The emerging role of microRNA-22 in the Leukemia: experimental and clinical implications.

MicroRNAs (miRNAs) are short noncoding RNAs, approximately 20-24 nucleotides long that negatively regulate gene expression by either inhibiting translation or cleaving complementary mRNA to participate in various biological processes. Accumulating evidence has indicated that miRNAs are widely present in hematological cancers, particularly leukemia, exhibiting either upregulation or downregulation in leukemia patients compared with healthy controls. These miRNAs have a pivotal role in the development, progression and metastasis of leukemia, as well as in the prognosis and/or relapse of patients. miR-22 is one of the abnormally expressed miRNAs in a variety of leukemia diseases, and is considered to be one of the few cancer suppressors. Recent research has demonstrated that miR-22 is involved in the regulation of leukemia cell proliferation, differentiation and apoptosis, and could be a promising biomarker and prognostic indicator for leukemia. Here, we summarize all relevant findings that carry out experimental investigation and clinical analyses, aiming to elucidate the comprehensive implications of miR-22 in various types of leukemia for the development of new therapeutic and prognostic strategies and new drug targets for the treatment of leukemia.

miR-22-3p ameliorates the symptoms of premature ovarian failure in mice by inhibiting CMKLR1 expression.

Premature ovarian failure (POF) affects many adult women less than 40 years of age and leads to infertility. This study was aimed at exploring the improving effects of miR-22-3p on the symptoms of POF in mice by inhibiting chemokine-like receptor 1 (CMKLR1) expression. Female mice were intraperitoneally injected with cyclophosphamide to construct POF mice models. Lentiviral vectors containing miR-22-3p, short hairpin RNA (sh)-CMKLR1, and overexpression (oe)-CMKLR1, respectively, or in combination, were injected into the ovaries of both sides of POF mice. miR-22-3p and CMKLR1 expression in ovarian tissues of mice was assessed, and the targeting relationship between miR-22-3p and CMKLR1 was predicted and verified. Serum estradiol (E2), anti-Mullerian hormone, and follicle-stimulating hormone levels were assessed. Ovarian weight was weighed, and pathological changes and the number of primordial follicles, primary follicles, secondary follicles, and atresia follicles were observed. Apoptosis of ovarian tissues was determined. In ovarian tissues of POF mice, miR-22-3p expression was decreased while CMKLR1 expression was increased. miR-22-3p up-regulation or CMKLR1 down-regulation restored sex hormone levels, improved ovarian weight and the number of primordial follicles, primary follicles, and secondary follicles, and reduced the number of atresia follicle and ovarian granulosa cell apoptosis in POF mice. miR-22-3p targeted CMKLR1, and overexpressing CMKLR1 reversed the ameliorative effects of miR-22-3p overexpression on POF mice. Our research highlights that overexpressed miR-22-3p down-regulates CMKLR1 to ameliorate the symptoms of POF in mice. Therefore, the miR-22-3p/CMKLR1 axis could improve the symptoms of POF.

[MicroRNA-22-3p Regulates the Expression of Kruppel-like Factor 6 to Affect the Cardiomyocyte-like Differentiation of Bone Marrow Mesenchymal Stem Cell].

Objective To explore the effect of microRNA-22-3p (miR-22-3p) regulating the expression of Kruppel-like factor 6 (KLF6) on the cardiomyocyte-like differentiation of bone marrow mesenchymal stem cell (BMSC). Methods Rat BMSC was isolated and cultured,and the third-generation BMSC was divided into a control group,a 5-azacytidine(5-AZA)group,a mimics-NC group,a miR-22-3p mimics group,a miR-22-3p mimics+pcDNA group,and a miR-22-3p mimics+pcDNA-KLF6 group.Real-time fluorescent quantitative PCR (qRT-PCR) was carried out to determine the expression of miR-22-3p and KLF6 in cells.Immunofluorescence staining was employed to detect the expression of Desmin,cardiac troponin T (cTnT),and connexin 43 (Cx43).Western blotting was employed to determine the protein levels of cTnT,Cx43,Desmin,and KLF6,and flow cytometry to detect the apoptosis of BMSC.The targeting relationship between miR-22-3p and KLF6 was analyzed by dual luciferase reporter gene assay. Results Compared with the control group,5-AZA up-regulated the expression of miR-22-3p (q=7.971,P<0.001),Desmin (q=7.876,P<0.001),cTnT (q=10.272,P<0.001),and Cx43 (q=6.256,P<0.001),increased the apoptosis rate of BMSC (q=12.708,P<0.001),and down-regulated the mRNA (q=20.850,P<0.001) and protein (q=11.080,P<0.001) levels of KLF6.Compared with the 5-AZA group and the mimics-NC group,miR-22-3p mimics up-regulated the expression of miR-22-3p (q=3.591,P<0.001;q=11.650,P<0.001),Desmin (q=5.975,P<0.001;q=13.579,P<0.001),cTnT (q=7.133,P<0.001;q=17.548,P<0.001),and Cx43 (q=4.571,P=0.037;q=11.068,P<0.001),and down-regulated the mRNA (q=7.384,P<0.001;q=28.234,P<0.001) and protein (q=4.594,P=0.036;q=15.945,P<0.001) levels of KLF6.The apoptosis rate of miR-22-3p mimics group was lower than that of 5-AZA group (q=8.216,P<0.001).Compared with the miR-22-3p mimics+pcDNA group,miR-22-3p mimics+pcDNA-KLF6 up-regulated the mRNA(q=23.891,P<0.001) and protein(q=13.378,P<0.001)levels of KLF6,down-regulated the expression of Desmin (q=9.505,P<0.001),cTnT (q=10.985,P<0.001),and Cx43 (q=8.301,P<0.001),and increased the apoptosis rate (q=4.713,P=0.029).The dual luciferase reporter gene experiment demonstrated that KLF6 was a potential target gene of miR-22-3p. Conclusion MiR-22-3p promotes cardiomyocyte-like differentiation of BMSC by inhibiting the expression of KLF6.

MicroRNA-22 Level in Patients with Multiple Sclerosis and Its Relationship with Vitamin D and Vitamin D Receptor Levels.

INTRODUCTION: Multiple sclerosis (MS) is known to be a multifactorial disorder. Numerous observational studies have suggested the implication of multiple genetic and environmental factors in the pathogenesis of MS. The aim of this work was to evaluate expression of the microRNA-22 (miRNA-22) level, in relation to vitamin D (VD) and VD receptor (VDR) levels in patients with MS during remission state. METHODS: This case-control study was conducted in 50 patients with clinically definite MS and 50 age- and sex-matched healthy controls. miRNA-22 expression was assessed in both MS patients and controls using quantitative RT-PCR. The serum level of VD and VDR was assessed in both MS patients and controls using ELISA techniques. RESULTS: The miRNA-22 level was significantly downregulated in MS patients in comparison to controls (p value <0.001). MS patients had also significantly lower VD and VDR levels in comparison to controls (p value <0.001 and <0.001, respectively). Patients with secondary progressive MS (SPMS) have a significantly higher miRNA-22 level than patients with relapsing remitting MS (RRMS) (p value = 0.042). There was a statistically significant positive correlation between the miRNA-22 level and EDSS (p value = 0.033). There was also a statistically significant positive correlation between the miRNA-22 level and VDR level (p value = 0.002). CONCLUSION: The miRNA-22 level was significantly downregulated in MS patients, but it had a positive correlation with disability status. Patients with SPMS have a significantly higher miRNA-22 level than patients with RRMS. VD and VDR levels were significantly lower in MS patients than controls. The miRNA-22 level was positively correlated with the VDR level.

MicroRNA-22-3p alleviates spinal cord ischemia/reperfusion injury by modulating M2 macrophage polarization via IRF5.

Cell death after spinal cord ischemia/reperfusion (I/R) can occur through necrosis, apoptosis, and autophagy, resulting in changes to the immune environment. However, the molecular mechanism of this immune regulation is not clear. Accumulating evidence indicates that microRNAs (miRs) play a crucial role in the pathogenesis of spinal cord I/R injury. Here, we hypothesized miR-22-3p may be involved in spinal cord I/R injury by interacting with interferon regulatory factor (IRF) 5. Rat models of spinal cord I/R injury were established by 12-min occlusion of the aortic arch followed by 48-hr reperfusion, with L4-6 segments of spinal cord tissues collected. MiR-22-3p agomir, a lentivirus-delivered siRNA specific for IRF5, or a lentivirus expressing wild-type IRF5 was injected intrathecally to rats with I/R injury to evaluate the effects of miR-22-3p and IRF5 on hindlimb motor function. Macrophages isolated from rats were treated with miR-22-3p mimic or siRNA specific for IRF5 to evaluate their effects on macrophage polarization. The levels of IL-1ß and TNF-α in spinal cord tissues were detected by ELISA. miR-22-3p was down-regulated, whereas IRF5 was up-regulated in rat spinal cord tissues following I/R. IRF5 was a target gene of miR-22-3p and could be negatively regulated by miR-22-3p. Silencing IRF5 or over-expressing miR-22-3p relieved inflammation, elevated Tarlov score, and reduced the degree of severity of spinal cord I/R injury. Increased miR-22-3p facilitated M2 polarization of macrophages and inhibited inflammation in tissues by inhibiting IRF5, thereby attenuating spinal cord I/R injury. Taken together, these results demonstrate that increased miR-22-3p can inhibit the progression of spinal cord I/R injury by repressing IRF5 in macrophages, highlighting the discovery of a promising new target for spinal cord I/R injury treatment.

microRNA-22-3p plays a protective role in a murine asthma model through the inhibition of the NLRP3-caspase-1-IL-1ß axis.

NEW FINDINGS: What is the central question of this study? How does miR-22-3p exert a protective role in asthma? What is the main finding and its importance? Upregulation of miR-22-3p hampered airway inflammation and release of inflammatory cytokines through blocking the activation of the NLRP3-caspase-1-IL-1ß signalling pathway in asthma. ABSTRACT: Asthma, a great public health burden, is triggered by inflammatory responses in the airways and these are not addressed appropriately by current therapies. This study aims to investigate the regulatory mechanism of microRNA-22-3p (miR-22-3p) on the proliferation of bronchial epithelial cells exposed to lipopolysaccharide (LPS) and expression of pro-inflammatory cytokines in a murine asthma model challenged by ovalbumin. We first confirmed the downregulation of miR-22-3p in the murine asthma model and bronchial epithelial cells. miR-22-3p remarkably reversed the decline in bronchial epithelial cell viability, enhancement in apoptosis rate and release of inflammatory factors induced by LPS. miR-22-3p targeted and conversely regulated NACHT, LRR and PYD domains-containing protein 3 (NLRP3). Overexpression of NLRP3 counteracted the inhibitory effect of miR-22-3p on inflammatory damage in bronchial epithelial cells through activation of caspase-1/interleukin (IL)-1ß. In an in vivo model, overexpression of miR-22-3p significantly attenuated airway obstruction and tissue damage in mice. In summary, our study underscores that miR-22-3p serves both as a negative regulator of the NLRP3-caspase-1-IL-1ß axis and as a protective factor against the inflammatory response, suggesting a future therapeutic role in asthma.

MicroRNA-22 inhibition prevents doxorubicin-induced cardiotoxicity via upregulating SIRT1.

Oxidative stress and cardiomyocyte apoptosis contributed to the progression of doxorubicin (Dox)-induced cardiotoxicity. Recent studies identified microRNA-22 (miR-22) as a cardiac- and skeletal muscle-enriched microRNA that functioned as a key regulator in stress-induced cardiac injury. The present study aimed to investigate the role and possible mechanism of miR-22 on Dox-induced oxidative stress and cardiomyocyte apoptosis. Mice were exposed to reduplicative injections of Dox (i.p., 4 mg/kg) weekly for consecutive 4 weeks to generate Dox-induced cardiotoxicity. Herein, we found that miR-22 level was significantly increased in murine hearts subjected to chronic Dox treatment. MiR-22 inhibition attenuated oxidative stress and cardiomyocyte apoptosis in vivo and in vitro, thereby preventing Dox-induced cardiac dysfunction. Mechanistically, we observed that miR-22 directly bound to the 3'-UTR of Sirt1 and caused SIRT1 downregulation. Conversely, miR-22 antagomir upregulated SIRT1 expression and SIRT1 inhibitor abolished the beneficial effects of miR-22 antagomir. In conclusion, miR-22 inhibition prevented oxidative stress and cardiomyocyte apoptosis via upregulating SIRT1 and miR-22 might be a new target for treating Dox-induced cardiotoxicity.

miR-22 and miR-214 targeting BCL9L inhibit proliferation, metastasis, and epithelial-mesenchymal transition by down-regulating Wnt signaling in colon cancer.

The epithelial-mesenchymal transition (EMT) is crucial for cancer progression. Evidence has shown that miR-22 and miR-214 play a key role in colon cancer progression; however, the underlying mechanism remains to be known. The effects of miR-22 and miR-214 on EMT are contradictory in different cancers, and whether miR-22 and miR-214 are involved in the colon cancer EMT process needs to be elucidated. In this study, we evaluated the exact role and the regulation mechanism of miR-22 and miR-214 in colon cancer. After transfection with miR-22 expression vector, the cell proliferation and migration capacity of HCT116 and RKO cells were significantly suppressed. Also, E-cadherin was increased and vimentin was decreased by miR-22 overexpression. Similar effects were also observed after miR-214 expression vector transfection. Dual-luciferase reporter confirmed that BCL9L is the target gene of both miR-22 and miR-214. Silencing of BCL9L inhibits cell proliferation and migration, and the expression of E-cadherin and vimentin was also altered by BCL9L knockdown, which was consistent with miR-22 or miR-214 transfection. Furthermore, miR-22 and miR-214 inhibited tumor growth in nude mice. Moreover, although the association between BCL9L's lower expression and longer survival time was statistically nonsignificant, a trend existed; further studies in a larger cohort are needed. Collectively, these data suggest that miR-22 and miR-214 inhibit cell proliferation, migration, and EMT of colon cancer, most likely by targeting BCL9L.-Sun, R., Liu, Z., Han, L., Yang, Y., Wu, F., Jiang, Q., Zhang, H., Ma, R., Miao, J., He, K., Wang, X., Zhou, D., Huang, C. miR-22 and miR-214 targeting BCL9L inhibit proliferation, metastasis, and epithelial-mesenchymal transition by down-regulating Wnt signliang in colon cancer.

Inhibition of EZH2 attenuates coronary heart disease by interacting with microRNA-22 to regulate the TXNIP/nuclear factor-κB pathway.

NEW FINDINGS: What is the central question of this study? The relevance of microRNA-22 (miR-22) has been indicated in coronary heart disease (CHD). How does it exert a protective role in CHD? What is the main finding and its importance? EZH2 inhibited transcription of the miR-22 promoter, thus modulating cell proliferation in human umbilical vein endothelial cells and vascular smooth muscle cells to induce CHD. ABSTRACT: MicroRNA-22 (miR-22) was indicated to modulate cell proliferation in human umbilical vein endothelial cells (HUVECs) under exposure to environmental toxicants. In the present study, we investigated the involvement of miR-22 in the mediation of HUVEC and vascular smooth muscle cell (VSMC) function, hence in the development of coronary heart disease (CHD). miR-22 expression was reduced in serum of CHD patients. Restoration of miR-22 decreased the proliferation, migration and invasion of VSMCs and increased apoptotic cells and inflammatory factors. In contrast, upregulation of miR-22 led to opposite trends in HUVECs. Chromatin immunoprecipitation and dual-luciferase assays validated that enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2) inhibited transcription of miR-22 promoter. EZH2, overexpressed in serum from CHD patients, diminished VSMC apoptosis, but facilitated HUVEC apoptosis. Luciferase reporter assays confirmed that thioredoxin-interacting protein (TXNIP) was a new direct target of miR-22. Overexpression of TXNIP blocked the function of miR-22 in HUVECs and VSMCs. Taken together, these findings will shed light on the role and mechanism of EZH2 in viability, migration, invasion and apoptosis via the miR-22/TXNIP axis in VSMCs and HUVECs, which might provide new insights into the treatment of CHD.

Matrine triggers colon cancer cell apoptosis and G0/G1 cell cycle arrest via mediation of microRNA-22.

Matrine (MAT) is an alkaloid in the dried roots of Sophora flavescens. The antitumor activity has been testified in colon cancer. Howbeit, the latent mechanism is still indistinct. The research probed the antitumor mechanism of MAT in colon cancer cells. MAT (0.25, 0.5, 0.75, 1, and 1.25 mM) was utilized to stimulate SW480 and SW620 cells for 24, 48, and 72 hr. Cell viability, apoptosis, cell cycle, and the correlative proteins were assessed via Cell Counting Kit-8, flow cytometry, and Western blot. microRNA-22 (miR-22) in MAT-treated or miR-22-silenced cells was estimated via real-time quantitative polymerase chain reaction. The functions of miR-22 inhibition were reassessed. Western blot was conducted for quantifying ß-catenin, MEK, and ERK. Luciferase reporter assay was done for confirming the targeting relationship between miR-22 and ERBB3 or MECOM. MAT prohibited cell viability, accelerated apoptosis, and triggered cells cycle stagnation at G0/G1 phase. Additionally, miR-22 was elevated by MAT; meanwhile, the influences of MAT were all inverted by miR-22 inhibitor. MAT enhanced the expression of miR-22, thereby obstructing Wnt/ß-catenin and MEK/ERK pathways. miR-22 had a potential to target mRNA 3'UTR of ERBB3 and MECOM. These discoveries manifested that MAT could evoke colon cancer cell apoptosis and G0/G1 cell cycle arrest via elevating miR-22.

Quercetin suppresses the tumorigenesis of oral squamous cell carcinoma by regulating microRNA-22/WNT1/ß-catenin axis.

Quercetin has potential pharmacological values in various carcinomas including oral squamous cell carcinoma (OSCC). Moreover, the anti-tumor effect of quercetin is correlated with WNT/ß-catenin pathway and miRNA dysregulation. In the present study, we aimed to further investigate whether quercetin can exert its anti-tumor function by regulating miR-22 together with miR-22 downstream pathway WNT1/ß-catenin in OSCC. The results of Cell Counting Kit-8 (CCK-8) and flow cytometry analyses showed that quercetin treatment and miR-22 overexpression resulted in the reduction of cell viability and the increase of cell apoptotic rate in OSCC. WNT1 was a target of miR-22, which was confirmed by bioinformatics, luciferase reporter and RNA immunoprecipitation (RIP) assays. RT-qPCR assay showed that quercetin promoted miR-22 expression and suppressed WNT1 and ß-catenin expression in OSCC cells, whereas this effect was abrogated by miR-22 inhibitor. Moreover, miR-22 depletion weakened quercetin-mediated viability inhibition and apoptosis increase in OSCC cells. Quercetin inhibited the growth of OSCC xenograft tumors by inducing miR-22 expression and repressing WNT1/ß-catenin pathway in vivo. Taken together, quercetin hampered OSCC tumorigenesis by regulating miR-22/WNT1/ß-catenin pathway in OSCC, providing a deep insight into the molecular targets of quercetin in the treatment of OSCC.

A calcium-sensitive feed-forward loop regulating the expression of the ATP-gated purinergic P2X7 receptor via specificity protein 1 and microRNA-22.

Cells have developed complex transcriptional regulatory mechanisms to maintain intracellular homeostasis and withstand pathophysiological stressors. Feed-forward loops comprising transcription factors that drive expression of both target gene and a microRNA as negative regulator, are gaining increasing recognition as key regulatory elements of cellular homeostasis. The ATP-gated purinergic P2X7 receptor (P2X7R) is an important driver of inflammation and has been implicated in the pathogenesis of numerous brain diseases including epilepsy. Changes in P2X7R expression have been reported in both experimental models and in epilepsy patients but the mechanism(s) controlling P2X7R levels remain incompletely understood. The specificity protein 1 (Sp1) has been shown to induce P2X7R transcription in vitro and recent data has identified microRNA-22 as a post-transcriptional repressor of P2X7R expression after seizures. In the present study we show that Sp1 can induce the transcription of both microRNA-22 and P2X7R in vitro during increased neuronal activity and in vivo in a mouse model of status epilepticus. We further show that Sp1-driven microRNA-22 transcription is calcium-sensitive and Sp1 occupancy of the microRNA-22 promoter region is blocked under conditions of seizure activity sufficient to elicit neuronal death. Taken together, our results suggest a neuronal activity-dependent P2X7R expression which is induced by the transcription factor Sp1 and repressed in a calcium-dependent manner by microRNA-22.

miR-22-Notch Signaling Pathway Is Involved in the Regulation of the Apoptosis and Autophagy in Human Ovarian Cancer Cells.

microRNA-22 (miR-22) is a brain-enriched regulatory gene which has been reported to be involved in the development of cancers. The Notch signaling pathway exerts important functions in cell growth. This study is designed to investigate the mechanisms of miR-22-Notch signaling pathway in apoptosis and autophagy of human ovarian cancer cells. After over-expressing miR-22 in human ovarian cancer cell lines OVCAR-3 and SKOV3, cell viability is determined by 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) method, cell apoptosis is observed by Flow cytometry (FCM), mRNA expression of miR-22 is measured by RNA preparation and RT-PCR, protein expression of Notch1, Hes1, Beclin1 and LC3B-II is analyzed by Western blot. It is suggested that miR-22 expression is heavily decreased in human ovarian cancer cell lines OVCAR-3 and SKOV3. Over-expression of miR-22 potently suppresses cell viability and authophagy while promotes the percentage of apoptotic cancer cells. In addition, the decreased expression level of Notch1 and its targeted gene is detected in miR-22-over-expressed cells. Moreover, followed by the block of the Notch signaling pathway using Notch1 small interference RNA (siRNA), the effects of miR-22 on the apoptosis and autophagy of human ovarian cancer cell lines OVCAR-3 and SKOV3 are obviously blocked. Together, miR-22 inhibits apoptosis and promotes autophagy of human ovarian cancer cells through the suppression of the Notch signaling pathway, indicating a potential use of miR-22 in the ovarian cancer treatment.

MiR-22-3p Regulates Cell Proliferation and Inhibits Cell Apoptosis through Targeting the eIF4EBP3 Gene in Human Cervical Squamous Carcinoma Cells.

Background: MicroRNAs (miRNAs) are non-coding small RNAs that function as negative regulators of gene expression and are involved in tumour biology. The eIF4E-binding proteins (eIF4EBPs) play essential roles in preventing translation initiation and inhibiting protein synthesis at a global or message-specific level in a variety of tumours. Methods: According to comparative miRNA profiles of clinical cervical cancer and non-cancerous cervical tissue specimens, several miRNAs were aberrantly expressed in the cervical cancer samples. C33a and SiHa cell proliferation and apoptosis were detected using methyl thiazolyl tetrazolium (MTT) and flow cytometry assays, respectively. Results: Among the aberrantly expressed miRNAs, miR-22-3p was significantly differentially expressed in cervical cancer tissues and was highly associated with cervical cancer cell growth regulation. In addition, bioinformatic predictions and experimental validation were used to identify whether eIF4E-binding protein 3 (eIF4EBP3) was a direct target of miR-22-3p; eIF4EBP3 protein levels were generally low in the cervical cancer tissues. Furthermore, functional studies revealed that either a miR-22-3p inhibitor or eIF4EBP3 overexpression could induce apoptosis in cervical cancer cells in vitro. Importantly, we found that eIF4EBP3 accumulation could significantly attenuate cervical cancer cell proliferation triggered by a miR-22-3p mimic as well as enhance apoptosis in cervical cancer cells. Conclusion: Taken together, our data provide primary proof that miR-22-3p can induce cervical cancer cell growth at least in part by up-regulating its expression to decrease eIF4EBP3 expression levels; miR-22-3p thus holds promise as a prognostic biomarker and potential therapeutic target for treating cervical cancer.

MicroRNA-22 Promoted Osteogenic Differentiation of Human Periodontal Ligament Stem Cells by Targeting HDAC6.

Stem cells transplantation is a promising therapy strategy for accelerating periodontal regeneration and reconstruction. Genetic modification could induce stem cells directional differentiation to facilitate recovery of physiological functions. In this study, we investigated the role and mechanism of miR-22 on human periodontal ligament stem cells (PDLSCs). First, a cellular model of osteogenic differentiation was first established by osteogenic inductive cocktail. Real-time PCR determined that expression of miR-22 was significantly increased during PDLSCs osteogenic differentiation. Alizirin red staining showed that overexpression of miR-22 in PDLSCs induced better mineralized nodule formation. Real-time PCR and Western blot further confirmed up-regulation of osteogenic genes Runx2 and OPN in miR-22-overexpressing PDLSCs. Conversely, inhibition of miR-22 delayed the process of PDLSCs osteogenic differentiation. Furthermore, Histone deacetylase 6 (HDAC6) was identified as a target gene of miR-22. Overexpression of miR-22 not only reduced the luciferase activity of the reporter containing the 3' untranslated region of HDAC6 mRNA, but also suppressed the endogenous protein expression of HDAC6. Rescue experiment showed that the promotion role of miR-22 in osteogenic differentiation could be relieved by overexpression of HDAC6. Meanwhile, overexpression of HDAC6 alone could also delay the osteogenic differentiation process. The results demonstrated that miR-22 promoted PDLSCs osteogenic differentiation by inhibiting HDAC6 expression, suggesting that miR-22 might be developed as a target of genetic modified stem cells therapy for periodontal diseases. J. Cell. Biochem. 118: 1653-1658, 2017. © 2017 Wiley Periodicals, Inc.

Protective Effects of Microrna-22 Against Endothelial Cell Injury by Targeting NLRP3 Through Suppression of the Inflammasome Signaling Pathway in a Rat Model of Coronary Heart Disease.

BACKGROUND/AIMS: This study aimed to identify the role of microRNA-22 (miR-22) in endothelial cell (EC) injury in coronary heart disease (CHD) by targeting NLRP3 through the inflammasome signaling pathway. METHODS: A total of 24 healthy male Sprague-Dawley (SD) rats were divided into normal and atherosclerosis groups. The atherosclerosis rats were assigned into blank, negative control (NC), miR-22 mimic, miR-22 inhibitor and miR-22 inhibitor + siNLRP3 groups. A luciferase reporter gene assay was used to detect the relationship between miR-22 and NLRP3. MiR-22 expression as well as NLRP3 and caspase-1 mRNA and protein expression were measured using quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting. The activity and apoptosis of coronary arterial endothelial cells (CAECs) were determined by MTT and Hoechst 33258. CAEC lumen formation was detected by a lumen formation assay. An enzyme-linked immunosorbent assay (ELISA) was used to detect IL-1ß, IL-6, IL-10 and IL-18 levels. RESULTS: The results indicated that the atherosclerosis group significantly decreased miR-22 expression but increased NLRP3 and caspase-1 mRNA and protein expression. The cell survival rate was significantly increased in the miR-22 mimic group and significantly reduced in the miR-22 inhibitor group. The miR-22 mimic group displayed a lower apoptosis rate and more cells with obvious lumen walls and numerous tubular structures, while cells in the miR-22 inhibitor group were unable to form lumen walls and had a scattered distribution compared to the blank group. The ELISA showed that IL-1ß, IL-6 and IL-18 levels were markedly decreased, while IL-10 was clearly increased in the miR-22 mimic group. In contrast, in the miR-22 inhibitor group, IL-1ß, IL-6 and IL-18 levels were significantly increased, and IL-10 levels were decreased. CONCLUSION: Our findings indicated that miR-22 could lower the levels of pro-inflammatory cytokines by inhibiting the NLRP3 inflammasome pathway, which suppresses CAEC apoptosis and protects CAECs in rats with CHD.

Effect of 1,25-(OH)2D3 on Proliferation of Fibroblast-Like Synoviocytes and Expressions of Pro-Inflammatory Cytokines through Regulating MicroRNA-22 in a Rat Model of Rheumatoid Arthritis.

OBJECTIVE: This study aims to investigate the regulatory mechanism of 1,25-(OH)2D3 on the proliferation of fibroblast-like synoviocytes (FLS) and expressions of pro-inflammatory cytokines in rheumatoid arthritis (RA) rats via microRNA-22 (miR-22). METHODS: A rat model of RA was established with a subcutaneous injection of type II collagen. After treated with different concentrations of 1,25-(OH)2D3 the proliferation of FLS was estimated by the MTT method, and the optimal concentration of 1,25-(OH)2D3 was selected for further experiments. Cell proliferation was detected by MTT. Cell cycle and apoptosis were analyzed by FCM. The IL-1ß, IL-6, IL-8, and PGE2 protein expressions were determined by ELISA, and MMP-3, INOS, and Cox-2 mRNA expressions were measured by qRT-PCR. RESULTS: The rat model of RA was successfully established. Compared with the blank group, the 1,25-(OH)2D3 and miR-22 inhibitors groups exhibited higher proliferation inhibition and apoptosis rates, lower levels of pro-inflammatory cytokines (IL-1ß, IL-6, IL-8, and PGE2), and decreased mRNA expressions of MMP-3, INOS, and Cox-2. The miR-22 mimics group had lower proliferation inhibition and apoptosis rates, elevated expressions of pro-inflammatory cytokines and MMP-3, INOS, and Cox-2 than the blank group. In contrast to the 1,25-(OH)2D3 group, the proliferation inhibition and apoptosis rates were down-regulated, and the expressions of pro-inflammatory cytokines and MMP-3, INOS, and Cox-2 were up-regulated in the 1,25-(OH)2D3 + miR-22 mimics group. CONCLUSION: Our study demonstrated that 1,25-(OH)2D3 inhibits the proliferation of FLS and alleviates inflammatory response in RA rats by down-regulating miR-22.

Predicative values of serum microRNA-22 and microRNA-126 levels for non-small cell lung cancer development and metastasis: a case-control study.

Presented study aims to explore the predictive values of serum microRNA-22 (miR-22) and miR-126 levels for non-small cell lung cancer (NSCLC) development and metastasis.A total of 127 NSCLC patients who were admitted in the First People's Hospital of Yancheng City from May, 2013 to May, 2015 were selected as the case group, including 71 cases of adenocarcinoma and 56 cases of squamous cell carcinoma. There were 112 healthy individuals selected as the control group. The qRT-PCR was performed to testify the serum miR-22 and miR-126 levels. Logistic regression analysis was conducted to analyze independent factors influencing NSCLC metastasis and receiver operating characteristic (ROC) curve was drawn to analyze the sensitivity and specificity of serum miR-22 and miR-126 levels in predicting NSCLC developments and metastasis.The serum miR-22 level was significantly higher in the case group than that in the control group, while the serum miR-126 level was lower in the case group as compared with that in the control group. Compared with squamous cell carcinoma patients, serum miR-22 level significantly increased, while serum miR-126 level decreased in patients with adenocarcinoma. Patients at III + IV stage showed increased serum miR-22 level and relatively decreased serum miR-126 level as compared to patients at I + II stage. Serum miR-22 level elevated in patients with metastasis; in contrast serum miR-126 level reduced in comparison to those without metastasis. In patients with familial inheritance, serum miR-22 level increased but serum miR-126 level decreased as compared to those without familial inheritance. The specificity and sensitivity of serum miR-22 and miR-126 levels in predicting NSCLC development were 99.11%, 84.30%, 82.68% and 96.40%, respectively. The specificity and sensitivity of serum miR-22 and miR-126 levels in predicting NSCLC metastasis were 59.74%, 96.00%, 84.00% and 62.30%, respectively.Results indicated that serum miR-22 and miR-126 levels may be used as the predicative biomarkers for NSCLC development and metastasis.

Neuroprotective effects of viral overexpression of microRNA-22 in rat and cell models of cerebral ischemia-reperfusion injury.

Several studies have reported that microRNA (MIR) is involved in the pathogenesis and progression of ischemic diseases, including cerebral ischemia, and that MIR-22 may inhibit the inflammatory response and cell apoptosis, which contribute to ischemia/reperfusion (I/R) injury. However, the specific function of MIR-22 in cerebral I/R injury remains far from clear. This study aimed to examine the potential protective effect of MIR-22 against cerebral I/R injury and its mechanism. As predicted, adenovirus-mediated MIR-22 overexpression markedly reduced the neurological score and infarct size (P < 0.05). We demonstrated that MIR-22 overexpression resulted in a reduction in inflammatory cytokines TNF-α, IL-6, COX-2, and iNOS, whereas the level of IL-10 was enhanced. MIR-22 overexpression significantly inhibited NF-κB activity by decreasing NF-κB coactivator NCOA1 expression. Furthermore, we found that MIR-22 could reduce the apoptotic rate of cortical neurons. Caspase-3 activity was inhibited by MIR-22, and the expression of the anti-apoptosis gene Bcl-2 in neurons was increased and that of the pro-apoptosis gene Bax decreased following MIR-22 overexpression. Our results suggest that MIR-22 could be used to treat cerebral I/R injury and that its neuroprotective effect may be attributed to a reduction in inflammation and apoptosis.

Advances in the study of the mechanism of action of miR­22 in liver lesions (Review).

Globally, nearly 2 million deaths annually are attributed to the development of liver diseases, with liver cancer and cirrhosis being particularly prominent, which makes liver disease a significant global health concern. Cirrhosis is closely linked to the evolution of hepatitis, hepatic fibrosis and fatty liver. However, most liver diseases have an insidious onset, are challenging to treat and the prognosis and efficacy of current therapies are unsatisfactory, which can result in irreversible functional damage to the liver. Therefore, there is an urgent need to explore the molecular mechanisms underlying liver disease and identify new biomarkers and therapeutic targets. In previous years, microRNAs (miRs), a class of short non-coding RNAs comprising 17-25 nucleotides, have attracted attention for their roles in various types of liver diseases. Among them, miR-22 serves a unique role in mediating multiple pathway mechanisms and epigenetic modifications and can act both as an inhibitor of liver cancer and a metabolic blocker. Given its close association with the liver, several studies have reported that the differential expression of miR-22 regulates the metabolic process of liver cancer and is involved in the evolution of hepatic fibrosis and steatohepatitis, making it a potential target for early diagnosis and treatment. The present manuscript aimed to comprehensively review the key role of miR-22 in the evolution of liver diseases and offer valuable references and guidance for subsequent studies by identifying its specific mechanism of action and future development prospects.