MiR-23a-3p promoted G1/S cell cycle transition by targeting protocadherin17 in hepatocellular carcinoma

MiR-23a-3p has been shown to promote liver cancer cell growth and metastasis and regulate that of chemosensitivity. Protocadherin17 (PCDH17) is a tumor suppressor gene and plays an essential part in cell cycle of hepatocellular carcinoma (HCC). This study aimed at evaluating the effects of miR-23a-3p and PCDH17 on HCC cell cycle and underlining the mechanism. The level of miR-23a-3p was up-regulated, while PCDH17 level was down-regulated in HCC tissues compared to adjacent tissues. For the in vitro studies, high expression of miR-23a-3p down-regulated PCDH17 level; increased cell viability; promoted G1/S cell cycle transition; up-regulated cyclin D1, cyclin E, CDK2, CDK4, p-p27, and p-RB levels; and down-regulated the expression of p27. Dual luciferase reporter assay suggested PCDH17 was a target gene of miR-23a-3p. MiR-23a-3p inhibitor and PCDH17 siRNA led to an increase in cell viability and the number of cells in the S phase and up-regulated cyclin D1 and cyclin E levels, compared with miR-23a-3p inhibitor and NC siRNA group. For the in vivo experiments, high expression of miR-23a-3p promoted tumor growth and reduced PCDH17 level in the cytoplasm. These results indicated that high expression of miR-23a-3p might promote G1/S cell cycle transition by targeting PCDH17 in HCC cells. The miR-23a-3p could be considered as a molecular target for HCC detection.

     

miR-30a attenuates drug sensitivity to 5-FU by modulating cell proliferation possibly by downregulating cyclin E2 in oral squamous cell carcinoma

We aimed to determine the functional role of the miRNA, which affects drug sensitivity to 5-FU in oral squamous cell carcinoma (OSCC), using two types of 5-FU-resistant and parental OSCC cell lines. MiRNA microarray data showed that miR-30a was significantly upregulated in two resistant cell lines. Therefore, we investigated the effects and molecular mechanism of miR-30a on 5-FU sensitivity. Stable overexpression of miR-30a in parental OSCC cells decreased cell proliferation and attenuated drug sensitivity to 5-FU. Cell cycle analysis indicated that miR-30a overexpression increased the proportion of G1 phase cells and decreased the proportion of S phase cells. MiR-30a knockdown using siRNA reversed the effects of miR-30a overexpression. DNA microarray analysis using miR-30a-overexpressing cell lines and a TargetScan database search showed that cyclin E2 (CCNE2) is a target of miR-30a. A luciferase reporter assay confirmed that a miR-30a mimic interacted with the specific binding site in the 3' UTR of CCNE2. CCNE2 knockdown with siRNA in OSCC cells yielded decreased drug sensitivity to 5-FU, similar to miR-30a overexpressing cells. These findings suggest that miR-30a in OSCC may be a novel biomarker of 5-FU-resistant tumors, as well as a therapeutic target for combating resistance.     

Overexpressed MicroRNA-182 Promotes Proliferation and Invasion in Prostate Cancer PC-3 Cells by Down-Regulating N-myc Downstream Regulated Gene 1 (NDRG1)

MicroRNAs, non-coding 20–22 nucleotide single-stranded RNAs, result in translational repression or degradation and gene silencing of their target genes, and significantly contribute to the regulation of gene expression. In the current study, we report that miR-182 expression was significantly upregulated in prostate cancer tissues and four cell lines, compared to benign prostatic hyperplasia tissues and normal prostatic epithelial (RWPE-1) cells. Ectopic overexpression of miR-182 significantly promotes the proliferation, increases the invasion, promotes the G1/S cell cycle transition and reduces early apotosis of PC-3 cells, while suppression of miR-182 decreased the proliferation and invasion, inhibits the G1/S cell cycle transition and increase early apotosis of PC-3 cells. Additionally, we demonstrated that miR-182 could downregulate expression of NDRG1 by directly targeting the NDRG1 3′-untranslated region. In conclusion, our results suggest that miR-182 plays an important role in the proliferation of human prostate cancer cells by directly suppressing the tumor supressor gene NDRG1. We uncovered a new epigenetic regulation of NDRG1.     

Tumour-suppressive microRNA-424-5p directly targets CCNE1 as potential prognostic markers in epithelial ovarian cancer

An accumulated evidence supports that MicroRNAs (miRNAs) have shown a prominent role in pathological processes and different tumor onset. However, to date, the potential functional roles and molecular mechanisms by how microRNA-424-5p(miR-424-5p) affects cancer cell proliferation are greatly unclear, especially in epithelial ovarian cancer(EOC).In this study, we demonstrated that miR-424-5p was significantly down-regulated in EOC tissues and cell lines. The level of miR-424-5p was negatively correlated with tumor size, TNM stage, pathological grade, lymphatic metastasis of EOC. Restoring miR-424-5p expression in EOC cells dramatically suppressed cell proliferation and caused an accumulation of cells in G1 phase, and thus contributed to better prognosis of EOC patients. Mechanistically, miR-424-5p inhibits CCNE1 expression through targeting CCNE1 3′UTR, and subsequent arrest cell cycle in G1/G0 phase by inhibiting E2F1-pRb pathway. This study revealed functional and mechanistic links between miR-424-5p and CCNE1 in the progression of EOC and provide an important insight into that miR-424-5p may serve as a therapeutic target in EOC.     

miR-9 inhibits the metastatic ability of hepatocellular carcinoma via targeting beta galactoside alpha-2,6-sialyltransferase 1

Glycosylation of cell surface proteins regulates critical cellular functions, including invasion and metastasis in cancer cells. Emerging evidence has shown that microRNAs (miRNAs) are involved in regulating both the glycosylation modifications on cell surface and the progression of cancer. In this study, we investigated the role of miR-9 in α-2,6-linked sialylation and the metastasis of mouse hepatocellular carcinoma (HCC). According to array-based miRNA expression profiling data of HCC cell lines Hepa1–6, Hca-P, and Hca-F with different lymphatic metastatic capacities, reverse correlation was found between miR-9 expression levels and the metastatic potential in these HCC cells. Additionally, β-galactoside α-2,6-sialyltransferase 1 (St6gal1) expression level is associated negatively with miR-9 and positively with metastatic potential. Bioinformatics analysis indicated that miR-9 could target St6gal1, which was verified by luciferase reporter assays. miR-9 overexpression reduced expression of St6gal1, which subsequently suppressed HCC cells metastatic potential. Moreover, upregulation of miR-9 could inhibit integrin-β1/FAK-mediated cell motility and migration signaling in mouse HCC cells. Together, our results suggest that miR-9 could act as a tumor suppressor and regulate mouse HCC cells migration and invasion by inhibiting the α-2,6-linked sialylation. This finding may provide insight into the relationship between abnormal miRNA expression and aberrant cell surface glycosylation during tumor lymphatic metastasis.     

miR-214-5p targets KLF5 and suppresses proliferation of human hepatocellular carcinoma cells

MicroRNAs (miRNAs) are small endogenous conserved RNAs regulating genes expression through base pairing with the 3′-untranslated region (3′-UTR) of target messenger RNAs. MiR-214-5p is a newly identified miRNA with its biological role largely unknown. In this study, we explored miR-214-5p expression status in 78 paired tumor and nontumor tissues obtained from patients with hepatocellular carcinoma (HCC) by RT-qPCR. The effects of miR-214-5p expression on HCC cell proliferation, cell cycle progression, and cell migration were measured by CCK-8 assay, flow cytometry, and wound-healing assay. A dual-luciferase activity assay was performed to identify whether KLF5 was a target of miR-214-5p. Kaplan-Meier curve and log-rank test were used to investigate the effects of miR-214-5p and KLF5 on overall survival and disease-free survival of patients with HCC. We found miR-214-5p expression was sharply reduced in HCC tissues and cell lines compared with the normal tissues and cell lines. Functional assay revealed that miR-214-5p overexpression could downregulate cell proliferation, cell migration, and arrested cell cycle at G0/G1 phase. Further, we validated Krüppel-like factor 5 (KLF5) as a direct target of miR-214-5p, and was upregulated in HCC and inversely correlated with the expression of miR-214-5p. Moreover, we found the low expression of miR-214-5p and high expression of KLF5 were correlated with tumor size, tumor stage, and poorer 5-year overall survival and disease-free survival of patients with HCC. In conclusion, our results suggested miR-214-5p functions as a tumor suppressor through targeting KLF5 in HCC. Also, miR-214-5p and KLF5 were identified as potential prognostic markers and might be therapeutic targets in HCC.     

MiR-15b regulates cell differentiation and survival by targeting CCNE1 in APL cell lines

MicroRNAs have been shown to be involved in various cell processes, including proliferation, apoptosis and differentiation. However, little is known about their function in granulopoiesis. In the present study, overexpression and knockdown experiments revealed that miR-15b was required to block the proliferation of NB4 and HL60 cells and induce them differentiated to granulocyte lineage. Moreover, we identified CCNE1 as a direct target of miR-15b, and demonstrated that CCNE1 was involved in cell differentiation and proliferation in acute promyelocytic leukemia cells. In addition, we demonstrated a novel pathway in which miR-15b regulated cells arrested in the G0/G1 phase and promoted terminal differentiation of cells by targeting CCNE1, which could modulate the cell cycle effort pRb in APL cells. These events blocked cell proliferation and promoted granulocyte differentiation. In conclusion, our data highlighted, for the first time, the important role of miR-15b in myeloid differentiation and suggested the potential role of miR-15b in cancer therapy.     

MARVELD1调控内含子miR-186、miR-335表达的研究

目的:前期研究发现,MARVELD1(具有囊泡运输和膜连接功能的MAL及相关蛋白1)在多种肿瘤细胞中表达下调,许多micro RNAs也随其发生变化。本研究探讨了MARVELD1调控mi R-186和mi R-335的表达方式。方法:本研究选取了多种肺癌细胞系,运用q RT-PCR的方法检测了MARVELD1以及mi R-186和mi R-335的表达情况,通过MARVELD1过表达体系和si RNA干扰MARVELD1表达的体系分析mi R-186和mi R-335表达变化情况,运用生物信息学网站对mi R-186和mi R-335进行分析,确认其是否为内含子mi RNA,并进一步应用MARVELD1过表达和RNAi体系检测MARVELD1对mi R-186和mi R-335的宿主基因的影响。结果:在肺癌细胞中,MARVELD1与mi R-186、mi R-335的表达呈现明显的正相关。在过表达MARVELD1之后,mi R-186、mi R-335出现高表达;当下调MARVELD1表达时,mi R-186、mi R-335则表现低表达。生物信息学分析发现mi R-186和mi R-335均为内含子mi RNA。进一步分析MARVELD1与mi R-186和mi R-335宿主基因的表达关系显示,MARVELD1可以上调它们的宿主基因的表达。结论:上述结果表明,MARVELD1可通过影响内含子mi R-186和mi R-335的宿主基因进而调控二者的表达,为进一步研究MARVELD1影响肿瘤细胞的发生机制奠定了基础。     

Ectopic expression of microRNA-874 represses epithelial mesenchymal transition through the NF-κB pathway via CCNE1 in cholangiocarcinoma

Cholangiocarcinoma (CC) is a devastating disease associated with poor survival rate. microRNAs (miRNAs) have recently been reported to assume a great role in CC development. This research aims to explore the functions of miR-874 in regulating epithelial mesenchymal transition (EMT) in CC. In obtained CC tissues and cells, miR-784 expression was assessed by RT-qPCR, and CCNE1 expression by RT-qPCR or immunohistochemistry. Dual-luciferase reporter assay was implemented for relationship between miR-784 and CCNE1. The roles of miR-784, CCNE1 and the NF-κB pathway in CC were investigated on human CC cell lines. CCNE1 was found to be highly expressed in CC while miR-874 expression was lowered in CC tissues and cells, thereby suggesting a negative regulatory effect of CCNE1. In QBC939 and RBE cells, overexpressing miR-874 or silencing CCNE1 led to augmented IκBα and E-cadherin expression, but diminished CCNE1, NF-κB, N-cadherin, and Vimentin expression. Moreover, overexpression of miR-874 or CCNE1 silencing led to reduced cell proliferation, invasion, and migration capabilities. In conclusion, we demonstrated that miR-874 negatively regulated CCNE1 to inhibit the NF-κB pathway, thus consequently suppressing EMT in CC. Therefore, the overexpression of miR-874 might bring favorable outcomes for the treatment of CC.     

Decrease of Let-7f in Low-Dose Metronomic Paclitaxel Chemotherapy Contributed to Upregulation of Thrombospondin-1 in Breast Cancer

Low-dose metronomic (LDM) paclitaxel therapy displayed a stronger anti-angiogenic activity on breast tumors with fewer side effects. Upregulation of anti-angiogenic factor Thrombospondin-1 (TSP-1) accords for therapeutic potency of LDM paclitaxel, but its molecular mechanism has not been elucidated yet. microRNAs (miRNAs) have emerged as new important regulators of tumor growth and metastasis. Here, we hypothesize that miRNAs are involved in TSP-1 overexpression in paclitaxel LDM therapy of breast tumors. The miRNA profile of tumor tissues from control, LDM and MTD groups in 4T1 mouse breast cancer model was detected by microarray, and then verified by quantitative real-time PCR (qRT-PCR). Luciferase assay and western blot were employed to explore the mechanisms of miRNAs involved in this process. We found that let-7f, let-7a, miR-19b and miR-340-5p were reduced by >2 fold, and miR-543* and miR-684 were upregulated by at least 50% in paclitaxel LDM therapy. qRT-PCR verification revealed that let-7f level was reduced most significantly in LDM therapy. Computational prediction using TargetScan and miRanda suggested THBS1 which encodes TSP-1 as a potential target for let-7f. Luciferase activity assay further confirmed that let-7f may bind to 3''UTR of THBS1 gene and inhibit its activity. Moreover, forced expression of let-7f led to a decrease of TSP-1 at both mRNA and protein levels in MCF-7 cells. Contrastly, let-7f inhibition induced an increased expression of THBS1 mRNA and TSP-1 protein, but did not affect the proliferation and apoptosis of MCF-7 cells. Paclitaxel LDM therapy led to a decrease of let-7f and the elevation of TSP-1 protein expression in MCF-7 cells, while overexpression of let-7f may abolish LDM-induced the upregulation of TSP-1 in MCF-7 cells. In summary, let-7f inhibition contributed to the upregulation of TSP-1 in paclitaxel LDM therapy, independently of proliferation, cell cycle arrest and apoptosis of breast cancer. This study indicates let-7f as a potential therapeutic target for breast tumor.     

MiR-219-5p inhibits hepatocellular carcinoma cell proliferation by targeting glypican-3

MicroRNAs (miRNAs) have been linked to the molecular pathogenesis of many cancers. In this study, we found that miR-219-5p was significantly downregulated in 83 HCC tissues and three HCC cell lines, compared to their non-tumor counterparts. MiR-219-5p expression correlated with tumor size, histological differentiation, and overall survival time in HCC patients. We also found that miR-219-5p could inhibit cell proliferation in vitro and arrest cell cycle at the G1 to S transition. Further studies identified that miR-219-5p reduced both the mRNA and protein levels of glypican-3 (GPC3). These findings indicate that miR-219-5p exerts tumor-suppressive effects in hepatic carcinogenesis through negative regulation of GPC3 expression.     

CircCCNB1 silencing acting as a miR-106b-5p sponge inhibited GPM6A expression to promote HCC progression by enhancing DYNC1I1 expression and activating the AKT/ERK signaling pathway

Background: Circular RNAs (circRNAs), which generally act as microRNA (miRNA) sponges to competitively regulate the downstream target genes of miRNA, play an essential role in cancer biology. However, few studies have been reported on the role of circRNA based competitive endogenous RNA (ceRNA) network in hepatocellular carcinoma (HCC). Herein, we aimed to screen and establish the circRNA/miRNA/mRNA networks related to the prognosis and progression of HCC and further explore the underlying mechanisms of tumorigenesis.Methods: GEO datasets {"type":"entrez-geo","attrs":{"text":"GSE97332","term_id":"97332"}}GSE97332, {"type":"entrez-geo","attrs":{"text":"GSE108724","term_id":"108724"}}GSE108724, and {"type":"entrez-geo","attrs":{"text":"GSE101728","term_id":"101728"}}GSE101728 were utilized to screen the differentially expressed circRNAs (DE-circRNAs), DE-miRNAs, and DEmRNAs between HCC and matched para-carcinoma tissues. After six RNA-RNA predictions and five intersections between DE-RNAs and predicted RNAs, the survival-related RNAs were screened by the ENCORI analysis tool. The ceRNA networks were constructed using Cytoscape software, based on two models of up-regulated circRNA/down-regulated miRNA/up-regulated mRNA and down-regulated circRNA/up-regulated miRNA/down-regulated mRNA. The qRT-PCR assay was utilized for detecting the RNA expression levels in HCC cells and tissues. The apoptosis, Edu, wound healing, and transwell assays were performed to evaluate the effect of miR-106b-5p productions on the proliferation, invasion, and metastasis of HCC cells. In addition, the clone formation, cell cycle, and nude mice xenograft tumor assays were used to investigate the influence of hsa_circ_0001495 (circCCNB1) silencing and overexpression on the proliferation of HCC cells in vitro and in vivo. Furthermore, the mechanism of downstream gene DYNC1I1 and AKT/ERK signaling pathway via the circCCNB1/miR-106b-5p/GPM6A network in regulating the cell cycle was also explored.Results: Twenty DE-circRNAs with a genomic length less than 2000bp, 11 survival-related DE-miRNAs, and 61 survival-related DE-mRNAs were screened out and used to construct five HCC related ceRNA networks. Then, the circCCNB1/miR-106b-5p/GPM6A network was randomly selected for subsequent experimental verification and mechanism exploration at in vitro and in vivo levels. The expression of circCCNB1 and GPM6A were significantly down-regulated in HCC cells and cancer tissues, while miR-106b-5p expression was up-regulated. After transfections, miR-106b-5p mimics notably enhanced the proliferation, invasion, and metastasis of HCC cells, while the opposite was seen with miR-105b-5p inhibitor. In addition, circCCNB1 silencing promoted the clone formation ability, the cell cycle G1-S transition, and the growth of xenograft tumors of HCC cells via GPM6A downregulation. Subsequently, under-expression of GPM6A increased DYNC1I1 expression and activated the phosphorylation of the AKT/ERK pathway to regulate the HCC cell cycle.Conclusions: We demonstrated that circCCNB1 silencing promoted cell proliferation and metastasis of HCC cells by weakening sponging of oncogenic miR-106b-5p to induce GPM6A underexpression. DYNC1I1 gene expression was up-regulated and further led to activation of the AKT/ERK signaling pathway.     

microRNA-489 Plays an Anti-Metastatic Role in Human Hepatocellular Carcinoma by Targeting Matrix Metalloproteinase-7

Dysregulation of microRNAs (miRNAs) is actively involved in the pathogenesis and tumorigenicity of hepatocellular carcinoma (HCC). miR-489 was found to play either oncogenic or tumor suppressive roles in human cancers. Recent study reported that the levels of miR-489 in late recurrent HCC patients were evidently higher than that in early recurrent cases, suggesting that miR-489 may function as a tumor suppressive miRNA in HCC. Yet, the clinical value and biological function of miR-489 remain rarely known in HCC. Here, we presented that miR-489 level in HCC tissues was notably reduced compared to matched non-cancerous specimens. Its decreased level was evidently correlated with adverse clinical parameters and poor prognosis of HCC patients. Accordingly, the levels of miR-489 were obviously down-regulated in HCC cells. Ectopic expression of miR-489 in HCCLM3 and MHCC97H cells prominently inhibits the migration and invasion of tumor cells and reduced lung metastases in vivo, while miR-489 knockdown increased these behaviors of HepG2 and MHCC97L cells. Mechanically, miR-489 negatively regulated matrix metalloproteinase-7 (MMP7) abundance in HCC cells. Herein, MMP7 was found to be a downstream molecule of miR-489 in HCC. An inversely correlation between miR-489 and MMP7 was confirmed in HCC specimens. MMP7 knockdown prohibited cell migration and invasion while MMP7 overexpression showed opposite effects on HCC cells. Furthermore, restoration of MMP7 expression could abrogate the anti-metastatic effects of miR-489 on HCCLM3 cells with enhanced cell migration and invasion. Altogether, miR-489 potentially acts as a prognostic predictor and a drug-target for HCC patients.     

miR-142-3p is a tumor suppressor that inhibits estrogen receptor expression in ER-positive breast cancer

Estrogen receptors (ERs) are involved in the development of many types of malignant tumors, in particular, breast cancer. Among others, ERs affect cell growth, proliferation, and differentiation. The microRNA (miRNA) miR-142-3p has been shown to inhibit carcinogenesis by regulating various cellular processes, including cell cycle progression, cell migration, apoptosis, and invasion. It does so via targeting molecules involved in a range of signaling pathways. We surgically collected 20 ER-positive breast cancer samples, each with matched adjacent normal breast tissue, and measured the expression of miR-142-3p via quantitative real-time polymerase chain reaction (qRT-PCR). Bioinformatics methods, luciferase reporter assay, qRT-PCR, and western blot analysis were used to assess whether miR-142-3p could target ESR1, which encodes the estrogen receptor, in ER-positive breast cancer cells and patient samples. We also restored miRNA expression and performed cell viability, cytotoxicity, and colony formation assays. Western blot analysis and qRT-PCR were used to study the expression of apoptosis and stemness markers. We found that miR-142-3p is downregulated in ER-positive breast cancers. Restoration of miR-142-3p expression in ER-positive breast cancer cells reduced cell viability, induced apoptosis via the intrinsic pathway and decreased both colony formation and the expression of stem cell markers. Bioinformatic analysis predicted miR-142-3p could bind to 3′-untranslated region ESR1 messenger RNA (mRNA). Consistently, we demonstrated that miR-142-3p reduced luciferase activity in ER-positive breast cancer cells, and decreased ESR1 expression in both mRNA and protein levels. The results revealed miR-142-3p and ESR1 expression correlated negatively in ER-positive breast cancer samples. The results suggest miR-142-3p acts as a tumor suppressor via multiple mechanisms. Thus, restoration of miR-142-3p expression, for example, via miRNA replacement therapy, may represent an effective strategy for the treatment of ER-positive breast cancer patients.     

Targeting miR-21 enhances the sensitivity of human colon cancer HT-29 cells to chemoradiotherapy in vitro

5-Fluorouracil (5-FU) is a classic chemotherapeutic drug that has been widely used for colorectal cancer treatment, but colorectal cancer cells are often resistant to primary or acquired 5-FU therapy. Several studies have shown that miR-21 is significantly elevated in colorectal cancer. This suggests that this miRNA might play a role in this resistance. In this study, we investigated this possibility and the possible mechanism underlying this role. We showed that forced expression of miR-21 significantly inhibited apoptosis, enhanced cell proliferation, invasion, and colony formation ability, promoted G1/S cell cycle transition and increased the resistance of tumor cells to 5-FU and X radiation in HT-29 colon cancer cells. Furthermore, knockdown of miR-21 reversed these effects on HT-29 cells and increased the sensitivity of HT-29/5-FU to 5-FU chemotherapy. Finally, we showed that miR-21 targeted the human mutS homolog2 (hMSH2), and indirectly regulated the expression of thymidine phosphorylase (TP) and dihydropyrimidine dehydrogenase (DPD). These results demonstrate that miR-21 may play an important role in the 5-FU resistance of colon cancer cells.     

XB130, a New Adaptor Protein,Regulates Expression of Tumor Suppressive MicroRNAs in Cancer Cells

XB130, a novel adaptor protein, promotes cell growth by controlling expression of many related genes. MicroRNAs (miRNAs), which are frequently mis-expressed in cancer cells, regulate expression of targeted genes. In this present study, we aimed to explore the oncogenic mechanism of XB130 through miRNAs regulation. We analyzed miRNA expression in XB130 short hairpin RNA (shRNA) stably transfected WRO thyroid cancer cells by a miRNA array assay, and 16 miRNAs were up-regulated and 22 miRNAs were down-regulated significantly in these cells, in comparison with non-transfected or negative control shRNA transfected cells. We chose three of the up-regulated miRNAs (miR-33a, miR-149 and miR-193a-3p) and validated them by real-time qRT-PCR. Ectopic overexpression of XB130 suppressed these 3 miRNAs in MRO cells, a cell line with very low expression of XB130. Furthermore, we transfected miR mimics of these 3 miRNAs into WRO cells. They negatively regulated expression of oncogenes (miR-33a: MYC, miR-149: FOSL1, miR-193a-3p: SLC7A5), by targeting their 3′ untranslated region, and reduced cell growth. Our results suggest that XB130 could promote growth of cancer cells by regulating expression of tumor suppressive miRNAs and their targeted genes.