MicroRNA-148a-3p enhances cisplatin cytotoxicity in gastric cancer through mitochondrial fission induction and cyto-protective autophagy suppression.
ABSTRACT: Cisplatin (CDDP) resistance is a major clinical problem associated with poor prognosis in gastric cancer (GC) patients. In this study, we performed integrated analysis of TCGA data from microRNAs (miRNAs) expression matrix of GC patients who received CDDP-based chemotherapy with GEO dataset which contains differential miRNAs expression profiles in CDDP-resistant and -sensitive cell lines. We identified miR-148a-3p downregulation as a key step involved in CDDP resistance. Using a cohort consisting 105 GC patients who received CDDP-based therapy, we found that miR-148a-3p downregulation was associated with a decrease in patients' disease-free survival (DFS, P = 0.0077). A series of experiment data demonstrated that: 1) miR-148a-3p was downregulated in CDDP-resistant GC cell lines; 2) miR-148a-3p reconstitution sensitized CDDP-resistant cells to CDDP treatment through promoting mitochondrial fission and decreasing AKAP1 expression level; 3) AKAP1 played a novel role in CDDP resistance by inhibiting P53-mediated DRP1 dephosphorylation; 4) miR-148a-3p reconstitution in CDDP-resistant cells inhibits the cyto-protective autophagy by suppressing RAB12 expression and mTOR1 activation. Taken together, our study demonstrates that miR-148a-3p could be a promising prognostic marker or therapeutic candidate for overcoming CDDP resistance in GC.
Project description:BACKGROUND:Resistance towards chemotherapy is a major obstacle in the treatment of esophageal squamous cell carcinoma (ESCC). We investigated the role of specific microRNAs in chemotherapy resistance and tumor biology. METHODS:We selected three microRNAs from characteristic microRNA signatures of resistant ESCC (hsa-miR-125a-5p, hsa-miR-130a-3p, hsa-miR-1226-3p), and hsa-miR-148a-3p. Effects on chemotherapy, adhesion, migration, apoptosis and cell cycle were assessed in six ESCC cell lines. Target analyses were performed using Western blotting and luciferase techniques. RESULTS:MiR-130a-3p sensitized cells towards cisplatin in 100% of cell lines, miR-148a-3p in 83%, miR-125a-5p in 67%, miR-1226-3p in 50% (p ? 0.04). MiR-130a-3p sensitized 83% of cell lines towards 5-FU, miR-148a-3p/miR-125a-5p/miR-1226-3p only 33% (p ? 0.015). Several resistance-relevant pathways seem to be targeted on various levels. Bcl-2 was confirmed as a direct target of miR-130a-3p and miR-148a-3p, and p53 as a target of miR-125a-5p. All microRNAs decreased migration and adhesion, except miR-130a-3p, and increased apoptosis. Simultaneous manipulation of two microRNAs exhibited additive sensitizing effects towards cisplatin in 50% (miR-125a-5p/miR-148a-3p), and 75% (miR-148a-3p/miR-130a-3p) of cell lines (p ? 0.016) [corrected] CONCLUSION:Our data present strong evidence that specific microRNA signatures are responsible for drug resistance and aggressiveness of ESCC. Final functional readout of these complex processes appears to be more important than single microRNA-target interactions.
Project description:Background Cisplatin (CDDP) is the first-line chemotherapy for gastric cancer (GC). The poor prognosis of GC patients is partially due to the development of CDDP resistance. Circular RNAs (circRNAs) are a subclass of noncoding RNAs that function as microRNA (miRNA) sponges. The role of circRNAs in CDDP resistance in GC has not been evaluated. Methods RNA sequencing was used to identify the differentially expressed circRNAs between CDDP-resistant and CDDP-sensitive GC cells. qRT-PCR was used to detect the expression of circMCTP2 in GC tissues. The effects of circMCTP2 on CDDP resistance were investigated in vitro and in vivo. Pull-down assays and luciferase reporter assays were performed to confirm the interactions among circMCTP2, miR-99a-5p, and myotubularin-related protein 3 (MTMR3). The protein expression levels of MTMR3 were detected by western blotting. Autophagy was evaluated by confocal microscopy and transmission electron microscopy (TEM). Results CircMCTP2 was downregulated in CDDP-resistant GC cells and tissues compared to CDDP-sensitive GC cells and tissues. A high level of circMCTP2 was found to be a favorable factor for the prognosis of patients with GC. CircMCTP2 inhibited proliferation while promoting apoptosis of CDDP-resistant GC cells in response to CDDP treatment. CircMCTP2 was also found to reduce autophagy in CDDP-resistant GC cells. MiR-99a-5p was verified to be sponged by circMCTP2. Inhibition of miR-99a-5p could sensitize GC cells to CDDP. MTMR3 was confirmed to be a direct target of miR-99a-5p. Knockdown of MTMR3 reversed the effects of circMCTP2 on the proliferation, apoptosis and autophagy of CDDP-resistant GC cells. CircMCTP2 was also confirmed to inhibit CDDP resistance in vivo in a nude mouse xenograft model. Conclusions CircMCTP2 sensitizes GC to CDDP through the upregulation of MTMR3 by sponging miR-99a-5p. Overexpression of CircMCTP2 could be a new therapeutic strategy for counteracting CDDP resistance in GC. Supplementary Information The online version contains supplementary material available at 10.1186/s13046-020-01758-w.
Project description:BACKGROUND:MicroRNAs (miRNAs) are widely studied non-coding RNAs that modulate gene expression. MiRNAs are deregulated in different tumors including gastric cancer (GC) and have potential diagnostic and prognostic implications. The aim of our study was to determine miRNA profile in GC tissues, followed by evaluation of deregulated miRNAs in plasma of GC patients. Using available databases and bioinformatics methods we also aimed to evaluate potential target genes of confirmed differentially expressed miRNA and validate these findings in GC tissues. METHODS:The study included 51 GC patients and 51 controls. Initially, we screened miRNA expression profile in 13 tissue samples of GC and 12 normal gastric tissues with TaqMan low density array (TLDA). In the second stage, differentially expressed miRNAs were validated in a replication cohort using qRT-PCR in tissue and plasma samples. Subsequently, we analyzed potential target genes of deregulated miRNAs using bioinformatics approach, determined their expression in GC tissues and performed correlation analysis with targeting miRNAs. RESULTS:Profiling with TLDA revealed 15 deregulated miRNAs in GC tissues compared to normal gastric mucosa. Replication analysis confirmed that miR-148a-3p, miR-204-5p, miR-223-3p and miR-375 were consistently deregulated in GC tissues. Analysis of GC patients' plasma samples showed significant down-regulation of miR-148a-3p, miR-375 and up-regulation of miR-223-3p compared to healthy subjects. Further, using bioinformatic tools we identified targets of replicated miRNAs and performed disease-associated gene enrichment analysis. Ultimately, we evaluated potential target gene BCL2 and DNMT3B expression by qRT-PCR in GC tissue, which correlated with targeting miRNA expression. CONCLUSIONS:Our study revealed miRNA profile in GC tissues and showed that miR-148a-3p, miR-223-3p and miR-375 are deregulated in GC plasma samples, but these circulating miRNAs showed relatively weak diagnostic performance as sole biomarkers. Target gene analysis demonstrated that BCL2 and DNMT3B expression in GC tissue correlated with their targeting miRNA expression.
Project description:BACKGROUND:Breast cancer is the leading cause of death among women. Cisplatin is an effective drug for breast cancer, but resistance often develops during long term chemotherapy. While the mechanism of chemotherapy resistance is still not fully understood. METHODS:Survival analyses of ATP7A and ATP7B were used to evaluate their effects on the development of Breast invasive carcinoma (BRCA). Immunostaining, flow cytometry, and IC50 assay were utilized to examine the effects of ATP7A-siRNA combined with cisplatin on apoptosis in breast cancer cells. Q-PCR, western blotting, and dual-luciferase assay were employed to confirm ATP7A is a novel target gene of miR-148a-3p. RESULTS:In this current study, we identified knocking-down ATP7A could enhance cytotoxicity treatment of cisplatin in breast cancer cells. We also demonstrated miR-148a-3p overexpression in BRCA cells increased the sensitivity to cisplatin, and subsequently enhanced DNA damage and apoptosis. Moreover, we found ATP7A is a novel target gene of miR-148a-3p. In brief, our results showed miR-148a could accelerate chemotherapy induced-apoptosis in breast cancer cells by inhibiting ATP7A expression. CONCLUSIONS:Our results highlight that inhibition of ATP7A is a potential strategy for targeting breast cancer resistant to cisplatin, and we provided an interesting method to compare the involvement of various genes in the assessment of cisplatin resistance.
Project description:BACKGROUND:Cisplatin (CDDP) treatment is one of the most predominant chemotherapeutic strategies for patients with gastric cancer (GC). A better understanding of the mechanisms of CDDP resistance can greatly improve therapeutic efficacy in patients with GC. Circular RNAs (circRNAs) are a class of noncoding RNAs whose functions are related to the pathogenesis of cancer, but, in CDDP resistance of GC remains unknown. METHODS:circAKT3 (hsa_circ_0000199, a circRNA originating from exons 8, 9, 10, and 11 of the AKT3 gene) was identified by RNA sequencing and verified by quantitative reverse transcription PCR. The role of circAKT3 in CDDP resistance in GC was assessed both in vitro and in vivo. Luciferase reporter assay, biotin-coupled RNA pull-down and fluorescence in situ hybridization (FISH) were conducted to evaluate the interaction between circAKT3 and miR-198. Functional experiments were measured by western blotting, a cytotoxicity assay, clonogenic assay and flow cytometry. RESULTS:The expression of circAKT3 was higher in CDDP-resistant GC tissues and cells than in CDDP-sensitive samples. The upregulation of circAKT3 in GC patients receiving CDDP therapy was significantly associated with aggressive characteristics and was an independent risk factor for disease-free survival (DFS). Our data indicated that circAKT3 promotes DNA damage repair and inhibits the apoptosis of GC cells in vivo and in vitro. Mechanistically, we verified that circAKT3 could promote PIK3R1 expression by sponging miR-198. CONCLUSIONS:circAKT3 plays an important role in the resistance of GC to CDDP. Thus, our results highlight the potential of circAKT3 as a therapeutic target for GC patients receiving CDDP therapy.
Project description:OBJECTIVE:Micro RNAs (miRNAs) are a class of non-coding regulatory RNAs. We performed a transcriptome-wide analysis of subcutaneous adipose tissue and in vitro studies to identify miRNAs and co-regulated target transcripts associated with insulin sensitivity (SI) and obesity in human. METHODS:We selected 20 insulin-resistant (IR, SI=2.0±0.7) and 20 insulin-sensitive (IS, SI=7.2±2.3) subjects from a cohort of 117 metabolically characterized non-diabetic Caucasians for comparison. RESULTS:After global profiling, 3 miRNAs had marginally different expressions between IR and IS subjects. A total of 14 miRNAs were significantly correlated with %fat mass, body mass index (BMI), or SI. The qRT-PCR validated the correlation of miR-148a-3p with BMI (r=-0.70, P=2.73X10(-6)). MiRNA target filtering analysis identified DNA methyltransferase 1 (DNMT1) as one of the target genes of miR-148a-3p. DNMT1 expression in adipose tissue was positively correlated with BMI (r=0.47, p=8.42X10(-7)) and was inversely correlated with miR-148a-3p (r=-0.34). Differentiation of SGBS preadipocytes showed up-regulation of miR-148a-3p and down-regulation of DNMT1 in differentiated adipocytes. After transfecting miR-148a-3p mimics into HeLa-S3 cells, DNMT1 was down-regulated, while transfection of adipose stem cells with miR-148a-3p inhibitor up-regulated DNMT1. CONCLUSIONS:Our results indicate that miR-148a-3pmediated regulation of DNMT1 expression may play a mechanistic role in obesity.
Project description:PURPOSE:Dysregulation of miR-148a-3p in gastric cancer was reported. However, the diagnostic potential and biological function of miR-148a-3p in gastric cancer progression is not fully studied. METHODS:Bioinformatics analysis and RT-qPCR assay were performed to analyze the expression of miR-148a-3p in gastric cancer tissues and plasma of gastric cancer patients. Receiver operating characteristic curve analysis was performed to analyze the diagnostic value of miR-148a-3p. In vitro proliferation, apoptosis, migration, invasion, sphere formation assay and Western blotting assay were performed to evaluate the biological function of miR-148a-3p in gastric cancer progression. RESULTS:miR-148a-3p was significantly down-regulated in both gastric cancer patients' tissue and plasma samples. Plasma miR-148a-3p showed promising efficacy for gastric cancer diagnosis. Overexpression of miR-148a-3p could inhibit the proliferative phenotype, metastatic phenotype, and cancer stem-like properties of gastric cancer cells. CONCLUSIONS:miR-148a-3p inhibits cancer progression and is a novel diagnostic biomarker for gastric cancer.
Project description:Although miR-148a-3p has been reported to function as a tumour suppressor in various cancers, the molecular mechanism of miR-148a-3p in regulating epithelial-to-mesenchymal transition (EMT) and stemness properties of pancreatic cancer (PC) cells remains to be elucidated. In the present study, we demonstrated that miR-148a-3p expression was remarkably down-regulated in PC tissues and cell lines. Moreover, low expression of miR-148a-3p was associated with poorer overall survival (OS) in patients with PC. In vitro, gain-of-function and loss-of-function experiments showed that miR-148a-3p suppressed EMT and stemness properties as well as the proliferation, migration and invasion of PC cells. A dual-luciferase reporter assay demonstrated that Wnt1 was a direct target of miR-148a-3p, and its expression was inversely associated with miR-148a-3p in PC tissues. Furthermore, miR-148a-3p suppressed the Wnt/?-catenin pathway via down-regulation of Wnt1. The effects of ectopic miR-148a-3p were rescued by Wnt1 overexpression. These biological functions of miR-148a-3p in PC were also confirmed in a nude mouse xenograft model. Taken together, these findings suggest that miR-148a-3p suppresses PC cell proliferation, invasion, EMT and stemness properties via inhibiting Wnt1-mediated Wnt/?-catenin pathway and could be a potential prognostic biomarker as well as a therapeutic target in PC.
Project description:miR-148a-3p downregulation has emerged as a critical factor in cancer progression yet, the underlying mechanisms of miR-148a-3p expression pattern and its function in bladder cancer remains to be elucidated. Here, we illustrate that miR-148a-3p is frequently downregulated in bladder cancer and that its expression may be regulated by DNA methylation. DNA methyltransferase 1 (DNMT1) and miR-148a-3p function in a positive feedback loop in bladder cancer. miR-148a-3p overexpression functions as a tumor suppressor in bladder cancer cells. miR-148a-3p inhibits bladder cancer cell proliferation and epithelial-mesenchymal transition (EMT) by regulating ERBB3/AKT2/c-myc and ERBB3/AKT2/Snail signaling. ERBB3, DNMT1 and AKT2 are downstream miR-148a-3p target genes. Furthermore, the miR-148a-3p/ERBB3/AKT2/c-myc signaling axis establishes a positive feedback loop in the regulation of bladder cancer. Taken together, our study demonstrates novel regulatory circuits involving miR-148a-3p/ERBB3/AKT2/c-myc and DNMT1 that controls bladder cancer progression, which may be useful in the development of more effective therapies against bladder cancer.
Project description:Gastric cancer (GC) has been classified as the fourth leading cause of cancer-related deaths worldwide. Due to their ability to suppress the expression of target genes, microRNAs (miRNAs) are listed as one of the key elements involved in the formation and development of tumors. This study was therefore conducted to investigate the effects of microRNA-135b (miR-135b) on cisplatin [ cis-diamminedichloroplatinum (CDDP)] resistance of GC cells through the MAPK signaling pathway by targeting mammalian ste20-like kinase 1 (MST1). A microarray-based gene expression analysis was performed to screen the GC-related differentially expressed genes. The 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide assay was performed to determine the sensitivity of GC cells to CDDP. The bioinformatics database and dual luciferase reporter gene assay were used to check whether MST1 was a direct target gene of miR-135b. GC cell lines were prepared with high CDDP resistance, after which they were cultured and transfected respectively, followed by the administration of transfected cells into nude mice and subsequent treatment with CDDP in an attempt to identify the underlying mechanisms and functions of miR-135b in relation to MST1 in GC progression. The results were highly indicative of the crucial role played by MST1 in the development of GC and the sensitivity of GC to CDDP. miR-135b was found to regulate MST1, which in turn had an impact on the development of GC. MKN28 was observed to be most sensitive to CDDP, whereas MKN45 presented with the poorest sensitivity to CDDP. Furthermore, the down-regulation of miR-135b resulted in inactivation of the MAPK signaling pathway; increased the expression of MST1 and Bax; and decreased expression of p-p38MAPK, p-ERK1/2, P-glycoprotein, p38MAPK, ERK1/2, multidrug resistance protein 1, multidrug resistance-associated protein 1, lung resistance-related protein, and Bcl-2, thus inhibiting CDDP resistance of GC cells. The down-regulation of miR-135b also restrained cell proliferation and induced the apoptosis rate of GC cells. In summary, the results of this study showed that the down-regulation of miR-135b induced apoptosis, and it inhibited proliferation and CDDP resistance of GC cells by inactivating the MAPK signaling pathway and increasing the expression of MST1.-Zhou, J., Chen, Q. Poor expression of microRNA-135b results in the inhibition of cisplatin resistance and proliferation and induces the apoptosis of gastric cancer cells through MST1-mediated MAPK signaling pathway.