Bit1-a novel regulator of astrocyte function during retinal development: proliferation, migration, and paracrine effects on vascular endothelial cell Abstract Studies have shown that astrocyte plays an important role in the formation of retinal vasculature during development. For our study, we investigated the role of Bcl2 inhibitor of transcription 1 (Bit1) in regulating astrocyte function from developing retina and its paracrine effects on vascular endothelial cell. Expression pattern of Bit1 was analyzed by immunofluorescent staining of whole mount rat retina. Astrocytes and retinal microvascular endothelial cells (RMECs) were isolated from rat retina for cultural studies. The proliferation and migration of astrocytes and RMECs were evaluated by CCK-8 assay, scratch assay, and transwell migration assay. Cell apoptosis was detected by anoikis assay. Angiogenesis assay was used to measure the ability of RMECs to form tube-like microvascular structure. siRNA knockdown assay was employed to regulate Bit1 expression in astrocytes. Immunofluorescent staining showed Bit1 expression in migrating retinal astrocytes co-localized with the marker glial fibrillary acidic protein (GFAP). Isolated retinal astrocytes from post-natal rat eyes have an elevated expression of Bit1 and show increased cell survival and decreased anoikis as compared with retinal astrocytes from embryo. Suppressing Bit1 by siRNA assay leads to decreased cell proliferation, migration, and increased anoikis of astrocytes. Meanwhile, Bit1 knockdown could decrease the astrocytic vascular endothelial growth factor (VEGF) expression leading to inhibitory paracrine effects on RMECs angiogenesis. Our findings reveal that Bit1 promotes cell survival, proliferation, migration, and maintains VEGF expression of retinal astrocytes, leading to enhanced paracrine effects on angiogenesis of vascular endothelial cells. Bit1 may serve as a novel regulator of astrocyte biological behaviors interplaying with vascular endothelial cell during retinal development.

Up-regulation of long non-coding RNA AWPPH inhibits proliferation and invasion of gastric cancer cells via miR-203a/DKK2 axis Abstract AWPPH is a newly discovered long non-coding RNA (lncRNA). However, the expression and function of AWPPH in gastric cancer (GC) have not yet been clarified. This study tries to assess the expression and biological roles of AWPPH in GC and the underlying mechanism. The expression of lncRNA AWPPH was evaluated in GC tissues and adjacent normal tissues from 40 patients. Cell Counting Kit-8 (CCK8) and transwell assays were applied to assess cell proliferation and invasion capabilities. Bioinformatics tool was employed to predict AWPPH’s sponging miRNA, while luciferase reporter assays were used to verify the target. LncRNA AWPPH was remarkably downregulated in GC and associated with metastasis. CCK8 and transwell assays proved that AWPPH inhibited cell proliferation and invasion in GC cells. MiR-203a was a predicted and further verified target of AWPPH. DKK2 was verified as a direct target of miR-203a. Upregulation of miR-203a attenuated the repressive effects of AWPPH on GC cell proliferation and invasion. AWPPH inhibited GC cell proliferation and invasion via miR-203a/DKK2 axis. This finding might provide new insight for the potential therapeutic strategies for GC in the future.

The clinical and prognostic significance of paraoxonase-2 in gastric cancer patients: immunohistochemical analysis Abstract Paraoxonase-2 (PON2) belongs to the paraoxonase (PON) protein family. Unlike paraoxonase-1 (PON1), the expression and significance of PON2 remained largely unknown in gastric cancer (GC). Thus, the purpose of our study was to investigate the role of PON2 in GC. First, we found PON2 expression was obviously increased in GC samples compared with paired normal tissue samples at The Cancer Genome Atlas (TCGA) database. Then the high expression status of PON2 mRNA and protein in GC tissues was confirmed by RT-qPCR and immunohistochemistry. Furthermore, we performed the immunohistochemical analysis to study the correlation between PON2 expression and clinicopathological parameters of GC patients, and found high PON2 expression had significantly positive association with diffuse type, clinical stage, tumor invasion, lymph node metastasis and distant metastasis in GC patients. Moreover, survival analysis suggested GC patients with high PON2 expression resulted in a remarkably shorter overall survival compared with GC patients with low PON2 expression, and high expression of PON2 acted as an unfavorable predictor for overall survival. The in vitro studies indicated that silencing of PON2 expression inhibited GC cell proliferation, migration and invasion. In conclusion, our findings give first evidence that PON2 serves as oncogene in GC.

LncRNA terminal differentiation-induced ncRNA (TINCR) sponges miR-302 to upregulate cyclin D1 in cervical squamous cell carcinoma (CSCC) Abstract This study aimed to investigate the role of lncRNA terminal differentiation-induced ncRNA (TINCR) in cervical squamous cell carcinoma (CSCC). By informatics analysis, we found that miR-302 may bind TINCR. Expression analysis showed that miR-302 was downregulated, while TINCR was upregulated in CSCC. Correlation analysis showed that they were not significantly correlated. In CSCC cells, miR-302 and TINCR failed to affect the expression of each other. However, miR-302 overexpression led to downregulated and TINCR overexpression led to upregulated cyclin D1 expression in CSCC cells. Interestingly, overexpression of cyclin D1 led to upregulated miR-302 and TINCR. Cell proliferation analysis showed that TINCR and cyclin D1 overexpression led to increased, while miR-302 overexpression led to decreased rate of cell proliferation. Moreover, miR-302 overexpression reduced the effects of TINCR overexpression. Therefore, TINCR sponges miR-302 to upregulate cyclin D1 in CSCC, thereby promoting cell proliferation.

Interaction between cancer cells and cancer-associated fibroblasts after cisplatin treatment promotes cancer cell regrowth Abstract Regrowth of cancer cells following chemotherapy is a significant problem for cancer patients. This study examined whether cancer-associated fibroblasts (CAFs), a major component of a tumor microenvironment, promote cancer cell regrowth after chemotherapy. First, we treated human lung adenocarcinoma cell line A549 and CAFs from four patients with cisplatin. Cisplatin treatment inhibited the viable cell number of A549 cells and induced epithelial–mesenchymal transition. After cisplatin was removed, A549 cells continued to manifest the mesenchymal phenotype and proliferated 2.2-fold in 4 days (regrowth of A549 cells). Cisplatin treatment inhibited the viable cell number of CAFs from four patients also. The CM (derived from cisplatin-pretreated CAFs from two patients) significantly enhanced the regrowth of cisplatin-pretreated A549 cells, and the CM derived from cisplatin-naïve CAFs marginally enhanced A549 regrowth. By contrast, the CM derived from either cisplatin-pretreated CAFs or cisplatin-naïve CAFs failed to enhance the growth of cisplatin-naïve A549 cells. The CM derived from cisplatin-pretreated CAFs did not enhance the proliferation of A549 cells in which epithelial–mesenchymal transition was induced by TGFβ-1. Our findings indicate the possibility that humoral factors from cisplatin-pretreated CAFs promote the regrowth of cisplatin-pretreated A549 cells. These results suggest that interactions between cancer cells and CAFs may significantly enhance cancer cell regrowth within the tumor microenvironment after cisplatin treatment.

MiR-202-5p/MATN2 are associated with regulatory T-cells differentiation and function in allergic rhinitis Abstract Regulatory T cells (Tregs) play a crucial role in allergic rhinitis (AR). However, the mechanism of how Tregs are regulated in AR is poorly understood. Here, we aimed to explore the role of Tregs in AR and how Tregs were regulated by miR-202-5P, which was demonstrated to be important in AR. Peripheral blood mononuclear cells (PBMC) were isolated from collected blood samples. Tregs were purified using Regulatory T Cell Isolation Kit, and differentiated from isolated CD4 T cells using recombinant human interleukin-2 (rhIL-2) and transforming growth factor beta (TGF-β). mRNA expression levels of miR-202-5p, matrilin-2 (MATN2), TGF-β1 and interleukin-10 (IL-10) were detected by real-time PCR. The concentrations of IL-4, interleukin-17 (IL-17), IL-10, interferon gamma (IFN-γ) and TGF-β1 were detected by enzyme-linked immunosorbent assay (ELISA). MATN2 protein level was detected by Western blot. MiR-202-5p expression dramatically elevated in PBMCs, CD4+ T cells and Tregs of AR patients. In vitro, miR-202-5p promoted Tregs differentiation via targeting MATN2. MiR-202-5p/MATN2 axis mediated Tregs proliferation and functions. MiR-202-5p/MATN2 are associated with regulatory T-cells differentiation and function in allergic rhinitis.

miRNA-21 promotes cell proliferation and invasion via VHL/PI3K/AKT in papillary thyroid carcinoma Abstract Papillary thyroid carcinoma (PTC) is the main kind of thyroid carcinoma, most of which are diagnosed in women. MiR-21 has been reported to be upregulated in multiple cancers to effect tumor growth. However, the role of miR-21 in PTC development remains unclear. In this present study, miR-21 and VHL expressions in PTC tissues and cells were evaluated by RT-qPCR and/or western blot. MTT assay and transwell assay were employed to assess cell proliferative and invasive abilities, respectively. Luciferase reporter assay was carried out to identify the target of miR-21and explore its roles in PTC. MiR-21 was upregulated in PTC tissues and cells. Ectopic of miR-21 expression promoted cell proliferative and invasive abilities, while knockdown miR-21 suppressed these in TPC-1 and BCPAP cells. Overexpression of miR-21 predicted poor prognosis in PTC. What is more, luciferase reporter assays showed miR-21 can directly target VHL in PTC cells. Knockdown of miR-21 expression inhibited TPC-1 and BCPAP cell invasion-mediated EMT and proliferation through the PI3K/AKT pathway. In addition, VHL reverses partial function of miR-21 on PTC cell proliferation and invasion. MiR-21 can inhibit cell proliferation and invasion by regulated VHL in PTC cells. The newly identified miR-21/VHL axis might provide a novel insight into the pathogenesis and therapy of PTC.

LncRNA HEIH regulates cell proliferation and apoptosis through miR-4458/SOCS1 axis in triple-negative breast cancer Abstract Hepatocellular carcinoma up-regulated EZH2-associated long non-coding RNA (HEIH) is a newly discovered lncRNA and has been suggested to be dysregulated in human cancers. However, the role of HEIH in triple-negative breast cancer (TNBC) was still unknown. Thus, the aim of our study was to investigate the clinical significance and biological function of HEIH in TNBC. In our study, we found that HEIH was overexpressed in TNBC tissues and cell lines compared with adjacent normal mammary tissues and normal mammary epithelial cell line, respectively. In addition, we conducted bioinformatic analysis, and found that HEIH harbors a potential miR-4458-binding site. Furthermore, we observed that HEIH and miR-4458 had a high correlation score in TNBC tissues, and HEIH directly binds to miR-4458, and negatively regulates miR-4458 expression in TNBC cells. The in vitro cell proliferation and apoptosis assays suggested down-regulation of HEIH inhibited TNBC cell proliferation and promoted apoptosis through regulating miR-4458/SOCS1 axis. Finally, we found that TNBC patients with tumor size ≥ 5 cm or advanced clinical stage had higher levels of HEIH expression than patients with tumor size < 5 cm or early clinical stage. In conclusion, HEIH functions as an oncogenic lncRNA that is overexpressed in TNBC and associated with clinical progression, and regulates TNBC cell proliferation and apoptosis through miR-4458/SOCS1 axis.

Involvement of the MDR1 gene and glycolipids in anticancer drug-resistance of human ovarian carcinoma-derived cells Abstract We previously reported that anti-paclitaxel-resistant ovarian carcinoma cells characteristically expressed the MDR1 (multidrug resistance 1) gene with enhanced synthesis of glycolipids, i.e., LacCer, Gb3Cer, Leb and GM3, and that anti-cisplatin-resistant cells lost GM3. To further examine the involvement of glycolipids and the MDR1 gene in the anticancer drug-resistance, we determined their expression and the sensitivity to anticancer drugs of several ovarian carcinoma-derived cells, i.e., serous KF28, mucinous HMKOA, endometrioid HNOA and clear cell RMG-1 cells. The MDR1 gene was only detected in RMG-1 cells, in which the amounts of Gb4Cer, Leb and GM3 were higher than in the other cells, which reflected their much higher resistance to paclitaxel and docetaxel compared to the other cells. Among HNOA, HMKOA and KF28 cells, all of which did not express the MDR1 gene, the HNOA and HMKOA cells were relatively more resistant to paclitaxel and docetaxel than KF28 cells, and contained more than sevenfold Gb4Cer and Leb in KF28 cells, indicating that cells containing glycolipids with longer carbohydrate chains, even without expression of the MDR1 gene, have the resistance property as to hydrophobic drugs. On the contrary, RMG-1 cells with the highest amount of GM3 were relatively more sensitive to cisplatin than the other cells, which probably due to a negative charge for binding with cisplatin. Thus, MDR1, and increased amounts of Gb4Cer, Leb and GM3 were suggested to be involved in the anticancer drug-resistance to hydrophobic paclitaxel and docetaxel, and GM3 was to basic cisplatin.

Sulforaphane inhibits the activation of hepatic stellate cell by miRNA-423-5p targeting suppressor of fused Abstract Liver fibrosis, a common pathological process in chronic liver diseases, is characterized by excessive accumulation of extracellular matrix proteins and considered as a wound healing response to chronic liver injury. Hepatic stellate cell (HSC) activation plays a key role in liver fibrosis development. Previous studies showed that sulforaphane (SFN) has wide protective effects against tissue injury and inflammation. Accumulating evidence has shown that microRNAs play important roles in the development of hepatic fibrosis, some of which have been identified as potential therapeutic targets. This study was conducted to explore the role of SFN in the suppression of HSC activation. Quantitative real-time PCR showed that HSC miR-423-5p levels were up-regulated during HSC activation and down-regulated after SFN administration. Further, transfection of a miR-423-5p mimic demonstrated that inhibition of HSC activation by SFN required down-regulation of miR-423-5p. We showed that suppressor of fused is the direct target of miR-423-5p. SFN may play a role in inhibiting hepatic fibrosis by downregulating miRNA-423-5p. MiRNA-423-5p may be useful as a therapeutic target for treating hepatic fibrosis.