Exosomal DLX6-AS1 from hepatocellular carcinoma cells induces M2 macrophage polarization to promote migration and invasion in hepatocellular carcinoma through microRNA-15a-5p/CXCL17 axis.

BACKGROUND: Hepatocellular carcinoma (HCC) cells-secreted exosomes (exo) could stimulate M2 macrophage polarization and promote HCC progression, but the related mechanism of long non-coding RNA distal-less homeobox 6 antisense 1 (DLX6-AS1) with HCC-exo-mediated M2 macrophage polarization is largely ambiguous. Thereafter, this research was started to unearth the role of DLX6-AS1 in HCC-exo in HCC through M2 macrophage polarization and microRNA (miR)-15a-5p/C-X-C motif chemokine ligand 17 (CXCL17) axis. METHODS: DLX6-AS1, miR-15a-5p and CXCL17 expression in HCC tissues and cells were tested. Exosomes were isolated from HCC cells with overexpressed DLX6-AS1 and co-cultured with M2 macrophages. MiR-15a-5p/CXCL17 down-regulation assays were performed in macrophages. The treated M2 macrophages were co-cultured with HCC cells, after which cell migration, invasion and epithelial mesenchymal transition were examined. The targeting relationships between DLX6-AS1 and miR-15a-5p, and between miR-15a-5p and CXCL17 were explored. In vivo experiment was conducted to detect the effect of exosomal DLX6-AS1-induced M2 macrophage polarization on HCC metastasis. RESULTS: Promoted DLX6-AS1 and CXCL17 and reduced miR-15a-5p exhibited in HCC. HCC-exo induced M2 macrophage polarization to accelerate migration, invasion and epithelial mesenchymal transition in HCC, which was further enhanced by up-regulated DLX6-AS1 but impaired by silenced DLX6-AS1. Inhibition of miR-15a-5p promoted M2 macrophage polarization to stimulate the invasion and metastasis of HCC while that of CXCL17 had the opposite effects. DLX6-AS1 mediated miR-15a-5p to target CXCL17. DLX6-AS1 from HCC-exo promoted metastasis in the lung by inducing M2 macrophage polarization in vivo. CONCLUSION: DLX6-AS1 from HCC-exo regulates CXCL17 by competitively binding to miR-15a-5p to induce M2 macrophage polarization, thus promoting HCC migration, invasion and EMT.

Circ_SPECC1 enhances the inhibition of miR-526b on downstream KDM4A/YAP1 pathway to regulate the growth and invasion of gastric cancer cells.

Gastric cancer (GC) is a common malignant tumor, and many studies have shown that circular RNAs (circRNAs) play important roles in the progress of GC. This study showed that circ_SPECC1 was down-regulated in various GC cell lines, significantly inhibited GC cell proliferation and invasion, and promote apoptosis, which might play an anti-oncogene role. Circ_SPECC1 was mainly located in the cytoplasm, and its sequence contained multiple potential binding sites of miR-526b. Pull-down experiments with biotinylated miR-526b mimics and circ_SPECC1 probe showed that they could enrich each other. RIP experiments found hat anti-AGO2 antibody could significantly enrich circ_SPECC1. Further dual luciferase reporter gene assay also confirmed that miR-526b could bind directly to circ_SPECC1. miR-526b was also down-regulated in GC cells, and one of its important target genes was KDM4A. Both circ_SPECC1 and miR-526b inhibited the expression of KDM4A and its downstream effector YAP1, but miR-526b inhibitors terminated the above-mentioned inhibition of circ_SPECC1, and KDM4A overexpression reversed the inhibition of circ_SPECC1 and miR-526b on YAP1 expression. Both miR-526b and KDM4A siRNA inhibited GC cell proliferation and invasion, and promote apoptosis; KDM4A overexpression had the opposite effects, and significantly blocked the regulation of miR-526b on cell growth and invasion. Therefore, circ_SPECC1 can enhance miR-526b inhibitory effect on downstream KDM4A/YAP1 pathway by adsorbing it, thus inhibiting GC cell growth and invasion. These findings enrich the mechanism of circRNAs in GC and will provide more new targets for the prevention and treatment of GC.

Role of Exercise Activity in Alleviating Neuropathic Pain in Diabetes via Inhibition of the Pro-Inflammatory Signal Pathway.

Hyperalgesia and allodynia are commonly observed in patients with diabetic neuropathy. The treatment and management of painful peripheral neuropathy is important in these patients. The purpose of this study was to examine the role of exercise in modulating neuropathic pain induced by diabetes. Diabetes was induced in rats by an intraperitoneal injection of streptozotocin (STZ). Control rats received saline injections. Groups included control rats without exercise (NT-control, n = 12), control rats with exercise (EX-control, n = 16), STZ rats without exercise (NT-STZ, n = 18), and STZ rats with exercise (EX-STZ, n = 22). Rats in EX groups ran on a treadmill 4 days/week for 5 weeks beginning from the week of STZ administration. Mechanical hypersensitivity (mechanical paw withdrawal thresholds [PWTs]) and glucose levels were tested weekly. Then, enzyme-linked immunoassay and Western blot analysis were used to determine the levels of pro-inflammatory cytokines (PICs) and their receptors in sensory nerves. PWTs were significantly increased after 4-5 weeks of exercise in STZ rats ( p < .05 vs. NT-STZ rats). Inhibition of neuropathic pain by exercise in STZ rats was accompanied by decreases in interleukin (IL)-1ß, IL-6, and tumor necrosis factor (TNF)-α levels and downregulated expression of their receptors. Furthermore, blocking individual PIC receptors elevated PWTs to a greater degree in STZ rats ( p < .05 vs. control rats). Overall, our data suggest that exercise can play a role in improving neuropathic pain induced by STZ and that PIC signaling is a part of the mechanism involved in this effect.

MicroRNA-145 exerts tumor-suppressive and chemo-resistance lowering effects by targeting CD44 in gastric cancer.

AIM: To determine the potential roles of CD4 and microRNA (miR)-145 in gastric cancer. METHODS: The levels of CD44 and miR-145 were determined in gastric cancer cells. Quantitative real-time polymerase chain reaction was used to measure to the level of CD44 mRNA. A luciferase reporter assay and western blotting were performed to examine the effect of miR-145 on CD44 expression. Tumor sphere and MTT assays were carried out to evaluate the self-renewal and chemo-resistance properties of gastric cancer cells. RESULTS: The expression of CD44 was greatly increased and miR-145 was decreased in gastric cancer cells that were highly enriched in cancer stem cells (CSCs). The results demonstrated that miR-145 regulated CD44 by targeting directly the CD44 3'-untranslated region (3'-UTR). In gastric cancer cells, overexpression of miR-145 repressed the activity of the CD44 3'-UTR, and disruption of miR-145/CD44 3'-UTR interactions abrogated the silencing effects. In addition, miR-145 inhibition stimulated CD44 3'-UTR activity and disruption of miR-145/CD44 3'-UTR interactions abrogated this stimulatory effect. Enforced CD44 expression greatly increased tumor sphere formation and chemo-resistance in gastric cancer cells. Furthermore, the inhibition of CSCs and the chemo-sensitivity of gastric cancer cells treated with miR-145 were significantly abrogated by overexpression of CD44. CONCLUSION: miR-145 targeting of CD44 plays critical roles in the regulation of tumor growth and chemo-resistance in gastric cancer.

[Relationship between the expression level of miR-29c and biological behavior of gastric cancer].

OBJECTIVE: To study the function and clinicopathological significance of RNA-29c (miR-29c) in the carcinogenesis and development of gastric cancer. METHODS: MicroRNA microarray was applied to assess the miRNAs expression profile of gastric cancer. Quantitative real-time PCR was used to detect the expression of miR-29c in 64 cases of gastric cancer tissues and corresponding normal gastric epithelium, as well as cell lines GES-1, BGC-823 and SGC-7901 cells. MTT assay and flow cytometry were applied to detect the effects of forced expression of miR-29c in gastric cancer BGC-823 cells including cell proliferation, apoptosis, cell cycle and drug sensitivity. Quantitative real-time PCR, Western blot and luciferase reporter assay were used to explore the targeted relationship between miR-29c and myeloid cell leukemia-1 (Mcl-1). RESULTS: Compared with normal gastric epithelium, seven microRNAs (miR-374b*, miRPlus-E1212, miR-338-5p, miR-297, miR-21, miR-135b, miR-18a) were significantly up-regulated more than 2-folds, and nine microRNAs (miR-29b-2*, miR-1260, miRPlus-E1241, miR-S1-5p, miR-148a, miR-29c, miR-647, miR-196b*, ebv-miR-BART5) were significantly down-reguated in gastric cancer tissues. The average expression level of miR-29c in gastric cancer tissues was 0.70 ± 0.34 and in corresponding normal epithelium was 1.00 ± 0.06 (P < 0.05). miR-29c expression was related to tumor size, lymph node metastasis, clinical stage, Laurén classification, Borrmann classification and Ming classification (P < 0.05). The poorer differentiation degree of gastric cell lines, the lower was miR-29c expression level (P < 0.05). Overexpression of miR-29c in gastric cancer BGC-823 cells suppressed cell proliferation, stimulated cell apoptosis, induced cell cycle arrest in S phase and increased the chemotherapy sensitivity to drug docetaxel (all were P < 0.05). The average expression level of Mcl-1 mRNA in gastric cancer tissues was 3.47 ± 1.34 and corresponding epithelialium was 1.00 ± 0.20 (P < 0.05). The expression level of miR-29c was negatively related with that of Mcl-1 mRNA in gastric cancer tissues. miR-29c directly targeted to regulation of Mcl-1 expression. CONCLUSIONS: There are special miRNA expression profile in gastric cancer. The expression of miR-29c is closely related to biological behavior of human gastric cancer. miR-29c is involved in targeted regulation of Mcl-1, and may be one of mechanisms of the carcinogenesis of gastric cancer.

[Clinicopathological significance and function of miR-135b in the occurrence and development of gastric cancer].

OBJECTIVE: To explore the function and clinicopathological significance of miR-135b in the occurrence and development of gastric cancer. METHODS: Seventy-two pairs of fresh gastric cancer tissues and corresponding normal gastric epithelium were collected at our department from September 2007 to March 2011. Six of them were randomly selected and miRNA microarray was applied to study the miRNA expression profiles of gastric cancer. Quantitative real-time PCR was used to validate the reliability of microarray and detect miR-135b expression in the above clinical samples, as well as cell lines GES-1, BGC-823 and SGC-7901. The methods of cell transfection, thiazolyl blue tetrazolium bromide (MTT) and flow cytometry were used to study the impact of miR-135 on cell proliferation, cell cycle and apoptosis of gastric cancer cells. Real-time quantitative PCR and Western blot were used to explore the relationship between miR-135b and adenomatous polyposis coli (APC). RESULTS: Compared with normal gastric tissues, 7 miRNA were significantly up-regulated and 9 miRNA significantly down-regulated for over 2 folds in gastric cancer tissues (P < 0.05). The results of microarray were verified by quantitative real-time PCR. MiR-135b expression was up-regulated in most clinical samples compared with their corresponding epithelium (n = 66, 91.67%). The average expression level of miR-135b in gastric cancer tissues was significantly higher than normal epithelium (3.42 ± 2.62 vs 1.00 ± 0.07, P < 0.05). MiR-135b was related to Laurén classification, tumor differentiation, invasion and pathologic tumor-node-metastasis (pTNM) stage of gastric cancer (all P < 0.05). The worse differentiation degree of gastric cell lines, the higher miR-135b expression level (P < 0.05). MiR-135b promoted gastric cancer cell proliferation, inhibited its apoptosis and directly regulated the expression of APC in gastric cancer cell. CONCLUSIONS: Special miRNA expression profiles of gastric cancer are found. MiR-135b is closely correlated with the biological behavior of human gastric cancer. And its regulation of APC may be one of the pathogenic mechanisms of gastric cancer.

[Evaluation of clinical effect of hysterectomy preserving the uterine artery].

OBJECTIVE: To discuss the clinical feasibility of hysterectomy that preserves the ascending branch and the ovarian branch of the uterine artery, and the effect on the ovary. METHODS: Fifty-two women who received hysterectomy with preservation of the ovaries, the so called divest due to benign gynecological disease were selected as the study group. It consisted of 35 patients who received subtotal-divest and 17 patients who received total-divest. Meanwhile, 38 cases who received traditional hysterectomy served as the control group. Two milliliter venous blood was taken and estradiol, follicle stimulating hormone (FSH), luteinizing hormone (LH), prolactin (PRL) were measured at 1 month preoperation, 1 month, 6 months, and 12 months postoperation from 30 women with age ranging from 36 - 45 years of the two groups each. All the patients in the study group received ultrasonography via vagina at 1 month preoperation and postoperation, respectively. RESULTS: Postoperatively, through vagina color ultrasound examination, in the 52 cases of study group, bilateral uterine arteries were not retained in 1 case, and unilateral uterine artery was not retained in 6 cases. The postoperative peak blood flow of uterine artery during systolic phase (Va) was (20 +/- 9) cm/s, and during diastolic phase (Vb) was (3.8 +/- 3.2) cm/s. Compared with the preoperative values, (29 +/- 8) cm/s, (7.1 +/- 3.4) cm/s, the velocity of blood was slowed obviously (P < 0.01). The postoperative resistance index (RI) (0.79 +/- 0.08), was not significantly different compared with the preoperative value (0.80 +/- 0.08) (P > 0.05). The operative time, bleeding volume, and the postoperative exhaust time had no significant difference between the two groups (P > 0.05). Comparing the hormone assay of two groups, there was a significant change at 6 months postoperation in the control group, but postoperation hormone assay had no significant change compared with the pre-operative result in study group. It indicated that the new approach did not affect the ovarian functions. CONCLUSION: The divest is applicable in clinics, and it can preserve the completeness of blood-supply and the function of the ovary.