Identification and functional characterization of microRNAs reveal a potential role in gastric cancer progression

Purpose

To investigate the potential candidate microRNA (miRNA) biomarkers for the clinical diagnosis, classification, and prognosis of gastric cancer (GC).

Methods

We use bioinformatics overlapping subclasses analysis to find the tumor grade and lymphatic metastasis-related GC specific miRNAs from the Cancer Genome Atlas (TCGA) database. Then, we further investigated these GC specific miRNAs distributions in different GC clinical features and their correlations overall survival on the basis of GC patients’ information and their related RNA sequencing profile from TCGA. Finally, we randomly selected some of key miRNAs use qRT-PCR to confirm the reliability and validity.

Results

22 GC specific key miRNAs were identified (Fold-change >2, P < 0.05), 11 of them were discriminatively expressed with tumor size, grade, TNM stage and lymphatic metastasis (P < 0.05). In addition, nine miRNAs (miR-196b-5p, miR-135b-5p, miR-183-5p, miR-182-5p, miR-133a-3p, miR-486-5p, miR-144-5p, miR-129-5p and miR-145-5p) were found to be significantly associated with overall survival (log-rank P < 0.05). Finally, four key miRNAs (miR-183-5p, miR-486-5p, miR-30c-2-3p and miR-133a-3p) were randomly selected to validation and their expression levels in 53 newly diagnosed GC patients by qRT-PCR. Results showed that the fold-changes between TCGA and qRT-PCR were 100 % in agreement. We also found miR-183-5p and miR-486-5p were significantly correlated with tumor TNM stage (P < 0.05), and miR-30c-2-3p and miR-133a-3p were associated with tumor differentiation degree and lymph-node metastasis (P < 0.05). These verified miRNAs clinically relevant, and the bioinformatics analysis results were almost the same.

Conclusion

These key miRNAs may functions as potential candidate biomarkers for the clinical diagnosis, classification and prognosis for GC.

     

Differential expression of miR-139, miR-486 and miR-21 in breast cancer patients sub-classified according to lymph node status

Purpose

Therapeutic decisions in breast cancer are increasingly guided by prognostic and predictive biomarkers. Non-protein-coding microRNAs (miRNAs) have recently been found to be deregulated in breast cancers and, in addition, to be correlated with several clinico-pathological features. One of the most consistently up-regulated miRNAs is miR-21. Here, we specifically searched for differentially expressed miRNAs in high-risk breast cancer patients as compared to low-risk breast cancer patients. In the same patients, we also compared miR-21 expression with the expression of its presumed target PTEN.

Methods

Both microarray and RT-qPCR techniques were used to assess miRNA expression levels in lymph node-positive and -negative human invasive ductal carcinoma tissues. Simultaneously, PTEN protein expression levels were assessed using immunohistochemistry.

Results

miR-486-5p and miR-139-5p were found to be down-regulated in patients with lymph node metastases, whereas miR-21 was found to be up-regulated in patients with a positive lymph node status. miR-21 expression levels were found to significantly correlate with tumour size (r?=?0.403, p?=?0.009; Spearman’s rank), whereas no relation was found between miR-21 and PTEN expression levels (Kruskal-Wallis test).

Conclusion

Down-regulation of miR-486-5p and miR-139-5p, in conjunction with up-regulation of miR-21, may represent a useful signature for the identification of high-risk breast cancer patients.     

MicroRNA-27b,microRNA-101 and microRNA-128 inhibit angiogenesis by down-regulating vascular endothelial growth factor C expression in gastric cancers

Vascular Endothelial Growth Factor C (VEGF-C) has critical roles in angiogenesis in human cancers; however, the underlying mechanisms regulating VEGF-C expression remain largely unknown. In the present study, VEGF-C protein expression and the density of blood vessels or lymphatic vessels were determined by immunohistochemistry in 103 cases of gastric cancer tissues. Suppression of VEGF-C by miR-27b, miR-101 and miR-128 was investigated by luciferase assays, Western blot and ELISA. The miRNAs expression levels were detected in human gastric cancers by real-time quantitative PCR. Cell proliferation, migration and invasion assays were performed to assess the effect of miRNAs on gastric cancer cells and human umbilical vascular endothelial cells (HUVECs). Our data showed that high VEGF-C expression was significantly associated with increased tumor size, advanced TNM classification and clinical stage, higher microvessel density (MVD) and lymphatic density (LVD), as well as poor survival in patients with gastric cancer. Furthermore, VEGF-C was found to be a direct target gene of miR-27b, miR-101, and miR-128. The expression levels of the three miRNAs were inversely correlated with MVD. Overexpression of miR-27b, miR-101, or miR-128 suppressed migration, proliferation activity, and tube formation in HUVECs by repressing VEGF-C secretion in gastric cancer cells. We conclude that miR-27b, miR-101 and miR-128 inhibit angiogenesis by down-regulating VEGF-C expression in gastric cancers.     

TGF-β1 alters microRNA profile in human gastric cancer cells

Objective: MicroRNAs (miRNAs) are important regulators that play a key role in tumorigenesis and tumor progression. Transforming growth factor-β1 (TGF-β1) is involved in invasion and metastasis in many tumors. In this study, we investigated the microRNAs (miRNA) profiles altered by TGF-β1 in gastric cancer (GC) cells. Methods: We detected the expression profiles of miRNA by miRNA microarray and quantitative real-time polymerase chain reaction. Migration and invasion, wound-healing assay, prediction of miRNA targets, Western blot and qRT-PCR analysis were carried out to determine the role of one selected miRNA, namely miR-193b, in affecting the biological behaviors of GC BGC823 cells. Results: Among 847 human miRNAs in the microarray, three miRNAs (miR-27a, miR-29b-1 and miR-194) were up-regulated and three (miR-574-3p, miR-193b and miR-130b) were down-regulated in BGC823 cells treated with TGF-β1 compared with control. miR-193b suppressed the invasion and metastasis of GC cells in vivo and in vitro, and down-regulated urokinase-type plasminogen activator (uPA) protein in GC cells. Conclusions: TGF-β1 altered miRNA expression profile in BGC823 cells. Among the altered miRNAs, TGF-β1 induced the down-regulation of miR-193b, which inhibited cell invasion and metastasis in vivo and in vitro, and down-regulated uPA protein in GC cells.     

Kallistatin inhibits lymphangiogenesis and lymphatic metastasis of gastric cancer by downregulating VEGF-C expression and secretion

Background

Tumor-induced lymphangiogenesis and lymphatic metastasis are predominant during the metastasis of many types of cancers. However, the endogenous inhibitors that counterbalance the lymphangiogenesis and lymphatic metastasis of tumors have not been well evaluated. Kallistatin has been recognized as an endogenous angiogenesis inhibitor.

Methods and results

Our recent study showed for the first time that the lymphatic vessel density (LVD) was reduced in lung and stomach sections from kallistatin-overexpressing transgenic mice. Kallistatin expresses anti-lymphangiogenic activity by inhibiting the proliferation, migration, and tube formation of human lymphatic endothelial cells (hLECs). Therefore, the present study focuses on the relationships of changes in kallistatin expression with the lymphangiogenesis and lymphatic metastasis of gastric cancer and its underlying mechanisms. Our results revealed that the expression of kallistatin in cancer tissues, metastatic lymph nodes, and plasma of gastric cancer patients was significantly downregulated and that the plasma level of kallistatin was negatively associated with the phase of lymph node metastasis. Furthermore, treatment with kallistatin recombinant protein decreased LVD and lymph node metastases in the implanted gastric xenograft tumors of nude mice. Mechanically, kallistatin suppressed the lymphangiogenesis and lymphatic metastasis by downregulating VEGF-C expression and secretion through the LRP6/IKK/I?B/NF-?B signaling pathway in gastric cancer cells.

Conclusions

These findings demonstrated that kallistatin functions as an endogenous lymphangiogenesis inhibitor and has an important part in the lymphatic metastasis of gastric cancer.

     

Deregulated microRNAs in gastric cancer tissue-derived mesenchymal stem cells: novel biomarkers and a mechanism for gastric cancer

Background:

MicroRNAs (miRNAs) are involved in gastric cancer development and progression. However, the expression and role of miRNAs in gastric cancer stromal cells are still unclear.

Methods:

The miRNAs differentially expressed in gastric cancer tissue-derived mesenchymal stem cells (GC-MSCs) relative to adjacent non-cancerous tissue-derived MSCs (GCN-MSCs) and in cancer tissues relative to adjacent non-cancerous tissues were screened using miRNA microarray and validated by quantitative RT–PCR. The impact of GC-MSCs on HGC-27 cells was observed in vitro using colony formation and transwell assays, and these cells were subcutaneously co-injected into mice to assess tumour growth in vivo. Exogenous downregulation of miR-221 expression in cells was achieved using an miRNA inhibitor.

Results:

miR-214, miR-221 and miR-222 were found to be commonly upregulated in GC-MSCs and cancer tissues. Their levels were tightly associated with lymph node metastasis, venous invasion and the TNM stage. Gastric cancer tissue-derived mesenchymal stem cells significantly promoted HGC-27 growth and migration and increased the expression of miR-221 via paracrine secretion, and the targeted inhibition of miR-221 in GC-MSCs could block its tumour-supporting role. GC-MSC-derived exosomes were found to deliver miR-221 to HGC-27 cells and promoted their proliferation and migration.

Conclusions:

Gastric cancer tissue-derived mesenchymal stem cells favour gastric cancer progression by transferring exosomal miRNAs to gastric cancer cells, thus providing a novel mechanism for the role of GC-MSCs and new biomarkers for gastric cancer.     

Identifying microRNAs regulating B7-H3 in breast cancer: the clinical impact of microRNA-29c

Background:

B7-H3, an immunoregulatory protein, is overexpressed in several cancers and is often associated with metastasis and poor prognosis. Here, our aim was to identify microRNAs (miRNAs) regulating B7-H3 and assess their potential prognostic implications in breast cancer.

Methods:

MicroRNAs targeting B7-H3 were identified by transfecting two breast cancer cell lines with a library of 810 miRNA mimics and quantifying changes of B7-H3 protein levels using protein lysate microarrays. For validations we used western immunoblotting and 3′-UTR luciferase assays. Clinical significance of the miRNAs was assayed by analysing whether their expression levels correlated with outcome in two cohorts of breast cancer patients (142 and 81 patients).

Results:

We identified nearly 50 miRNAs that downregulated B7-H3 protein levels. Western immunoblotting validated the impact of the 20 most effective miRNAs. Thirteen miRNAs (miR-214, miR-363*, miR-326, miR-940, miR-29c, miR-665, miR-34b*, miR-708, miR-601, miR-124a, miR-380-5p, miR-885-3p, and miR-593) targeted B7-H3 directly by binding to its 3′-UTR region. Finally, high expression of miR-29c was associated with a significant reduced risk of dying from breast cancer in both cohorts.

Conclusions:

We identified miRNAs efficiently downregulating B7-H3 expression. The expression of miR-29c correlated with survival in breast cancer patients, suggesting a tumour suppressive role for this miRNA.     

Circulating microRNAs from the miR-106a–363 cluster on chromosome X as novel diagnostic biomarkers for breast cancer

Purpose

Novel noninvasive biomarkers with high sensitivity and specificity for the diagnosis of breast cancer (BC) are urgently needed in clinics. The aim of this study was to explore whether miRNAs from the miR-106a–363 cluster can be detected in the circulation of BC patients and whether these miRNAs can serve as potential diagnostic biomarkers.

Methods

The expression of 12 miRNAs from the miR-106a–363 cluster was evaluated using qRT-PCR in 400 plasma samples (from 200 BC patients and 200 healthy controls (HCs)) and 406 serum samples (from 204 BC patients and 202 HCs) via a three-phase study. The identified miRNAs were further examined in tissues (32 paired breast tissues), plasma exosomes (from 32 BC patients and 32 HCs), and serum exosomes (from 32 BC patients and 32 HCs).

Results

Upregulated levels of four plasma miRNAs (miR-106a-3p, miR-106a-5p, miR-20b-5p, and miR-92a-2-5p) and four serum miRNAs (miR-106a-5p, miR-19b-3p, miR-20b-5p, and miR-92a-3p) were identified and validated in BC. A plasma 4-miRNA panel and a serum 4-miRNA panel were constructed to discriminate BC patients from HCs. The areas under the receiver-operating characteristic curves of the plasma panel were 0.880, 0.902, and 0.858, and those of the serum panel were 0.910, 0.974, and 0.949 for the training, testing, and external validation phases, respectively. Two overlapping miRNAs (miR-106a-5p and miR-20b-5p) were consistently upregulated in BC tissues. Except for the expression of the plasma-derived exosomal miR-20b-5p, the expression patterns of exosomal miRNAs were concordant between plasma and serum, indicating the potential use of exosomal miRNAs as biomarkers.

Conclusion

We identified four plasma miRNAs and four serum miRNAs from the miR-106a–363 cluster as promising novel biomarkers for the diagnosis of BC.

     

Loss of has-miR-337-3p expression is associated with lymph node metastasis of human gastric cancer

Background

Metastasis is the major cause of cancer-related death in patients with gastric cancer, and aberrant expression of various microRNAs (miRNAs) is associated with cancer metastasis.

Methods

Profiling of differentially expressed miRNAs was performed in three cases of primary gastric cancer and the corresponding metastatic lymph node tissues. Then, the five most altered miRNAs were further verified in 16 paired samples. Two of these five miRNAs were further assessed for their effects on the regulation of gastric cancer cell growth and invasion.

Results

The miRNA profile data showed 151 upregulated miRNAs (≥ 1.5-fold) and 285 downregulated miRNAs (≤ 0.67-fold) in the metastatic tissues compared to the primary gastric cancer tissues. Among these five miRNAs (i.e., hsa-miR-508-5p, hsa-miR-30c, hsa-miR-337-3p, hsa-miR-483-5p, and hsa-miR-134), expression of hsa-miR-337-3p and hsa-miR-134 was significantly downregulated in these 16 lymph node metastatic tissues compared to their primary tumor tissues (P<0.05) and in nine gastric cancer cell lines compared to the nonmalignant GES cell line. Furthermore, induction of hsa-miR-134 or hsa-miR-337-3p expression did not dramatically affect gastric cancer cell proliferation, but transfection of the hsa-miR-337-3p mimic did reduce gastric cancer cell invasion capacity.

Conclusions

These findings indicate that hsa-miR-337-3p plays a role in the reduction of gastric cancer cell invasion capacity, and further studies on the mechanism of hsa-miR-337-3p in gastric cancer metastasis are warranted.     

miR-589-5p inhibits MAP3K8 and suppresses CD90<Superscript>+</Superscript> cancer stem cells in hepatocellular carcinoma

Background

Cancer stem cells (CSCs) are important in the tumorigenesis and progression of hepatocellular carcinoma (HCC). MicroRNAs (miRNAs) play crucial roles regulating CD133⁺ and EpCAM⁺ CSCs in HCC, although it is unclear whether miRNAs regulate CD90⁺ CSCs in HCC.

Methods

The miRNA profiles of CD90⁺ and CD90^- HCC cells were analyzed using a miRNA microarray and quantitative real-time PCR (qRT-PCR). CSC characteristics were examined by qRT-PCR and Western blot of pluripotency-associated genes, clone and sphere formation assay, transwell migration assay, and nude mice tumorigenicity assay. miR-589-5p mimic transfection was used to overexpress miR-589-5p in vitro. The CD90 and miR-589-5p expressions of HCC samples were detected by immunohistochemistry and qRT-PCR, respectively.

Results

miR-589-5p and miR-33b-5p were down-regulated in CD90⁺ cells. Overexpression of miR-589-5p suppressed CD90⁺ CSC characteristics such as Oct4, Sox2 and Nanog expression, a high likelihood of forming cell spheres, high invasiveness and high tumorigenicity. Luciferase reporter assays demonstrated that miR-589-5p directly binds to the 3?-untranslated region of mitogen-activated protein kinase kinase kinase 8 (MAP3K8) mRNA, and exogenous miR-589-5p down-regulated MAP3K8 expression. In addition, siRNA inhibition of MAP3K8 also suppressed CD90⁺ CSC characteristics, even in the absence of miR-589-5p overexpression. In HCC tissues, miR-589-5p expression was inversely correlated with CD90 expression, and high CD90 expression and low miR-589-5p expression were positively correlated with vascular invasion and recurrence and significantly decreased disease-free and overall survival by clinical analysis.

Conclusion

In HCC, miR-589-5p down-regulates the stemness characteristics of CD90⁺ CSCs in part by silencing MAP3K8. CD90 and miR-589-5p expression predict HCC outcomes and might be novel molecular targets for HCC treatment.

     

Circulating microRNA-203 predicts metastases,early recurrence,and poor prognosis in human gastric cancer

Background

Metastasis is a major cause of death in patients with gastric cancer (GC). MicroRNAs (miRNAs) relating to the epithelial–mesenchymal transition (EMT) control GC progression and metastasis. The aim of this study was to evaluate serum EMT-associated miRNAs for metastatic and prognostic noninvasive biomarkers in GC.

Methods

In the first step of this study (preliminary experiments), we selected candidate miRNAs associated with metastasis by analyzing the expression of the miR-200 family (miR-200a, miR-200b, miR-200c, miR-141, and miR-429) and miR-203 in serum samples from stage I (n = 12) and stage IV (n = 12) GC patients. The second phase involved the independent validation of candidate miRNAs in serum specimens from 130 patients with GC and 22 controls.

Results

Based on the preliminary experiments, miR-203 was selected as the candidate serum miRNA that was most closely associated with metastasis. Validation analysis revealed that serum miR-203 levels were significantly lower in stage IV than stage I–III GC patients. Serum miR-203 expression was significantly lower in GC patients with a higher T stage, vessel invasion, and lymph node, peritoneal, and distant metastases. Low expression of serum miR-203 was significantly associated with poor disease-free and overall survival. Multivariate analysis revealed that low serum miR-203 expression was an independent predictive marker for lymph node, peritoneal, and distant metastases and a poor prognosis in patients with GC.

Conclusions

Serum miR-203 has the potential to serve as a noninvasive biomarker for prognosis and to predict metastasis in patients with GC.

     

miRNA profiling in metastatic renal cell carcinoma reveals a tumour-suppressor effect for miR-215

Background:

Renal cell carcinoma (RCC) is the most common neoplasm of the adult kidney. Metastatic RCC is difficult to treat. The 5-year survival rate for metastatic RCC is ⩽10%. Recently, microRNAs (miRNAs) have been shown to have a role in cancer metastasis and potential as prognostic biomarkers in cancer.

Method:

We performed a miRNA microarray to identify a miRNA signature characteristic of metastatic compared with primary RCCs. We validated our results by quantitative real-time PCR. We performed experimental and bioinformatic analyses to explore the involvement of miR-215 in RCC progression and metastasis.

Results:

We identified 65 miRNAs that were significantly altered in metastatic compared with primary RCCs. We validated our results by examining the expression of miR-10b, miR-126, miR-196a, miR-204 and miR-215, in two independent cohorts of patients. We showed that overexpression of miR-215 decreased cellular migration and invasion in an RCC cell line model. In addition, through gene expression profiling, we identified direct and indirect targets of miR-215 that can contribute to tumour metastasis.

Conclusion:

Our analysis showed that miRNAs are altered in metastatic RCCs and can contribute to kidney cancer metastasis through different biological processes. Dysregulated miRNAs represent potential prognostic biomarkers and may have therapeutic applications in kidney cancer.     

The effect of the expression of vascular endothelial growth factor (VEGF)-C and VEGF receptor-3 on the clinical outcome in patients with gastric carcinoma

Aims

We aimed to investigate the relationship among VEGF-C/VEGFR-3 expression, lymphatic metastasis and patient prognosis in gastric carcinoma.

Material and methods

VEGF-C and VEGFR-3 expression in gastric carcinoma tissues obtained from 204 patients who underwent curative gastrectomy (105 cases presented with lymph node metastasis and 99 cases without metastasis) was examined immunohistochemically. There was no significant difference in the other clinicopathologic variables except for postoperative pathological tumor stage (pT) and TNM stage between the two groups. The results were statistically processed.

Results

The results showed that VEGF-C was located mainly in the cytoplasm of tumor cells and VEGFR-3 was found predominantly in the endothelium of lymphatic vessels. VEGF-C and VEGFR-3 expression was more frequent in gastric carcinoma tissues than that in normal gastric tissues, 54.90% and 35.29% respectively, which revealed that the expression of VEGF-C and VEGFR-3 was significantly stronger in patients with lymph node metastasis than in those without metastasis. Patients who had positive staining for VEGF-C showed significantly less favorable survival rates compared with patients who had negative staining for VEGF-C. The survival rates of patients who had positive staining for VEGFR-3 also were significantly lower compared with patients who had negative staining for VEGFR-3. Patients who had positive staining for both VEGF-C and VEGFR-3 exhibited the most unfavorable prognosis. Multivariate analysis demonstrated that the expression of VEGF-C and VEGFR-3 was an independent prognostic determinant. In addition, faint to moderate VEGF-C expression was detected in normal gastric epithelial cells (18/204, 8.9%).

Conclusions

VEGF-C and VEGFR-3 expression could serve as a prognostic biomarker in patients with gastric carcinoma.     

Identification of serum miRNAs as novel non-invasive biomarkers for detection of high risk for early gastric cancer

Background:

Many micro-RNAs (miRNAs) are differentially expressed in Helicobacter pylori-infected gastric mucosa and in gastric cancer tissue and previous reports have suggested the possibility of serum miRNAs as complementary tumour markers. The aim of the study was to investigate serum miRNAs and pepsinogen levels in individuals at high risk for gastric cancer both before and after H. pylori eradication.

Methods:

Patients with recent history of endoscopic resection for early gastric cancer and the sex- and age-matched controls were enrolled. Serum was collected from subjects before or after eradication and total RNA was extracted to analyse serum levels of 24 miRNAs. Serum pepsinogen (PG) I and II levels were measured using enzyme-linked immunosorbent assay kits.

Results:

Using miR-16 as an endogenous control, the relative levels of miR-106 and let-7d before and after H. pylori eradication and miR-21 after eradication were significantly higher in the high-risk group than in the controls. H. pylori eradication significantly decreased miR-106b levels and increased let-7d only in the control group. After eradication, the combination MiR-106b with miR-21 was superior to serum pepsinogen and the most valuable biomarker for the differentiating high-risk group from controls.

Conclusion:

Serum miR-106b and miR-21 may provide a novel and stable marker of increased risk for early gastric cancer after H. pylori eradication.     

Effect of exosome biomarkers for diagnosis and prognosis of breast cancer patients

Purpose

Exosomes are gradually detected as an indicator for diagnosis and prognosis of breast cancer in clinic and a systematic review was conducted.

Methods

A search for clinical studies published before July 1, 2017 was performed. Methods of exosome purification and identification from all studies were extracted. For diagnosis evaluation, the comparison of exosome biomarkers expression between breast cancer patients and healthy women was obtained; for prognosis prediction, the correlation between exosome biomarkers expression and chemotherapy resistance, overall survival (OS), disease-free survival (DFS), recurrence and metastasis of breast cancer was also extracted.

Results

A total of 11 studies with 921 breast cancer patients were included. Ultracentrifugation is the most frequent method to purify exosomes and transmission electron microscopy is commonly used to identify exosomes. Exosome biomarkers (such as HER2, CD47, Del-1, miR-1246 and miR-21) in breast cancer patients are significantly higher than those in healthy controls, exosomal GSTP1 and TRPC5 are related to chemotherapy resistance, exosome-carrying TRPC5, NANOG, NEUROD1, HTR7, KISS1R and HOXC are correlated to PFS, DFS or OS, and some exosomal proteins (HER2, KDR, CD49d, CXCR4 and CD44) as well as miRNAs (miR-340-5p, miR-17-5p, miR-130a-3p, miR-93-5p) are associated with tumor recurrence or distant organ metastasis.

Conclusions

Exosome biomarkers can be used for early diagnosis and prognosis of breast cancer patients in clinic.