MicroRNA‐425‐5p regulates chemoresistance in colorectal cancer cells via regulation of Programmed Cell Death 10

Acquired chemoresistance represents a major obstacle in cancer treatment, the underlying mechanism of which is complex and not well understood. MiR‐425‐5p has been reported to be implicated tumorigenesis in a few cancer types. However, its role in regulating chemoresistance has not been investigated in colorectal cancer (CRC) cells. Microarray analysis was performed in isogenic chemosensitive and chemoresistant HCT116 cell lines to identify differentially expressed miRNAs. miRNA quantitative real‐time PCR was used to detect miR‐425‐5p expression levels between drug resistant and parental cancer cells. MiR‐425‐5p mimic and inhibitor were transfected, followed by CellTiter‐Glo^® assay to examine drug sensitivity in these two cell lines. Western Blot and luciferase assay were performed to investigate the direct target of miR‐425‐5p. Xenograft mouse models were used to examine in vivo function of miR‐425‐5p. Our data showed that expression of miR‐425‐5p was significantly up‐regulated in HCT116‐R compared with parental HCT116 cells. Inhibition of miR‐425‐5p reversed chemoresistance in HCT116‐R cells. Programmed cell death 10 (PDCD10) is the direct target of miR‐425‐5p which is required for the regulatory role of miR‐425‐5p in chemoresistance. MiR‐425‐5p inhibitor sensitized HCT116‐R xenografts to chemo drugs in vivo. Our study demonstrated that miR‐425‐5p regulates chemoresistance of CRC cells by modulating PDCD10 expression level both in vitro and in vivo. MiR‐425‐5p may represent a new therapeutic target for the intervention of CRC.     

Hsa_circ_0128846 promotes tumorigenesis of colorectal cancer by sponging hsa‐miR‐1184 and releasing AJUBA and inactivating Hippo/YAP signalling

Hsa_circ_0128846 was found to be the most significantly up‐regulated circRNA in our bioinformatics analysis. However, the role of hsa_circ_0128846 in colorectal cancer has not been explored. We thus aim to explore the influence and mechanism of hsa_circ_0128846 in colorectal cancer by sponging its downstream miRNA target miR‐1184. We collected 40 colorectal cancer patients’ tumour tissues to analyse the expression of hsa_circ_0128846, miR‐1184 and AJUBA using qRT‐PCR and Western blot where needed. Then, we constructed stably transfected SW480 and HCT116 cells to study the influence of hsa_circ_0128846, miR‐1184 and AJUBA on colorectal cancer cell phenotypes. To obtain reliable results, a plethora of experiments including RNA immunoprecipitation assay, flow cytometry, EdU incorporation assay, wound healing migration assay, transwell invasion assay and live imaging of nude mice xenograft assay were performed. The binding relationship between hsa_circ_0128846, miR‐1184 and AJUBA mRNA in colorectal cancer was validated by reported gene assay. In colorectal cancer tissues, circ_0128846 and AJUBA were both significantly up‐regulated, while miR‐1184 was significantly down‐regulated compared with healthy tissues. Meanwhile, hsa_circ_0128846 can absorb miR‐1184 to promote the progression of CRC in vivo and SW480 and HCT116 cell phenotypes in vitro. The knockdown of AJUBA, a downstream target of miR‐1184, reversed the effect of miR‐1184 in CRC cells via enhancing the phosphorylation of the Hippo/YAP signalling pathway proteins MST1, LATS1 and YAP. This study revealed that hsa_circ_0128846 contributed to the development of CRC by decreasing the expression of miR‐1184, thereby increasing AJUBA expression and inactivating Hippo/YAP signalling.     

miR-301a promotes pancreatic cancer cell proliferation by directly inhibiting bim expression

It is well known that microRNAs (miRNAs) play an important role in many diseases, including tumorigenesis. However, the mechanisms by which miRNAs regulate pancreatic cancer (PC) development remain poorly understood. In the present study, we assayed expression level of miR‐301a in PC tissues by real‐time PCR, and defined the target gene and biological function by luciferase reporter assay and Western blot analysis. We first verified that the expression level of miR‐301a was significantly increased in PC tissues. Moreover, miR‐301a overexpression promoted PC cell proliferation, whereas its depletion decreased cell proliferation. We further demonstrated that miR‐301a directly targeted 3′‐UTR of Bim gene, and inhibited its protein expression in vitro and in vivo. Importantly, Bim re‐expression reduced PC cell proliferation induced by miR‐301a. These data suggest an important role of miR‐301a in the molecular etiology of PC and implicate the potential application of miR‐301a in PC therapy. J. Cell. Biochem. 113: 3229–3235, 2012. © 2012 Wiley Periodicals, Inc.     

Atractylenolide II reverses the influence of lncRNA XIST/miR‐30a‐5p/ROR1 axis on chemo‐resistance of colorectal cancer cells

This investigation was conducted to elucidate whether atractylenolide II could reverse the role of lncRNA XIST/miR‐30a‐5p/ROR1 axis in modulating chemosensitivity of colorectal cancer cells. We totally collected 294 pairs of colorectal cancer tissues and adjacent normal tissues and also purchased colorectal cancer cell lines and human embryonic kidney cell line. 5‐fluorouracil, cisplatin, mitomycin and adriamycin were designated as the chemotherapies for colorectal cell lines, and atractylenolides were arranged as the Chinese drug. The expressions of XIST, miR‐30a‐5p and ROR1 were quantified with aid of qRT‐PCR or Western blot, and luciferase reporter gene assay was implemented to determine the relationships among XIST, miR‐30a‐5p and ROR1. Our results demonstrated that XIST and ROR1 expressions were dramatically up‐regulated, yet miR‐30a‐5p expression was down‐regulated within colorectal cancer tissues (P < 0.05). The overexpressed XIST and ROR1, as well as under‐expressed miR‐30a‐5p, were inclined to promote viability and proliferation of colorectal cells under the influence of chemo drugs (P < 0.05). In addition, XIST could directly target miR‐30a‐5p, and ROR1 acted as the targeted molecule of miR‐30a‐5p. Interestingly, atractylenolides not only switched the expressions of XIST, miR‐30a‐5p and ROR1 within colorectal cancer cells but also significantly intensified the chemosensitivity of colorectal cancer cells (P < 0.05). Finally, atractylenolide II was discovered to slow down the viability and proliferation of colorectal cancer cells (P < 0.05). In conclusion, the XIST/miR‐30a‐5p/ROR1 axis could be deemed as pivotal markers underlying colorectal cancer, and administration of atractylenolide II might improve the chemotherapeutic efficacy for colorectal cancer.     

Long intergenic noncoding RNA 00908 promotes proliferation and inhibits apoptosis of colorectal cancer cells by regulating KLF5 expression

Long intergenic noncoding RNAs (lincRNAs) play a vital role in the occurrence and progression of cancer. The mechanism of lincRNAs in colorectal cancer (CRC) has not been fully elucidated. In this context, an integrated comparative long noncoding RNA (lncRNA) microarray technology was used to determine the expression profile of lncRNAs in CRC. The roles of LINC00908 are unclear. We found that LINC00908 was significantly upregulated in CRC. Inhibition of LINC00908 resulted in reduced cell proliferation and G1 cell cycle arrest, which was mediated by cyclin D1, cyclin‐dependent kinase 4, and phosphorylated retinoblastoma. Moreover, inhibition of LINC00908‐induced apoptosis through the intrinsic apoptosis signaling pathway, as shown by the activation of caspase‐9 and caspase‐3. Mechanistically, miR‐143‐3p directly bound to LINC00908. miR‐143‐3p expression was negatively correlated with LINC00908 expression in CRC tissue. Functional experiments revealed opposing roles for miR‐143‐3p and LINC00908, suggesting that LINC00908 negatively regulates miR‐143‐3p. Mechanistically, miR‐143‐3p directly targets LINC00908. The KLF5 inhibitor ML264 affected proliferation and apoptosis, indicating that LINC00908 may act as a competing endogenous RNA to facilitate the expression of the miR‐143‐3p target gene KLF5. Thus, LINC00908 has an important proliferative and antiapoptotic role in CRC by regulating the cell cycle and intrinsic apoptosis. LINC00908 could be a potential biomarker and a new therapeutic target for CRC.     

Extracellular vesicle‐derived circ_SLC19A1 promotes prostate cancer cell growth and invasion through the miR‐497/septin 2 pathway

The occurrence and development of prostate cancer (PCa) is complex, and the related mechanism is not fully understood. Current studies have found that extracellular vesicles (EVs) and circular RNAs (circRNAs) have important functions in various tumours and other diseases. In this study, the detection of circRNAs in PCa showed that circ_SLC19A1 was increased in PCa cells and their secreted EVs. EVs with high expression of circ_SLC19A1 could be taken up by PCa cells, which promoted cell proliferation and invasion. The sequence of circ_SLC19A1 contained multiple binding sites for miR‐497, and circ_SLC19A1 could bind directly to miR‐497 in cells. The expression of miR‐497 was downregulated in PCa cells, while the expression of its target gene septin 2 (SEPT2) was upregulated significantly. Transfection of circ_SLC19A1 small interfering RNA (siRNA) or miR‐497 mimics could significantly inhibit the expression of SEPT2 and the phosphorylation of extracellular signal‐regulated kinase 1 and 2 (ERK1/2). After co‐transfection of circ_SLC19A1 siRNA and miR‐497 inhibitors or SEPT2 overexpression vector, the expression of SEPT2 and ERK1/2 phosphorylation levels showed no significant changes. Similar results were obtained with co‐transfection of miR‐497 mimics and the SEPT2 overexpression vector. Therefore, cancer cells can regulate the expression of SEPT2 through miR‐497 by secreting EVs with high expression of circ_SLC19A1, thus affecting the activation of the downstream ERK1/2 pathway and ultimately regulating PCa cell growth and invasion. Therefore, EV‐derived circ_SLC19A1 plays an important regulatory role in PCa and may be an important target for PCa prevention and treatment.     

Long non‐coding RNA NNT‐AS1 sponges miR‐424/E2F1 to promote the tumorigenesis and cell cycle progression of gastric cancer

Long non‐coding RNAs (lncRNAs) have been illustrated to function as important regulators in carcinogenesis and cancer progression. However, the roles of lncRNA NNT‐AS1 in gastric cancer remain unclear. In the present study, we investigate the biological role of NNT‐AS1 in gastric cancer tumorigenesis. Results revealed that NNT‐AS1 expression level was significantly up‐regulated in GC tissue and cell lines compared with adjacent normal tissue and normal cell lines. The ectopic overexpression of NNT‐AS1 indicated the poor prognosis of GC patients. In vitro experiments validated that NNT‐AS1 knockdown suppressed the proliferation and invasion ability and induced the GC cell cycle progression arrest at G0/G1 phase. In vivo xenograft assay, NNT‐AS1 silencing decreased the tumour growth of GC cells. Bioinformatics online program predicted that miR‐424 targeted the 3′‐UTR of NNT‐AS1. Luciferase reporter assay, RNA‐immunoprecipitation (RIP) and RNA pull‐down assay validated the molecular binding within NNT‐AS1 and miR‐424, therefore jointly forming the RNA‐induced silencing complex (RISC). Moreover, E2F1 was verified to act as the target gene of NNT‐AS1/miR‐424, indicating the NNT‐AS1/miR‐424/E2F1 axis. In conclusion, our study indicates that NNT‐AS1 sponges miR‐424/E2F1 to facilitate GC tumorigenesis and cycle progress, revealing the oncogenic role of NNT‐AS1 for GC.     

MicroRNA‐760 Inhibits Doxorubicin Resistance in Hepatocellular Carcinoma through Regulating Notch1/Hes1‐PTEN/Akt Signaling Pathway

Accumulating studies have suggested that microRNA‐760 (miR‐760) plays an important role in chemoresistance of various cancer cells. However, whether miR‐760 regulates the chemoresistance of hepatocellular carcinoma (HCC) remains unclear. In this study, we found that miR‐760 was decreased in HCC cell lines, and doxorubicin (Dox) treatment significantly decreased miR‐760 expression in HCC cells. Overexpression of miR‐760 sensitized HCC cells to Dox‐induced cytotoxicity and apoptosis, whereas miR‐760 inhibition showed the opposite effects. Notch1 was predicted as a target gene of miR‐760. miR‐760 negatively regulated Notch1 expression and Notch1/Hes1 signaling. Overexpression of miR‐760 increased PTEN expression and decreased the phosphorylation of Akt. Activation of Notch signaling significantly reversed the inhibitory effect of miR‐760 on Dox‐resistance and abrogated the effect of miR‐760 on the PTEN/Akt signaling pathway in HCC cells. Overall, our results demonstrate that miR‐760 inhibits Dox‐resistance in HCC cells through inhibiting Notch1 and promoting PTEN expression.     

microRNA‐577 suppresses tumor growth and enhances chemosensitivity in colorectal cancer

In this work, we aimed to determine the expression and biological functions of microRNA (miR)‐577 in colorectal cancer (CRC). The results showed that miR‐577 was downregulated in CRC specimens and cell lines. Restoration of miR‐577 significantly suppressed the proliferation and colony formation and induced a G0/G1 cell cycle arrest in CRC cells. 5‐Fluorouracil (5‐FU)‐resistant SW480 cells (SW480/5‐FU) were found to have elevated levels of miR‐577. Ectopic expression of miR‐577 enhanced 5‐FU sensitivity in SW480/5‐FU cells. Heat shock protein 27 (HSP27) was identified as a target gene of miR‐577. Enforced expression of HSP27 reversed the effects of miR‐577 on CRC cell growth and 5‐FU sensitivity. Xenograft tumors derived from miR‐577‐overexpressing SW480 cells exhibited significantly slower growth than control tumors. In conclusion, our results support that miR‐577 acts as a tumor suppressor in CRC likely through targeting HSP27. Therefore, miR‐577 may have therapeutic potential in the treatment of CRC.     

Epigenetic silenced miR‐125a‐5p could be self‐activated through targeting Suv39H1 in gastric cancer

Emerging evidence suggests that microRNAs (miRNAs) serve an important role in tumorigenesis and development. Although the low expression of miR‐125a‐5p in gastric cancer has been reported, the underlying mechanism remains unknown. In the current study, the low expression of miR‐125a‐5p in gastric cancer was verified in paired cancer tissues and adjacent non‐tumour tissues. Furthermore, the GC islands in the miR‐125a‐5p region were hypermethylated in the tumour tissues. And the hypermethylation was negatively correlated with the miR‐125a‐5p expression. Target gene screening showed that the histone methyltransferase Suv39H1 was one of the potential target genes. In vitro studies showed that miR‐125a‐5p could directly suppress the Suv39H1 expression and decrease the H3K9me3 levels. On the other hand, the Suv39H1 could induce demethylation of miR‐125a‐5p, resulting in re‐activation of miR‐125a‐5p. What is more, overexpessing miR‐125a‐5p could also self‐activate the silenced miR‐125a‐5p in gastric cancer cells, which suppressed cell migration, invasion and proliferation in vitro and inhibited cancer progression in vivo. Thus, we uncovered here that the epigenetic silenced miR‐125a‐5p could be self‐activated through targeting Suv39H1 in gastric cancer, suggesting that miR‐125a‐5p might be not only the potential prognostic value as a tumour biomarker but also potential therapeutic targets in gastric cancer.     

CircRNA WHSC1 targets the miR‐646/NPM1 pathway to promote the development of endometrial cancer

Circular RNAs (circRNAs) play important roles in human cancer progression. Their high stability and tissue specificity make circRNAs important molecular targets for clinical diagnosis, treatment and prognosis. However, the functions and molecular mechanisms of circRNA WHSC1 in endometrial cancer are unknown. CircWHSC1 expression in normal endometrial and endometrial cancer tissues was detected using PCR. Overexpression or knockdown of circWHSC1 in endometrial cancer cell lines HEC‐1B or Ishikawa, respectively, cell function experiments were used to detect the impact of circWHSC1 on endometrial cancer cells. A nude mouse xenograft model was used to detect changes in tumorigenesis of HEC‐1B cells after circWHSC1 overexpression. Bioinformatics and dual luciferase reporter gene technology were used to predict and validate the sponging ability of circWHSC1 on microRNAs. Gene expression changes were detected by using Western blotting. CircWHSC1 expression was increased in endometrial cancer tissues. CircWHSC1 overexpression promoted the proliferation, migration and invasion of endometrial cancer cells and decreased apoptosis. CircWHSC1 knockdown had the opposite effect. CircWHSC1 overexpressed nude mice showed increased tumorigenicity. Bioinformatics predicted that circWHSC1 binds to miR‐646, which was confirmed using luciferase reporter gene assays. High expression of miR‐646 could reverse the effect of circWHSC1 on endometrial cancer cells. Western blotting showed increased or decreased levels of nucleophosmin 1 (NPM1), an miR‐646 downstream target, after circWHSC1 overexpression or knockdown, respectively. CircWHSC1 promotes endometrial cancer development through sponging miR‐646 and targeting NPM1.     

KITLG is a novel target of miR‐34c that is associated with the inhibition of growth and invasion in colorectal cancer cells

MiR‐34c is considered a potent tumour suppressor because of its negative regulation of multiple target mRNAs that are critically associated with tumorigenesis and metastasis. In the present study, we demonstrated a novel target of miR‐34c, KITLG, which has been implicated in colorectal cancer (CRC). First, we found a significant negative relationship between miR‐34c and KITLG mRNA expression levels in CRC cell lines, including HT‐29, HCT‐116, SW480 and SW620 CRC cell lines. In silico analysis predicted putative binding sites for miR‐34c in the 3′ untranslated region (3′UTR) of KITLG mRNA. A dual‐luciferase reporter assay further confirmed that KITLG is a direct target of miR‐34c. Then, the cell lines were infected with lentiviruses expressing miR‐34c or a miR‐34c specific inhibitor. Restoration of miR‐34c dramatically reduced the expression of KITLG mRNA and protein, while silencing of endogenous miR‐34c increased the expression of KITLG protein. The miR‐34c‐mediated down‐regulation of KITLG was associated with the suppression on proliferation, cellular transformation, migration and invasion of CRC cells, as well as the promotion on apoptosis. Knockdown of KITLG by its specific siRNA confirmed a critical role of KITLG down‐regulation for the tumour‐suppressive effects of miR‐34c in CRC cells. In conclusion, our results demonstrated that miR‐34c might interfere with KITLG‐related CRC and could be a novel molecular target for CRC patients.     

Human embryonic stem cells and metastatic colorectal cancer cells shared the common endogenous human microRNA‐26b

The increase in proliferation and the lack of differentiation of cancer cells resemble what occur in the embryonic stem cells during physiological process of embryogenesis. There are also striking similarities in the behaviour between the invasive placental cells and invasive cancer cells. In the present study, microarrays were used to analyse the global expression of microRNAs in a human embryonic stem cell line (i.e. HUES‐17) and four colorectal cancer (CRC) cell lines (i.e. LoVo, SW480, HT29 and Caco‐2) with different metastatic potentialities. Only the expression of miR‐26b was significant decreased in HUES‐17s and LoVo cells, compared with other three cell lines (P < 0.01). The quantitative real‐time PCR analysis confirmed the results of the microarray analysis. Overexpression of miR‐26b expression by miR‐26 mimics transfection and led to the significant suppression of the cell growth and the induction of apoptosis in LoVo cells in vitro, and the inhibition of tumour growth in vivo. Moreover, the potential targets of miR‐26b was predicted by using bioinformatics, and then the predicted target genes were further validated by comparing gene expression profiles between LoVo and NCM460 cell lines. Four genes (TAF12, PTP4A1, CHFR and ALS2CR2) with intersection were found to be the targets of miR‐26b. MetaCore network analysis further showed that the regulatory pathways of miR‐26b were significantly associated with the invasiveness and metastasis of CRC cells. These data suggest that miR‐26b might serve as a novel prognostic factor and a potential therapeutic target for CRC.     

MiR‐376a suppresses the proliferation and invasion of non‐small‐cell lung cancer by targeting c‐Myc

It has been reported that miR‐376a is involved in the formation and progression of several types of cancer. However, the expression and function of miR‐376a is still unknown in non‐small cell lung carcinomas (NSCLC). In this study, the expression of miR‐376a in NSCLC tissues and cell lines were examined by real‐time PCR, the effects of miR‐376a on cell proliferation, apoptosis and invasion were evaluated in vitro. Luciferase reporter assay was performed to identify the targets of miR‐376a. The results showed that miR‐376a was significantly downregulated in NSCLC tissues and cell lines. Restoration of miR‐376a in NSCLC cell line A549 significantly inhibited cell proliferation, increased cell apoptosis and suppressed cell invasion, compared with control‐transfected A549 cells. Luciferase reporter assay showed that c‐Myc, an oncogene that regulating cell survival, angiogenesis and metastasis, was a direct target of miR‐376a. Over‐expression of miR‐376a decreased the mRNA and protein levels of c‐Myc in A549 cells. In addition, upregulation of c‐Myc inhibited miR‐376a‐induced inhibition of cell proliferation and invasion in A549 cells. Therefore, our results indicate a tumor suppressor role of miR‐376a in NSCLC by targeting c‐Myc. miR‐376a may be a promising therapeutic target for NSCLC.     

Long non‐coding RNA SNHG15 promotes CDK14 expression via miR‐486 to accelerate non‐small cell lung cancer cells progression and metastasis

Long non‐coding RNAs (lncRNAs) have been validated to play important role in multiple cancers, including non‐small cell lung cancer (NSCLC). In present study, our team investigate the biologic role of SNHG15 in the NSCLC tumorigenesis. LncRNA SNHG15 was significantly upregulated in NSCLC tissue samples and cells, and its overexpression was associated with poor prognosis of NSCLC patients. In vitro, loss‐of‐functional cellular experiments showed that SNHG15 silencing significantly inhibited the proliferation, promoted the apoptosis, and induced the cycle arrest at G0//G1 phase. In vivo, xenograft assay showed that SNHG15 silencing suppressed tumor growth of NSCLC cells. Besides, SNHG15 silencing decreased CDK14 protein expression both in vivo and vitro. Bioinformatics tools and luciferase reporter assay confirmed that miR‐486 both targeted the 3′‐UTR of SNHG15 and CDK14 and was negatively correlated with their expression levels. In summary, our study conclude that the ectopic overexpression of SNHG15 contribute to the NSCLC tumorigenesis by regulating CDK14 protein via sponging miR‐486, providing a novel insight for NSCLC pathogenesis and potential therapeutic strategy for NSCLC patients.     

miR‐516a‐3p inhibits breast cancer cell growth and EMT by blocking the Pygo2/Wnt signalling pathway

miR‐516a‐3p has been reported to play a suppressive role in several types of human tumours. However, the expression level, biological function and fundamental mechanisms of miR‐516a‐3p in breast cancer remain unclear. In the present study, we found that miR‐516a‐3p expression was down‐regulated and Pygopus2 (Pygo2) expression was up‐regulated in human breast cancer tissues and cells. Through analysing the clinicopathological characteristics, we demonstrated that low miR‐516a‐3p expression or positive Pygo2 expression was a predictor of poor prognosis for patients with breast cancer. The results of a dual luciferase reporter assay and Western blot analysis indicated that Pygo2 was a target gene of miR‐516a‐3p. Moreover, overexpression of miR‐516a‐3p inhibited cell growth, migration and invasion as well as epithelial‐mesenchymal transition (EMT) of breast cancer cells, whereas reduced miR‐516a‐3p expression promoted breast cancer cell growth, migration, invasion and EMT. Furthermore, we showed that miR‐516a‐3p suppressed cell proliferation, metastasis and EMT of breast cancer cells by inhibiting Pygo2 expression. We confirmed that miR‐516a‐3p exerted an anti‐tumour effect by inhibiting the activation of the Wnt/β‐catenin pathway. Finally, xenograft tumour models were used to show that miR‐516a‐3p inhibited breast cancer cell growth and EMT via suppressing the Pygo2/Wnt signalling pathway. Taken together, these results show that miR‐516a‐3p inhibits breast cancer cell growth, metastasis and EMT by blocking the Pygo2/ Wnt/β‐catenin pathway.