Novel role of miR-133a-3p in repressing gastric cancer growth and metastasis via blocking autophagy-mediated glutaminolysis.
ABSTRACT: BACKGROUND:Autophagy plays a crucial role in sustaining the homeostasis in various malignant diseases. It has also been reported to promote tumor development in multiple cancers. Glutaminolysis instead of Warburg Effect produce adequate ATP and provide nitrogen and carbon to replenish the TCA cycle which has been discovered to be a new energy source for tumor cells recently. By means of degrading intracellular particles including amino acids, nucleotides, fatty acids, sugars and aged organisms, autophagy can recycle the aforementioned particles into bioenergetics and biosynthesis pathways, finally favoring tumor cells. MicroRNA is a kind of noncoding RNA that regulates the targeting gene expression mostly at post-transcription level. Among these miRNAs, microRNA-133a-3p is reported to be a tumor suppressor in numerous cancers. METHODS:We characterized the down-regulated expression level of microRNA-133a-3p in gastric cancer via TCGA database. Subsequently, we verified the tumor suppressor role of microRNA-133a-3p in gastric cancer cells through a series biological function assay. We used immunofluorescence and transmission electron microscope to observe the negative effect of microRNA-133a-3p on autophagy and used dual-luciferase report assay to identify the candidate gene GABARAPL1 of microRNA-133A-3p.Then we used high performance liquid phase mass spectrometry and seahorse analysis to detect whether miR-133a-3p could block the glutaminolysis metabolism through autophagy. At last, we confirmed the tumor suppressor role of microRNA-133a-3p in vivo on PDX mice model. RESULTS:We demonstrated that microRNA-133a-3p overexpression could block the activation of autophagy to ruin the abnormal glutaminolysis and further inhibit the growth and metastasis of gastric cancer cells. We successfully proved gastric cancer cells can replenish glutaminolysis via autophagy and microRNA-133a-3p could block aforementioned pathway by targeting core autophagy participants GABARAPL1 and ATG13.We then verified the negative function of microRNA-133a-3p on autophagy-mediated glutaminolysis both in PDX model and human gastric cancer organoid model. CONCLUSIONS:MicroRNA-133a-3p targets GABARAPL1 to block autophagy-mediated glutaminolysis, further repressing gastric cancer growth and metastasis.
Project description:Increasing evidence has demonstrated that microRNA (miR)?133a?3p is an important regulator of hepatocellular carcinoma (HCC). In the present study, the diagnostic role of miR?133a?3p in HCC, and the potential functional pathways, were both explored based on publicly available data. Eligible microarray datasets were collected from NCBI Gene Expression Omnibus (GEO) database and ArrayExpress database. The data related to HCC and matched adjacent normal tissues were also downloaded from The Cancer Genome Atlas (TCGA). Published studies reporting the association between miR?133a?3p expression and HCC were reviewed from multiple databases. By combining the data derived from three sources (GEO, TCGA and published studies), the authors analyzed the comprehensive relationship between miR?133a?3p expression and clinicopathological features of HCC. Eventually, putative targets of miR?133a?3p in HCC were selected for further bioinformatics prediction. A total of eight published microarray datasets were gathered, and the pooled results demonstrated that the expression of miR?133a?3p in the tumor group was lower than that in normal groups [standardized mean difference (SMD)=?0.54; 95% confidence interval (CI), ?0.74 to ?0.35; P<0.001]. Consistently, the level of miR?133a?1 in HCC was reduced markedly compared to normal tissues (P<0.001) based on TCGA data, and the AUC value of low miR?133a?1 expression for HCC diagnosis was 0.670 (P<0.001). Furthermore, the combined SMD of all datasets (GEO, TCGA and literature) suggested that significant difference was observed between the HCC group and the normal control group, and lower miR?133a?3p expression in HCC group was noted (SMD=?0.69; 95% CI, ?1.10 to ?0.29; P=0.001). In addition, the authors discovered five key genes of the calcium signaling pathway (NOS1, ADRA1A, ADRA1B, ADRA1D and TBXA2R) that may probably be targeted by miR?133a?3p in HCC. The study reveals that miR?133a?3p may function as a tumor suppressor in HCC. The prospective novel pathways and key genes of miR?133a?3p could offer potential biomarkers for HCC; however, the predictions require further confirmation.
Project description:BACKGROUND:Breast cancer (BC) is the most common malignancy among females with dismal quality of life in patients. It has been proven that epigenetic factors, especially microRNAs, are involved in breast carcinogenesis and progression. This study aimed to assess the expression and clinical performances of a five-microRNA signature (miR-127-3p, miR-133a-3p, miR-155-5p, miR-199b-5p, and miR-342-5p) in breast cancer and adjacent normal tissues to identify a potential biomarker for BC and investigate the relationship between their expression and clinicopathological features of BC patients as well. METHODS:In this case-control investigation, we recruited 50 pairs of tumor and matched non-tumor surgical specimens from patients diagnosed with BC. Expression levels of miR-127-3p, miR-133a-3p, miR-155-5p, miR-199b-5p, and miR-342-5p were measured in BC and adjacent normal tissues by RT-qPCR. RESULTS:We found that miR-127-3p, miR-133a-3p, miR-199b-5p, and miR-342-5p were significantly down-regulated, while miR-155-5p was significantly up-regulated in BC tumor tissues compared with the corresponding adjacent normal tissues. The decreased expression of miR-127-3p, miR-133a-3p, miR-342-5p, and up-regulation of miR-155-5p showed a significant correlation with disease stage. We also found a significant down-regulation of miR-127-3p, miR-199b-5p, and miR-342-5p compared in HER-2-negative patients. Our results indicated that miR-155-5p had a higher expression level in HER-2-positive patients. Receiver operating characteristic (ROC) curve analysis demonstrated that all these five microRNAs can serve as potential biomarkers to distinguish between tumor and non-tumor breast tissue samples. CONCLUSIONS:The present findings suggested that dysregulation of this five-miRNA signature might be considered as a promising and functional biomarker for BC diagnosis.
Project description:The GABARAPL1 protein belongs to the ATG8 family whose members are involved in autophagy. Our laboratory previously demonstrated that GABARAPL1 associates with autophagic vesicles, regulates autophagic flux and acts as a tumor suppressor protein in breast cancer. In this study, we aimed to determine whether GABARAPL1 conjugation to autophagosomes is necessary for its tumor suppressive functions using the MCF-7 breast cancer cell line overexpressing GABARAPL1 or a G116A mutant, which is unable to be lipidated and associated to autophagosomes. We show that the G116A mutation impaired GABARAPL1 function in autophagosome/lysosome fusion and inhibited lysosome activity but did not alter MTOR and ULK1 activities or tumor growth in vivo. Our results demonstrate for the first time that GABARAPL1 plays different regulatory functions during early and late stages of autophagy, independently or not of its conjugation to autophagosomes, but its tumor suppressive function appeared to be independent of its conjugation to autophagic vesicles.
Project description:MicroRNA has been recently recognized as playing a prominent role in tumorigenesis and metastasis. Here, we report that miR-338-3p was epigenetically silenced in gastric cancer, and its down-regulation was significantly correlated with gastric cancer clinicopathological features. Strikingly, restoring miR-338-3p expression in SGC-7901 gastric cancer cells inhibited proliferation, migration, invasion and tumorigenicity in vitro and in vivo, at least partly through inducing apoptosis. Furthermore, we demonstrate the oncogene SSX2IP is a target of miR-338-3p. We propose that miR-338-3p functions as a tumor suppressor in gastric cancer, and the methylation status of its CpG island could serve as a potential diagnostic marker for gastric cancer.
Project description:Background:In this study, we investigated the molecular mechanisms of human long non-coding RNA (lncRNA) FYVE RhoGEF And PH Domain Containing 5 Antisense RNA 1 (FGD5-AS1) and its downstream epigenetic axis, human microRNA-153-3p (hsa-miR-153-3p)/Cbp/P300-interacting transactivator with Glu/Asp-rich carboxy-terminal domain 2 (CITED2) in human gastric cancer. Methods:Gastric cancer cell lines and clinical tumor samples were used to assess FGD5-AS1 expression levels. Lentivirus containing FGD5-AS1 small interfering RNA (sh-FGD5AS1) was applied to knockdown FGD5-AS1 expression. Cancer cells in vitro and in vivo proliferation, and 5-FU chemoresistance were assessed, respectively. Expressions of hsa-miR-153-3p/CITED2 were also assessed in FGD5-AS1-downregulated gastric cancer cells. Hsa-miR-153-3p was knocked down and CITED2 was upregulated to assess their direct functional correlations with FGD5-AS1 in gastric cancer. Results:Both gastric cancer cell lines and human tumor samples showed aberrant FGD5-AS1 upregulation. Lentiviral-induced FGD5-AS1 knockdown reduced cancer proliferation, 5-FU chemoresistance in vitro, and tumorigenicity in vivo. Hsa-miR-153-3p/CITED2 axis was confirmed to be downstream of FGD5-AS1 in gastric cancer. Hsa-miR-153-3p inhibition or CITED2 upregulation reversed the tumor-suppressing effects of FGD5-AS1 downregulation on gastric cancer proliferation and 5-FU chemoresistance. Conclusion:We demonstrated that FGD5-AS1 can regulate human gastric cancer cell functions, possibly through its downstream epigenetic axis of hsa-miR-153-3p/CITED2.
Project description:It has been discovered that miR-133a-3p acts as a tumor suppressor in bladder cancer (BC). Nevertheless, the function of miR-133a-3p in BC remains unclarified. Thus, we carried out this study to validate the expression of miR-133a-3p in BC and provide insights into the molecular mechanism underlying it. To assess the expression of miR-133a-3p in BC, we searched eligible studies from literature and Gene expression Omnibus (GEO) to perform a meta-analysis. We also plotted the summary receiver operating characteristic (SROC) curve to evaluate the diagnostic ability of miR-133a-3p in BC. Additionally, the potential target genes of miR-133a-3p were acquired from 14 online software programs and GEO database. Protein-protein interaction (PPI) network was created to identify the hub genes. Then, Gene Ontology (GO) functional annotation analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were carried out to investigate the regulatory network of the target genes. From the meta-analysis, miR-133a-3p was remarkably downregulated in BC tissues compared with that in non-cancer tissues (standard mean difference =-3.84, 95% confidence interval =-6.99-0.29). Moreover, results from SROC suggested that miR-133a-3p exhibited the ability to diagnose BC (area under curve =0.8418). As for the bioinformatics study, 488 genes were chosen as the potential targets of miR-133a-3p in BC, among which 10 genes were defined as hub genes (all degrees >5). Further GO and KEGG pathway analysis indicated that the target genes of miR-133a-3p aggregated in specific biological process and pathways. In conclusion, miR-133a-3p possessed great diagnostic potential with its downregulation in BC, and miR-133a-3p might serve as a novel biomarker for BC.
Project description:Bone metastasis is a leading cause of morbidity and mortality in advanced prostate cancer (PCa). Downexpression of miR-133a-3p has been found to contribute to the progression, recurrence and distant metastasis in PCa. However, clinical significance of miR-133a-3p in bone metastasis of PCa, and the biological role of miR-133a-3p and its molecular mechanisms underlying bone metastasis of PCa remain unclear.miR-133a-3p expression was evaluated in 245 clinical PCa tissues by real-time PCR. Statistical analysis was performed to evaluate the clinical correlation between miR-133a-3p expression and clinicopathological features, and overall and bone metastasis-free survival in PCa patients. The biological roles of miR-133a-3p in the bone metastasis of PCa were investigated both in vitro and in vivo. Bioinformatics analysis, real-time PCR, western blot and luciferase reporter analysis were applied to demonstrate the relationship between miR-133a-3p and its potential targets. Western blotting and luciferase assays were examined to identify the underlying pathway involved in the anti-tumor role of miR-133a-3p. Clinical correlation of miR-133a-3p with its targets was verified in human PCa tissues.miR-133a-3p expression is reduced in PCa tissues compared with the adjacent normal tissues and benign prostate lesion tissues, particularly in bone metastatic PCa tissues. Low expression of miR-133a-3p is significantly correlated with advanced clinicopathological characteristics and shorter bone metastasis-free survival in PCa patients by statistical analysis. Moreover, upregulating miR-133a-3p inhibits cancer stem cell-like phenotypes in vitro and in vivo, as well as attenuates anoikis resistance in vitro in PCa cells. Importantly, administration of agomir-133a-3p greatly suppresses the incidence of PCa bone metastasis in vivo. Our results further demonstrate that miR-133a-3p suppresses bone metastasis of PCa via inhibiting PI3K/AKT signaling by directly targeting multiple cytokine receptors, including EGFR, FGFR1, IGF1R and MET. The negative clinical correlation of miR-133a-3p with EGFR, FGFR1, IGF1R, MET and PI3K/AKT signaling activity is determined in clinical PCa tissues.Our results unveil a novel mechanism by which miR-133a-3p inhibits bone metastasis of PCa, providing the evidence that miR-133a-3p may serve as a potential bone metastasis marker in PCa, and delivery of agomir-133a-3p may be an effective anti-bone metastasis therapeutic strategy in PCa.
Project description:Circulating microRNAs have drawn a great deal of attention as promising novel biomarkers for breast cancer. However, to date, the results are mixed. Here, we performed a three-stage microRNA analysis using plasma samples from breast cancer patients and healthy controls, with efforts taken to address several pitfalls in detection techniques and study design observed in previous studies. In the discovery phase with 122 Caucasian study subjects, we identified 43 microRNAs differentially expressed between breast cancer cases and healthy controls. When those microRNAs were compared with published data from other studies, we identified three microRNAs, including miR-148b, miR-133a and miR-409-3p, whose plasma levels were significantly higher in breast cancer cases than healthy controls and were also significant in previous independent studies. In the validation phase with 50 breast cancer cases and 50 healthy controls, we validated the associations with breast cancer detection for miR-148b and miR-133a (P = 1.5×10-6 and 1.3×10-10, respectively). In the in-vitro study phase, we found that both miR-148b and miR-133a were secreted from breast cancer cell lines, showing their secretory potential and possible tumor origin. Thus, our data suggest that both miR-148b and miR-133a have potential use as biomarkers for breast cancer detection.
Project description:PURPOSE:Human papillomavirus (HPV) infected oropharyngeal squamous cell carcinoma (OPSCC) patients have a better prognosis compared to HPV(-) counterparts. However, a subset of HPV(+) patients with a smoking history fail to respond to the standard of care treatments such as radiation and chemotherapy. To understand the underlying mechanism driving HPV(+) OPSCC patient resistance to treatment and recurrence, we sought to identify and characterize the differentially expressed miRNAs and their target genes in HPV(+) smokers and non-smokers. EXPERIMENTAL DESIGN:MicroRNA expression analysis was performed using Nanostring in tumor tissues isolated from a prospective cohort of HPV(+) smoking (n = 9) and HPV(+) (n = 13) non-smoking OPSCC patients. Identified miRNAs of interest were further validated using qRT-PCR in cigarette smoke extract (CSE) treated HPV(+) and E6/E7 overexpressing HPV(-) cells. RESULTS:In comparison to OPSCC HPV(+) non-smokers, 38 miRNAs were significantly altered in the HPV(+) smoker patients cohort and out of that 9 were downregulated. Altered miRNA expression was also detected in the serum and metastatic lymph nodes of HPV(+) smokers versus non-smokers. Expression of miR-133a-3p was significantly downregulated in OPSCC smokers, HPV(+) cells and E6/E7 overexpressing HPV(-) cells treated with CSE. Reduction of miR-133a-3p induced the upregulation of miR-133a-3p target mRNAs EGFR and HuR. CONCLUSIONS:Our results indicate that miR-133a-3p is a target of smoking-induced changes in HPV(+) patients and alters the expression of EGFR and HuR which may promote HPV associated oropharyngeal cancer. Therefore, future treatment strategies for HPV(+) OPSCC smokers should focus on EGFR inhibition and the development of selective therapies to target HuR.
Project description:BACKGROUND:miR-133a-3p has been recently discovered to be down-regulated in various human malignancies, including breast cancer, and reduced miR-133a-3p levels have been significantly associated with breast cancer cell growth and invasion. However, the regulatory mechanisms leading to abnormal expression of miR-133a-3p in breast cancer remain obscure. METHODS:qRT-PCR was applied to detect the expression of miR-133a-3p in breast cancer tissues and cell lines. Bisulfite sequencing was used to detect the degree of methylation of the miR-133a-3p promoter. The effects of miR-133a-3p on breast cancer in vitro were examined by cell proliferation assay, transwell assay, flow cytometry, and western blotting. Bioinformatic analysis, dual-luciferase assay and RIP assay were employed to identify the interaction between miR-133a-3p and MAML1. A xenograft model was used to show the metastasis of breast cancer cells. RESULTS:We confirmed that miR-133a-3p was silenced by DNA hypermethylation in breast cancer cell lines and tissues, which predicted poor prognosis in breast cancer patients, and reducing miR-133a-3p expression led to a significant increase in the migration, invasion, proliferation, and stemness of breast cancer cells in vitro. Mastermind-like transcriptional coactivator 1 (MAML1) was confirmed to be a target of miR-133a-3p involved in regulating breast cancer metastasis both in vitro and in vivo. Moreover, a series of investigations indicated that MAML1 initiated a positive feedback loop, which could up-regulate DNA methyltransferase 3A (DNMT3A) to promote hypermethylation of the miR-133a-3p promoter. CONCLUSION:Taken together, our findings revealed a novel miR-133a-3p/MAML1/DNMT3A positive feedback loop in breast cancer cells, which may become a potential therapeutic target for breast cancer.