Protective role of circRNA CCND1 in ulcerative colitis via miR-142-5p/NCOA3 axis.

BACKGROUND: Increasing research indicates that circular RNAs (circRNAs) play critical roles in the development of ulcerative colitis (UC). This study aimed to determine the role of circRNA CCND1 in UC bio-progression, which has been shown to be downregulated in UC tissues. METHODS: Reverse transcription quantitative polymerase chain reaction was used to determine the levels of circRNA CCND1, miR-142-5p, and nuclear receptor coactivator-3 (NCOA3) in UC tissues and in lipopolysaccharide (LPS)-induced Caco-2 cells. Target sites of circRNA CCND1 and miR-142-5p were predicted using StarBase, and TargetScan to forecast potential linkage points of NCOA3 and miR-142-5p, which were confirmed by a double luciferase reporter-gene assay. Cell Counting Kit 8 and flow cytometry assays were performed to assess Caco-2 cell viability and apoptosis. TNF-α, IL-1ß, IL-6, and IL-8 were detected using Enzyme-Linked Immunosorbent Assay kits. RESULTS: CircRNA CCND1 was downregulated in UC clinical samples and LPS-induced Caco-2 cells. In addition, circRNA CCND1 overexpression suppressed LPS-induced apoptosis and inflammatory responses in Caco-2 cells. Dual-luciferase reporter-gene assays showed that miR-142-5p could be linked to circRNA CCND1. Moreover, miR-142-5p was found to be highly expressed in UC, and its silencing inhibited LPS-stimulated Caco-2 cell apoptosis and inflammatory responses. Importantly, NCOA3 was found downstream of miR-142-5p. Overexpression of miR-142-5p reversed the inhibitory effect of circRNA CCND1-plasmid on LPS-stimulated Caco-2 cells, and the effects of miR-142-5p inhibitor were reversed by si-NCOA3. CONCLUSION: CircRNA CCND1 is involved in UC development by dampening miR-142-5p function, and may represent a novel approach for treating UC patients.

Tanshinone IIA reverses oxaliplatin resistance in colorectal cancer through microRNA-30b-5p/AVEN axis.

This research aims to explore the role of Tanshinone IIA (Tan IIA) and microRNA (miR)-30b-5p in chemoresistance of colorectal cancer (CRC). The expression levels of miR-30b-5p and apoptosis and caspase activation inhibitor (AVEN) was detected by reverse transcription-quantitative polymerase chain reaction assay. The cell proliferation and apoptosis were examined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and flow cytometry assays. The target relationship between miR-30b-5p and AVEN was confirmed by Dual-luciferase reporter assay. Transwell assay was performed to assess CRC cells' metastasis. Western blot was carried out to measure the apoptosis-related protein. The results showed that miR-30b-5p was lowly expressed in oxaliplatin-resistance CRC cells SW480 (SW480/R) compared to SW480 cells. Overexpression of miR-30b-5p significantly suppressed the malignant biological behaviors of SW480/R cells and significantly promoted the sensitivity of SW480/R cells to oxaliplatin by down-regulated AVEN expression. Besides, Tan IIA treatment upregulated miR-30b-5p expression in SW480/R cells. Moreover, miR-30b-5p upregulation strengthened the promoting effect of Tan IIA on the sensitivity of SW480/R cells to oxaliplatin. In conclusion, Tan IIA and miR-30b-5p could reverse oxaliplatin resistance of CRC cells and may thus be potential treatment strategies for treating patients with CRC.

Inhibition of miR-96 enhances the sensitivity of colorectal cancer cells to oxaliplatin by targeting TPM1.

Colorectal cancer (CRC) is one of the major threats to human health worldwide. In the treatment of CRC, chemoresistance affects the efficacy of platinum-based therapies. Oxaliplatin is one of the most commonly used first-line medications for the treatment of CRC; however, chemoresistance is common among patients receiving oxaliplatin treatment, which significantly decreases its therapeutic efficacy. The present study focused on the roles of microRNA (miR)-96 in the oxaliplatin resistance of CRC cells and the underlying mechanisms. First, the expression of miR-96 was compared between CRC and adjacent tissues. Furthermore, target genes of miR-96 were predicted, and a dual-luciferase reporter assay was employed to confirm whether the candidate tropomyosin 1 (TPM1) is a direct target of miR-96. In addition, CRC cells were transfected with miR-96 inhibitor, miR-negative control, small interfering RNA (siRNA) targeting TPM1 or siRNA NC, and then treated with oxaliplatin. CCK-8 assay and flow cytometry were performed to examine the proliferation and apoptosis of the CRC cell line SW480. Next, reverse transcription-quantitative PCR and western blot analysis were performed to determine the mRNA and/or protein levels of miR-96, Bcl-2, BAX and TPM1. The results indicated that miR-96 was upregulated in CRC compared with normal adjacent tissues, while TPM1 was downregulated. The luciferase activity was reduced following transfection with miR-96 mimics and luciferase reporter plasmid containing the wild-type sequence of the 3'-untranslated region of TPM1. Furthermore, knockdown of miR-96 combined with oxaliplatin reduced the viability and induced apoptosis of CRC cells, which was further verified by decreased expression of Bcl-2 and the increased expression of TPM1 and BAX. Taken together, the downregulation of miR-96 enhanced the sensitivity of CRC cells to oxaliplatin.

Tanshinone IIA Reverses Oxaliplatin Resistance In Human Colorectal Cancer Via Inhibition Of ERK/Akt Signaling Pathway.

BACKGROUND: Oxaliplatin (OXA)-based chemotherapy is generally used to treat human cancers, whereas OXA resistance is a main obstacle for the treatment of colorectal cancer (CRC). Evidence has shown that tanshinone IIA (Tan IIA) could induce apoptosis in CRC cells. However, the role of combination of OXA and Tan IIA on OXA-resistance CRC cells remains unknown. Thus, this study aimed to investigate the effects of Tan IIA in combination with OXA on OXA-resistance CRC cells. METHODS: MTT assay, Ki67 immunofluorescence staining and flow cytometry were used to detect viability, proliferation and apoptosis in OXA-resistant cell line SW480/OXA, respectively. The expressions of Bcl-2, Bax, active caspase 3, p-Akt and p-ERK in SW480/OXA cells were detected with Western blot. In vivo animal study was performed finally. RESULTS: In this study, the inhibitory effects of OXA on the proliferation and invasion of SW480/OXA cells were significantly enhanced by Tan IIA. In addition, Tan IIA obviously enhanced the anti-apoptosis effects of OXA on SW480/OXA cells via decreasing the levels of Bcl-2, p-Akt and p-ERK, and increasing the levels of Bax and active caspase 3. In vivo experiments confirmed that Tan IIA enhanced OXA sensitivity in SW480/OXA xenograft model. CONCLUSION: We found that Tan IIA could reverse OXA resistance in OXA-resistance CRC cells. Therefore, OXA combined with Tan IIA might be considered as a therapeutic approach for the treatment of OXA-resistant CRC.

Therapeutic effect of protease-activated receptor 2 agonist SLIGRL-NH2 on loperamide-induced Sprague-Dawley rat constipation model and the related mechanism.

PURPOSE: To investigate the therapeutic effects of protease-activated receptor 2 (PAR-2) agonist SLIGRL-NH2 on loperamide-induced Sprague-Dawley (SD) rat constipation animal models. MATERIALS AND METHODS: Loperamide was injected subcutaneously to induce constipation twice a day for 3 days. SD rats (n = 30) were randomly divided into five groups: non-constipation group (control, n = 6), constipation group (constipation, n = 6), constipation + SLIGRL-NH2 low-dosage group (SLIGRL-NH2 low, n=6), constipation + SLIGRL-NH2 high-dosage group (SLIGRL-NH2 high, n = 6), and constipation + prucalopride (positive control, n = 6). The SLIGRL-NH2 low group and SLIGRL-NH2 high group were administered with 2.5 µmol/kg and 5 µmol/kg SLIGRL-NH2, respectively, and the prucalopride group received 2 mg/kg prucalopride. The control and constipation group received 1× PBS under the same pattern. SLIGRL-NH2 and prucalopride were orally administrated once daily for 7 days. On the final day of oral administration, food intake, water intake, the number of stool pellets, weight, and fecal water content was calculated; moreover, the colons of rats in different groups were collected and histological features were examined by hematoxylin and eosin staining; furthermore, the expression of anoctamin-1 was determined by Immunohistochemical methods, and the expressions of c-kit and PAR-2 were examined using real-time quantitative polymerase chain reaction and Western blot methods; finally, the expressions of neurotransmitter vasoactive intestinal peptide (VIP) and substance P (SP) were examined using enzyme-linked immuno-sorbent assay methods. RESULTS: The feeding and excretion behaviors, intestinal transit ratio, and the histological feature of the colon in the constipated rats were all improved by SLIGRL-NH2 treatment; moreover, SLIGRL-NH2 treatment induced significant increase in the expression of PAR-2 and also increased number of interstitial Cajal cells. Furthermore, SLIGRL-NH2 also decreased the contents of the inhibitory neurotransmitter VIP and increased the expression of the excitatory neurotransmitter SP. High dose of SLIGRL-NH2 has shown similar anti-constipation effects as prucalopride. CONCLUSION: These results suggested that SLIGRL-NH2 can enhance gastrointestinal transit and alleviate in rats with loperamide-induced constipation.