Long non-coding RNA OIP5-AS1 promotes pancreatic cancer cell growth through sponging miR-342-3p via AKT/ERK signaling pathway

Opa-interacting protein 5 antisense RNA 1 (OIP5-AS1), a long non-coding RNA (lncRNA), has been reported to link with the progression of some cancers. However, its biological functions and underlying molecular mechanisms in pancreatic cancer are largely unknown. The aim of this study was to investigate the role of lncRNA OIP5-AS1 in pancreatic cancer. Quantitative real-time PCR analysis revealed that OIP5-AS1 is highly expressed in pancreatic cancer tissues versus adjacent non-tumor tissues. In vitro functional assays showed that downregulation of OIP5-AS1 or overexpression of miR-342-3p inhibited the proliferation, decreased Ki67 expression, and induced cell cycle arrest in pancreatic cancer cells. The expression of cyclinD1, CDK4, and CDK6 was decreased by knockdown of OIP5-AS1. Moreover, we found that OIP5-AS1 acted as a miR-342-3p sponge to suppress its expression and function. Dual-luciferase assay confirmed the interaction of OIP5-AS1 and miR-342-3p and verified anterior gradient 2 (AGR2) as a direct target of miR-342-3p. Results showed that depletion of miR-342-3p abolished the inhibitory effects of OIP5-AS1 knockdown on pancreatic cancer cell growth. The expression of Ki67, AGR2, cyclinD1, CDK4, CDK6, p-AKT, and p-ERK1/2 was reversed by silencing of miR-342-3p in pancreatic cancer cells with OIP5-AS1 knockdown. Further, knockdown of OIP5-AS1 suppressed tumor growth in a xenograft mouse model of pancreatic cancer. OIP5-AS1 induced pancreatic cancer progression via activation of AKT and ERK signaling pathways. Therefore, we demonstrate that OIP5-AS1 functions as oncogene in pancreatic cancer and its downregulation inhibits pancreatic cancer growth by sponging miR-342-3p via targeting AGR2 through inhibiting AKT/ERK signaling pathway.

     

DDX17 induces epithelial-mesenchymal transition and metastasis through the miR-149-3p/CYBRD1 pathway in colorectal cancer

DEAD box helicase 17 (DDX17) has been reported to be involved in the initiation and development of several cancers. However, the functional role and mechanisms of DDX17 in colorectal cancer (CRC) malignant progression and metastasis remain unclear. Here, we reported that DDX17 expression was increased in CRC tissues compared with noncancerous mucosa tissues and further upregulated in CRC liver metastasis compared with patient-paired primary tumors. High levels of DDX17 were significantly correlated with aggressive phenotypes and worse clinical outcomes in CRC patients. Ectopic expression of DDX17 promoted cell migration and invasion in vitro and in vivo, while the opposite results were obtained in DDX17-deficient CRC cells. We identified miR-149-3p as a potential downstream miRNA of DDX17 through RNA sequencing analysis, and miR-149-3p displayed a suppressive effect on the metastatic potential of CRC cells. We demonstrated that CYBRD1 (a ferric reductase that contributes to dietary iron absorption) was a direct target of miR-149-3p and that miR-149-3p was required for DDX17-mediated regulation of CYBRD1 expression. Moreover, DDX17 contributed to the metastasis and epithelial to mesenchymal transition (EMT) of CRC cells via downregulation of miR-149-3p, which resulted in increased CYBRD1 expression. In conclusion, our findings not only highlight the significance of DDX17 in the aggressive development and prognosis of CRC patients, but also reveal a novel mechanism underlying DDX17-mediated CRC cell metastasis and EMT progression through manipulation of the miR-149-3p/CYBRD1 pathway.Subject terms: Cell migration, Metastasis     

Circ-ERBB2 knockdown sensitized colorectal cancer cells to 5-FU via miR-181a-5p/PTEN/Akt pathway

Colorectal cancer (CRC) is the fourth most deadly cancer worldwide, drug resistance impedes treatment of CRC. It is still urgent to find new molecular targets to improve the sensitivity of chemotherapeutic drugs. In this study, circ-ERBB2 was upregulated in CRC cells. Upregulation of circ-ERBB2 promoted CRC cells proliferation and clone formation, but inhibited apoptosis. We identified miR-181a-5p as circ-ERBB2's target. The effect of miR-181a-5p on CRC cells was contrary to circ-ERBB2, miR-181a-5p downregulation abolished the function of circ-ERBB2 silencing in CRC cells. In addition, phosphatase and tensin homolog (PTEN) was verified as miR-181a-5p's downstream target, circ-ERBB2 activates the Akt pathway and inhibits cell apoptosis through modulating miR-181a-5p/PTEN. Circ-ERBB2 silencing significantly reduced CRC cell resistance to 5-FU. miR-181a-5p downregulation abolished the role of circ-ERBB2 knockdown in CRC cell resistance to 5-FU. In conclusion, upregulation of circ-ERBB2 promoted the malignancy of CRC and reduced CRC cell resistance to 5-FU. Besides, additional mechanism study provided a novel regulatory pathways that circ-ERBB2 knockdown promoted CRC cell sensitivity to 5-FU by regulating miR-181a-5p/PTEN/Akt pathway. This research indicated that circ-ERBB2 may be a valuable biomarker for the diagnosis and treatment of CRC.     

RFC2, a direct target of miR-744, modulates the cell cycle and promotes the proliferation of CRC cells

Colorectal cancer (CRC) is a common digestive tract malignancy, which is characterized by high mortality, morbidity, and poor prognosis. Replication factor C subunit 2 (RFC2), one RFC family member, was reported to be related to various malignancies and plays an important role in proliferation, invasion, and metastasis. Nonetheless, the RFC2 biological role within CRC is still unknown. RFC2 expression profiles in CRC tissues were collected based on The Cancer Genome Atlas database, whereas miR-744 and RFC2 expression levels were detected in human CRC tissues. miR-744 and RFC2 effects on the proliferation of CRC were assessed both in vivo and in vitro. RFC2 was recognized to be a direct miR-744 target through luciferase reporter assay. RFC2 upregulation was observed within CRC tissues, and a high RFC2 level showed a correlation with poor clinicopathological symptoms. RFC2 knockdown inhibited CRC cell proliferation through promoting cell cycle arrest at the G1 phase, which was achieved by cyclin E2 (CCNE2) downregulation in vivo and in vitro. miR-744 was identified to be the tumor suppressor microRNA, which targeted RFC2 directly for inhibiting the proliferation of CRC cells both in vivo and in vitro. miR-744 downregulation was detected within CRC tissue, and messenger RNA expression showed a negative correlation with RFC2 expression within CRC tissues. Our study demonstrates that the miR-744/RFC2/CCNE2 axis potentially provides a candidate for a treatment strategy for CRC.     

LncRNA CASC11 promoted gastric cancer cell proliferation,migration and invasion in vitro by regulating cell cycle pathway

In this study, we aimed to investigate the effects of lncRNA CASC11 on gastric cancer (GC) cell progression through regulating miR-340-5p and cell cycle pathway. Expressions of lncRNA CASC11 in gastric cancer tissues and cell lines were determined by qRT-PCR. Differentially expressed lncRNAs, mRNAs and miRNAs were screened through microarray analysis. The relationship among CASC11, CDK1 and miR-340-5p was predicted by TargetScan and validated through dual luciferase reporter assay. Western blot assay examined the protein level of CDK1 and several cell cycle regulatory proteins. GO functional analysis and KEGG pathway analysis were used to predict the association between functions and related pathways. Cell proliferation was determined by CCK-8 assays. Cell apoptosis and cell cycle were detected by flow cytometry assay. CASC11 was highly expressed in GC tissues and cell lines. Knockdown of CASC11 inhibited GC cell proliferation, promoted cell apoptosis and blocked cell cycle. KEGG further indicated an enriched cell cycle pathway involving CDK1. QRT-PCR showed that miR-340-5p was down-regulated in GC cells tissues, while CDK1 was up-regulated. Furthermore, CASC11 acted as a sponge of miR-340-5p which directly targeted CDK1. Meanwhile, miR-340-5p overexpression promoted GC cell apoptosis and induced cell cycle arrest, while CDK1 overexpression inhibited cell apoptosis and accelerated cell cycle. Our study revealed the mechanism of CASC11/miR-340-5p/CDK1 network in GC cell line, and suggested that CASC11 was a novel facilitator that exerted a biological effect by activating the cell cycle signaling pathway. This finding provides a potential therapeutic target for GC.     

Knockdown of CDK2AP1 in Primary Human Fibroblasts Induces p53 Dependent Senescence

Cyclin Dependent Kinase-2 Associated Protein-1 (CDK2AP1) is known to be a tumor suppressor that plays a role in cell cycle regulation by sequestering monomeric CDK2, and targeting it for proteolysis. A reduction of CDK2AP1 expression is considered to be a negative prognostic indicator in patients with oral squamous cell carcinoma and also associated with increased invasion in human gastric cancer tissue. CDK2AP1 overexpression was shown to inhibit growth, reduce invasion and increase apoptosis in prostate cancer cell lines. In this study, we investigated the effect of CDK2AP1 downregulation in primary human dermal fibroblasts. Using a short-hairpin RNA to reduce its expression, we found that knockdown of CDK2AP1in primary human fibroblasts resulted in reduced proliferation and in the induction of senescence associated beta-galactosidase activity. CDK2AP1 knockdown also resulted in a significant reduction in the percentage of cells in the S phase and an accumulation of cells in the G1 phase of the cell cycle. Immunocytochemical analysis also revealed that the CDK2AP1 knockdown significantly increased the percentage of cells that exhibited γ-H2AX foci, which could indicate presence of DNA damage. CDK2AP1 knockdown also resulted in increased mRNA levels of p53, p21, BAX and PUMA and p53 protein levels. In primary human fibroblasts in which p53 and CDK2AP1 were simultaneously downregulated, there was: (a) no increase in senescence associated beta-galactosidase activity, (b) decrease in the number of cells in the G1-phase and increase in number of cells in the S-phase of the cell cycle, and (c) decrease in the mRNA levels of p21, BAX and PUMA when compared with CDK2AP1 knockdown only fibroblasts. Taken together, this suggests that the observed phenotype is p53 dependent. We also observed a prominent increase in the levels of ARF protein in the CDK2AP1 knockdown cells, which suggests a possible role of ARF in p53 stabilization following CDK2AP1 knockdown. Altogether, our results show that knockdown of CDK2AP1 in primary human fibroblasts reduced proliferation and induced premature senescence, with the observed phenotype being p53 dependent.     

Retracted: Long noncoding RNA HAGLROS regulates apoptosis and autophagy in colorectal cancer cells via sponging miR-100 to target ATG5 expression

The aim of this study was to explore the relationship between the expression of HOXD antisense growth-associated long noncoding RNA (HAGLROS) and prognosis of patients with colorectal cancer (CRC), as well as the roles and regulatory mechanism of HAGLROS in CRC development. The HAGLROS expression in CRC tissues and cells was detected. The correlation between HAGLROS expression and survival time of CRC patients was investigated. Moreover, HAGLROS was overexpressed and suppressed in HCT-116 cells, followed by detection of cell viability, apoptosis, and the expression of apoptosis-related proteins and autophagy markers. Furthermore, the association between HAGLROS and miR-100 and the potential targets of miR-100 were investigated. Besides, the regulatory relationship between HAGLROS and PI3K/AKT/mTOR pathway was elucidated. The results showed that HAGLROS was highly expressed in CRC tissues and cells. Highly expression of HAGLROS correlated with a shorter survival time of CRC patients. Moreover, knockdown of HAGLROS in HCT-116 cells induced apoptosis by increasing the expression of Bax/Bcl-2 ratio, cleaved-caspase-3, and cleaved-caspase-9, and inhibited autophagy by decreasing the expression of LC3II/LC3I and Beclin-1 and increasing P62 expression. Furthermore, HAGLROS negatively regulated the expression of miR-100, and HAGLROS controlled HCT-116 cell apoptosis and autophagy through negatively regulation of miR-100. Autophagy related 5 (ATG5) was verified as a functional target of miR-100 and miR-100 regulated HCT-116 cell apoptosis and autophagy through targeting ATG5. Besides, HAGLROS overexpression activated phosphatidylinositol-3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/Akt/mTOR) pathway. In conclusion, a highly expression of HAGLROS correlated with shorter survival time of CRC patients. Downregulation of HAGLROS may induce apoptosis and inhibit autophagy in CRC cells by regulation of miR-100/ATG5 axis and PI3K/AKT/mTOR pathway.     

CDK11p110 plays a critical role in the tumorigenicity of esophageal squamous cell carcinoma cells and is a potential drug target

Esophageal squamous cell carcinoma (ESCC) is a serious malignancy with limited options for targeted therapy. The exploration of novel targeted therapies for combating ESCC is urgently needed. Cyclin-dependent kinases (CDKs) play important roles in the progression of cancers; however, the function of CDK11^p110 (cyclin-dependent kinase 11^p110) in ESCC is still unknown. Here, we investigated the effects and molecular mechanisms of CDK11^p110 in the proliferation and growth of ESCC by examining the expression of CDK11^p110 in ESCC tissues and by detecting phenotypic changes in ESCC cells after CDK11^p110 knockdown or overexpression in vitro and in vivo. According to the tissue microarray analysis, compared with its expression level in normal tissues, the expression level of CDK11^p110 was significantly elevated in ESCC tissues; this result was in concordance with the data in TCGA (The Cancer Genome Atlas) datasets. In addition, RNAi-mediated CDK11^p110 silencing exerted a substantial inhibitory effect on the proliferation, clonogenicity and migration ability of ESCC cells. Further study indicated that CDK11^p110 knockdown arrested ESCC cells in the G2/M phase of the cell cycle and induced cell apoptosis. Moreover, stable shRNA-mediated CDK11^p110 knockdown inhibited tumor growth in an ESCC xenograft model, and overexpression of CDK11^p110 enhanced tumor growth. In addition, the Ki67 proliferation index was closely associated with the elevation or depletion of CDK11^p110 in vivo. In summary, this study provides evidence that CDK11^p110 play a critical role in the tumorigenicity of ESCC cells, which suggests that CDK11^p110 may be a promising therapeutic target in ESCC.

Abbreviations: CDKs: cyclin-dependent kinases; CDK11: Cyclin-dependent kinase 11; CDK11^p110: Cyclin-dependent kinase 11^p110, the larger isomer of cyclin-dependent kinase 11; ESCC: esophageal squamous cell carcinoma; FACS: fluorescence-activated cell sorting; FDA: the Food and Drug Administration; TCGA: The Cancer Genome Atlas; TMA: tissue microarray.     

IKBIP,a novel glioblastoma biomarker,maintains abnormal proliferation of tumor cells by inhibiting the ubiquitination and degradation of CDK4

Sustained proliferative signaling is a crucial hallmark and therapeutic target in glioblastoma (GBM); however, new intrinsic regulators and their underlying mechanisms remain to be elucidated. In this study, I kappa B kinase interacting protein (IKBIP) was identified to be correlated with the progression of GBM by analysis of The Cancer Genome Atlas (TCGA) data. TCGA database analysis indicated that higher IKBIP expression was associated with high tumor grade and poor prognosis in GBM patients, and these correlations were subsequently validated in clinical samples. IKBIP knockdown induced G1/S arrest by blocking the Cyclin D1/CDK4/CDK6/CDK2 pathway. Our results showed that IKBIP may bind directly to CDK4, a key cell cycle checkpoint protein, and prevent its ubiquitination-mediated degradation in GBM cells. An in vivo study confirmed that IKBIP knockdown strongly suppressed cell proliferation and tumor growth and prolonged survival in a mouse xenograft model established with human GBM cells. In conclusion, IKBIP functions as a novel driver of GBM by binding and stabilizing the CDK4 protein. IKBIP could be a potential therapeutic target in GBM.     

Downregulation of beta1,4-galactosyltransferase 1 inhibits CDK11(p58)-mediated apoptosis induced by cycloheximide

Cyclin-dependent kinase 11 (CDK11; also named PITSLRE) is part of the large family of p34(cdc2)-related kinases whose functions appear to be linked with cell cycle progression, tumorigenesis, and apoptotic signaling. The mechanism that CDK11(p58) induces apoptosis is not clear. Some evidences suggested beta1,4-galactosyltransferase 1 (beta1,4-GT 1) might participate in apoptosis induced by CDK11(p58). In this study, we demonstrated that ectopically expressed beta1,4-GT 1 increased CDK11(p58)-mediated apoptosis induced by cycloheximide (CHX). In contrast, RNAi-mediated knockdown of beta1,4-GT 1 effectively inhibited apoptosis induced by CHX in CDK11(p58)-overexpressing cells. For example, the cell morphological and nuclear changes were reduced; the loss of cell viability was prevented and the number of cells in sub-G1 phase was decreased. Knock down of beta1,4-GT 1 also inhibited the release of cytochrome c from mitochondria and caspase-3 processing. Therefore, the cleavage of CDK11(p58) by caspase-3 was reduced. We proposed that beta1,4-GT 1 might contribute to the pro-apoptotic effect of CDK11(p58). This may represent a new mechanism of beta1,4-GT 1 in CHX-induced apoptosis of CDK11(p58)-overexpressing cells.     

Ran GTPase protein promotes human pancreatic cancer proliferation by deregulating the expression of Survivin and cell cycle proteins

Ran, a member of the Ras GTPase family, has important roles in nucleocytoplasmic transport. Herein, we detected Ran expression in pancreatic cancer and explored its potential role on tumour progression. Overexpressed Ran in pancreatic cancer tissues was found highly correlated with the histological grade. Downregulation of Ran led to significant suppression of cell proliferation, cell cycle arrest at the G1/S phase and induction of apoptosis. In vivo studies also validated that result. Further studies revealed that those effects were at least partly mediated by the downregulation of Cyclin A, Cyclin D1, Cyclin E, CDK2, CDK4, phospho-Rb and Survivin proteins and up regulation of cleaved Caspase-3.     

Oncogenic retinoic acid receptor γ knockdown reverses multi-drug resistance of human colorectal cancer via Wnt/β-catenin pathway

Retinoic acid receptor γ (RARγ), a unique member of the nuclear receptor superfamily, plays an important role in the progression of several cancers such as hepatocellular carcinoma, esophageal cancer, and cholangiocarcinoma. However, little is known about the regulatory mechanism of the RARγ expression in colorectal cancer (CRC) progression. In the present study, we found that RARγ was frequently overexpressed in human CRC specimens and CRC cell lines, and it mainly resided in the cytoplasm in CRC specimens. Tissue microarrays showed that RARγ indicated vital clinical significance in CRC. RARγ knockdown neither affected CRC cell proliferation nor blocked the cell cycle of CRC cells. However, RARγ knockdown increased the sensitivity of CRC cells to chemotherapeutics through downregulation of multi-drug resistance 1(MDR1). Further studies suggested that RARγ knockdown resulted in downregulation of MDR1, in parallel with suppression of the Wnt/β-catenin pathway. Moreover, a significantly positive association between RARγ and MDR1 was demonstrated in CRC tissue microarrays. Collectively, these results suggested that overexpression of RARγ contributed to the multidrug chemoresistance of CRC cells, at least in part due to upregulation of MDR1 via activation of the Wnt/β-catenin pathway, indicating that RARγ might serve as a potential therapeutic target for chemoresistant CRC patients.     

Phospholipase C delta 1 regulates cell proliferation and cell-cycle progression from G1- to S-phase by control of cyclin E-CDK2 activity

In the present study, we examined the role of PLC delta 1 (phospholipase C delta 1) in the regulation of cellular proliferation. We demonstrate that RNAi (RNA interference)-mediated knockdown of endogenous PLC delta 1, but not PLC beta 3 or PLC epsilon, induces a proliferation defect in Rat-1 and NIH 3T3 fibroblasts. The decreased proliferation was not due to an induction of apoptosis or senescence, but was associated with an approx. 60% inhibition of [(3)H]thymidine incorporation. Analysis of the cell cycle with BrdU (bromodeoxyuridine)/propidium iodide-labelled FACS (fluorescence-activated cell sorting) demonstrated an accumulation of cells in G(0)/G(1)-phase and a corresponding decrease in cells in S-phase. Further examination of the cell cycle after synchronization by serum-starvation demonstrated normal movement through G(1)-phase but delayed entry into S-phase. Consistent with these findings, G(1) cyclin (D2 and D3) and CDK4 (cyclin-dependent kinase 4) levels and associated kinase activity were not affected. However, cyclin E-associated CDK2 activity, responsible for G(1)-to-S-phase progression, was inhibited. This decreased activity was accompanied by unchanged CDK2 protein levels and paradoxically elevated cyclin E and cyclin E-associated CDK2 levels, suggesting inhibition of the cyclin E-CDK2 complex. This inhibition was not due to altered stimulatory or inhibitory phosphorylation of CDK2. However, p27, a Cip/Kip family CKI (CDK inhibitor)-binding partner, was elevated and showed increased association with CDK2 in PLC delta 1-knockdown cells. The result of the present study demonstrate a novel and critical role for PLC delta 1 in cell-cycle progression from G(1)-to-S-phase through regulation of cyclin E-CDK2 activity and p27 levels.     

PER3, a novel target of miR-103, plays a suppressive role in colorectal cancer in vitro

Colorectal cancer has become the third most common cancer and leads to high mortality worldwide. Although colorectal cancer has been studied widely, the underlying molecular mechanism remains unclear. PER3 is related to tumor differentiation and the progression of colorectal cancer. High expression of miR-103 is associated with poor prognosis in patients with colorectal cancer. However, the relationship between miR-103 and PER3 in CRC cells remains unclear. In this study, we found that PER3 was downregulated in CRC tissues and CRC cell lines, whereas miR-103 was upregulated in CRC cell lines. We also found that PER3 promoted CRC cells apoptosis. These results indicate that PER3 plays a suppressive role in CRC cells. Moreover, we found that PER3 was targeted, at least partially, by miR-103. Taken together, we provide evidence to characterize the role of PER3 in CRC, which may be a new therapeutic target for CRC. [BMB Reports 2014;47(9): 500-505]