scholarly journals miR-146a-5p Regulated Cell Proliferation and Apoptosis by Targeting SMAD3 and SMAD4

2020 ◽  
Vol 27 (5) ◽  
pp. 411-418 ◽  
Author(s):  
Meiyu Qiu ◽  
Tao Li ◽  
Binhu Wang ◽  
Hongbin Gong ◽  
Tao Huang
Keyword(s):  
Cell Proliferation ◽  
Protein Expression ◽  
Early Pregnancy ◽  
Caspase 3 ◽  
Target Genes ◽  
Luciferase Reporter ◽  
Bewo Cell ◽  
Regulation Mechanism ◽  
Placental Development ◽  
Qrt Pcr

Background: microRNAs (miRNAs) are a small, endogenous non-coding RNAs that are involved in post-transcriptional gene regulation of many biological processes, including embryo implantation and placental development. In our previous study, miR-146a-5p was found expressed higher in the serum exosomes of pregnant sows than non-pregnant. The research on miR-146a-5p has been mainly related to human diseases, but there are few studies on its effects on the reproduction of sows in early pregnancy. Objective: In this article, our motivation is to study the role of miR-146a-5p in the early pregnancy of sows on the cell proliferetion and apoptosis by targeting SMAD3 and SMAD4. Methods: Bioinformatics software was used to identify the target genes of miR-146a-5p. The wildtype and mutant-type recombinant plasmids of dual-luciferase reporter with 3'-UTR of Smad3 or 3'- UTR of Smad4 were constructed, and co-transfected in porcine kidney cell (PK-15 cell) with miR- 146a-5p mimic, mimic-NC(M-NC), inhibitor and inhibitor-NC(IN-NC), then dual-luciferase activity analysis, qRT-PCR and Western blot were performed to verify the target genes. After the transfection of BeWo choriocarcinoma cell (BeWo cell) with miR-146a-5p mimic, M-NC, inhibitor and IN-NC, the mRNA expression of Caspase-3, BAX and Bcl-2 was measured using qRT-PCR, and the cell proliferation was measured using CCK-8 kit. Results: The luciferase, mRNA and protein expression of Smad3 in PK-15 cells treated by Smad3- 3'-UTR-W co-transfected with miR-146a-5p mimic were significantly lower than that with miR- 146a-5p M-NC, and the results of Smad4 were similar to Smad3, but the protein expression had a trend to lower in mimic group. The expression level of Bcl-2 in the miR-146a-5p mimic group was significantly lower than that in the miR-146a-5p M-NC group, but the expression pattern of Caspase-3 was just opposite. The mimic of miR-146a-5p reduced the proliferation of BeWo cells, however the inhibitor increased. Conclusion: Smad3 and Smad4 are the direct target genes of miR-146a-5p. The expression of Smad3 and Smad4 were affected by the mimic and inhibitor of miR-146a-5p. miR-146a-5p affects cell apoptosis and proliferation by regulating their target genes. This study provided new data to understand the regulation mechanism of early pregnancy in sows.

scholarly journals Overexpression of NELFE contributes to gastric cancer progression via Wnt/β-catenin signaling-mediated activation of CSNK2B expression

2021 ◽  
Vol 40 (1) ◽  
Author(s):  
Shijun Yu ◽  
Li Li ◽  
Hui Cai ◽  
Bin He ◽  
Yong Gao ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Cell Proliferation ◽  
Cancer Progression ◽  
Target Genes ◽  
Mrna Level ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Loss Of Function ◽  
Mice Model ◽  
Qrt Pcr

Abstract Background Accumulating evidence has highlighted the importance of negative elongation factor complex member E (NELFE) in tumorigenesis. However, the relationship between NELFE and gastric cancer (GC) remains unclear. This study aimed to explore the expression pattern and specific function of NELFE in GC. Methods NELFE expression was evaluated by immunohistochemistry and qRT-PCR in GC tissues, respectively. Cell proliferation, migration and invasion were measured by CCK-8, colony formation, transwell assays, and nude mice model. Bioinformatics analysis was performed to search potential target genes of NELFE, and a Cignal Finder 10-Pathway Reporter Array was used to explore potential signaling pathways regulated by NELFE. Dual-luciferase reporter assays, qRT-PCR and western blotting were conducted to verify their regulatory relationship. The expression correlations among NELFE, β-catenin and CSNK2B were further explored by immunohistochemistry on consecutive resections. Results NELFE was significantly overexpressed in GC tissues both in protein and mRNA level and negatively correlated with the prognosis of GC patients. Gain- and loss-of-function experiments showed that NELFE potentiated GC cell proliferation and metastasis in vitro and in vivo. CSNK2B was identified as a downstream effector of NELFE. Wnt/β-catenin signaling may mediate the regulation of CSNK2B by NELFE. In addition, NELFE, β-catenin and CSNK2B were all remarkably upregulated in tumor tissues compared with adjacent normal tissues, and their expression levels in GC were positively correlated with each other. Conclusion Our findings reveal a new NELFE-Wnt/β-catenin-CSNK2B axis to promote GC progression and provide new candidate targets against this disease.

scholarly journals Mir-573 regulates cell proliferation and apoptosis by targeting Bax in human degenerative disc cells following hyperbaric oxygen treatment

2020 ◽  
Author(s):  
Song-Shu Lin ◽  
Chi-Chien Niu ◽  
Li-Jen Yuan ◽  
Tsung-Ting Tsai ◽  
Po-Liang Lai ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Hyperbaric Oxygen ◽  
Caspase 3 ◽  
Target Genes ◽  
Vital Role ◽  
Luciferase Reporter ◽  
Nucleus Pulposus Cells ◽  
Caspase 9 ◽  
Proliferation And Apoptosis ◽  
Reporter Assays

Abstract Background: MicroRNA (miRNA) plays a vital role in the intervertebral disc (IVD) degeneration. The expression level of miR-573 was downregulated whereas Bax was upregulated notably in human degenerative nucleus pulposus cells (NPCs). In this study, we aimed to investigate the role of miR-573 in human degenerative NPCs following hyperbaric oxygen (HBO) treatment. Methods: NPCs were separated from human degenerated IVD tissues. The control cells were maintained in 5% CO2/95% air and the hyperoxic cells were exposed to 100% O2 at 2.5 atmospheres absolute. MiRNA expression profiling was performed via microarray and confirmed by real-time PCR, and miRNA target genes were identified using bioinformatics and luciferase reporter assays. The mRNA and protein levels of Bax were measured. The proliferation of NPCs were detected using MTT assay. The protein expression levels of Bax, cleaved caspase 9, cleaved caspase 3, pro-caspase 9 and pro-caspase 3 were examined.Results: Bioinformatics analysis indicated that the 3′ untranslated region (UTR) of the Bax mRNA contained the “seed-matched-sequence” for hsa-miR-573, which was validated via reporter assays. MiR-573 was induced by HBO and simultaneous suppression of Bax was observed in NPCs. Knockdown of miR-573 resulted in upregulation of Bax expression in HBO-treated cells. In addition, overexpression of miR-573 by HBO increased cell proliferation and coupled with inhibition of cell apoptosis. The cleavage of pro‑caspase 9 and pro‑caspase 3 was suppressed while the levels of cleaved caspase 9 and caspase 3 were decreased in HBO-treated cells. Transfection with anti-miR-573 partly suppressed the effects of HBO. Conclusion: Mir-573 regulates cell proliferation and apoptosis by targeting Bax in human degenerative NPCs following HBO treatment.

scholarly journals MicroRNA-665 facilitates cell proliferation and represses apoptosis through modulating Wnt5a/β-Catenin and Caspase-3 signaling pathways by targeting TRIM8 in LUSC

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Tian-Jun Chen ◽  
Qi Zheng ◽  
Fei Gao ◽  
Tian Yang ◽  
Hui Ren ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Cell Growth ◽  
Signaling Pathway ◽  
Cell Lines ◽  
Caspase 3 ◽  
Luciferase Reporter Assay ◽  
Luciferase Reporter ◽  
Reporter Assay ◽  
Qrt Pcr

Abstract Background MicroRNAs (miRNAs) are involved in the oncogenesis, development and transformation of lung squamous cell carcinoma (LUSC). miR-665 is clinically significant and acts as a pivotal function in some cancers. Nevertheless, the effects and the potential mechanisms of miR-665 in human LUSC are still unknown. Methods To analyse the clinical significant of miR-665 in human LUSC, quantitative real-time PCR (qRT-PCR) was use to measure miR-665 expression in LUSC specimen tissues and cell lines. Tripartite motif 8 (TRIM8) was verified a target of miR-665 by performing bioinformatic prediction and luciferase reporter assay. The expression levels of TRIM8 were examined through qRT-PCR and Western blotting in LUSC specimen tissues. CCK8 assay was fulfilled for analyzing the function in LUSC cell proliferation. Flow cytometry was used to detect cell and apoptosis. TRIM8 silencing and overexpression further verified the biological effects as those caused by miR-665. Results Here we reported that miR-665 expression was upregulated in LUSC specimen tissues and cell lines. High miR-665 levels were related to differentiation, tumor size and TNM stage. miR-665 mimics facilitated LUSC cell growth and cell cycle G1-S transition and repressed apoptosis. miR-665 inhibitor suppressed cell proliferation and G1-S transition and promoted apoptosis. miR-665 expression was negatively correlated with TRIM8 mRNA expression in LUSC. Luciferase reporter assay confirmed that TRIM8 was a direct target gene of miR-665. miR-665 mimics downregulated the TRIM8 levels, and miR-665 inhibitor upregulated the TRIM8 levels in LUSC cells. Particularly, silencing TRIM8 led to the similar effects of miR-665 mimics in LUSC cells. Overexpression of TRIM8 inhibited LUSC cell proliferation in vitro and in vivo. Furthermore, miR-665 promoted LUSC cell proliferation through facilitating the Wnt5a/β-catenin signaling pathway and restrained apoptosis via inhibiting Caspase-3 signaling pathway, whereas TRIM8 suppressed cell growth by repressing the Wnt5a/β-catenin signaling pathway and induced apoptosis through activating Caspase-3 signaling pathway. Conclusions The current study demonstrates that miR-665 facilitates LUSC cell proliferation and cell cycle transition by regulation of the Wnt5a/β-Catenin signaling pathway and represses cell apoptosis via modulation of Caspase-3 signaling pathway by directly targeting TRIM8. These findings suggest that miR-665 might be a potential new target for LUSC therapy.

scholarly journals miR-339-3p regulated acute pancreatitis induced by caerulein through targeting TNF receptor-associated factor 3 in AR42J cells

2020 ◽  
Vol 15 (1) ◽  
pp. 912-922
Author(s):  
Qi Wang ◽  
Shaofeng Liu ◽  
Zhen Han
Keyword(s):  
Acute Pancreatitis ◽  
Protein Expression ◽  
Cell Apoptosis ◽  
Caspase 3 ◽  
Luciferase Reporter ◽  
Regulation Mechanism ◽  
Tnf Receptor ◽  
Associated Factor ◽  
Tnf Α ◽  
P38 Pathway

AbstractAcute pancreatitis (AP) is an inflammatory disease with high morbidity and mortality. The regulation mechanism of miRNA is involved in the production and development of various diseases, but the regulation mechanism of miRNA in AP is still not fully elucidated. The expression of miR-339-3p was detected using quantitative real-time PCR. The levels of TNF-α, IL-1β, and IL-6 were detected using enzyme-linked immunosorbent assay. Cell apoptosis was measured using flow cytometry. The protein expressions of TNF receptor-associated factor 3 (TRAF3), Bcl-2, C-caspase 3, Bax, p-p38, and p38 were measured using western blot. Luciferase reporter assay and RNA immunoprecipitation assay were applied to ensure that miR-399-3p targeted TRAF3. Caerulein promoted the expression of TNF-α, IL-1β, and IL-6, enhanced the expression of C-caspase 3 and Bax while inhibited Bcl-2 protein expression. Meanwhile, caerulein also reduced the expression of miR-339-3p and induced the expression of TRAF3 in rat pancreatic acinar cells. miR-399-3p transfection inhibited the levels of TNF-α, IL-1β, and IL-6 and C-caspase 3 and Bax protein expression as well as suppressed cell apoptosis, while increased Bcl-2 protein expression in caerulein-induced AP. TRAF3 has been verified as a target of miR-339-3p. Interestingly, the reduction of miR-399-3p inhibited the p38 pathway, which was impaired by the upregulation of TRAF3. In addition, the suppression effects of miR-339-3p on cell inflammation and apoptosis in caerulein-induced AP were reversed by enhancing TRAF3 expression. In this study, in vitro model of AP was characterized by strong inflammation and cell apoptosis. We have first demonstrated the regulatory network of miR-339-3p and TRAF3. Overexpression of miR-339-3p inhibited cell inflammation and cell apoptosis in caerulein-induced AP through modulating TRAF3 expression via the p38 pathway, providing a new therapeutic target in the treatment of AP.

scholarly journals miR-491-3p is Downregulated in Retinoblastoma and Inhibit Tumor Cells Growth and Metastasis by Targeting SNN

2020 ◽  
Author(s):  
Yang Hu ◽  
Ming Zhao ◽  
Li Li ◽  
Jie Ding ◽  
Yu-Min Gui ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Cell Lines ◽  
Target Genes ◽  
Expression Profiles ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Mesenchymal Transition ◽  
Qrt Pcr ◽  
Gene Assay ◽  
E Cadherin

Abstract Retinoblastoma (Rb) is the most common pediatric malignant tumor of the eyes. Previous studies demonstrated that miR-491-3p is downregulated in various cancers. However, its function in Rb remains unknown. A total of 15 pairs of primary Rb tissues and adjacent noncancerous tissues were collected. Quantitative real-time PCR (qRT-PCR) was used to investigate the expression profiles of miR-491-3p. qRT-PCR, western blotting and in situ immunocytochemistry were performed to investigate the expression profiles of epithelial–mesenchymal transition-related proteins (E-cadherin, Vimentin and N-cadherin) in Rb tissues and Rb cell lines as well as cell morphology. Cell proliferation was estimated by MTS and colony formation assays. Apoptosis was determined by FACS, cell migration and invasion were analyzed using transwell chambers. MiR-491-3p’s target genes were predicted using target gene prediction databases. The interplay between miR-491-3p and SNN was evaluated through dual luciferase reporter gene assay. MiR-491-3p was significantly downregulated in mixed collection of 15 pairs of Rb tissues and Rb cell lines. Overexpression of miR-491-3p enhanced apoptosis, and significantly suppressed proliferation, migration and invasion of Rb cells. In contrast, the present of miR-491-3p inhibitor showed reversed results which apoptosis decreased, while cell proliferation of ARPE-19 cells increased. In addition, miR-491-3p increased the expression of E-cadherin, and dramatically decreased the expression of Vimentin and N-cadherin in Rb tissues and Rb cell lines, noticeable changes in morphology, too, as cells became less cohesive and more adhering. We found out that SNN was the pairing target of miR-491-3p and result showed that miR-491-3p and SNN interacted with each other. We also found out that the effects of miR-491-3p were in Rb cells were almost entirely canceled out at the overexpression of SNN. Our findings collectively suggest that miR-491-3p is an important tumor suppressor in Rb, which inhibits tumor growth and metastasis in Rb. These implicate it may be explored as a new therapeutic target in Rb.

scholarly journals miR-99a-3p Targeting EIF4EBP1 Affects B Lymphocytes Function Through Autophagy and Aggravates SLE Disease Progression

2021 ◽  
Author(s):  
meng yang ◽  
Deng Danqi
Keyword(s):  
Cell Proliferation ◽  
B Cells ◽  
Protein Expression ◽  
Reporter Gene ◽  
Immune Cells ◽  
Target Genes ◽  
Kidney Tissue ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Mrna And Protein Expression

Abstract Background:Overactivation of immune cells plays a key role in the pathogenesis of systemic lupus erythematosus (SLE). The regulation of immune cells by miRNA is a research hotspot.In this study, the second-generation high-throughput sequencing found that the expression of miR-99a-3p in SLE decreased, but the specific mechanism is still unclear.The purpose of this study is to explore the potential target genes, target cells of miR-99a-3p and their potential mechanisms affecting the progression of SLE.Methods: Isolate PBMC from healthy individulas and SLE patients, transfect Ball-1, Jurkat, THP-1 and K562 cells with miR-99a-3p agomir and antagomir,detect miR-99a-3p, predict target genes and autophagy pathway mRNA and protein expression by RT-qPCR and Western blotting.CCK-8 method detects cell proliferation, PI method detects cell cycle, flow cytometry detects cell apoptosis, and luciferase reporter gene experiment determines miR-99a-3p target genes.With C57BL/6J mice as control,construct miR-99a-3p overexpression and interference model based on MRL/lpr mice,ELISA detects plasma ANA, dsDNA, IgE, IgM, IL-6, IL-10, BLyS.Pathological analysis of HE staining and C3 immunofluorescence(IF) deposition in mouse kidney tissue,Immunohistochemistry(IHC) detects changes in target genes and pathway proteins in kidney tissue,isolate B cells to verify the differential expression of miR-99a-3p, target genes, pathway mRNA and protein.Results: Compared with healthy individulas, miR-99a-3p in SLE was down-regulated, while EIF4EBP1, LC3II, LAMP-2A mRNA and protein expression were up-regulated.After Ball-1 was transfected with miR-99a-3p agomir, cell proliferation decreased and apoptosis increased.After transfected with miR-99a-3p antagomir, the effect was opposite;Luciferase reporter gene detection proved that miR-99a-3p directly targets EIF4EBP1. Rescue experiments confirmed the interaction model between miR-99a-3p and EIF4EBP1. Clinical, in vitro, and in vivo experiments further confirmed that miR-99a-3p agomir can reduce the expression of EIF4EBP1, LC3-Ⅱ, and LAMP-2A, while miR-99a-3p antagomir had the opposite effect.In vivo experiment antagomir group mice serum ANA, dsDNA, IgE, IgM, IL-6, IL-10, BLyS were higher than those in the MRL/lpr group, EIF4EBP1, LC3-Ⅱ, LAMP-2A mRNA, protein and IHC levels also increased, and the urinary protein and C3 IF deposition of mice in the antagomir group were increased, and the related indexes of mice in the agomir group were lower than those in the MRL/lpr group.Conclusion:The expression of miR-99a-3p in SLE PBMC was down-regulated. Up-regulation of miR-99a-3p by transfection can protect B cells from autophagy mediated by EIF4EBP1.The down-regulation of miR-99a-3p induces autophagy by regulating the autophagy signaling pathway mediated by EIF4EBP1 in SLE B cells. These results indicate that miR-99a-3p and EIF4EBP1 may be potential targets of SLE.

scholarly journals Mir-573 regulates cell proliferation and apoptosis by targeting Bax in human degenerative disc cells following hyperbaric oxygen treatment

2021 ◽  
Vol 16 (1) ◽  
Author(s):  
Song-Shu Lin ◽  
Chi-Chien Niu ◽  
Li-Jen Yuan ◽  
Tsung-Ting Tsai ◽  
Po-Liang Lai ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Nucleus Pulposus ◽  
Hyperbaric Oxygen ◽  
Caspase 3 ◽  
Target Genes ◽  
Luciferase Reporter ◽  
Nucleus Pulposus Cells ◽  
Caspase 9 ◽  
Proliferation And Apoptosis ◽  
Reporter Assays

Abstract Background MicroRNA (miRNA) plays a vital role in the intervertebral disc (IVD) degeneration. The expression level of miR-573 was downregulated whereas Bax was upregulated notably in human degenerative nucleus pulposus cells. In this study, we aimed to investigate the role of miR-573 in human degenerative nucleus pulposus (NP) cells following hyperbaric oxygen (HBO) treatment. Methods NP cells were separated from human degenerated IVD tissues. The control cells were maintained in 5% CO2/95% air and the hyperoxic cells were exposed to 100% O2 at 2.5 atmospheres absolute. MiRNA expression profiling was performed via microarray and confirmed by real-time PCR, and miRNA target genes were identified using bioinformatics and luciferase reporter assays. The mRNA and protein levels of Bax were measured. The proliferation of NPCs was detected using MTT assay. The protein expression levels of Bax, cleaved caspase 9, cleaved caspase 3, pro-caspase 9, and pro-caspase 3 were examined. Results Bioinformatics analysis indicated that the 3′ untranslated region (UTR) of the Bax mRNA contained the “seed-matched-sequence” for hsa-miR-573, which was validated via reporter assays. MiR-573 was induced by HBO and simultaneous suppression of Bax was observed in NP cells. Knockdown of miR-573 resulted in upregulation of Bax expression in HBO-treated cells. In addition, overexpression of miR-573 by HBO increased cell proliferation and coupled with inhibition of cell apoptosis. The cleavage of pro-caspase 9 and pro-caspase 3 was suppressed while the levels of cleaved caspase 9 and caspase 3 were decreased in HBO-treated cells. Transfection with anti-miR-573 partly suppressed the effects of HBO. Conclusion Mir-573 regulates cell proliferation and apoptosis by targeting Bax in human degenerative NP cells following HBO treatment.

scholarly journals METTL14 promotes tumorigenesis by regulating lncRNA OIP5-AS1/miR-98/ADAMTS8 signaling in papillary thyroid cancer

2021 ◽  
Vol 12 (6) ◽  
Author(s):  
Xiaoping Zhang ◽  
Dan Li ◽  
Chengyou Jia ◽  
Haidong Cai ◽  
Zhongwei Lv ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Thyroid Cancer ◽  
Papillary Thyroid Cancer ◽  
Xenograft Model ◽  
Luciferase Reporter ◽  
Papillary Thyroid ◽  
Qrt Pcr ◽  
The Relationship

Abstract Background Papillary thyroid cancer (PTC) is the most common type of cancer of the endocrine system. Long noncoding RNAs (lncRNAs) are emerging as a novel class of gene expression regulators associated with tumorigenesis. Through preexisting databases available for differentially expressed lncRNAs in PTC, we uncovered that lncRNA OIP5-AS1 was significantly upregulated in PTC tissues. However, the function and the underlying mechanism of OIP5-AS1 in PTC are poorly understood. Methods Expression of lncRNA OIP5-AS1 and miR-98 in PTC tissue and cells were measured by quantitative real-time PCR (qRT-PCR). And expression of METTL14 and ADAMTS8 in PTC tissue and cells were measured by qRT-PCR and western blot. The biological functions of METTL14, OIP5-AS1, and ADAMTS8 were examined using MTT, colony formation, transwell, and wound healing assays in PTC cells. The relationship between METTL14 and OIP5-AS1 were evaluated using RNA immunoprecipitation (RIP) and RNA pull down assay. And the relationship between miR-98 and ADAMTS8 were examined by luciferase reporter assay. For in vivo experiments, a xenograft model was used to investigate the effects of OIP5-AS1 and ADAMTS8 in PTC. Results Functional validation revealed that OIP5-AS1 overexpression promotes PTC cell proliferation, migration/invasion in vitro and in vivo, while OIP5-AS1 knockdown shows an opposite effect. Mechanistically, OIP5-AS1 acts as a target of miR-98, which activates ADAMTS8. OIP5-AS1 promotes PTC cell progression through miR-98/ADAMTS8 and EGFR, MEK/ERK pathways. Furthermore, RIP and RNA pull down assays identified OIP5-AS1 as the downstream target of METTL14. Overexpression of METTL14 suppresses PTC cell proliferation and migration/invasion through inhibiting OIP5-AS1 expression and regulating EGFR, MEK/ERK pathways. Conclusions Collectively, our findings demonstrate that OIP5-AS1 is a METTL14-regulated lncRNA that plays an important role in PTC progression and offers new insights into the regulatory mechanisms underlying PTC development.

scholarly journals miR-636 inhibits EMT, cell proliferation and cell cycle of ovarian cancer by directly targeting transcription factor Gli2 involved in Hedgehog pathway

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Jiong Ma ◽  
Chunxia Zhou ◽  
Xuejun Chen
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Protein Expression ◽  
Signaling Pathway ◽  
Luciferase Reporter ◽  
Cell Proliferation And Migration ◽  
Hh Signaling ◽  
And Migration ◽  
Proliferation And Migration

Abstract Background Hedgehog (Hh) signaling pathway, which is essential for cell proliferation and differentiation, is noted to be aberrantly activated in tumor from increasing studies in recent years. MicroRNAs (miRNAs) as an important non-coding RNA in cells have been proven to possess a regulatory role specific to the Hh signaling pathway. Here, in vitro and in vivo cellular/molecular experiments were adopted to clarify the regulatory mechanism linking miR-636 to the Hh signaling pathway in ovarian cancer (OVC). Methods Protein–protein interaction analysis was performed to identify the hub gene in the Hh pathway. TargetScan database was used to predict the potential upstream regulators for Gli2. qRT-PCR was performed to test the expression of miR-636, while Western blot was conducted to detect the expression of proteins related to the Hh pathway and epithelial-mesenchymal transition (EMT). For cell functional experiments, HO-8910PM OVC cell line was used. MTT assay and wound healing assay were used to measure the effect of miR-636 on cell proliferation and migration. Flow cytometry was carried out to examine the effect of miR-636 on cell cycle, and Western blot was used to identify the change in expression of Hh and EMT-related proteins. Dual-luciferase reporter gene assay was implemented to detect the targeting relationship between miR-636 and Gli2. Xenotransplantation models were established for in vivo examination. Results Gli2 was identified as the hub gene of the Hh pathway and it was validated to be regulated by miR-636 based on the data from TargetScan and GEO databases. In vitro experiments discovered that miR-636 was significantly lowly expressed in OVC cell lines, and overexpressing miR-636 significantly inhibited HO-8910PM cell proliferation, migration and induced cell cycle arrest in G0/G1 phase, while the inhibition of miR-636 caused opposite results. Dual-luciferase reporter gene assay revealed that Gli2 was the target gene of miR-636 in OVC. Besides, overexpressed miR-636 decreased protein expression of Gli2, and affected the expression of proteins related to the Hh signaling pathway and EMT. Rescue experiments verified that overexpression of Gli2 reversed the inhibitory effect of miR-636 on HO-8910PM cell proliferation and migration, and attenuated the blocking effect of miR-636 on cell cycle. The xenotransplantation experiment suggested that miR-636 inhibited cell growth of OVC by decreasing Gli2 expression. Besides, overexpressing Gli2 potentiated the EMT process of OVC cells via decreasing E-cadherin protein expression and increasing Vimentin protein expression, and it reversed the inhibitory effect of miR-636 on OVC cell proliferation in vivo. Conclusion miR-636 mediates the activation of the Hh pathway via binding to Gli2, thus inhibiting EMT, suppressing cell proliferation and migration of OVC. Trial registration: The experimental protocol was established, according to the ethical guidelines of the Helsinki Declaration and was approved by the Human Ethics Committee of The Second Affiliated hospital of Zhejiang University School of Medicine (IR2019001235). Written informed consent was obtained from individual or guardian participants.

LncRNA SNHG14 Promotes Progression of Ovarian Cancer by Regulating miR-206 Expression

2021 ◽  
Vol 7 (5) ◽  
pp. 3997-4004
Author(s):  
Zhibo Zou ◽  
Lin Peng
Keyword(s):  
Ovarian Cancer ◽  
Flow Cytometry ◽  
Cell Proliferation ◽  
Tumor Growth ◽  
Cancer Progression ◽  
Nude Mice ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Qrt Pcr ◽  
Research Objects

Objective: This study aimed to probe into the effect of LncRNA SNHG14 on ovarian cancer progression by regulating miR-206.Methods: Fifty-seven ovarian cancer (OC) patients who were treated in our hospital from December 2017 to December 2019 were collected as the research objects. During the operation, OC tissues and paracancerous tissues of patients were collected, and the effect of SNHG14 on OC tumor growth in nude mice was detected, and SNHG14 inhibitor was transfected into OC cells. The relative expression of SNHG14 in tissues and cells was detected by qRT-PCR, cell proliferation was testedvia CCK8, migration and invasion were detected through Transwell, apoptosis was assessedvia flow cytometry, and the targeted relationship between SNHG14 and miR-206 was detected by dual luciferase reporter gene.Results: SNHG14 is highly expressed in OC tissues, cells and nude mice. Down-regulating it can inhibit the biological ability of OC cells and inhibit the growth of nude mice tumors. It can directly target miR-206 to regulate CCND1 expression and promote OC progression.Conclusion: LncRNA SNHG14 can act as miR-206 sponge to regulate CCND1 expression downstream of miR-206 and promote OC progression.

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