miR-616-3p alleviates inflammatory response by targeting C-X-C motif chemokine ligand 5.

C/EBP homologous protein (CHOP) is a key regulator in ER stress-mediated signaling pathway via PERK-dependent unfolded protein response. It has been known that microRNA-616 (miR-616) is produced from the intron of the human DDIT3 gene encoding CHOP and increased by ER stress. However, the role of miR-616 and its targets are not fully addressed yet. Here we try to identify a novel target of miR-616 in human lung epithelial cells. Microarray analysis showed that CXCL5 is the most downregulated gene by miR-616 overexpression in A549 cells. We also found that CXCL5 mRNA and protein levels were significantly reduced by miR-616 mimic in the presence or absence of TNFα, while anti-miR-616 enhanced CXCL5 expression. In addition, miR-616-3p targeting sequence in 3'UTR of CXCL5 was confirmed by luciferase reporter assay suggesting that miR-616-3p directly binds to 3'UTR of CXCL5 and inhibits CXCL5 expression. Finally, we confirmed that conditioned medium from A549 cells treated with TNFα or Streptococcus pneumoniae lysates increased intra-alveolar neutrophil infiltration in a mouse model of pulmonary inflammation, while this induction was significantly reduced in a conditioned medium from cells transfected with miR-616-3p. These results suggest that miR-616-3p can alleviate CXCL5-induced pulmonary inflammatory response via targeting 3'UTR of CXCL5 gene.

UPR-Induced miR-616 Inhibits Human Breast Cancer Cell Growth and Migration by Targeting c-MYC.

C/EBP homologous protein (CHOP), also known as growth arrest and DNA damage-inducible protein 153 (GADD153), belongs to the CCAAT/enhancer-binding protein (C/EBP) family. CHOP expression is induced by unfolded protein response (UPR), and sustained CHOP activation acts as a pivotal trigger for ER stress-induced apoptosis. MicroRNA-616 is located within an intron of the CHOP gene. However, the regulation of miR-616 expression during UPR and its function in breast cancer is not clearly understood. Here we show that the expression of miR-616 and CHOP (host gene of miR-616) is downregulated in human breast cancer. Both miR-5p/-3p arms of miR-616 are expressed with levels of the 5p arm higher than the 3p arm. During conditions of ER stress, the expression of miR-616-5p and miR-616-3p arms was concordantly increased primarily through the PERK pathway. Our results show that ectopic expression of miR-616 significantly suppressed cell proliferation and colony formation, whereas knockout of miR-616 increased it. We found that miR-616 represses c-MYC expression via binding sites located in its protein coding region. Furthermore, we show that miR-616 exerted growth inhibitory effects on cells by suppressing c-MYC expression. Our results establish a new role for the CHOP locus by providing evidence that miR-616 can inhibit cell proliferation by targeting c-MYC. In summary, our results suggest a dual function for the CHOP locus, where CHOP protein and miR-616 can cooperate to inhibit cancer progression.

Hsa_circ_0007494 suppresses prostate cancer progression via miR-616/PTEN axis.

Circular RNAs (circRNAs) are important players in prostate cancer (PCa) development and progression, but their detailed mechanisms have not been elucidated. Herein, hsa_circ_0007494 was suppressed in the PCa cell lines and tissues. This resulted in metastasis of tumors to the lymph node and predicted tumor stage. Functionally, overexpression of hsa_circ_0007494 inhibited the proliferation and invasive capacity of the cells in vitro and blocked the growth of tumors in vivo. Hsa_circ_0007494 functioned as a "molecular sponge" for miR-616 and hence upregulated the target of miR-616, PTEN. In addition, rescue assays revealed that PTEN silencing (or miR-616 mimics) blocked the tumor-suppressing effects of hsa_circ_0007494 overexpression on PCa progression. Together, our findings indicate that hsa_circ_0007494 suppresses PCa by forming a negative regulatory network including hsa_circ_0007494/miR-616/PTEN. Thus, hsa_circ_0007494 may be a treatment target for PCa.

Circ_DCAF6 potentiates cell stemness and growth in breast cancer through GLI1-Hedgehog pathway.

Circular RNAs (circRNAs) are extensively revealed as a malignant activator or suppressor in multiple pathological processes including cancer cell stemness and growth. However, the association of circ_DCAF6 with breast cancer (BC) cell growth and stemness has not been well depicted. In this research, qRT-PCR clarified the high level of circ_DCAF6 in BC cells. In functional aspects, BC cells presented suppressed proliferation and stemness in the absence of circ_DCAF6. The potential correlation of circ_DCAF6 with Hedgehog (Hh) pathway was unveiled utilizing its specific inhibitor or agonist in qRT-PCR and functional assays. Circ_DCAF6 positively mediated the expression of GLI1 and its facilitating impacts on BC cell proliferation and stemness required the participation of GLI1-dependent Hh signaling pathway. In depth, circ_DCAF6 post-transcriptionally upregulated GLI1 expression through sequestering miR-616-3p. Rescue experiments verified that the suppressive influence of circ_DCAF6 depletion or miR-616-3p upregulation on BC progression was reversed by GLI1 upregulation. In summary, a probable contribution of circ_DCAF6 to BC cell growth and stemness was elaborated. Circ_DCAF6 assisted in Hh signal pathway activation via the up-regulation of GLI1 by sponging miR-616-3p, generating new thoughts on future direction of antagonizing BC tumorigenesis and stem-like property.

LOC285194 inhibits proliferation of human keratinocytes through regulating miR-616/GATA3 pathway.

LncRNA LOC285194 has been associated with the occurrence of psoriasis. However, the underlying mechanisms that lead to psoriasis remain unclear. In this study, the expression of LOC285194, miR-616, and GATA3 was determined by western blotting and quantitative real-time PCR, and their relationships were assessed using dual-luciferase reporter assays. The effects of LOC285194 on the proliferation and apoptosis of keratinocytes were investigated using cell counting kit-8 assays and flow cytometry, respectively. Reduced expression of LOC285194 was detected in the skin lesion samples from patients with psoriasis. Overexpression of LOC285194 led to reduced cell viability, cell cycle arrest, and increased cell apoptosis in keratinocytes, whereas LOC285194 silencing resulted in opposite effects. In addition, LOC285194 was found to negatively regulate miR-616, which modulated GATA3 expression through its direct binding to the 3'-untranslated region of GATA3. Knockdown of GATA3 rescued LOC285194 overexpression-mediated cell viability reduction, cell cycle arrest and apoptosis induction in keratinocytes. Taken together, LOC285194 was found to inhibit keratinocyte growth by sponging miR-616 that regulates GATA3.

MiR-616-3p promotes angiogenesis and EMT in gastric cancer via the PTEN/AKT/mTOR pathway.

Dysregulation of microRNAs has been demonstrated to be involved in a variety of biological events related to cancer, including proliferation, metastasis, angiogenesis and immune escape. MiR-616-3p is located on the chromosome region 12q13.3, however, its potential role and clinical implications in gastric cancer remain poorly understood. The current study aimed to investigate the potential role of miR-616-3p in gastric cancer. The results showed that miR-616-3p was up-regulated in cancer tissues. Higher expression of miR-616-3p in tumor tissues also predicted poor prognosis. Furthermore, loss- and gain-of-function in vitro revealed that miR-616-3p promoted angiogenesis and EMT in gastric cancer cells. Mechanistically, further analysis demonstrated that the effects of miR-616-3p on metastasis and angiogenesis occurred through the down-regulation of PTEN, a direct target of miR-616-3p. We propose that the restoration of PTEN expression may block miR-616-3p-induced EMT and angiogenesis. Collectively, our findings suggest that the miR-616-3p-PTEN signaling axis might be a potential therapeutic target for gastric cancer.

[Retracted] MicroRNA­616 promotes the growth and metastasis of non­small cell lung cancer by targeting SOX7.

Following the publication of this paper, it was drawn to the Editor's attention by a concerned reader that the cell formation assay data shown in Figs. 3D, 4D, 8D and 9D were strikingly similar to data that had already appeared in another article written by different authors at different research institutes [Wang Z, Jiang C, Chen W, Zhang G, Luo D, Cao Y Wu J, Ding Y and Liu B: Baicalein induces apoptosis and autophagy via endoplasmic reticulum stress in hepatocellular carcinoma. Biomed Res Int: 732516, 2014]. Owing to the fact that the contentious data in the above article had already been published prior to its submission to Oncology Reports, the Editor has decided that this paper should be retracted from the Journal. The authors were asked for an explanation to account for these concerns, but the Editorial Office did not receive a reply. The Editor apologizes to the readership for any inconvenience caused. [Oncology Reports 38: 2078­2086, 2017; DOI: 10.3892/or.2017.5854].

Hsa_circ_0015382 is involved in the pathogenesis of preeclampsia by mediating THBS2 expression.

BACKGROUND: Preeclampsia (PE) is a hypertensive disorder of pregnancy that causes significant maternal and perinatal morbidity and mortality. Circular RNA (circRNA) hsa_circ_0015382 is associated with the pathogenesis of PE, but its underlying regulatory mechanism remains to be explored. METHODS: Relative RNA levels of hsa_circ_0015382, microRNA-616-3p and thrombospondin-2 (THBS2) were detected by quantitative reverse transcription-polymerase chain reaction. In vitro regulatory effects of hsa_circ_0015382 on the proliferation, migration, invasion and angiogenesis of trophoblasts were evaluated by CCK-8, flow cytometry for cell cycle, EdU, transwell, wound healing and HUVEC tube formation assays, respectively. Targeting interaction was verified by dual-luciferase reporter and RNA immunoprecipitation assays. RESULTS: Hsa_circ_0015382 was highly expressed in placental tissues from PE patients. Upregulation of hsa_circ_0015382 repressed trophoblast proliferation, migration, invasion and lowered trophoblast-induced HUVEC tube formation. Hsa_circ_0015382 was validated as a miR-616-3p sponge and miR-616-3p targeted THBS2. Hsa_circ_0015382 could mediate trophoblast proliferation, migration, invasion and regulate trophoblast-induced HUVEC tube formation by sponging miR-616-3p and regulating THBS2 expression. CONCLUSION: Hsa_circ_0015382 is associated with the pathogenesis of PPE by regulating the miR-616-3p/THBS2 axis. HIGHLIGHTS: Hsa_circ_0015382 is overexpressed in preeclampsia patients. Hsa_circ_0015382 inhibits trophoblast proliferation, migration, invasion and decreases trophoblast-induced HUVEC tube formation. Hsa_circ_0015382 interacts with miR-616-3p to regulate THBS2 expression.

Apatinib combined with Keytruda treatment induces apoptosis of gastric carcinoma cells through CES4/miR-616-5p/DUSP2 axis.

Gastric carcinoma (GC) is a highly malignant and heterogeneous tumour. Long non-coding RNA CES4 is down-regulated in GC. However, whether CES4 can participate in GC remains unclear; we have carried out research on this topic. GC cells (HGC-27 and MKN-7) were treated with anti-tumour drugs: apatinib combined with Keytruda. Cell viability and apoptosis were detected by CCK-8 assay and flow cytometry. Gene and protein expression were examined by quantitative real-time PCR and western blot. Luciferase reporter assay was performed to verify the relationship among CES4, miR-616-5p and dual-specificity phosphatase-2 (DUSP2). CES4 was highly expressed in the apatinib combined with Keytruda-treated HGC-27 and MKN-7 cells. Apatinib combined with Keytruda treatment repressed cell viability and promoted apoptosis of HGC-27 and MKN-7 cells, which was abrogated by CES4 knockdown. Furthermore, CES4 promoted DUSP2 expression by sponging miR-616-5p in HGC-27 and MKN-7 cells. CES4 knockdown promoted cell viability and inhibited apoptosis of drug-treated HGC-27 and MKN-7 cells by regulating miR-616-5p/DUSP2 axis. In conclusion, these data demonstrate that apatinib combined with Keytruda treatment induces apoptosis of GC cells through CES4/miR-616-5p/DUSP2 axis. Thus, this work provides the experimental basis for the combination of apatinib and Keytruda as a treatment for GC.

miR-616-5p Promotes Invasion and Migration of Bladder Cancer via Downregulating NR2C2 Expression.

BACKGROUND: MicroRNAs, small non-coding RNA molecules with about 22 nucleotides in length, play a significant role in the development of bladder cancer. Previous studies found that miR-616-5p could promote the progress of cancers. However, its role in bladder cancer remains unclear. In the study, we aimed to demonstrate how miR-616-5p impacts the invasion and migration of bladder cancer and its potential downstream targets. METHODS: Firstly, qRT-PCR was used to detect the expression of miR-616-5p in normal bladder uroepithelial cell lines and bladder cancer cell lines. Then, chamber-transwell invasion and wound healing migration assays were used to detect the roles of miR-616-5p and NR2C2 in invasion and migration. Subsequently, Western blot was used to evaluate the regulation effects of miR-616-5p and NR2C2. Finally, luciferase assays were performed to manifest the mechanism of miR-616-5p and NR2C2 regulation. RESULTS: We found that miR-616-5p was upregulated in bladder cancer, and it could promote the invasion and migration of bladder cancer in vitro. Moreover, we demonstrated that NR2C2 was a downstream target of miR-616-5p. miR-616-5p could inhibit the expression of NR2C2 by binding to the 3'UTR of NR2C2 mRNA. Importantly, patients with a high expression of NR2C2 showed better prognoses in bladder cancer. CONCLUSIONS: This study identifies that miR-616-5p can promote bladder cancer progression via altering the expression of NR2C2. Therefore, identifying miR-616-5p expression levels might be a useful strategy for developing potential therapeutic targets in bladder cancer.

MiR-616 plays oncogenic role in hepatocellular carcinoma progression through suppressing PTEN expression and activating PI3K/AKT pathway.

Aims: Given their regulatory roles in gene expression, microRNAs play an important role in tumorigenesis. The current study aimed to explore the function and the related mechanisms of miR-616 in hepatocellular carcinoma (HCC).Methods: The expression of miR-616 was detected using quantitative real-time polymerase chain reaction (qRT-PCR). Chi-square test was applied to estimate the association of miR-616 with clinical characteristics of HCC patients. Cell transfection was performed by Lipofectamine® 2000. MTT assay was used to detect cell proliferation, whereas cell motility was estimated using Transwell assay. The protein expression was detected using western blot.Results: MiR-616 was significantly up-regulated in HCC tissues and cells (p < .05 for all). Moreover, its elevated expression was positively correlated with lymph node metastasis (p = .008) and TNM stage (p = .012). Knockdown of miR-616 resulted in decreased cell proliferation, migration and invasion. Moreover, the inhibition of miR-616 significantly suppressed PI3K/AKT pathway. The bioinformatics analysis and luciferase reporter assay suggested that PTEN was a targeted gene of miR-616. PTEN had the capacity to reverse the oncogenic function of miR-616 in HCC.Conclusion: MiR-616 activates PI3K/AKT pathway through suppressing PTEN expression, thus promoting the progression of HCC.

Linc-OIP5 working as a ceRNA of miR-616 promotes PON1 expression in HUEVC cells.

Coronary atherosclerosis affects human health all over the world. PON1 was found to be associated with coronary atherosclerosis but the specific mechanism is still unclear. Non-coding RNA plays an important role in many diseases. In recent years, studies have focused on non-coding RNA in coronary atherosclerosis. In this study, we investigated the effect of non-coding RNA Linc-OIP5 on PON1 expression. Results showed that in the serum of patients with coronary atherosclerosis, there was lower PON1 activity and PON1 level, the same trend in Linc-OIP5, and the opposite trend in miR-616 expression. Through further cell experiments, with up-regulation or down-regulation of Linc-OIP5 and miR-616, we found that Linc-OIP5 positively regulated the expression of PON1 gene and its protein level, while miR-616 negatively regulated the expression of PON1 gene and protein. Through further functional experiments, we also found that the levels of superoxide dismutase (SOD) and nitric oxide (NO) related to oxidative stress also had corresponding changes. Further dual luciferase reporter assay demonstrated that Linc-OIP5 regulated PON1 expression as a ceRNA of miR-616. Thus Linc-OIP5 working as a ceRNA of miR-616 apparently promotes PON1 expression in HUEVC cells and Linc-OIP5 and miR-616 may be an ideal target for the treatment of coronary atherosclerosis in the future.

Long noncoding RNA TUSC7 inhibits cell proliferation, migration and invasion by regulating SOCS4 (SOCS5) expression through targeting miR-616 in endometrial carcinoma.

BACKGROUND: Long non-coding RNA (lncRNA) is emerging as an important regulator in various physiological and pathological processes. Recently, it was found that lncRNA long non-coding RNA tumor suppressor candidate 7 (TUSC7) could play tumor suppressive roles in several cancers. However, the function and underlying regulatory mechanism of lncRNA TUSC7 in endometrial carcinoma (EC) remains largely unclear. METHODS: The expression levels of TUSC7 and microRNAs-616 (miR-616) were analyzed by real-time PCR and in situ hybridization. Cell cycle and cell metastasis associated protein expressions were determined by western blotting. Cell proliferation, cycle and metastasis were determined by CCK-8 cell viability, colony formation, flow cytometer, wound scratch and transwell assays respectively in vitro. RNA pull-down, luciferase and western blotting assays were used to examine the target relationship between TUSC7 and miR-616 or that between miR-616 and suppressors of cytokine signaling 4 (5) (SOCS4 (SOCS5)). The functional effects of TUSC7 through sponging miR-616 were further examined using a xenograft tumor mouse model in vivo. RESULTS: TUSC7 was downexpressed in EC tissues and cell lines, and TUSC7 upregulation could remarkably inhibit cell proliferation, cycle progression and metastasis in EC cells. Mechanistic investigations demonstrated that TUSC7 can interact with miR-616 and decrease its expression, thereby upregulating the expression of miR-616's targets SOCS4 (SOCS5). Additionally, in vivo experiments using a xenograft tumor mouse model revealed that TUSC7 can serve as a tumor suppressor through sponging miR-616, and upregulating SOCS4 (SOCS5) in EC. CONCLUSIONS: In this study, a newly identified regulatory mechanism of lncRNA TUSC7/miR-616/ SOCS4 (SOCS5) axis was systematically studied, which may hold promise as a promising target for EC treatment.