Protein phosphatase 2A-B55? enhances chemotherapy sensitivity of human hepatocellular carcinoma under the regulation of microRNA-133b.
ABSTRACT: Hepatocellular carcinoma (HCC) remains a major public health problem worldwide. The identification of effective chemotherapeutic targets for advanced HCC patients is urgently required. In this study, we investigated the role of protein phosphatase 2A-B55? subunit (PP2A-B55?, encoded by the PPP2R2D gene) and related mechanisms affecting chemotherapy sensitivity of HCC.Experimental approaches for measuring the levels of PPP2R2D mRNA and B55? protein in HCC included bioinformatics analyses, quantitative real-time polymerase chain reaction (qRT-PCR), western blotting (WB), immunofluorescence and immunohistochemistry assays. Cell cycle, migration, colony formation, apoptosis, and cell proliferation assays in stable PPP2R2D-knockdown and -overexpression cell lines in vitro, and tumorigenicity assays in vivo, were performed to explore the function of B55? in cisplatin (cDDP) chemotherapy of HCC. Bioinformatics prediction, luciferase reporter assays, qRT-PCR, WB, and cell cycle analyses were used to reveal the regulatory relationship between microRNA-133b (miR-133b) and PPP2R2D expression. miR-133b mimic and inhibitor were used to elucidate the regulatory mechanism.Our studies showed that PPP2R2D expression was down-regulated in both HCC tumors and HCC cell lines. Treatment with cDDP increased the amount of B55? protein. Artificially increasing the expression of B55? counteracted cyclin-dependent kinase 1 activation, modulated transitions of the cell cycle, and increased the suppressive effect of cDDP on cell migration, colony formation, apoptosis, and proliferation in vitro and tumor growth in vivo, thus enhancing therapeutic efficiency. In contrast, knockdown of B55? partially inhibited the effect of cDDP chemotherapy. miR-133b was shown to regulate PPP2R2D expression by binding to the 3'-untranslated region of PPP2R2D mRNA. The miR-133b/PPP2R2D signaling pathway affects the effectiveness of cDDP chemotherapy.PP2A-B55?, regulated by miR-133b, enhances the sensitivity of HCC to cDDP chemotherapy. Our data indicate that PP2A-B55? might be a novel and attractive target for increasing chemotherapy sensitivity of HCC.
Project description:BackgroundLong noncoding RNA taurine upregulated 1 (TUG1) has been reported to play an important role in human cancers. However, little is known about the role of TUG1 in drug resistance and its mechanism in tongue squamous cell carcinoma (TSCC).MethodsTwenty-one cisplatin-sensitive or resistant TSCC patients were enrolled in this study. Cisplatin-resistant cells (SCC25/CDDP and CAL27/CDDP) were used for experiments in vitro. Transfection was performed using Lipofectamine 2000 transfection reagent. The levels of TUG1, microRNA-133b (miR-133b) and cysteine-X-cysteine chemokine receptor 4 (CXCR4) were measured by quantitative real-time polymerase chain reaction or western blot. The cisplatin resistance was investigated by cell viability, transwell invasion and apoptosis assays. The interactions among TUG1, miR-133b and CXCR4 were evaluated by luciferase reporter assay and RNA immunoprecipitation. Murine xenograft model was established using the stably transfected CAL27/CDDP cells.ResultsTUG1 expression was elevated in cisplatin-resistant TSCC tissues and cells compared with that in sensitive group and its knockdown inhibited cisplatin resistance to SCC25/CDDP and CAL27/CDDP cells. miR-133b was targeted via TUG1 and its overexpression suppressed cisplatin resistance. Moreover, CXCR4 was a target of miR-133b. CXCR4 silence repressed cisplatin resistance, which was reversed by miR-133b knockdown. The level of CXCR4 protein was decreased by inhibition of TUG1 and recuperated by miR-133b knockdown. Besides, interference of TUG1 attenuated tumor growth by regulating miR-133b and CXCR4 in vivo.ConclusionDownregulation of TUG1 impeded cisplatin resistance in TSCC-resistant cells by mediating miR-133b and CXCR4, indicating TUG1 as a promising target for TSCC chemotherapy.
Project description:BACKGROUND Multi-drug resistance is one of the major problems limiting the efficacy of cisplatin (CDDP) in treatment of hepatocellular carcinoma (HCC), and abnormal microRNA (miRNA) expression in drug-resistant cell lines plays an important role in liver cancer chemotherapy resistance. MATERIAL AND METHODS We established stable Hep3B and 97L HCC cell strains resistant to CDDP, both in vitro and in vivo. A combination of microRNA microarray and RT-qPCR experiments were used to screen differentially expressed miRNAs in HCC cell strains. A CCK-8 assay was carried out to detect and calculate the survival rates and relative inhibitory rates. Oligonucleotide transfection was used to confirm the regulatory function of the miRNA in HCC drug resistance. RESULTS The IC50 of Hep3B/CDDP(v), 97L/CDDP(v), Hep3B/CDDP(s), and 97L/CDDP(s) were significantly higher than that of their parental cells. Moreover, the doubling time of drug-resistant cells increased compared with their parent cells. MiRCURYTM LNA Array (v 16.0) high-throughput tests of resistant cell models and their parent cells showed that there were 5 downregulated microRNAs in the 4 drug-resistant cell lines, and we chose hsa-miR-33a-5p as our target for further study. Oligonucleotide transfection showed that miR-33a-5p overexpression increased the cisplatin sensitivity of Hep3B/CDDP(v) and 97L/CDDP(v) drug-resistant cells and reduced their resistance. Additionally, inhibition of miR-33a-5p expression reduced cisplatin sensitivity in Hep3B and 97L and increased their drug resistance. CONCLUSIONS This study confirmed that the most downregulated microRNA, miR-33a-5p, can mediate the cisplatin resistance of HCC cells, providing a new and feasible direction for research into combatting liver cancer chemotherapy resistance.
Project description:Accumulating studies reveal that aberrant microRNA (miRNA) expression can affect the development of chemotherapy drug resistance by modulating the expression of relevant target proteins. The aim of this study was to investigate the role of miR-133b in the development of drug resistance in ovarian cancer cells.We examined the levels of miR-133b expression in ovarian carcinoma tissues and the human ovarian carcinoma cell lines (A2780, A2780/DDP and A2780/Taxol, respectively). We determined the cell viability of these cell lines treated with cisplatin or paclitaxel in the presence or absence of miR-133b or anti-miR-133b transfection using the MTT assay. Reverse transcription polymerase chain reaction and Western blotting were used to assess the mRNA and protein expression levels of two drug-resistance-related genes: glutathione S-transferase (GST)-π and multidrug resistance protein 1 (MDR1). The dual-luciferase reporter assay was used to detect the promoter activity of GST-π in the presence and absence of miR-133b.The expression of miR-133b was significantly lower in primary resistant ovarian carcinomas than in the chemotherapy-sensitive carcinomas (P<0.05), and the same results were found in primary resistant ovarian cell lines (A2780/Taxol and A2780/DDP) compared to the chemotherapy-sensitive cell line (A2780; P<0.05). Following miR-133b transfection, four cell lines showed increased sensitivity to paclitaxel and cisplatin, while anti-miR-133b transfection reduced cell sensitivity to paclitaxel and cisplatin. Dual-luciferase reporter assay showed that miR-133b interacted with the 3'-untranslated region of GST-π. Compared to controls, the mRNA and protein levels of MDR1 and GST-π were downregulated after miR-133b transfection and upregulated after anti-miR-133b transfection.MicroRNA-133b may reduce ovarian cancer drug resistance by silencing the expression of the drug-resistance-related proteins, GST-π and MDR1. In future, the combination of miR-133b with chemotherapy agents may prevent the development of drug resistance in ovarian cancers.
Project description:<h4>Background</h4>Hepatocellular carcinoma (HCC) is one of the most common cancers with high incidence and mortality. However, the underlying mechanisms of HCC still remain unclear. Eukaryotic translation initiation factors (eIFs) have a substantial effect on tumor development. In this study, we were aimed to investigate the role of eukaryotic translation initiation factor 4 gamma 2 (EIF4G2) in HCC.<h4>Methods</h4>Western blot (WB) of 30 paired HCC tissues and tissue microarrays (TMAs) conducted by immunohistochemistry (IHC) in 89 paired HCC samples were performed to assess EIF4G2 expression. Clone formation, real-time cell analysis (RTCA), wound healing and transwell assays were adopted to evaluate the role of EIF4G2 on HCC cell proliferation, migration and invasion abilities. The function of EIF4G2 in HCC tumor growth was assessed in a xenograft nude mouse model in vivo. The regulation of EIF4G2 by miR-144-3p was performed by luciferase reporter assay and WB.<h4>Results</h4>The EIF4G2 protein was clearly upregulated in HCC tissues, and high EIF4G2 expression was closely related to HCC prognosis. EIF4G2 silencing could inhibit HCC cell growth and metastasis in vitro, and suppress tumorigenesis in vivo by repressing the ERK signaling pathway. The results of luciferase reporter assays, WB and IHC staining verified that EIF4G2 was negatively regulated by miR-144. And re-expression of EIF4G2 could partially reverse the inhibiting effect of miR-144 in HCC.<h4>Conclusion</h4>In summary, our study revealed the role of EIF4G2 in HCC development via the activation of the ERK pathway. We also found that EIF4G2 could be negatively regulated by the tumor suppressor miR-144. Our investigations indicated that EIF4G2 might be a promising therapeutic target in HCC.
Project description:BACKGROUND:A number of hepatocellular carcinoma (HCC) patients have developed resistance against transcatheter arterial chemoembolization (TACE) treatment. In this study, we aimed to develop a panel of microRNAs (miRs) biomarkers to predict clinical outcomes in HCC patients after TACE treatment. METHODS:The expression level of twenty miRs was evaluated in FFPE tissues collected from 33 HCC patients. We selected four differentially expressed miRs in TACE-responders versus non-responders and re-assessed their expression in 51 serum samples. The expressions of miRs associated with overall survival (OS), progression-free survival (PFS), and treatment outcomes were investigated. The diagnostic accuracy of these miRs in predicting patients' response to TACE was also evaluated. RESULTS:The baseline of miR-106b, miR-107 and miR-133b was significantly elevated (p?<?.001) in sera of TACE-responders while miR-26a was elevated (p?<?.001) in non-responders. miR-26a and miR-133b recorded the highest diagnostic performance as individual classifiers in response to TACE (AUC?=?1.0 and 100% sensitivity and specificity). Intriguingly, miR-133b distinguished complete responders from partial responders and non-responders (AUC???0.90). The PFS was improved (p?<?.05) in the high expression group of miR-31, miR-200b, miR-133b and miR-181a over their low expression group. CONCLUSION:Circulating miR-133b, miR-26a, miR-107 and miR-106 in serum are potential candidates to be utilized as prognostic biomarkers for predication of TACE treatment outcomes in HCC patients.
Project description:Osteosarcoma (OS) is a primary malignant tumor of bone tissue. Effective chemotherapy may improve the survival of patients with OS. MicroRNAs (miRs) serve significant roles in the regulatory function of tumorigenesis and chemosensitivity of different types of cancer. miR-22 has been revealed to inhibit the proliferation and migration of OS cells, as well as increasing their sensitivity to cisplatin (CDDP). The mechanisms of action behind the functions of miR-22 in OS drug resistance require investigation. Therefore, in the present study, the human OS cell lines (MG-63, U2OS, Saos2 and OS9901) and a drug-resistant cell line (MG-63/CDDP) were cultured. Cell proliferation, apoptosis and autophagy assays were performed to investigate the proliferation, apoptosis and autophagy of cell lines transfected with miR-22 mimic. Reverse transcription-quantitative polymerase chain reaction and western blot analysis were performed to investigate the expression levels of associated genes. The results revealed that miR-22 inhibited the proliferation of MG-63 cells and MG-63/CDDP cells, and enhanced the anti-proliferative ability of CDDP. miR-22 induced apoptosis and inhibited autophagy of MG-63 cells and MG-63/CDDP cells. Apoptosis-related genes, including caspase-3 and Bcl-2-associated X protein were upregulated, while B-cell lymphoma-2 was downregulated in both cell lines transfected with the miR-22 mimic. Autophagy protein 5, beclin1 and microtubules-associated protein 1 light chain 3 were downregulated in both cell lines transfected with miR-22 mimic. Furthermore, the in vitro and in vivo expression levels of metadherin (MTDH) in the OS/OS-CDDP-resistant models were downregulated following transfection with the miR-22 mimic. Therefore, the results of the present study suggested that miR-22 promoted CDDP sensitivity by inhibiting autophagy and inducing apoptosis in OS cells, while MTDH may serve a positive role in inducing CDDP resistance of OS cells.
Project description:BACKGROUND: MicroRNA-133b (miR-133b), which is a muscle-specific microRNA, has been reported to be downregulated in human colorectal carcinoma (CRC) when compared to adjacent non-tumor tissue. However, its diagnostic value and role in CRC have yet to be described. CXC chemokine receptor-4 (CXCR4), which participates in multiple cell processes such as cell invasion-related signaling pathways, was predicted to be a potential target of miR-133b. The aim of this study was to investigate the associations and functions of miR-133b and CXCR4 in CRC initiation and invasion. METHODS: Mature miR-133b and CXCR4 expression levels were detected in 31 tumor samples and their adjacent, non-tumor tissues from patients with CRC, as well as in 6 CRC cell lines, using real-time quantitative RT-PCR (qRT-PCR). Luciferase reporter assays and Western blots were used to validate CXCR4 as a putative target gene of miR-133b. Regulation of CXCR4 expression by miR-133b was assessed using qRT-PCR and Western blot analysis, and the effects of exogenous miR-133b and CXCR4 on cell invasion and migration were evaluated in vitro using the SW-480 and SW-620 CRC cell lines. RESULTS: A significant downregulation of miR-133b was observed in 93.55% of CRC tissues, and the expression of miR-133b was much lower in metastatic tumors (stage C and D, stratified by the Modified Dukes Staging System) than in primary tumors (stage A and B). In contrast, CXCR4 protein expression significantly increased in 52.63% of CRC samples, and increased CXCR4 expression in CRC was associated with advanced tumor stage. CXCR4 was shown to be a direct target of miR-133b by luciferase reporter assays, and transfection of miR-133b mimics inhibited invasion and stimulated apoptosis of SW-480 and SW-620 CRC cells. CONCLUSIONS: Our study demonstrated that downregulated miR-133b contributed to increased cell invasion and migration in CRC by negatively regulating CXCR4. These findings may be significant for the development of therapy target for CRC.
Project description:Recent studies have shown that long non-coding RNAs (lncRNAs) play a pivotal role in the pathogenesis and progression of hepatocellular carcinoma (HCC). However, the biological action and potential mechanism of liver cancer cell drug resistance have not been clearly clarified. In this study, lncRNAs were screened and differentially expressed in parental and cisplatin-resistant cell lines (HepG2 and HepG2/CDDP). A novel lncRNA, termed NRAL (Nrf2 regulation-associated lncRNA), was identified, and the initial results indicated that it was highly expressed in HepG2 cisplatin resistant cell lines compared to their parental counterparts. Functionally, NRAL depletion significantly enhanced CDDP-mediated cytotoxicity and apoptosis in two cisplatin-resistant HCC cell lines. Mechanistically, the results indicated that NRAL regulates Nrf2 expression through miR-340-5p serving as a competing endogenous RNA (ceRNA), thus influencing the CDDP-induced phenotype in HCC. Collectively, the present investigation suggest that the NRAL/miR-340-5p/Nrf2 axis mediates cisplatin resistance in HCC, which may provide novel targets for overcoming cisplatin resistance in hepatocellular carcinoma cells.
Project description:Hepatocellular carcinoma (HCC) is a lethal malignancy with poor prognosis and limited treatment options. Transarterial chemoembolization (TACE) using chemotherapy agents-doxorubicin and cisplatin-is an accepted treatment option for locally advanced hepatocellular carcinoma. In the current study, we analyzed the expression pattern of a selected panel of 94 miRNAs in archival samples that were collected prior to treatment from 15 Egyptian patients diagnosed with advanced hepatocelleular carcinoma. We observed an overall increase in miRNA expression in HCC samples compared with normal subjects. Out of 94 examined miRNAs, 53 were significantly upregulated while 3 miRNAs were downregulated in HCC samples compared to normal liver samples. Comparing the pretreatment miRNA expression profiles in HCC patients and the patients response to TACE treatment resulted in the identification of a set of 12 miRNAs that are significantly upregulated in nonresponders group. This miRNA panel includes miR-10a-1, miR-23a-1, miR-24, miR-26a, miR-27a, miR-30c, miR-30e, miR-106b, miR-133b, miR-199a, miR-199-3p, and miR-200b. Furthermore, we observed that a panel of 10 miRNAs was significantly associated with patients' survival status at 1 year. These results highlight the potential implications of pretreatment miRNAs expression profiling in prediction of the patients' response to TACE treatment in liver cancer.
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.