Use of recombinant microRNAs as antimetabolites to inhibit human non-small cell lung cancer

During the development of therapeutic microRNAs (miRNAs or miRs), it is essential to define their pharmacological actions. Rather, miRNA research and therapy mainly use miRNA mimics synthesized in vitro. After experimental screening of unique recombinant miRNAs produced in vivo, three lead antiproliferative miRNAs against human NSCLC cells, miR-22-3p, miR-9-5p, and miR-218-5p, were revealed to target folate metabolism by bioinformatic analyses. Recombinant miR-22-3p, miR-9-5p, and miR-218-5p were shown to regulate key folate metabolic enzymes to inhibit folate metabolism and subsequently alter amino acid metabolome in NSCLC A549 and H1975 cells. Isotope tracing studies further confirmed the disruption of one-carbon transfer from serine to folate metabolites by all three miRNAs, inhibition of glucose uptake by miR-22-3p, and reduction of serine biosynthesis from glucose by miR-9-5p and -218-5p in NSCLC cells. With greater activities to interrupt NSCLC cell respiration, glycolysis, and colony formation than miR-9-5p and -218-5p, recombinant miR-22-3p was effective to reduce tumor growth in two NSCLC patient-derived xenograft mouse models without causing any toxicity. These results establish a common antifolate mechanism and differential actions on glucose uptake and metabolism for three lead anticancer miRNAs as well as antitumor efficacy for miR-22-3p nanomedicine, which shall provide insight into developing antimetabolite RNA therapies.

Effect of miR-22 Targeting FMNL2 on Cell Migration and Apoptosis in Childhood Acute Myeloid Leukemia / 中国实验血液学杂志

OBJECTIVE@#To investigate the effect of miR-22 targeting formin-like protein 2 (FMNL2) on the migration and apoptosis of childhood acute myeloid leukemia (AML) cells.@*METHOD@#Peripheral blood samples from 11 children with AML, 10 children with immune thrombocytopenia, human AML cell lines TF-1a, HL-60, THP-1 and human bone marrow stromal cells HS-5 were used as the research objects. UniCel DxH 800 automatic hematology analyzer detected platelet count, hemoglobin, and white blood cell count in peripheral blood samples, and RT-qPCR detected miR-22 expression in peripheral blood samples and AML cells. HL-60 cells were transfected with LipofectamineTM 2000 kit, the experiments were divided into seven groups: blank (no cells transfected), miR-NC, miR-22 mimics, si-NC, si-FMNL2 , miR-22 mimics+OE-NC and miR-22 mimics+OE-FMNL2 . RT-qPCR was used to detect the expression of miR-22 in each group. Transwell was used to detect cell migration. Flow cytometry was used to detect cell apoptosis. Dual-luciferase reporter gene detection experiments verified the targeting relationship between miR-22 and FMNL2 . Western blot was used to detect the expression of FMNL2 protein.@*RESULTS@#Compared with the control group, the number of leukocytes in the peripheral blood of children with AML was significantly increased (P <0.001), while the concentration of hemoglobin and the number of platelets were significantly decreased P <0.001). The expression level of miR-22 in peripheral blood of children with AML was significantly lower than that in control group (P <0.001). Compared with HS-5 cells, the expression levels of miR-22 in TF-1a, HL-60, and THP-1 cells were significantly decreased (P <0.05), and in HL-60 cells was the lowest. Therefore, HL-60 cells were selected for subsequent experiments. Up-regulation of miR-22 or silencing of FMNL2 could reduce the number of migrating cells and increase apoptosis rate (P <0.05). MiR-22 targeted and negatively regulated the expression of FMNL2 . FMNL2 overexpression reversed the effects of up-regulated miR-22 on migration and apoptosis of HL-60 cells.@*CONCLUSION@#MiR-22 can inhibit the migration and promote apoptosis of HL-60 cells by down regulating the expression of FMNL2 .

Pan-cancer analysis of the expression pattern of long non-coding RNA MIR22HG / 南方医科大学学报

OBJECTIVE@#To conduct a pan-cancer analysis of the expression of long non-coding RNA (lncRNA) MIR22HG and explore its association with clinical characteristics.@*METHODS@#We analyzed the expression of MIR22HG in different tumors and its association with clinical staging, lymph node metastasis, tumor mutation burden (TMB) and microsatellite instability (MSI) using R package based on the Cancer Genome Atlas (TCGA) datasets. The relationship between MIR22HG expression and infiltrating immune cells was analyzed using TIMER algorithm. The association of MIR22HG gene alteration frequency with the clinical outcomes was examined using cBioPortal online software. Data form Genomics of Drug Sensitivity in Cancer (GDSC) were used to analyze the relationship between MIR22HG and the sensitivity of chemotherapy drugs. We specifically analyzed MIR22HG expression in hepatocellular carcinoma (HCC) and its correlation with sorafenib treatment using GEO database and verified the results in 12 pairs of HCC specimens. Kaplan-Meier analysis was performed to analyze the correlation of MIR22HG with the outcomes of sorafenib treatment. We also tested the effects of MIR22HG overexpression and knockdown on IC50 of sorafenib in HCC cells.@*RESULTS@#MIR22HG was downregulated in most tumors (P < 0.05), where its deletion mutations were frequent, and associated with a poor prognosis (P < 0.05). In many tumors, MIR22HG expression level was correlated with clinical stage, lymph node metastasis, TMB, MSI, immune cell infiltration, immune checkpoint-related genes, and sensitivity to common chemotherapeutic drugs (P < 0.05). Among the 6 common infiltrating immune cells in cancers, neutrophil infiltration had the strongest correlation with MIR22HG expression level, especially in breast cancer, rectal cancer and kidney renal papillary cell carcinoma (P < 0.05). MIR22HG was downregulated in HCC in association with HCC progression (P < 0.05). In HCC patients, a low MIR22HG expression was associated with a favorable outcome after sorafenib treatment (HR=2.94, P=0.075) and was capable of predicting the response to sorafenib treatment (AUC=0.8095). Compared with the negative control, MIR22HG overexpression obviously reduced sorafenib sensitivity (with IC50 of 7.731 vs 15.61) while MIR22HG knockdown increased sorafenib sensitivity of HCC cells (with IC50 of 7.986 vs 5.085).@*CONCLUSION@#MIR22HG expression level is correlated with clinical stage, lymph node metastasis, TMB, MSI, immune cell infiltration, and chemosensitivity in most cancer, suggesting its potential as an immunotherapeutic target and also a prognostic biomarker for tumors.

Effect of recombinant adenovirus Ad-mir-22 on glucose uptake in HepG2 cells / 生物工程学报

The recombinant adenoviruses expressing miR-22 (Ad-miR-22) was constructed and the effect of Ad-miR-22 on insulin signal pathway and glucose uptake in HepG2 cells was analyzed. MiR-22 gene was amplified by PCR from human hepatocytes and cloned into the pAdTrack-CMV vector to generate the shuttle plasmid pAdT-22. The positive colonies were confirmed by PCR and sequencing. The resultant shuttle plasmid was linearized with Pme I, followed by co-transformation into competent BJ5183 cells containing an adenoviral backbone plasmid (pAdEasy-1) to create the recombinant plasmid pAd-miR-22. After digested with Pac I, the linearized pAd-miR-22 was transfected into 293A packaging cell line to generate recombinant adenoviruses Ad-miR-22. HepG2 cells were infected with Ad-miR-22 or control Ad-GFP (adenoviruses expressing green fluorescent protein), and then the miR-22 expression levels were analyzed by qPCR. The result shows that adenovirus-mediated overexpression of miR-22 significantly decreased insulin-induced glucose uptake in HepG2 cells. Moreover, overexpression of miR-22 markedly decreased insulin-induced phosphorylation of GSK-3β. miR-22 also increased the mRNA levels of gluconeogenic genes in HepG2 cells. Furthermore, Western blotting results indicate that the protein expression of SIRT1 decreased in Ad-miR-22 infected HepG2 cells as compared with Ad-GFP infected HepG2 cells. In summary, overexpressing of miR-22 significantly increased gluconeogenesis while decreased glucose uptake in HepG2 cells. The effect of miR-22 on glucose metabolism may be mediated by SIRT1.

The subsequent biological effects of simulated microgravity on endothelial cell growth in HUVECs / 中华创伤杂志(英文版)

<p><b>PURPOSE</b>Microgravity is known to cause endothelium dysfunction in astronauts returning from spaceflight. We aimed to reveal the regulatory mechanism in alterations of human endothelial cells after simulated microgravity (SMG).</p><p><b>METHODS</b>We utilized the rotary cell culture system (RCCS-1) to explore the subsequent effects of SMG on human umbilical vein endothelial cells (HUVECs).</p><p><b>RESULTS</b>SMG-treated HUVECs appeared obvious growth inhibition after return to normal gravity, which might be attributed to a set of responses including alteration of cytoskeleton, decreased cell adhesion capacity and increased apoptosis. Expression levels of mTOR and its downstream Apaf-1 were increased during subsequent culturing after SMG. miR-22 was up-regulated and its target genes SRF and LAMC1 were down-regulated at mRNA levels. LAMC1 siRNAs reduced cell adhesion rate and inhibited stress fiber formation while SRF siRNAs caused apoptosis.</p><p><b>CONCLUSION</b>SMG has the subsequent biological effects on HUVECs, resulting in growth inhibition through mTOR signaling and miR-22-mediated mechanism.</p>

5-Fluorouracil suppresses human hepatocellular carcinoma cell growth via promoting miR-22 expression / 基础医学与临床

Objective To investigate the impact of 5-fluorouracil (5-FU) on miR-22 expression in human hepato-cellular carcinoma (HCC) cell lines and to elucidate the molecular mechanism of 5-fluorouracil for HCC chemo-therapy. Methods Real-time PCR analysis was conducted to determine the expression levels of miR-22 in HCC tissue specimens and HCC cell lines. The expression of miR-22 and pri-miR-22 (primary miR-22) was evaluated in HepG2 and Huh7 cells with 5-FU treatment by using real-time PCR and we also performed Western blot analysis to detect the protein level of HDAC4 in HCC cells with the same treatment. A rescue assay was employed by using 5-FU treatment in combination with miR-22 inhibitor(Anti-22) to further investigate the correlation among 5-FU, miR-22,and HCC cell growth. Results miR-22 expression depicted a significant downregulation in HCC tissues and cell lines (P<0.01). 5-FU treatment led to an augment of miR-22 expression in HepG2 and Huh7 cells(P<0.001) and resulted in a decrease of HDAC4 protein levels, which was verified as a direct target of miR-22 in HCC cells (P<0.01). Conclusions 5-FU has suppressive effect on HCC growth which could be potentially ex-plained by miR-22-mediated HDAC4 axis.

5-Fluorouracil suppresses human hepatocellular carcinoma cell growth via promoting miR-22 expression / 基础医学与临床

Objective To investigate the impact of 5-fluorouracil (5-FU) on miR-22 expression in human hepato-cellular carcinoma (HCC) cell lines and to elucidate the molecular mechanism of 5-fluorouracil for HCC chemo-therapy. Methods Real-time PCR analysis was conducted to determine the expression levels of miR-22 in HCC tissue specimens and HCC cell lines. The expression of miR-22 and pri-miR-22 (primary miR-22) was evaluated in HepG2 and Huh7 cells with 5-FU treatment by using real-time PCR and we also performed Western blot analysis to detect the protein level of HDAC4 in HCC cells with the same treatment. A rescue assay was employed by using 5-FU treatment in combination with miR-22 inhibitor(Anti-22) to further investigate the correlation among 5-FU, miR-22,and HCC cell growth. Results miR-22 expression depicted a significant downregulation in HCC tissues and cell lines (P<0.01). 5-FU treatment led to an augment of miR-22 expression in HepG2 and Huh7 cells(P<0.001) and resulted in a decrease of HDAC4 protein levels, which was verified as a direct target of miR-22 in HCC cells (P<0.01). Conclusions 5-FU has suppressive effect on HCC growth which could be potentially ex-plained by miR-22-mediated HDAC4 axis.

miR-22 suppresses tumorigenesis and improves radiosensitivity of breast cancer cells by targeting Sirt1

BACKGROUND: miR-22 has been shown to be frequently downregulated and act as a tumor suppressor in multiple cancers including breast cancers. However, the role of miR-22 in regulating the radioresistance of breast cancer cells, as well as its underlying mechanism is still not well understood. METHODS: The expressions of miR-22 and sirt1 at mRNA and protein levels were examined by qRT-PCR and Western Blot. The effects of miR-22 overexpression and sirt1 knockdown on cell viability, apoptosis, radiosensitivity, γ-H2AX foci formation were evaluated by CCK-8 assay, flow cytometry, colony formation assay, and γ-H2AX foci formation assay, respectively. Luciferase reporter assay and qRT-PCR analysis were performed to confirm the interaction between miR-22 and sirt1. RESULTS: miR-22 was downregulated and sirt1 was upregulated at both mRNA and protein levels in breast cancer cells. miR-22 overexpression or sirt1 knockdown significantly suppressed viability, induced apoptosis, reduced survival fraction, and increased the number of γ-H2AX foci in breast cancer cells. Sirt1 was identified as a target of miR-22 and miR-22 negatively regulated sirt1 expression. Ectopic expression of sirt1 dramatically reversed the inhibitory effect of miR-22 on cell viability and promotive effect on apoptotic rates and radiosensitivity in breast cancer cells. CONCLUSIONS: miR-22 suppresses tumorigenesis and improves radiosensitivity of breast cancer cells by targeting sirt1, providing a promising therapeutic target for breast cancer.

MiR-22-3p suppresses cell proliferation by targeting AEG-1 in NSCLC / 实用医学杂志

Objective To investigate the expression and effect of miR-22-3p in non-small cell lung cancer (NSCLC). Methods The miR-22-3p expression level in seventy-six NSCLC tissues and para-cancer tissues was detected by qRT-PCR. The relationship between the expression of miR-22-3p and gender,age,tumor size,histolo-gy grade,pathological type and lymph node metastasis was analyzed. The function of miR-22-3p on the prolifera-tion of NSCLC cells was tested by growth curve assay. Target genes of miR-22-3p were predicted by online software Targetscan. Luciferase reporter assay and qRT-PCR was used to certificate the prediction. Results The expression of miR-22-3p was increased in NSCLC tissues than the para-cancer tissues and was correlated to lymph node metas-tasis. Overexpression of miR-22-3p could suppress the proliferation of A549 cells. Astrocyte-Elevated Gene-1(AEG-1) was predicted to be a target of miR-22-3p. MiR-22-3p was revealed to bind to AEG-13′UTR by luciferase report-er assay. Overexpression of miR-22-3p could inhibit the expression of AEG-1 in A549 cells. Suppression of miR-22-3p could increase AEG-1 expression. Conclusion MiR-22-3p could inhibit the proliferation of NSCLC by tar-geting AEG-1.

Up-regulation of miR-22 through Wnt pathway suppresses proliferation, migration and invasion in human gastric MGC803 cells by DADS / 中国药理学通报

Aim To investigate the up-regulation of miR-22 through Wnt pathway inhibits the proliferation,migration and invasion in human gastric MGC803 cells induced by diallyl disulfide(DADS).Methods The effects of proliferation,migration,and invasion of gastric cancer cells were evaluated by MTT,wound-healing and invasion assays.Online prediction software was applied to search the target gene of miR-22.Luciferase report gene assay was used to assess the target genes Wnt-1 of miR-22.The expressions of Wnt-1,β-catenin and TCF-4 were tested by qRT-PCR and Western blot,respectively.Results MTT showed that DADS and miR-22 notably decreased the proliferation compared with control group(P<0.05).Wound-healing assay showed that DADS and miR-22 could significantly inhibit the migration of MGC803 cells compared with the control group, especially in miR-22+DADS(P<0.05). Invasion assay showed that DADS and miR-22 could markedly inhibit the invasion of MGC803 cells compared with the control group, especially in miR-22+DADS(P<0.05). Online prediction software to search the target gene exhibited that Wnt-1 may be a target gene of miR-22. Luciferase report gene assay disclosed that Wnt-1 was identified as a direct target of miR-22. Qrt-PCR showed that the expression of Wnt-1 Mrna was respectively down-regulated by DADS and miR-22 compared withcontrol group, especially in miR-22+DADS(P<0.05). Western blot exhibited that DADS and miR-22 obviously suppressed the expressions of Wnt-1, β-catenin and TCF-4 proteins, especially in miR-22+DADS(P<0.05).Conclusion Up-regulation of miR-22 through Wnt pathway can remarkably suppress the proliferation, migration and invasion in MGC803 cells by DADS.

Detection of Serum miR-122 and miR-22 in Patients with Chronic Hepatitis B by Using of a Quantitative Real-time PCR / 医学研究杂志

Objective To establish a real -time quantitative PCR ( RT-PCR) assay for detecting serum miR -122, miR-22, and evaluate the clinical significance of miR -122 and miR-22 in patients with chronic hepatitis B ( CHB) by using of this assay .Meth-ods The mature miRNAs were reversely transcripted by using of stem -loop primers .SYBR GreenⅠquantitative real-time PCR ( qRT-PCR) was used for quantification of the miRNAs .The sensitivity of this assay was evaluated by using of the 10-fold-diluted miRNA-122 cDNA standards and the specificity was verified by using of melting curve assay .The accuracy was assessed by intra -assay coeffi-cient of variation (CV) of threshold cycle (Ct value), which were calculated from a 20-times-repeat detection of the miR -122 cDNA (2 ×105 , 2 ×106 , 2 ×107 copies/μl) standards.Using the established qRT -PCR assay, we detected the expression of serum miR -122 and miR-22 in the patients with CHB and healthy controls .Results The qRT-PCR assay exhibited good performances in the linear range, sensitivity and reproducibility while detecting miR -122 and miR-22.The relative level of miR -122 and miR-22 was 17.88 vs 5.35 in the CHB patients and 1.80 vs 1.67 in the controls (P=0.000).Conclusion Using of stem-loop primers, we established a qRT-PCR assay for detection of serum miR -122 and miR-22.Serum miR-122 and miR-22 increased significantly in the CHB pa-tients.

miR-22 inhibited glioma cells proliferation by targeting / 中国癌症杂志

Background and purpose:miR-22 has been reported to be down-regulated in gastric cancer, lung cancer, colorectal cancer, and breast cancer. However, its expression in glioma was still poorly known. This study aimed to explicit whether miR-22 suppresses cell proliferation by targeting MTDH, thus to reveal molecular mechanism that miR-22 functions as a tumor suppressor in glioma. Methods:Quantitative real-time polymerase chain reaction (qRT-PCR) was conducted for detecting the expression of miR-22 in gliomas and normal brain tissues. MTDH 3’UTR-luciferase vector was constructed and dual-luciferase reporter gene assay was employed to examine the effect of miR-22 on luciferase activity. U251 cells were transfected with miR-22 mimics, and MTDH siRNA as for postive control, then Western blot was performed to detect the expressions of MTDH protein. The proliferation ability of U251 cells was evaluated by MTT assay. Results:miR-22 was down-regulated in glioma tissues. Glioma patients with relatively high expression of miR-22 showed lower mortality compared with low expression of miR-22 by using Kaplan-Meier survival curves. We demonstrated miR-22 could bind to the 3’ untranslated region (UTR) of MTDH and inhibited the luciferase activity. Western blot showed that the expression of MTDH protein was inhibited by restored miR-22 or siR MTDH in U251 cells. Overexpression of miR-22 or siR MTDH inhibited the proliferation of U251 cells. Conclusion:miR-22 suppresses cell proliferation by targeting MTDH in glioma.

Diagnostic Value of Circulating Extracellular miR-134, miR-185, and miR-22 Levels in Lung Adenocarcinoma-Associated Malignant Pleural Effusion / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: The accurate and timely diagnosis of malignant pleural effusion (MPE) in lung cancer patients is important because MPE has a poor prognosis and is classified as stage IV disease. Molecular biomarkers for pleural effusion, such as circulating extracellular microRNAs (miRNAs) isolated from pleural fluid, may help in the diagnosis of MPE. The present study examined whether miRNAs that are deregulated in lung cancer (miR-134, miR-185, and miR-22) can serve as diagnostic markers for lung adenocarcinoma-associated MPE (LA-MPE). MATERIALS AND METHODS: Real-time reverse transcription quantitative polymerase chain reaction was used to measure the expression of the three miRNAs in samples from 87 patients with pleural effusion comprising 45 LA-MPEs and 42 benign pleural effusions (BPEs). The area under the receiver operating characteristic curve (AUC) was then used to evaluate the diagnostic performance of each of the three miRNAs and compare it with that of the common tumor marker, carcinoembryonic antigen (CEA). RESULTS: The expression of all three miRNAs was significantly lower in LA-MPE than in BPE (p <0.001). The AUCs for miR-134, miR-185, miR-22, and CEA were 0.721, 0.882, 0.832, and 0.898, respectively. Combining CEA with the three miRNAs increased the diagnostic performance, yielding an AUC of 0.942 (95% confidence interval, 0.864 to 0.982), with a sensitivity of 91.9% and a specificity of 92.5%. CONCLUSION: The present study suggests that the expression levels of circulating extracellular miR-134, miR-185, and miR-22 in patients with pleural effusion may have diagnostic value when differentiating between LA-MPE and BPE.