Tumor‐suppressive microRNA‐1291 directly regulates glucose transporter 1 in renal cell carcinoma

Our recent studies of microRNA (miRNA) expression signatures demonstrated that microRNA‐1291 (miR‐1291) was significantly downregulated in renal cell carcinoma (RCC) clinical specimens and was a putative tumor‐suppressive miRNA in RCC. The aim of the present study was to investigate the functional significance of miR‐1291 in cancer cells and to identify novel miR‐1291‐mediated cancer pathways and target genes in RCC. Expression of miR‐1291 was significantly downregulated in RCC tissues compared with adjacent non‐cancerous tissues. Restoration of mature miR‐1291 in RCC cell lines (A498 and 786‐O) revealed significant inhibition of cell proliferation, migration and invasion, suggesting that miR‐1291 functioned as a tumor suppressor. To identify miR‐1291‐mediated molecular pathways and targets, we used gene expression analysis (expression of RCC clinical specimens and miR‐1291‐transfected A498 cells) and in silico database analysis. Our data demonstrated that 79 signaling pathways were significantly regulated by tumor‐suppressive miR‐1291 in RCC cells. Moreover, solute career family 2 member 1 (SLC2A1) was a candidate target of miR‐1291 regulation. The SLC2A1 gene provides instructions for producing glucose transporter protein type 1 (GLUT1). Luciferase reporter assays showed that miR‐1291 directly regulated SLC2A1/GLUT1. In RCC clinical specimens, the expression of SLC2A1/GLUT1 mRNA was significantly higher in cancer tissues than in non‐cancerous tissues. A significant inverse correlation was recognized between SLC2A1/GLUT1 and miR‐1291 expression (r = ?0.55, P < 0.0001). Loss of tumor‐suppressive miR‐1291 enhanced RCC cell proliferation, migration and invasion through targeting SLC2A1/GLUT1. The identification of novel tumor‐suppressive miR‐1291‐mediated molecular pathways and targets has provided new insights into RCC oncogenesis and metastasis.     

Regulation of spindle and kinetochore‐associated protein 1 by antitumor miR‐10a‐5p in renal cell carcinoma

Analysis of our original microRNA (miRNA) expression signature of patients with advanced renal cell carcinoma (RCC) showed that microRNA‐10a‐5p (miR‐10a‐5p) was significantly downregulated in RCC specimens. The aims of the present study were to investigate the antitumor roles of miR‐10a‐5p and the novel cancer networks regulated by this miRNA in RCC cells. Downregulation of miR‐10a‐5p was confirmed in RCC tissues and RCC tissues from patients treated with tyrosine kinase inhibitors (TKI). Ectopic expression of miR‐10a‐5p in RCC cell lines (786‐O and A498 cells) inhibited cancer cell migration and invasion. Spindle and kinetochore‐associated protein 1 (SKA1) was identified as an antitumor miR‐10a‐5p target by genome‐based approaches, and direct regulation was validated by luciferase reporter assays. Knockdown of SKA1 inhibited cancer cell migration and invasion in RCC cells. Overexpression of SKA1 was observed in RCC tissues and TKI‐treated RCC tissues. Moreover, analysis of The Cancer Genome Atlas database demonstrated that low expression of miR‐10a‐5p and high expression of SKA1 were significantly associated with overall survival in patients with RCC. These findings showed that downregulation of miR‐10a‐5p and overexpression of the SKA1 axis were highly involved in RCC pathogenesis and resistance to TKI treatment in RCC.     

Tumor‐suppressive microRNA‐135a inhibits cancer cell proliferation by targeting the c‐MYC oncogene in renal cell carcinoma

Recently, many studies have suggested that microRNAs (miRNAs) are involved in cancer cell development, invasion, and metastasis of various types of human cancers. In a previous study, miRNA expression signatures from renal cell carcinoma (RCC) revealed that expression of microRNA‐135a (miR‐135a) was significantly reduced in cancerous tissues. The aim of this study was to investigate the functional significance of miR‐135a and to identify miR‐135a‐mediated molecular pathways in RCC cells. Restoration of mature miR‐135a significantly inhibited cancer cell proliferation and induced G₀/G₁ arrest in the RCC cell lines caki2 and A498, suggesting that miR‐135a functioned as a potential tumor suppressor. We then examined miR‐135a‐mediated molecular pathways using genome‐wide gene expression analysis and in silico analysis. A total of 570 downregulated genes were identified in miR‐135a transfected RCC cell lines. To investigate the biological significance of potential miR‐135a‐mediated pathways, we classified putative miR‐135a‐regulated genes according to the Kyoto Encyclopedia of Genes and Genomics pathway database. From our in silico analysis, 25 pathways, including the cell cycle, pathways in cancer, DNA replication, and focal adhesion, were significantly regulated by miR‐135a in RCC cells. Moreover, based on the results of this analysis, we investigated whether miR‐135a targeted the c‐MYC gene in RCC. Gain‐of‐function and luciferase reporter assays showed that c‐MYC was directly regulated by miR‐135a in RCC cells. Furthermore, c‐MYC expression was significantly upregulated in RCC clinical specimens. Our data suggest that elucidation of tumor‐suppressive miR‐135a‐mediated molecular pathways could reveal potential therapeutic targets in RCC.     

Tumor‐suppressive microRNA‐218 inhibits cancer cell migration and invasion via targeting of LASP1 in prostate cancer

Our recent studies of the microRNA (miRNA) expression signature in prostate cancer (PCa) indicated that miRNA‐218 (miR‐218) was significantly downregulated in clinical specimens, suggesting that miR‐218 might act as a tumor‐suppressive miRNA in PCa. The aim of the present study was to investigate the functional significance of miR‐218 in PCa and to identify novel miR‐218‐regulated cancer pathways and target genes involved in PCa oncogenesis and metastasis. Restoration of miR‐218 in PCa cell lines (PC3 and DU145) revealed that this miRNA significantly inhibited cancer cell migration and invasion. Gene expression data and in silico analysis demonstrated that LIM and SH3 protein 1 (LASP1) is a potential target of miR‐218 regulation. LASP1 is a cytoskeletal scaffold protein that plays critical roles in cytoskeletal organization and cell migration. Luciferase reporter assays showed that miR‐218 directly regulated expression of LASP1. Moreover, downregulating the LASP1 gene significantly inhibited cell migration and invasion in cancer cells, and the expression of LASP1 was upregulated in cancer tissues. We conclude that loss of tumor‐suppressive miR‐218 enhanced cancer cell migration and invasion in PCa through direct regulation of LASP1. Our data on pathways regulated by tumor‐suppressive miR‐218 provide new insight into the potential mechanisms of PCa oncogenesis and metastasis.     

MicroRNA expression profiles distinguish liposarcoma subtypes and implicate miR‐145 and miR‐451 as tumor suppressors

Liposarcomas are rare, heterogeneous and malignant tumors that can be divided into four histological subtypes with different characteristics and clinical behavior. Treatment consists of surgery in combination with systemic chemotherapy, but nevertheless mortality rates are high. More insight into the biology of liposarcoma tumorigenesis is needed to devise novel therapeutic approaches. MicroRNAs (miRNAs) have been associated with carcinogenesis in many tumors and may function as tumor suppressor or oncogene. In this study we examined miRNA expression in an initial series of 57 human liposarcomas (including all subtypes), lipomas and normal fat by miRNA microarrays. Supervised hierarchical clustering of the most differentially expressed miRNAs (p < 0.0002) distinguished most liposarcoma subtypes and control tissues. The distinction between well differentiated liposarcomas and benign lipomas was blurred, suggesting these tumor types may represent a biological continuum. MiRNA signatures of liposarcoma subtypes were established and validated in an independent series of 58 liposarcomas and control tissues. The expression of the miR‐143/145 and miR‐144/451 cluster members was clearly reduced in liposarcomas compared to normal fat. Overexpression of miR‐145 and miR‐451 in liposarcoma cell lines decreased cellular proliferation rate, impaired cell cycle progression and induced apoptosis. In conclusion, we show that miRNA expression profiling can be used to discriminate liposarcoma subtypes, which can possibly aid in objective diagnostic decision making. In addition, our data indicate that miR‐145 and miR‐451 act as tumor suppressors in adipose tissue and show that re‐expression of these miRNAs could be a promising therapeutic strategy for liposarcomas.     

MicroRNA‐92b‐3p is a prognostic oncomiR that targets TSC1 in clear cell renal cell carcinoma

Although several studies have reported that microRNA (miR)‐92b‐3p is involved in various cellular processes related to carcinogenesis, its physiological role in clear cell renal cell carcinoma (ccRCC) remains unclear. To clarify the role of miR‐92b‐3p in ccRCC, we compared miR‐92b‐3p expression levels in ccRCC tissues and adjacent normal renal tissues. Significant upregulation of miR‐92b‐3p was observed in ccRCC tissues. Overexpression of miR‐92b‐3p using a miRNA mimic promoted proliferation, migration, and invasion activities of ACHN cells. Functional inhibition of miR‐92b‐3p by a hairpin miRNA inhibitor suppressed Caki‐2 cell growth and invasion activities in vitro. Mechanistically, it was found that miR‐92b‐3p directly targeted the TSC1 gene, a known upstream regulator of mTOR. Overexpression of miR‐92b‐3p decreased the protein expression of TSC1 and enhanced the downstream phosphorylation of p70S6 kinase, suggesting that the mTOR signaling pathway was activated by miR‐92b‐3p in RCC cells. Importantly, a multivariate Cox proportion hazard model, based on TNM staging and high levels of miR‐92b‐3p, revealed that miR‐92b‐3p expression (high vs. low hazard ratio, 2.86; 95% confidence interval, 1.20‐6.83; P = .018) was a significant prognostic factor for overall survival of ccRCC patients with surgical management. Taken together, miR‐92b‐3p was found to act as an oncomiR, promoting cell proliferation by downregulating TSC1 in ccRCC.     

Tumor‐suppressive microRNA‐223 inhibits cancer cell migration and invasion by targeting ITGA3/ITGB1 signaling in prostate cancer

Analysis of microRNA (miRNA) expression signatures in prostate cancer (PCa) and castration‐resistant PCa has revealed that miRNA‐223 is significantly downregulated in cancer tissues, suggesting that miR‐223 acts as a tumor‐suppressive miRNA by targeting oncogenes. The aim of this study was to investigate the functional roles of miR‐223 and identify downstream oncogenic targets regulated by miR‐223 in PCa cells. Functional studies of miR‐223 were carried out to investigate cell proliferation, migration, and invasion using PC3 and PC3M PCa cell lines. Restoration of miR‐223 significantly inhibited cancer cell migration and invasion in PCa cells. In silico database and genome‐wide gene expression analyses revealed that ITGA3 and ITGB1 were direct targets of miR‐223 regulation. Knockdown of ITGA3 and ITGB1 significantly inhibited cancer cell migration and invasion in PCa cells by regulating downstream signaling. Moreover, overexpression of ITGA3 and ITGB1 was observed in PCa clinical specimens. Thus, our data indicated that downregulation of miR‐223 enhanced ITGA3/ITGB1 signaling and contributed to cancer cell migration and invasion in PCa cells. Elucidation of the molecular pathways modulated by tumor‐suppressive miRNAs provides insights into the mechanisms of PCa progression and metastasis.     

miR‐367 promotes proliferation and stem‐like traits in medulloblastoma cells

In medulloblastoma, abnormal expression of pluripotency factors such as LIN28 and OCT4 has been correlated with poor patient survival. The miR‐302/367 cluster has also been shown to control self‐renewal and pluripotency in human embryonic stem cells and induced pluripotent stem cells, but there is limited, mostly correlational, information about these pluripotency‐related miRNA in cancer. We evaluated whether aberrant expression of such miRNA could affect tumor cell behavior and stem‐like traits, thereby contributing to the aggressiveness of medulloblastoma cells. Basal expression of primary and mature forms of miR‐367 were detected in four human medulloblastoma cell lines and expression of the latter was found to be upregulated upon enforced expression of OCT4A. Transient overexpression of miR‐367 significantly enhanced tumor features typically correlated with poor prognosis; namely, cell proliferation, 3‐D tumor spheroid cell invasion and the ability to generate neurosphere‐like structures enriched in CD133 expressing cells. A concurrent downregulation of the miR‐367 cancer‐related targets RYR3, ITGAV and RAB23, was also detected in miR‐367‐overexpressing cells. Overall, these findings support the pro‐oncogenic activity of miR‐367 in medulloblastoma and reveal a possible mechanism contributing to tumor aggressiveness, which could be further explored to improve patient stratification and treatment of this important type of pediatric brain cancer.     

Dual tumor‐suppressors miR‐139‐5p and miR‐139‐3p targeting matrix metalloprotease 11 in bladder cancer

Our recent study of the microRNA (miRNA) expression signature of bladder cancer (BC) by deep‐sequencing revealed that two miRNA, microRNA‐139‐5p/microRNA‐139‐3p were significantly downregulated in BC tissues. The aim of this study was to investigate the functional roles of these miRNA and their modulation of cancer networks in BC cells. Functional assays of BC cells were performed using transfection of mature miRNA or small interfering RNA (siRNA). Genome‐wide gene expression analysis, in silico analysis and dual‐luciferase reporter assays were applied to identify miRNA targets. The associations between the expression of miRNA and its targets and overall survival were estimated by the Kaplan–Meier method. Gain‐of‐function studies showed that miR‐139‐5p and miR‐139‐3p significantly inhibited cell migration and invasion by BC cells. The matrix metalloprotease 11 gene (MMP11) was identified as a direct target of miR‐139‐5p and miR‐139‐3p. Kaplan–Meier survival curves showed that higher expression of MMP11 predicted shorter survival of BC patients (P = 0.029). Downregulated miR‐139‐5p or miR‐139‐3p enhanced BC cell migration and invasion in BC cells. MMP11 was directly regulated by these miRNA and might be a good prognostic marker for survival of BC patients.     

Long non‐coding RNA urothelial cancer‐associated 1 promotes bladder cancer cell migration and invasion by way of the hsa‐miR‐145–ZEB1/2–FSCN1 pathway

Numerous studies suggest that several long non‐coding RNAs (lncRNAs) play critical roles in bladder cancer development and progression. Long non‐coding RNA urothelial cancer‐associated 1 (lncRNA‐UCA1) is highly expressed in bladder cancer tissues and cells, and it has been shown to play an important role in regulating aggressive phenotypes of bladder cancer cells. However, little is known about the molecular mechanism of lncRNA‐UCA1‐mediated bladder cancer cell migration and invasion. Here, we show that overexpression of lncRNA‐UCA1 could induce EMT and increase the migratory and invasive abilities of bladder cancer cells. Mechanistically, lncRNA‐UCA1 induced EMT of bladder cancer cells by upregulating the expression levels of zinc finger E‐box binding homeobox 1 and 2 (ZEB1 and ZEB2), and regulated bladder cancer cell migration and invasion by tumor suppressive hsa‐miR‐145 and its target gene the actin‐binding protein fascin homologue 1 (FSCN1). Furthermore, we also observed a positive correlation between lncRNA‐UCA1 and ZEB1/2 expression, and a negative correlation between lncRNA‐UCA1 and hsa‐miR‐145 expression in bladder cancer specimens. Importantly, we found that lncRNA‐UCA1 repressed hsa‐miR‐145 expression to upregulate ZEB1/2, whereas the suppression of hsa‐miR‐145 could upregulate lncRNA‐UCA1 expression in bladder cancer cells. Moreover, the binding site for hsa‐miR‐145 within exons 2 and 3 of lncRNA‐UCA1 contributed to the reciprocal negative regulation of lncRNA‐UCA1 and hsa‐miR‐145. Taken together, our results identified that lncRNA‐UCA1 enhances bladder cancer cell migration and invasion in part through the hsa‐miR‐145/ZEB1/2/FSCN1 pathway. Therefore, lncRNA‐UCA1 might act as a promising therapeutic target for the invasion and metastasis of bladder cancer.     

MicroRNA‐143 regulates collagen type III expression in stromal fibroblasts of scirrhous type gastric cancer

Gastric cancer (GC) is one of the most common malignancies worldwide. In particular, scirrhous type GC is highly metastatic and is characterized clinically by rapid disease progression and poor prognosis. MicroRNAs (miRNAs) play crucial roles in cancer development and progression. In the present study, we identified several miRNAs that are expressed at higher levels in scirrhous type GC than in non‐scirrhous type GC by miRNA microarray analysis. Among these, microRNA‐143 (miR‐143) expression was higher in scirrhous type GC than in non‐scirrhous types of GC. In situ hybridization and quantitative RT‐PCR analysis showed that miR‐143 is expressed by stromal fibroblasts but not by cancer cells. In stromal cells, miR‐143 enhanced collagen type III expression in normal gastric fibroblasts and cancer‐associated fibroblasts through activation of transforming growth factor‐β)/SMAD signaling. Furthermore, high miR‐143 expression in GC was associated with worse cancer‐specific mortality (P = 0.0141). Multivariate analysis revealed that miR‐143 was an independent prognostic factor. Treatment of GC cell lines with 5‐aza‐2′‐deoxycytidine restored the expression of miR‐143, and precursor miR‐143 caused the inhibition of cancer cell invasion. These data suggest that miR‐143 regulates fibrosis of scirrhous type GC through induction of collagen expression in stromal fibroblasts and that miR‐143 expression serves as a prognostic marker of GC.     

Tumor‐suppressive microRNA‐145 induces growth arrest by targeting SENP1 in human prostate cancer cells

Prostate cancer (PCa) prevails as the most commonly diagnosed malignancy in men and the third leading cause of cancer‐related deaths in developed countries. One of the distinct characteristics of prostate cancer is overexpression of the small ubiquitin‐like modifier (SUMO)‐specific protease 1 (SENP1), and the upregulation of SENP1 contributes to the malignant progression and cell proliferation of PCa. Previous studies have shown that the expression of microRNA‐145 (miRNA‐145) was extensively deregulated in PCa cell lines and primary clinical prostate cancer samples. Independent target prediction methods have indicated that the 3′‐untranslated region of SENP1 mRNA is a potential target of miR‐145. Here we found that low expression of miR‐145 was correlated with high expression of SENP1 in PCa cell line PC‐3. The transient introduction of miR‐145 caused cell cycle arrest in PC‐3 cells, and the opposite effect was observed when miR‐145 inhibitor was transfected. Further studies revealed that the SENP1 3′‐untranslated region was a regulative target of miR‐145 in vitro. MicroRNA‐145 also suppressed tumor formation in vivo in nude mice. Taken together, miR‐145 plays an important role in tumorigenesis of PCa through interfering SENP1.     

Cluster microRNAs miR‐194 and miR‐215 suppress the tumorigenicity of intestinal tumor organoids

Tumor stem cells with self‐renewal and multipotent capacity play critical roles in the initiation and progression of cancer. Recently, a new 3‐D culture system known as organoid culture has been developed, allowing Lgr5‐positive stem cells to form organoids that resemble the properties of original tissues. Here we established organoids derived from intestinal tumors of Apc^min/+ mice and normal intestinal epithelia of C57BL/6J mice and investigated the roles of microRNA (miRNA) in intestinal tumor organoids. The results of microarray analyses revealed that expression of the cluster miRNAs, miR‐194 and miR‐215 was markedly suppressed in intestinal tumor organoids in comparison with organoids derived from normal intestinal epithelia. Enforced expression of miR‐194 resulted in inhibition of E2f3, a positive regulator of the cell cycle and growth suppression of intestinal tumor organoids. In addition, enforced expression of miR‐215 suppressed the cancer stem cell signature through downregulation of intestinal stem cell markers including Lgr5. These findings indicate that the miRNA cluster including miR‐194 and miR‐215 plays important roles in suppressing the growth and attenuating the stemness of intestinal tumor organoids.     

Impact of novel oncogenic pathways regulated by antitumor miR‐451a in renal cell carcinoma

Recent analyses of our microRNA (miRNA) expression signatures obtained from several types of cancer have provided novel information on their molecular pathology. In renal cell carcinoma (RCC), expression of microRNA‐451a (miR‐451a) was significantly downregulated in patient specimens and low expression of miR‐451a was significantly associated with poor prognosis of RCC patients (P = .00305) based on data in The Cancer Genome Atlas. The aims of the present study were to investigate the antitumor roles of miR‐451a and to identify novel oncogenic networks it regulated in RCC cells. Ectopic expression of miR‐451a significantly inhibited cancer cell migration and invasion by RCC cell lines, suggesting that miR‐451a had antitumor roles. To identify oncogenes regulated by miR‐451a in RCC cells, we analyzed genome‐wide gene expression data and examined information in in silico databases. A total of 16 oncogenes and were found to be possible targets of miR‐451a regulation. Interestingly, high expression of 9 genes (PMM2, CRELD2, CLEC2D, SPC25, BST2, EVL, TBX15, DPYSL3, and NAMPT) was significantly associated with poor prognosis. In this study, we focused on phosphomannomutase 2 (PMM2), which was the most strongly associated with prognosis. Overexpression of PMM2 was detected in clinical specimens and Spearman's rank test indicated a negative correlation between the expression levels of miR‐451a and PMM2 (P = .0409). Knockdown of PMM2 in RCC cells inhibited cancer cell migration and invasion, indicating overexpression of PMM2 could promote malignancy. Analytic strategies based on antitumor miRNAs is an effective tool for identification of novel pathways of cancer.     

miR‐335 inhibited cell proliferation of lung cancer cells by target Tra2β

Accumulating evidence has suggested that the dysregulation of miRNA is an important factor in the pathogenesis of lung cancer. Here, we demonstrate that miR‐335 expression is reduced in non‐small cell lung cancer (NSCLC) tumors relative to non‐cancerous adjacent tissues, while the expression of Tra2β is increased. In addition, clinical data revealed that the increased Tra2β and decreased miR‐335 expression observed in NSCLC cells was associated with poor patient survival rates. In vitro experimentation showed that the overexpression of miR‐335 inhibited the growth, invasion and migration capabilities of A459 lung cancer cells, by targeting Tra2β. In contrast, inhibition of miR‐335 or overexpression of the Tra2β target gene stimulated the growth, invasion and migratory capabilities of A459 lung cancer cells in vitro. Furthermore, overexpression of miR‐335 or inhibition of Tra2β decreased the phosphorylation of Rb‐S780 and Rb‐AKT. Overall, these findings suggest that the downregulation of miR‐335 in A459 lung cancer cells promoted cell proliferation through upregulation of Tra2β, mediated via activation of the AKT/mTOR signaling pathway, and suggest that miR‐335 may have potential as a novel therapeutic target for NSCLC.     

Feline sarcoma‐related protein expression correlates with malignant aggressiveness and poor prognosis in renal cell carcinoma

Feline sarcoma‐related protein (Fer) is a ubiquitously expressed non‐receptor protein tyrosine kinase associated with proliferation in various cancer cells. However, no reports have described the pathological roles and prognostic value of Fer expression in renal cell carcinoma (RCC). We investigated Fer expression in three RCC cell lines (ACHN, Caki‐1, and Caki‐2) and in normal tubule cells (HK‐2) by immunoblotting. Fer expression was highest in ACHN cells, with Caki‐1 showing intermediate levels and Caki‐2 showing low levels, and was undetectable in HK‐2. RNA interference was therefore used to assess the effects of Fer knockdown in ACHN. Knockdown of Fer expression was found to inhibit RCC cell proliferation and colony formation. Immunohistochemical analysis of 131 human RCC tissues (110 conventional, 11 chromophobe, and 10 papillary) investigated relationships between Fer expression and clinicopathological features, including cancer cell proliferation, apoptosis, and prognostic value for survival. In human tissues, Fer expression was significantly higher in cancer cells than in normal tubules. In addition, expression levels correlated with cancer cell proliferation, but not with apoptosis. Multivariate analysis indicated associations of Fer expression with pT stage, tumor grade, and metastasis (P < 0.001). Fer expression was also prognostic for cause‐specific survival according to multivariate analysis (hazard ratio, 3.89; 95% confidence interval, 1.02–14.84, P = 0.047). Fer expression correlates with RCC cell proliferation both in vitro and in vivo, and with tumor progression and survival. This represents useful information for discussing the pathological and clinical significance of Fer in RCC.     

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.     

Downregulation of ULK1 by microRNA‐372 inhibits the survival of human pancreatic adenocarcinoma cells

Dysregulation of microRNA (miRNA) expression in various cancers and their role in cancer progression is well documented. The purpose of this study was to investigate the biological role of miR‐372 in human pancreatic adenocarcinoma (HPAC). We collected 20 pairs of HPAC tissues and adjacent non‐cancerous tissues to detect miR‐372 expression levels. We transfected BXPC‐3 and PANC‐1 cells with miR‐372 inhibitor/mimics to study their effect on cell proliferation, apoptosis, invasion, migration and autophagy. In addition, miR‐372 mimics and a tumor protein UNC51‐like kinase 1 (ULK1) siRNA were co‐transfected into BXPC‐3 and PANC‐1 cells to explore the mechanism of miR‐372 and ULK1 on HPAC tumorigenesis. We found that the expression of miR‐372 was markedly downregulated in HPAC cells compared to adjacent normal tissues. Furthermore, functional assays showed that miR‐372 inhibited cell proliferation, invasion, migration and autophagy in BXPC‐3 and PANC‐1 cells. An inverse correlation between miR‐372 expression and ULK1 expression was observed in HPAC tissues. Downregulation of ULK1 inhibited the overexpression effects of miR‐372, and upregulation of ULK1 reversed the effects of overexpressed miR‐372. Finally, we found that silencing ULK1 or inhibiting autophagy partly rescued the effects of miR‐372 knockdown in HPAC cells, which may explain the influence of miR‐372/ULK1 in HPAC development. Taken together, these results revealed a significant role of the miR‐372/ULK1 axis in suppressing HPAC cell proliferation, migration, invasion and autophagy.     

Regulation of ITGA3 by the anti‐tumor miR‐199 family inhibits cancer cell migration and invasion in head and neck cancer

For patients with head and neck squamous cell carcinoma (HNSCC), survival rates have not improved due to local recurrence and distant metastasis. Current targeted molecular therapies do not substantially benefit HNSCC patients. Therefore, it is necessary to use advanced genomic approaches to elucidate the molecular mechanisms underlying the aggressiveness of HNSCC cells. Analysis of our microRNA (miRNA) expression signature by RNA sequencing showed that the miR‐199 family (miR‐199a‐5p, miR‐199a‐3p, miR‐199b‐5p and miR‐199b‐3p) was significantly reduced in cancer tissues. Ectopic expression of mature miRNA demonstrated that all members of the miR‐199 family inhibited cancer cell migration and invasion by HNSCC cell lines (SAS and HSC3). These findings suggested that both passenger strands and guide strands of miRNA are involved in cancer pathogenesis. In silico database and genome‐wide gene expression analyses revealed that the gene coding for integrin α3 (ITGA3) was regulated by all members of the miR‐199 family in HNSCC cells. Knockdown of ITGA3 significantly inhibited cancer cell migration and invasion by HNSCC cells. Moreover, overexpression of ITGA3 was confirmed in HNSCC specimens, and high expression of ITGA3 predicted poorer survival of the patients (P = 0.0048). Our data revealed that both strands of pre‐miR‐199a (miR‐199a‐5p and miR‐199a‐3p) and pre‐miR‐199b (miR‐199b‐5p and miR‐199b‐3p) acted as anti‐tumor miRNA in HNSCC cells. Importantly, the involvement of passenger strand miRNA in the regulation of cellular processes is a novel concept in RNA research. Novel miRNA‐based approaches for HNSCC can be used to identify potential targets for the development of new therapeutic strategies.     

MicroRNA‐143‐3p inhibits colorectal cancer metastases by targeting ITGA6 and ASAP3

MicroRNAs, which regulate mRNAs, operate through a variety of signaling pathways to participate in the development of colorectal cancer (CRC). In this study, we found that microRNA (miR)‐143‐3p expression was significantly lower in both CRC and liver metastatic CRC tissues from liver compared with normal colonic tissues. Functional assays showed that miR‐143‐3p inhibited CRC cell invasion and migration in vitro. Using a bioinformatics approach, we identified miR‐143‐3p target mRNAs. Among the candidate targets, only the expression of integrin alpha 6 (ITGA6) and ArfGAP with the SH3 domain and ankyrin repeat and PH domain 3 (ASAP3) were significantly reduced by miR‐143‐3p mimics as examined by western blot, and the metastasis potential of CRC cells was attenuated by endogenous ITGA6 and ASAP3 knockdown, determined by migration and invasion assays. Both ITGA6 and ASAP3 were upregulated in CRC tissues compared to normal tissues. Analysis of the relationship between clinicopathological features and ITGA6/ASAP3 protein expression in 200 patients with CRC showed a significant difference in positive ITGA6 expression between the early stage (I + II) and the advanced stage (III + IV), and ASAP3 expression levels positively correlated with metastasis in the lymph nodes. These results indicate that miR‐143‐3p acts as an anti‐oncogene by downregulating ITGA6/ASAP3 protein expression and could offer new insight into potential therapeutic targets for CRC.