Overexpression of lncRNA-MEG3 inhibits endometrial cell proliferation and invasion via miR-21-5p/DNMT3B/Twist

Abstract Recent studies have found that lncRNA-MEG3(MEG3) plays an important role in the development of EMs (Endometriosis), but the specific mechanism needs to be further explored. This study aimed to investigate the effect of MEG3 on the proliferation, invasion of EMs cells. The authors used RT-qPCR to detect the expression of MEG3 and miR-21-5p in EMs tissues and hESCs cells, MTT and Transwell to detect cell proliferation and invasion, western blotting assay to detect the expression of DNMT3B and Twist, MSP to detect the methylation of Twist. The present study's detection results showed that MEG3 was lowly expressed in EMs tissues and hESCs cells, and overexpression of MEG3 could down-regulate miR-21-5p and inhibit endometrial cell proliferation and invasion. In addition, overexpression of MEG3 upregulated the expression of DNMT3B and promoted the methylation of TWIST. In conclusion, the present findings suggest that MEG3 is downregulated in EMs tissues, and overexpression of MEG3 can promote the activity of DNA methyltransferase DNMT3B by downregulating miR-21-5p, thereby promoting the methylation of Twist, downregulating Twist level to inhibits hESCs cells proliferation and invasion.

Overexpression of lncRNA MEG3 inhibits proliferation and invasion of glioblastoma U251 cells / 南方医科大学学报

OBJECTIVE@#To investigate the effects of overexpression of long noncoding RNA (lncRNA) MEG3 on the proliferation and invasion of glioblastoma U251 cells by suppressing the expression of hypoxia inducible factor 1@*METHODS@#The expression of lncRNA MEG3 and HIF1@*RESULTS@#The expression of MEG3 was significantly lower and HIF1@*CONCLUSIONS@#MEG3 overexpression inhibits the proliferation and invasion of U251 cells through suppressing the expression of HIF1

Effect of lncRNA MEG3 on radiosensitivity of nasopharyngeal carcinoma cells by down-regulating miR-7-5p expression / 中华放射肿瘤学杂志

Objective To investigate the effect and underlying mechanism of lncRNA MEG3 on the radiosensitivity of nasopharyngeal carcinoma cells.Methods this experiment,overexpression control group,MEG3 overexpression group,miR-NC inhibition group,miR-7-5p inhibition group,overexpression control+4 Gy group,MEG3 overexpression+4 Gygroup,miR-NC inhibition+4 Gy group,miR-7-5p inhibition+4 Gy group,MEG3 overexpression + miR-NC overexpression group,MEG3 overexpression + miR-7-Sp overexpression group were established.The expression of miR-7-5p and MEG3 was detected by qRT-PCR.The radiosensitivity of nasopharyngeal carcinoma cells was measured by clone formation assay.Cell apoptosis was assessed by flow cytometry.The fluorescence activity was evaluated by dual luciferase reporter assay.Results MEG3 was lowly expressed in nasopharyngeal carcinoma tissues and cells.Overexpression of MEG3 and inhibition of miR-7-5p expression increased the radiosensitivity of nasopharyngeal carcinoma cells and promoted radiation-induced cell apoptosis.MEG3 could targetedly regulate the miR-7-5p expression.Overexpression of miR-7-5p reversed the effect of overexpression of MEG3 on the sensitization of nasopharyngeal carcinoma cells and the promotion of apoptosis induced by radiation exposure.Conclusions Overexpression of MEG3 increases the radiosensitivity of nasopharyngeal carcinoma cells and promotes radiation-induced cell apoptosis.The mechanism may be related to the down-regulation of miR-7-5p expression.

The effects of long non-coding RNA MEG3 on endoplasmic reticulum stress-induced colon cancer cell apoptosis and the growth of transplanted human colonic carcinoma in nude mice / 西安交通大学学报(医学版)

Objective: To investigate the effects of maternally expressed gene 3 (MEG3) on endoplasmic reticulum stress-induced colon cancer cell apoptosis and the growth of transplanted human colonic carcinoma in nude mice. Methods:Colon cancer cell lines with the lowest expression level of MEG3 were screened by RT-PCR; negative control cell lines were constructed and stable transfectants were screened. RT-PCR was performed for measuring the lncRNA level of MEG3, cell growth was measured by CCK8, cell apoptosis was determined by Hoechst. Western blot was used to determine the protein levels of Bcl-2, Bax, cleaved Caspase-9, p-PERK, ATF-4, p-EIF2α, CHOP, cleaved Caspase-12, p-AMPK, AMPK, p-mTOR, and mTOR. The expression of CHOP was silenced by si-CHOP, and then cell apoptosis was determined by Hoechst. Transplanted human colonic carcinoma in nude mice was established and tumor volume was measured, tumor growth was measured by IHC Ki67, cell apoptosis was determined by TUNNEL, Western blot was used to determine the protein levels of CHOP, cleaved Caspase-12, and cleaved Caspase-3. Results: The SW480 cell line had the lowest expression level of MEG3 and was used in subsequent experiments. In cytological experiments, compared with those in control group, the cell growth, protein levels of Bcl-2 and ratio of p-mTOR/mTOR were decreased significantly; the apoptosis rate, protein levels of Bax, cleaved Caspase-9, p-PERK, ATF-4, p-eIFα, CHOP and cleaved Caspase-12 and ratio of p-AMPK/AMPK increased notably; the expression of CHOP was silenced; the apoptosis induced by pc-MEG3 could be significantly decreased. In animal experiments, compared with control group, the tumor volume and number of positive cells of Ki67 decreased significantly; the apoptosis rate, protein levels of CHOP, cleaved Caspase-12 and cleaved Caspase-3 increased markedly. Conclusion: MEG3 can promote colon cancer cell apoptosis by endoplasmic reticulum stress, and CHOP may play a crucial role in the process. Furthermore, AMPK/mTOR is also involved in the regulation of apoptosis by MEG3.

DNMT1 protein promotes retinoblastoma proliferation by silencing MEG3 gene / 南方医科大学学报

OBJECTIVE@#To investigate whether DNMT1 protein induces retinoblastoma proliferation by silencing MEG3 gene.@*METHODS@#Two retinoblastoma cell lines (HXO-RB44 and SO-RB50) and a normal human retinal pigment epithelial (RPE) cell line were transfected with the plasmid pcDNA-DNMT1 or si-DNMT1 for up-regulating or interference of DNMT1 expression, and with pcDNA-MEG3 or si-MEG3 for up-regulating or interference of MEG3 expression. Western blotting was used to detect the changes in the expression of DNMT1 protein in the transfected cells, and CCK-8 and EdU assays were used to detect the changes in cell proliferation. Real-time quantitative PCR (qRT-PCR) was performed to detect MEG3 expression in SO-RB50 and HXO-RB44 cells after transfection, and the methylation level of MEG3 gene promoter after interference of DNMT1 expression was detected using methylation-specific PCR.@*RESULTS@#SO-RB50 and HXO-RB44 cells showed significantly increased expression of DNMT1 protein as compared with normal RPE cells ( < 0.05). In HXO-RB44 cells, transfection with pcDNADNMT1 resulted in significantly increased expression of DNMT1 protein, enhanced cell proliferation ability, and significantly reduced expression of MEG3 ( < 0.05). In SO-RB50 cells, transfection with si-DNMT1 significantly reduced the expression of DNMT1 protein, suppressed the cell proliferation, and increased MEG3 expression ( < 0.05). Interference of DNMT1 significantly reduced the methylation level of MEG3 gene promoter. After reversing the regulatory effect of DNMT1 on MEG3 gene, DNMT1 protein showed significantly weakened ability to regulate retinoblastoma cell proliferation ( < 0.05).@*CONCLUSIONS@#In retinoblastoma cells, the up-regulation of DNMT1 protein induces promoter methylation and inactivation of MEG3 gene and eventually leads to abnormal cell proliferation.

Effect of lncRNA MEG3 on the malignant biological behaviors of cervical cancer standard regimen for advanced colorectal cancer / 中国肿瘤生物治疗杂志

@#[Abstract] Objective: To explore the regulatory effect of lncRNA maternal imprinting gene 3 (MEG3) on proliferation, migration, invasion and epithelial-mesenchymal transition (EMT) of cervical cancer cells via miR-9-5p/SOCS5 axis. Methods: A total of 20 pairs of cancer and para-cancerous tissue specimens resected from cervical cancer patients in Chongqing Hospital of Traditional Chinese Medicine from January 2017 to June 2019 were collected for this study. Using liposome transfection technology, pcDNA3.1-MEG3,si-MEG3, miR-9-5p mimics, miR-9-5p inhibitor and their control plasmids were transfected into cervical cancer HeLa and SiHa cells respectively to construct overexpression and silence cell model. qPCR was used to detect the expression levels of MEG3, miR-9-5p and SOCS5 in cervical cancer tissues and cell lines. CCK-8 method and Transwell chamber method were used to detect cell proliferation, migration and invasion ability. The expression levels of E-cadherin and vimentin in cells were detected by cellular immunofluorescence experiments. Target genes were predicted through the Online Bioinformatics TargetScan database. Dual luciferase reporter gene assay was used to verify the targeting relationship between miR-9-5p and MEG3, SOCS5, respectively. Results: Compared with para-cancerous tissues and cervical epithelial HcerEpic cells, the expressions of MEG3 and SOCS5 were significantly down-regulated and the expression of miR-9-5p was significantly up-regulated in cervical cancer tissues and cell lines (all P<0.01). TargetScan database analysis and Dual luciferase reporter gene assay confirmed the targeting relationship between miR-9-5p and MEG3 or SOCS5. MEG3 and SOCS5 significantly inhibited while miR-9-5p significantly promoted cell proliferation, migration and invasion ability (all P<0.01). MEG3 and SOCS5 promoted E-cadherin expression and inhibited vimentin expression, while miR-9-5p inhibited E-cadherin expression and promoted vimentin expression (P<0.05 or P<0.01). Conclusion: lncRNA MEG3 regulates proliferation, migration, invasion and EMT of cervical cancer cells via miR-9-5p/SOCS5 axis.

lncRNA MEG3 inhibits the growth of hepatocellular carcinoma cells by sponging miR-9-5p to upregulate SOX11

The long non-coding RNA (lncRNA) maternally expressed gene 3 (MEG3), a tumor suppressor, is critical for the carcinogenesis and progression of different cancers, including hepatocellular carcinoma (HCC). To date, the roles of lncRNA MEG3 in HCC are not well illustrated. Therefore, this study used western blot and qRT-PCR to evaluate the expression of MEG3, miR-9-5p, and Sex determining Region Y-related HMG-box 11 (SOX11) in HCC tissues and cell lines. RNA pull-down and luciferase reporter assay were used to evaluate these molecular interactions. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and flow cytometry detected the viability and apoptosis of HCC cells, respectively. The results showed that MEG3 and SOX11 were poorly expressed but miR-9-5p was highly expressed in HCC. The expression levels of these molecules suggested a negative correlation between MEG3 and miR-9-5p and a positive correlation with SOX11, confirmed by Pearson's correlation analysis and biology experiments. Furthermore, MEG3 could combine with miR-9-5p, and SOX11 was a direct target of miR-9-5p. Moreover, MEG3 over-expression promoted cell apoptosis and growth inhibition in HCC cells through sponging miR-9-5p to up-regulate SOX11. Therefore, the interactions among MEG3, miR-9-5p, and SOX11 might offer a novel insight for understanding HCC pathogeny and provide potential diagnostic markers and therapeutic targets for HCC.

Promoter methylation of MEG3 gene in hepatocellular carcinoma and its clinical significance / 肿瘤研究与临床

Objective To investigate promoter methylation state of MEG3 gene in hepatocellular carcinoma and its clinical significance. Methods Methylation-specific polymerase chain reaction (MSP) was performed to detect methylation of MEG3 promoter in 58 cases of hepatocellular carcinoma tissues and 20 cases of normal control from December 2014 to December 2016 in Shanxi Provincial Cancer Hospital, and to analyze the relationship between methylation and the clinicopathological features of the patients. Results The methylation incidence rate of MEG3 promoter in hepatocellular carcinoma tissues (55.2 %, 32/58) was higher than that in normal liver tissue (25.0 %, 5/20), and there was a significant statistical difference (χ2=6.72,P =0.02).There was no significant difference in the incidence of MEG3 methylation of the patients with different age, gender, α-fetoprotein levels, tumor number and differentiation (all P> 0.05). There was a significant difference in the incidence of MEG3 methylation of the patients with different tumors diameter, liver cirrhosis, hepatitis B surface antigen (HBsAg), TNM staging and distant metastasis (all P< 0.05). Conclusion Aberrant promoter methylation in MEG3 genes may be associated with the occurrence of hepatocellular carcinoma,tumor diameter,liver cirrhosis,HBsAg,TNM staging and distant metastasis.

Melatonin prevents endothelial cell pyroptosis via regulation of long noncoding RNA MEG3/miR-223/NLRP3 axis / 中国药理学与毒理学杂志

OBJECTIVE To investigate the anti-pyroptotic effects of melatonin in atherosclerotic endothelium and to elucidate the potential mechanisms.METHODS ApoE-/-mice were randomly divid-ed into four groups (n=8): the normal-diet group (ND), the normal-diet group treated with melatonin (10 mg·kg-1)(ND+MLT),the high-fat-diet group(HFD),and the high-fat-diet group treated with melatonin (HFD+MLT).After 12 weeks,the expression levels of pyroptosis related genes including NLRP3,ASC, cleaved-caspase 1,GSDMD-N,IL-1β and IL-18 were examined in aortic endothelium by Western blotting, qRT-PCR and immunofluorescent staining.Besides,levels of MEG3 and miR-223 were also tested by qRT-PCR.The interaction between MEG3 and miR-223 was detected by luciferase assay.For in vitro study,human aortic endothelial cells(HAECs)were transiently transfected with miR-223 mimic,miR-223 inhibitor (AMO-223), MEG3-overexpressing plasmid or negative controls. After 6 h of transfection, the medium was replaced by fresh medium with or without ox-LDL(25 μg·mL-1)for 24 h and then treated with or without melatonin (10 μmol·L-1) for 48 h. Cell pyroptosis was evaluated by Hoechst 33342/PI staining and differentially expressed pyroptosis related genes. RESULTS Melatonin markedly reduced the atherosclerotic plaque in aorta of ApoE-/- mice. Meanwhile, melatonin also attenuated the expression NLRP3, ASC, cleaved-caspase1, NF-κB/GSDMD, GSDMD-N termini, IL-1β, and IL-18 in aortic endo-thelium.Consistent anti-pyroptotic effects were also observed in ox-LDL-treated HAECs.We found that lncRNAMEG3 enhanced pyroptosis in HAECs. Moreover, MEG3 acted as an endogenous sponge by sequence complementarity to suppress the function of miR-223 and to increase NLRP3expression and enhance endothelial cell pyroptosis. Furthermore, knockdown of miR-223 blocked the anti-pyroptotic actions of melatonin in ox-LDL-treated HAECs. CONCLUSION Melatonin prevents endothelial cell pyroptosis via MEG3/miR-223/NLRP3 axis in atherosclerosis and therefore melatonin replacement might be considered a new strategy for protecting endothelium against pyroptosis thereby for the treat-ment of atherosclerosis associated with pyroptosis.

Expression and clinical significance of MEG3 and GAS5 in the colorectal cancer / 中国现代普通外科进展

Objective:To detect the expression of long non-coding RNA (Long non-coding RNA,IncRNA) maternally expressed gene 3 (maternally expressed gene 3,MEG3),specific growth inhibitor 5 (Growth arrest-specific 5,GAS5) and to analyze the correlation between GAS5 and cyclin P21,E2F1,so as to investigate the mechanism of GAS5.Methods:From December 2014 to December 2015,63 consecutive Patients with colorectal cancer admitted to Qingdao Municipal Hospital for surgical treatment were included into our study.We collect colorectal cancer tissues and paired normal tissues.We tested the relative expression of MEG3,GAS5 genes by real-time quantitative PCR (RT-PCR),and Western blot was used to detect the expression of P21 and E2F1 in colorectal cancer and to evaluate their correlations with GAS5.Results:The expression of MEG3 in cancer (7.76 ± 1.38) was lower than that in normal tissues(9.52 ± 1.31)P＜0.052.The expression of GAS5 was decreased in cancer tissues (3.98 ± 1.15) relative to the normal (6.21 ± 1.33)P＜0.05.3.It showed that there were positive relationships between P21 and GAS5(r=0.410,P=0.001),and there were negative relationships between E2F1 and GAS5(r=-0.366,P=0.003).Conclusions:MEG3 and GAS5 were decreased in colorectal cancer,suggesting that they play inhibiting effects on the cancer.P21 and E2F1 are important target spots that GAS5 give play to the role of tumor suppressor.

LncRNA-MEG3 mediated apoptosis of retinoblastoma by regulating P53 pathway / 眼科新进展

Objective To investigate whether MEG3 involved in the development of retinoblastoma by down-regulating the expression of P53 protein.Methods The MEG3 expression of retinoblastoma tissues and corresponding non-tumor tissues were detected by quantitative real-time PCR (qRT-PCR).Retinoblastoma cell lines SO-RB50 or HXO-RB44 were transfected with pcDNA-MEG3 or siRNA-MEG3,after which cell apoptosis was tested by flow cytometry and P53 protein expression was tested by Western blot.Results MEG3 expression of retinoblastoma tissues was significantly reduced compared with corresponding non-tumor tissues(P =0.014).MEG3 level was significantly increased in pcDNA-MEG3 transfected SO-RB50 cells (P =0.002) and significantly decreased in siRNA-MEG3 transfected HXO-RB44 cells (P =0.004).Flow cytometry showed that the SO-RB50 cells apoptosis was significantly increased with the MEG3 over-expression(P ＜ 0.05),as well as the HXO-RB44 cells apoptosis was significantly decreased with the MEG3 knockdown(P ＜ 0.05),compared with the control group,respectively.Furthermore,Western blot showed that P53 protein level was significantly increased after SO-RB50 transfected with pcDNA-MEG3 (P ＜ 0.05),while significantly decreased after HXO-RB44 transfected with siRNA-MEG3 (P ＜ 0.05),compared with the control group,respectively.Conclusion MEG3 is down-regulated in retinoblastoma,affect the development of retinoblastoma,and may induce the retinoblastoma cell apoptosis by promoting the expression of P53 protein.

LncRNA MEG3 Regulates Imatinib Resistance in Chronic Myeloid Leukemia via Suppressing MicroRNA-21

Imatinib resistance has become a major clinical problem for chronic myeloid leukemia. The aim of the present study was to investigate the involvement of MEG3, a lncRNA, in imatinib resistance and demonstrate its underlying mechanisms. RNAs were extracted from CML patients’ peripheral blood cells and human leukemic K562 cells, and the expression of MEG3 was measured by RT-qPCR. Cell proliferation and cell apoptosis were evaluated. Western blotting was used to measure the protein expression of several multidrug resistant transporters. Luciferase reporter assay was performed to determine the binding between MEG3 and miR-21. Our results showed that MEG3 was significantly decreased in imatinib-resistant CML patients and imatinib-resistant K562 cells. Overexpression of MEG3 in imatinib-resistant K562 cells markedly decreased cell proliferation, increased cell apoptosis, reversed imatinib resistance, and reduced the expression of MRP1, MDR1, and ABCG2. Interestingly, MEG3 binds to miR-21. MEG3 and miR-21 were negatively correlated in CML patients. In addition, miR-21 mimics reversed the phenotype of MEG3-overexpression in imatinib-resistant K562 cells. Taken together, MEG3 is involved in imatinib resistance in CML and possibly contributes to imatinib resistance through regulating miR-21, and subsequent cell proliferation, apoptosis and expression of multidrug resistant transporters.

Methylation state of MEG3 gene in epithelial ovarian cancer and its clinical significanc research / 实用医学杂志

Objective To study the relationship between the methylation status of CPG islands in MEG3 gene promoter region of epithelial ovarian cancer and its clinical and pathological features. Methods The promoter methylation status was evaluated by MSP (methylation-specific polymerase chain reaction ) in 47 cases of ovarian cancer tissue and 15 cases of normal control. Results The methylation ratio (42.6%) of the MEG3 genes in the ovarian cancer was statistically significantly higher (P = 0.035 ) than that (13.3%) in the normal control. The methyation rate of the group with an age > 60 years old was slightly higher than that of the group with an age≤60 years old, without statistically significant (P > 0.05), so was observed in ovarian cancers of stage Ⅰ andⅡ than that in stage Ⅲ and Ⅳ. There were also no significant differences in MEG3 gene methylation positive rate neither in different pathological grading nor in various ovarian cancer tissues (P > 0.05). Conclusion Abnormal methylation in MEG3 gene may be associated with epithelial ovarian cancer , but no relation to its clinical pathology.

Expression of DLK1 and MEG3 genes in porcine tissues during postnatal development

The Drosophila-like homolog 1 (DLK1), a transmembrane signal protein similar to other members of the Notch/Delta/Serrate family, regulates the differentiation process in many types of mammalian cells. Callipyge sheep and DLK1 knockout mice are excellent examples of a fundamental role of the gene encoding DLK1 in muscle growth and fat deposition. DLK1 is located within co-regulated imprinted clusters (the DLK1/DIO3 domain), along with other imprinted genes. Some of these, e.g. the RNA coding MEG3 gene, presumedly interfere with DLK1 transcription. The aim of our study was to analyze DLK1 and MEG3 gene expression in porcine tissues (muscle, liver, kidney, heart, brain stem) during postnatal development. The highest expression of both DLK1 and MEG3 variant 1 (MEG3 var.1) was observed in the brain-stem and muscles, whereas that of MEG3 variant 2 (MEG3 var.2) was the most abundant in muscles and the heart. During development (between 60 and 210 days of age) expression of analyzed genes was down-regulated in all the tissues. An exception was the brain-stem, where there was no significant change in MEG3 (both variants) mRNA level, and relatively little decline (2-fold) in that of DLK1 transcription. This may indicate a distinct function of the DLK1 gene in the brain-stem, when compared with other tissues.

Effect of 5-aza-2’-deoxycytidine on MEG3 gene expression and proliferation of human mammary cancer line MCF7 / 中国癌症杂志

Background and purpose:The MEG3 gene is a imprinted gene, whose loss may be associated with the pathogenesis and progression of several tumor types. This study was done to investigate the transcription of MEG3 mRNA in human mammary cancer cell line MCF7 and cell proliferation, in order to explore the effect of the methylation inhibitor, 5-aza-2'-deoxycytidine (5-aza-CdR) on MEG3 gene expression and proliferation in MCF7. Methods:MCF7 was treated with 5 ?mol/L 5-aza-CdR for 2, 4, 6 days, then the alteration of MEG3 gene expression was detected by RT-PCR and Northern blot technology and the proliferation difference in cell growth of MCF7 was observed by MTT. Results:After treated with 5-aza-CdR, the transcription of MEG3 mRNA in MCF7 was increased and the growth of MCF7 was reduced. MCF7 was treated with 5 ?mol/L 5-aza-CdR for 2, 4, 6 days, the inhibitory rates were (23.16?3.93), (49.39?2.38), (64.73?2.24), there were signifi cant differences between them. Conclusion:The growth of MCF7 was possibly inhibited by MEG3 gene, and the downregulation of MEG3 gene might result from the methylation, which was involved in the mammary cancer pathogenesis.