0.047). In SCC patients with lymph node metastasis, however, miR-155 had a positive prognostic impact on survival in univariate (P = 0.034) as well as in multivariate (HR 0.45, CI 95% 0.21-0.96, P = 0.039) analysis.
The prognostic impact of miR-155 depends on histological subtype and nodal status in NSCLC.
The role of microRNAs in small-cell lung carcinoma (SCLC) is largely unknown. miR-34a is known as a p53 regulated tumor suppressor microRNA in many cancer types. However, its therapeutic implication has never been studied in SCLC, a cancer type with frequent dysfunction of p53. We investigated the expression of a panel of 7 microRNAs (miR-21, miR-29b, miR-34a/b/c, miR-155, and let-7a) in 31 SCLC tumors, 14 SCLC cell lines, and 26 NSCLC cell lines. We observed significantly lower miR-21, miR-29b, and miR-34a expression in SCLC cell lines than in NSCLC cell lines. The expression of the 7 microRNAs was unrelated to SCLC patients' clinical characteristics and was neither prognostic in term of overall survival or progression-free survival nor predictive of treatment response. Overexpression or downregulation of miR-34a did not influence SCLC cell viability. The expression of these 7 microRNAs also did not predict in vitro sensitivity to cisplatin or etoposide in SCLC cell lines. Overexpression or downregulation of miR-34a did not influence sensitivity to cisplatin or etoposide in SCLC cell lines. In contrast to downregulation of the miR-34a target genes cMET and Axl by overexpression of miR-34a in NSCLC cell lines, the intrinsic expression of cMET and Axl was low in SCLC cell lines and was not influenced by overexpression of miR-34a. Our results suggest that the expression of the 7 selected microRNAs are not prognostic in SCLC patients, and miR-34a is unrelated to the malignant behavior of SCLC cells and is unlikely to be a therapeutic target.
Background: The IALT, JBR.10, ANITA and Cancer and Leukemia Group B 9633 trials compared adjuvant chemotherapy with observation for patients with resected non-small-cell lung cancer (R-NSCLC). Data from the metastatic setting suggest high tumor class III beta-tubulin (TUBB3) expression is a determinant of insensitivity to tubulin-targeting agents (e.g. vinorelbine, paclitaxel). In 265 patients from JBR.10 (vinorelbine–cisplatin versus observation), high TUBB3 was an adverse prognostic factor and was associated (nonsignificantly) with ‘greater’ survival benefit from chemotherapy. We explored this further in additional patients from JBR.10 and the other three trials.
Patients and methods: TUBB3 immunohistochemical staining was scored for 1149 patients on the four trials. The original JBR.10 cut-off scores were used to classify tumors as TUBB3 high or low. The prognostic and predictive value of TUBB3 on disease-free survival (DFS) and overall survival (OS) was assessed by Cox models stratified by trial and adjusted for clinical factors.
Results: High TUBB3 expression was prognostic for OS [hazard ratio (HR) = 1.27 (1.07–1.51), P = 0.008) and DFS [HR = 1.30 (1.11–1.53), P = 0.001). TUBB3 was not predictive of a differential treatment effect [interaction P = 0.20 (OS), P = 0.23 (DFS)]. Subset analysis (n = 420) on vinorelbine–cisplatin gave similar results.
Conclusions: The prognostic effect of high TUBB3 expression in patients with R-NSCLC has been validated. We were unable to confirm a predictive effect for TUBB3.
chemotherapy; meta-analysis; predictive value; prognostic; randomized trial; tubulin
AIM: To compare the microRNA (miR) profiles in the primary tumor of patients with recurrent and non-recurrent gastric cancer.
METHODS: The study group included 45 patients who underwent curative gastrectomies from 1995 to 2005 without adjuvant or neoadjuvant therapy and for whom adequate tumor content was available. Total RNA was extracted from formalin-fixed paraffin-embedded tumor samples, preserving the small RNA fraction. Initial profiling using miR microarrays was performed to identify potential biomarkers of recurrence after resection. The expression of the differential miRs was later verified by quantitative real-time polymerase chain reaction (qRT-PCR). Findings were compared between patients who had a recurrence within 36 mo of surgery (bad-prognosis group, n = 14, 31%) and those who did not (good-prognosis group, n = 31, 69%).
RESULTS: Three miRs, miR-451, miR-199a-3p and miR-195 were found to be differentially expressed in tumors from patients with good prognosis vs patients with bad prognosis (P < 0.0002, 0.0027 and 0.0046 respectively). High expression of each miR was associated with poorer prognosis for both recurrence and survival. Using miR-451, the positive predictive value for non-recurrence was 100% (13/13). The expression of the differential miRs was verified by qRT-PCR, showing high correlation to the microarray data and similar separation into prognosis groups.
CONCLUSION: This study identified three miRs, miR-451, miR-199a-3p and miR-195 to be predictive of recurrence of gastric cancer. Of these, miR-451 had the strongest prognostic impact.
MicroRNA; Prognosis; Recurrence; Gastric cancer
MicroRNAs (miRNAs) play an important role in carcinogenesis through the regulation of their target genes. miRNA-related single nucleotide polymorphisms (miR-SNPs) can affect miRNA biogenesis and target sites and can alter microRNA expression and functions. We examined 11 miR-SNPs, including 5 in microRNA genes, 3 in microRNA binding sites and 3 in microRNA-processing machinery components, and evaluated time to recurrence (TTR) according to miR-SNP genotypes in 175 surgically resected non-small-cell lung cancer (NSCLC) patients. Significant differences in TTR were found according to KRT81 rs3660 (median TTR: 20.3 months for the CC genotype versus 86.8 months for the CG or GG genotype; P = 0.003) and XPO5 rs11077 (median TTR: 24.7 months for the AA genotype versus 73.1 months for the AC or CC genotypes; P = 0.029). Moreover, when patients were divided according to stage, these differences were maintained for stage I patients (P = 0.002 for KRT81 rs3660; P<0.001 for XPO5 rs11077). When patients were divided into sub-groups according to histology, the effect of the KRT81 rs3660 genotype on TTR was significant in patients with squamous cell carcinoma (P = 0.004) but not in those with adenocarcinoma. In the multivariate analyses, the KRT81 rs3660 CC genotype (OR = 1.8; P = 0.023) and the XPO5 rs11077 AA genotype (OR = 1.77; P = 0.026) emerged as independent variables influencing TTR. Immunohistochemical analyses in 80 lung specimens showed that 95% of squamous cell carcinomas were positive for KRT81, compared to only 19% of adenocarcinomas (P<0.0001). In conclusion, miR-SNPs are a novel class of SNPs that can add useful prognostic information on the clinical outcome of resected NSCLC patients and may be a potential key tool for selecting high-risk stage I patients. Moreover, KRT81 has emerged as a promising immunohistochemical marker for the identification of squamous cell lung carcinoma.
Breast cancer is the most common malignancy with the highest incidence rates among women worldwide. Triple-negative breast cancer (TNBC) represents the major phenotype of basal-like molecular subtype of breast cancer, characterized by higher incidence in young women and a very poor prognosis. MicroRNAs (miRNAs) are small non-coding RNAs playing significant role in the pathogenesis of many cancers including breast cancer. Therefore, miRNAs are also potential prognostic and/or predictive biomarkers in triple-negative breast cancer patients.
Thirty-nine TNBC patients with available formalin-fixed paraffin-embedded (FFPE) tissues were enrolled in the study. MiR-34a, miR-34b, and miR-34c were analyzed using qRT-PCR and correlated to clinico-pathological features of TNBC patients.
Expression levels of miR-34b significantly correlate with disease free survival (DFS) (p = 0.0020, log-rank test) and overall survival (OS) (p = 0.0008, log-rank test) of TNBC patients. No other significant associations between miR-34a, miR-34b, and miR-34c with available clinical pathological data were observed.
MiR-34b expression negatively correlates with disease free survival and overall survival in TNBC patients. Thus, miR-34b may present a new promising prognostic biomarker in TNBC patients, but independent validations are necessary.
triple-negative breast cancer; miR-34a; miR-34b; miR-34c; prognosis
MicroRNAs (miRNAs) are small non-coding RNAs (~22 nucleotides) that regulate gene expression at a post-transcriptional level via imperfect base pairing to the 3’-UTR of their target mRNAs. Previous studies from our group identified a number of deregulated miRNAs due to the loss of p53 tumor suppressor in colon cancer cell lines. To further investigate the in vivo biological significance of these miRNAs, the expressions of hsa-let-7g, hsa-miR-143, hsa-miR-145, hsa-miR-181b and hsa-miR-200c were investigated using formalin-fixed paraffin-embedded (FFPE) colon cancer specimens to evaluate the potential relationship with chemosensitivity and tumorigenesis.
Patients and Methods
Forty-six patients with recurrent or residual colon cancer lesions were treated with the 5-fluorouracil-based antimetabolite S-1. This includes twenty-one pairs of tumor and normal samples. Total RNAs were isolated and the expression level of each particular miRNA was quantified using real time qRT-PCR analysis.
The expression levels of hsa-let-7g, hsa-miR-181b and hsa-miR-200c were over-expressed in tumor tissues compared to normal tissues. The expression levels of hsa-let-7g (p=0.03; Mann-Whitney test) and hsa-miR-181b (p=0.02; Mann-Whitney test) were strongly associated with clinical response to S-1. Although hsa-let-7g and hsa-miR-181b are strongly associated with patient’s response to S-1 treatment, they are not significant prognostic factors for predicting survival.
hsa-let-7g, hsa-miR-181b and hsa-miR-200c may be associated with tumorigenesis in colon cancer. In addition, hsa-let-7g and hsa-miR-181b may be potential indicators for chemoresponse to S-1 based chemotherapy.
miRNA; colon cancer; S-1
There is increasing evidence that altered microRNA expression is associated with tumor progression and survival in cancer patients. We tested if the expression of specific microRNAs was associated with prognosis and disease progression in early stage lung adenocarcinoma.
The expression of miR-21, miR-17 and miR-155 was measured by quantitative RT-PCR in tissues from 317 non small cell lung cancer (NSCLC) patients that originated from Maryland, Norway and Japan. Kaplan Meier and Cox regression analysis evaluated associations of microRNA expression with cancer-specific mortality and disease free survival.
Elevated miR-21 (hazard ratio [HR] 2.06, 1.13–3.75), miR-17 (HR 2.00, 1.10–3.61), miR-155 (HR 2.37, 1.27–4.42) was associated with worse cancer-specific mortality in the Maryland cohort. These were evaluated in two additional cohorts and only miR-21 was associated with worse cancer-specific mortality in the Norwegian cohort (HR 2.78, 1.22–6.31) and worse relapse free survival in the Japanese cohort (HR 2.82, 1.57–5.07). More advanced stage tumors expressed significantly higher levels of miR-21 compared to TNM stage I tumors. TNM stage I patients were evaluated separately and high levels of miR-21 was associated with worse cancer-specific mortality (HR 2.16, 1.11–4.21) and relapse-free survival (3.40, 1.57–7.36) independent of other clinical factors.
Conclusions and Summary
This is the first study to report that increased miR-21 expression is associated with disease progression and survival in stage I lung cancer. This suggests that expression of miR-21 may contribute to lung carcinogenesis and serve as a therapeutic target or early stage prognostic biomarker for lung adenocarcinoma.
Angiogenesis is regarded as a hallmark in cancer development, and anti-angiogenic treatment is presently used in non-small cell lung cancer (NSCLC) patients. MicroRNAs (miRs) are small non-coding, endogenous, single stranded RNAs that regulate gene expression. In this study we aimed to identify significantly altered miRs related to angiogenesis in NSCLC.
From a large cohort of 335 NSCLC patients, paraffin-embedded samples from 10 patients with a short disease specific survival (DSS), 10 with a long DSS and 10 normal controls were analyzed. The miRs were quantified by microarray hybridization and selected miRs were validated by real-time qPCR. The impacts of different pathways, including angiogenesis, were evaluated by Gene Set Enrichment Analysis (GSEA) derived from Protein ANalysis THrough Evolutionary Relationship (PANTHER). One of the most interesting candidate markers, miR-155, was validated by in situ hybridization (ISH) in the total cohort (n = 335) and correlation analyses with several well-known angiogenic markers were done.
128 miRs were significantly up- or down-regulated; normal versus long DSS (n = 68) and/or normal versus short DSS (n = 63) and/or long versus short DSS (n = 37). The pathway analysis indicates angiogenesis-related miRs to be involved in NSCLC. There were strong significant correlations between the array hybridization and qPCR validation data. The significantly altered angiogenesis-related miRs of high interest were miR-21, miR-106a, miR-126, miR-155, miR-182, miR-210 and miR-424. miR-155 correlated significantly with fibroblast growth factor 2 (FGF2) in the total cohort (r = 0.17, P = 0.002), though most prominent in the subgroup with nodal metastasis (r = 0.34, P<0.001).
Several angiogenesis-related miRs are significantly altered in NSCLC. Further studies to understand their biological functions and explore their clinical relevance are warranted.
Although notable progress has been made in the treatment of non-small-cell lung cancer (NSCLC) in recent years, this disease is still associated with a poor prognosis. Despite early-stage NSCLC is considered a potentially curable disease following complete resection, the majority of patients relapse and eventually die after surgery. Adjuvant chemotherapy prolongs survival, altough the absolute improvement in 5-year overall survival is only approximately 5%.
Trying to understand the role of genes which could affect drug activity and response to treatment is a major challenge for establishing an individualised chemotherapy according to the specific genetic profile of each patient. Among genes involved in the DNA repair system, the excision repair cross-complementing 1 (ERCC1) is a useful markers of clinical resistance to platinum-based chemotherapy. In the International Lung Cancer Trial (IALT) adjuvant chemotherapy significantly prolonged survival among patients with ERCC1 negative tumors but not among ERCC1-positive patients. BRCA1 and ribonucleotide reductase M1 (RRM1), two other key enzymes in DNA synthesis and repair, appear to be modulators of drug sensitivity and may provide additional information for customizing adjuvant chemotherapy.
Several clinical trials suggest that overexpression of class III β-tubulin is an adverse prognostic factor in cancer since it could be responsible for resistance to anti-tubulin agents. A retrospective analysis of NCIC JBR.10 trial showed that high tubulin III expression is associated with a higher risk of relapse following surgery alone but also with a higher probability of benefit from adjuvant cisplatin plus vinorelbine chemotherapy.
Finally, the use of gene expression patterns such as the lung metagene model could provide a potential mechanism to refine the estimation of a patient’s risk of disease recurrence and could affect treatment decision in the management of early stage of NSCLC.
In this review we will discuss the potential role of pharmacogenomic approaches to guide the medical treatment of early stage NSCLC.
NSCLC; adjuvant treatment; molecular markers; ERCC1; RRM1; β-tubulin; EGFR.
Analysis of molecular genetic markers in biological fluids has been proposed as a useful tool for cancer diagnosis. MicroRNAs (miRNAs) are small regulatory RNAs that are frequently dysregulated in lung cancer and have shown promise as tissue-based markers for its prognostication. The aim of this study was to determine whether aberrant miRNA expression can be used as a marker in sputum specimen for the diagnosis of non-small cell lung cancer (NSCLC). Experimental Design: Expressions of mature miRNAs, mir-21 and mir-155, were examined by real-time reverse transcription polymerase chain reaction (RT-PCR) and normalized to that of control miRNA, U6B, in sputum of 23 patients with NSCLC and 17 cancer-free subjects. The data was compared with conventional sputum cytology for the diagnosis of lung cancer. All endogenous miRNAs were present in sputum in a remarkably stable form and sensitively and specifically detected by real-time RT-PCR. Mir-21 expression in the sputum specimens was significantly higher in cancer patients (76.32 ± 9.79) than cancer-free individuals (62.24±3.82) (p<0.0001). Furthermore, overexpression of mir-21 showed highly discriminative receiver-operator characteristic (ROC) curve profile, clearly distinguishing cancer patients from cancer-free subjects with areas under the ROC curve at 0.902 ± 0.054. Detection of mir-21 expression produced 69.66% sensitivity and 100.00% specificity in diagnosis of lung cancer, as compared with 47.82% sensitivity and 100.00% specificity by sputum cytology. The measurement of altered miRNA expression in sputum could be a useful noninvasive approach for the diagnosis of lung cancer.
MicroRNA; sputum; lung cancer; real-time RT-PCR; diagnosis
microRNAs have emerged as key regulators of gene expression, and their altered expression has been associated with tumorigenesis and tumor progression. Thus, microRNAs have potential as both cancer biomarkers and/or potential novel therapeutic targets. Although accumulating evidence suggests the role of aberrant microRNA expression in endometrial carcinogenesis, there are still limited data available about the prognostic significance of microRNAs in endometrial cancer. The goal of this study is to investigate the prognostic value of selected key microRNAs in endometrial cancer by the analysis of archival formalin-fixed paraffin-embedded tissues.
Total RNAs were extracted from 48 paired normal and endometrial tumor specimens using Trizol based approach. The expression of miR-26a, let-7g, miR-21, miR-181b, miR-200c, miR-192, miR-215, miR-200c, and miR-205 were quantified by real time qRT-PCR expression analysis. Targets of the differentially expressed miRNAs were quantified using immunohistochemistry. Statistical analysis was performed by GraphPad Prism 5.0.
The expression levels of miR-200c (P<0.0001) and miR-205 (P<0.0001) were significantly increased in endometrial tumors compared to normal tissues. Kaplan-Meier survival analysis revealed that high levels of miR-205 expression were associated with poor patient overall survival (hazard ratio, 0.377; Logrank test, P = 0.028). Furthermore, decreased expression of a miR-205 target PTEN was detected in endometrial cancer tissues compared to normal tissues.
miR-205 holds a unique potential as a prognostic biomarker in endometrial cancer.
MicroRNA plays an important role in human diseases and cancer. We seek to investigate the expression status, clinical relevance, and functional role of microRNA in non-small cell lung cancer.
We performed miRNA expression profiling in matched lung adenocarcinoma and uninvolved lung using 56 pairs of fresh-frozen (FF) and 47 pairs of formalin-fixed, paraffin-embedded (FFPE) samples from never smokers. The most differentially expressed miRNA genes were evaluated by Cox analysis and Log-Rank test. Among the best candidate, miR-708 was further examined for differential expression in two independent cohorts. Functional significance of miR-708 expression in lung cancer was examined by identifying its candidate mRNA target and through manipulating its expression levels in cultured cells.
Among the 20 miRNAs most differentially expressed between tested tumor and normal samples, high expression level of miR-708 in the tumors was most strongly associated with an increased risk of death after adjustments for all clinically significant factors including age, sex, and tumor stage (FF cohort: HR, 1.90; 95% CI, 1.08-3.35; P=.025 and FFPE cohort: HR, 1.93; 95% CI, 1.02-3.63; P=.042). The transcript for TMEM88 gene has a miR-708 binding site in its 3′ UTR and was significantly reduced in tumors high of miR-708. Forced miR-708 expression reduced TMEM88 transcript levels and increased the rate of cell proliferation, invasion, and migration in culture.
MicroRNA-708 acts as an oncogene contributing to tumor growth and disease progression by directly down regulating TMEM88, a negative regulator of the Wnt signaling pathway in lung cancer.
NSCLC; adenocarcinoma; miR-708; never smoker; survival; TMEM88; Wnt signaling
Lung cancer is the leading cause of cancer-related deaths in the world, and non-small cell lung cancer (NSCLC) accounts for 80% of cases. MicroRNA-21 (miR-21) expression is increased and predicts poor survival in NSCLC. Although miR-21 function has been studied in vitro using cancer cell lines, the role of miR-21 in tumor development in vivo is unknown. We utilize transgenic mice with loss-of-function and gain-of-function miR-21 alleles combined with a model of NSCLC to determine the role of miR-21 in lung cancer. We show that over-expression of miR-21 enhances tumorigenesis and genetic deletion of miR-21 partially protects against tumor formation. MiR-21 drives tumorigenesis through inhibition of negative regulators of the Ras/MEK/ERK pathway and inhibition of apoptosis.
MicroRNAs are small noncoding RNA molecules that negatively regulate gene expression via degradation or translational repression of their targeted mRNAs. It is known that aberrant microRNA expression can play important roles in cancer, but the role of microRNAs in autoimmune diseases is only beginning to emerge. In this study, the expression of selected microRNAs is examined in rheumatoid arthritis.
Total RNA was isolated from peripheral blood mononuclear cells obtained from patients with rheumatoid arthritis, and healthy and disease control individuals, and the expression of miR-146a, miR-155, miR-132, miR-16, and microRNA let-7a was analyzed using quantitative real-time PCR.
Rheumatoid arthritis peripheral blood mononuclear cells exhibited between 1.8-fold and 2.6-fold increases in miR-146a, miR-155, miR-132, and miR-16 expression, whereas let-7a expression was not significantly different compared with healthy control individuals. In addition, two targets of miR-146a, namely tumor necrosis factor receptor-associated factor 6 (TRAF6) and IL-1 receptor-associated kinase 1 (IRAK-1), were similarly expressed between rheumatoid arthritis patients and control individuals, despite increased expression of miR-146a in patients with rheumatoid arthritis. Repression of TRAF6 and/or IRAK-1 in THP-1 cells resulted in up to an 86% reduction in tumor necrosis factor-α production, implicating that normal miR-146a function is critical for the regulation of tumor necrosis factor-α production.
Recent studies have shown that synovial tissue and synovial fibroblasts from patients with rheumatoid arthritis exhibit increased expression of certain microRNAs. Our data thus demonstrate that microRNA expression in rheumatoid arthritis peripheral blood mononuclear cells mimics that of synovial tissue/fibroblasts. The increased microRNA expression in rheumatoid arthritis patients is potentially useful as a marker for disease diagnosis, progression, or treatment efficacy, but this will require confirmation using a large and well defined cohort. Our data also suggest a possible mechanism contributing to rheumatoid arthritis pathogenesis, whereby miR-146a expression is increased but unable to properly function, leading to prolonged tumor necrosis factor-α production in patients with rheumatoid arthritis.
The high mortality rate of lung cancer patients is mainly due to the late stage at which lung cancer is diagnosed. For effective cancer prevention programs and early diagnosis, better blood-based markers are needed. Hence, blood-based microarray profiling of microRNA (miR) expression was performed in preoperative serum of 21 non-small cell lung cancer (NSCLC) patients and 11 healthy individuals by microfluid biochips containing 1158 different miRs. Two out of the 30 most dysregulated miRs were further validated in serum of 97 NSCLC patients, 20 patients with benign lung diseases and 30 healthy individuals by TaqMan MicroRNA Assays. Microarray profiling showed that miR-361-3p and miR-625* were significantly down-regulated in serum of lung cancer patients. Their further evaluation by quantitative RT-PCR showed that the levels of miR-361-3p and miR-625* were lower in NSCLC than in benign disease (p = 0.0001) and healthy individuals (p = 0.0001, p = 0.0005, respectively). Moreover, the levels of miR-625* were significantly lower in patients with large cell lung cancer (LCLC, p = 0.014) and smoking patients (p = 0.030) than in patients with adenocarcinoma and non-smoking patients, respectively. A rise in the levels of both miRs was observed in the postoperative samples compared with the preoperative levels (p = 0.0001). Functional analyses showed that Smad2 and TGFß1 are not dysregulated by miR-361-3p and miR-625* in the lung cell line A549, respectively. Our present pilot study suggests that miR-361-3p and miR-625* might have a protective influence on the development of NSCLC, and the quantitative assessment of these miRs in blood serum might have diagnostic potential to detect NSCLC, in particular in smokers.
NSCLC (non-small cell lung cancer) often exhibits resistance to paclitaxel treatment. Identifying the elements regulating paclitaxel response will advance efforts to overcome such resistance in NSCLC therapy. Using in vitro approaches, we demonstrated that over-expression of the microRNA miR-337-3p sensitizes NCI-H1155 cells to paclitaxel, and that miR-337-3p mimic has a general effect on paclitaxel response in NSCLC cell lines, which may provide a novel adjuvant strategy to paclitaxel in the treatment of lung cancer. By combining in vitro and in silico approaches, we identified STAT3 and RAP1A as direct targets that mediate the effect of miR-337-3p on paclitaxel sensitivity. Further investigation showed that miR-337-3p mimic also sensitizes cells to docetaxel, another member of the taxane family, and that STAT3 levels are significantly correlated with taxane resistance in lung cancer cell lines, suggesting that endogenous STAT3 expression is a determinant of intrinsic taxane resistance in lung cancer. The identification of a miR-337-3p as a modulator of cellular response to taxanes, and STAT3 and RAP1A as regulatory targets which mediate that response, defines a novel regulatory pathway modulating paclitaxel sensitivity in lung cancer cells, which may provide novel adjuvant strategies along with paclitaxel in the treatment of lung cancer and may also provide biomarkers for predicting paclitaxel response in NSCLC.
MicroRNAs (miRs) are conserved small non-coding RNAs that negatively regulate gene expression. The miR profiles are markedly altered in cancers and some of them have a causal role in tumorigenesis. Here, we report changes in miR expression profile in hepatocellular carcinomas (HCCs) developed in male Fisher rats-fed folic acid, methionine, and choline-deficient (FMD) diet. Comparison of the miR profile by microarray analysis showed altered expression of some miRs in hepatomas compared to the livers from age-matched rats on the normal diet. While let-7a, miR-21, miR-23, miR-130, miR-190, and miR-17-92 family of genes was upregulated, miR-122, an abundant liver-specific miR, was downregulated in the tumors. The decrease in hepatic miR-122 was a tumor-specific event because it did not occur in the rats switched to the folate and methyl-adequate diet after 36 weeks on deficient diet, which did not lead to hepatocarcinogenesis. miR-122 was also silent in a transplanted rat hepatoma. Extrapolation of this study to human primary HCCs revealed that miR-122 expression was significantly (P = 0.013) reduced in 10 out of 20 tumors compared to the pair-matched control tissues. These findings suggest that the downregulation of miR-122 is associated with hepatocarcinogenesis and could be a potential biomarker for liver cancers.
folate/methyl-deficient diet; hepatocellular carcinoma; microRNA; miR-122; miR-17-92; miR-21
MicroRNAs (miRNAs) have prognostic and therapeutic value for colorectal cancers (CRCs). Although formalin-fixed paraffin-embedded (FFPE) tissues are available for biomarker studies, the stability of miRNAs in these tissues stored for long periods (>20 years) is unknown. The present effort involved analysis of 345 FFPE CRC tissues, stored for 6 to 28 years (1982-2004), for the expression of six miRNAs (miR-20a, miR-21, miR-106a, miR-181b, miR-203, and miR-324-5p) using TaqMan® microRNA assays and quantitative real-time PCR (qRT-PCR). Evaluation, by linear regression analysis, of miRNA expression among archived CRC tissues found similar levels of all six miRNAs in tissues stored over this period (correlation coefficients, R2, ranged from <0.0001-0.009; and t-test p-values were ≥ 0.05). Thus, miRNAs are stable in FFPE tissues stored for long periods of time, and such samples can be used for discovery of biomarkers.
miRNA; Colorectal Cancer; FFPE; Stability; Biomarkers
MicroRNA (miRNA) expression is deregulated in many types of human cancers. We sought to investigate the expression patterns of the miRNAs, miR-21, miR-145 and miR-155 in sporadic gastric cancer in a Chinese population.
Total RNA was extracted from archived gastric cancer tissues and adjacent non-cancerous tissues from 20 pairs of paraffin-embedded specimens. Expression levels of miR-21, miR-145 and miR-155 were detected by quantitative reverse transcriptase PCR using a specific stem-loop primer, with U6 as the internal reference gene.
The expression of miR-21 and miR-155 in gastric cancer samples was significantly higher than in paired non-cancerous samples (P < 0.05). There was no statistically significant difference in expression levels of miR-145 between cancerous and non-cancerous tissues (P > 0.05).
In Chinese sporadic gastric cancer tissues, the expressions of the oncogenic miR-21 and miR-155 were significantly up-regulated, while the expression of the tumor suppressor miR-145 was decreased, although this decrease was not statistically significant. Thus there is specificity in the miRNA expression pattern in gastric cancers in the Chinese population.
MicroRNA; expression; gastric cancer
MicroRNAs (mirs) are small non-coding RNA molecules (~22 nucleotides) that regulate post-transcriptional gene expression. Currently, there has not been a comprehensive study of their role in primary HNSCC. To determine the role of mirs in head and neck squamous cell carcinoma (HNSCC), we screened for altered microRNA expression in HNSCC primary tissue and cell lines. We then further tested the functional impact of alterations of specific mirs.
An initial screening of 4 primary HNSCC, 4 normal mucosal controls and 4 HNSCC cell lines were analyzed for mature microRNA expression by microarray. Significance was determined using Significance Analysis of Microarrays (SAM). Nine microRNAs were found by SAM to be up-regulated or down-regulated in tumor tissue including mir-21,let-7,18,29c,142-3p,155,146b(over-expressed) and 494(under-expressed). Mir-21 was validated by qRT-PCR.
Functional validation by growth assays was performed, further validating mir-21. Transfection of mir-21 into JHU-011 and JHU-012 cell lines showed a 39% increase in cell growth at 72 hrs relative to controls (p<.05). Transfection of the inhibitor into JHU-O12 cell lines showed a 92% decrease in cell growth relative to controls at 72hrs (p<.05). In addition, flow cytometry analysis of JHU-012 cells 48 hrs after mir-21 inhibitor transfection showed a statistically significantly increase in cytochrome c release and increased apoptosis.
These differentially expressed microRNAs may be of interest as potential novel oncogenes and tumor suppressor genes in HNSCC. Mir-21 is a putative oncogenic microRNA in head and neck cancer.
MicroRNA; HNSCC; Mir-21; Mir-494; Mir; Squamous Cell Cancer
Pancreatic ductal adenocarcinoma (PDAC) has a dismal prognosis. The high risk of recurrence following surgical resection provides the rationale for adjuvant therapy. However, only a subset of patients benefit from adjuvant therapy. Identification of molecular markers to predict treatment outcome is therefore warranted. The aim of the present study was to evaluate whether expression of novel candidate biomarkers, including microRNAs, can predict clinical outcome in PDAC patients treated with adjuvant therapy.
Formalin-fixed paraffin embedded specimens from a cohort of 82 resected Korean PDAC cases were analyzed for protein expression by immunohistochemistry and for microRNA expression using quantitative Real-Time PCR. Cox proportional hazards model analysis in the subgroup of patients treated with adjuvant therapy (N = 52) showed that lower than median miR-21 expression was associated with a significantly lower hazard ratio (HR) for death (HR = 0.316; 95%CI = 0.166–0.600; P = 0.0004) and recurrence (HR = 0.521; 95%CI = 0.280–0.967; P = 0.04). MiR-21 expression status emerged as the single most predictive biomarker for treatment outcome among all 27 biological and 9 clinicopathological factors evaluated. No significant association was detected in patients not treated with adjuvant therapy. In an independent validation cohort of 45 frozen PDAC tissues from Italian cases, all treated with adjuvant therapy, lower than median miR-21 expression was confirmed to be correlated with longer overall as well as disease-free survival. Furthermore, transfection with anti-miR-21 enhanced the chemosensitivity of PDAC cells.
Low miR-21 expression was associated with benefit from adjuvant treatment in two independent cohorts of PDAC cases, and anti-miR-21 increased anticancer drug activity in vitro. These data provide evidence that miR-21 may allow stratification for adjuvant therapy, and represents a new potential target for therapy in PDAC.
Polo-like kinase 1 (PLK1) is highly expressed in many human cancers and regulates critical steps in mitotic progression. Previously, we have reported that PLK1 was overexpressed in non-small cell lung cancer (NSCLC), but the underlying molecular mechanisms are not well understood. By using microRNA (miR) target prediction algorithms, we identified miR-100 that might potentially bind the 3’-untranslated region of PLK1 transcripts. The purpose of this study was to investigate the roles of miR-100 and its association with PLK1 in NSCLC development.
Taqman real-time quantitative RT-PCR assay was performed to detect miR-100 expression 10 NSCLC tissues and corresponding nontumor tissues. Additionally, the expression of miR-100 in 110 NSCLC tissues and its correlation with clinicopathological factors or prognosis of patients was analyzed. Finally, the effects of miR-100 expression on growth, apoptosis and cell cycle of NSCLC cells by posttranscriptionally regulating PLK1 expression were determined.
MiR-100 was significantly downregulated in NSCLC tissues, and low miR-100 expression was found to be closely correlated with higher clinical stage, advanced tumor classification and lymph node metastasis of patients. The overall survival of NSCLC patients with low miR-100 was significantly lower than that of those patients with high miR-100, and univariate and multivariate analyses indicated that low miR-100 expression might be a poor prognostic factor. Also, miR-100 mimics could lead to growth inhibition, G2/M cell cycle arrest and apoptosis enhancement in NSCLC cells. Meanwhile, miR-100 mimics could significantly inhibit PLK1 mRNA and protein expression and reduce the luciferase activity of a PLK1 3’ untranslated region-based reporter construct in A549 cells. Furthermore, small interfering RNA (siRNA)-mediated PLK1 downregulation could mimic the effects of miR-100 mimics while PLK1 overexpression could partially rescue the phenotypical changes of NSCLC cells induced by miR-100 mimics.
Our findings indicate that low miR-100 may be a poor prognostic factor for NSCLC patients and functions as a tumor suppressor by posttranscriptionally regulating PLK1 expression.
MicroRNAs (miRNAs, miRs) are small non-coding RNAs that negatively regulate gene expression at the post-transcriptional level. MicroRNAs are dysregulated in cancer and may play essential roles in tumorigenesis. Additionally, miRNAs have been shown to have prognostic and diagnostic value in certain types of cancer. The objective of this study was to identify dysregulated miRNAs in endometrioid endometrial adenocarcinoma (EEC) and the precursor lesion, complex atypical hyperplasia (CAH).
We compared the expression profiles of 723 human miRNAs from 14 cases of EEC, 10 cases of CAH, and 10 normal proliferative endometria controls using Agilent Human miRNA arrays following RNA extraction from formalin-fixed paraffin-embedded (FFPE) tissues. The expression of 4 dysregulated miRNAs was validated using real time reverse transcription-PCR.
Forty-three miRNAs were dysregulated in EEC and CAH compared to normal controls (p<0.05). The entire miR-200 family (miR-200a/b/c, miR-141, and miR-429) was up-regulated in cases of EEC.
This information contributes to the candidate miRNA expression profile that has been generated for EEC and shows that certain miRNAs are dysregulated in the precursor lesion, CAH. These miRNAs in particular may play important roles in tumorigenesis. Examination of miRNAs that are consistently dysregulated in various studies of EEC, like the miR-200 family, will aid in the understanding of the role that miRNAs play in tumorigenesis in this tumour type.
Adjuvant chemotherapy has been shown to improve survival rates of postoperative patients with non-small cell lung cancer (NSCLC). Biomarkers could help select an appropriate chemotherapy for NSCLC patients or predict the efficacy of chemotherapy. The objective of this study was to explore the possible prognostic and predictive role of topoisomerase II alpha (TopIIα) expression level in postoperative NSCLC patients who received adjuvant chemotherapy.
Patients with stage I-III NSCLC, who underwent surgery in our hospital from January 2004 to December 2007 and who also received adjuvant chemotherapy after surgery, were analyzed in this study. Expression of TopIIα and Ki67 in paraffin-embedded tissues was detected by immunohistochemistry (IHC). The relationships between clinicopathological characteristics, chemotherapy regimens, the expression of biomarkers and disease free survival (DFS) were analyzed.
TopIIα and Ki67 were highly expressed in 22.5% and 36.4% of the 151 patients, respectively. Univariate survival analysis showed that male sex (P = 0.036), non-adenocarcinoma (P = 0.004), earlier pathological TNM stage (P = 0.001) or pathological N stage (P < 0.001), and high expression of TopIIα (P = 0.012) were correlated with better DFS, whereas age, smoking history, different chemotherapy regimens, T stage and expression level of Ki67 were of no prognostic significance. Further stratified analysis showed that vinorelbine (NVB)-containing adjuvant regimens were generally associated with better DFS than regimens without NVB in patients with low TopIIα expression, though the difference was not statistically significant (P = 0.065). Pairwise comparisons for patients with low TopIIα expression indicated that the NVB-containing regimen was associated with better DFS than the docetaxel (TXT)-containing regimen (P = 0.047). COX multivariate analysis showed that pathological TNM stage, histological subtype and expression level of TopIIα to be independent of risk factors affecting DFS in postoperative NSCLC patients who received chemotherapy.
High TopIIα expression was discovered to be correlated with better DFS for postoperative NSCLC patients who received adjuvant chemotherapy. The NVB-containing chemotherapy regimen was more effective than the TXT-containing regimen in improving DFS in patients with low TopIIα expression. TopIIα could be considered to be an independent prognostic biomarker of DFS in postoperative NSCLC patients who received adjuvant chemotherapy.