Journal: American Journal of Translational Research

Article Title: LINC01619 promotes non-small cell lung cancer development via regulating PAX6 by suppressing microRNA-129-5p

doi:

Figure Lengend Snippet: LINC01619 served as a competing endogenous LncRNA (ceRNA) to sponge miR-129-5p in NSCLC. A. LINC01619 WT and MUT fragments containing the binding site for miR-129-5p was designed. B. Luciferase reporter assay indicated that miR-129-5p was directly inhibited by LINC01619. C and D. RIP assay revealed that LINC01619 was binding to miR-129-5p. E. RNA FISH experiment showed that LINC01619 and miR-129-5p were mainly colocalized in the cytoplasm. F. LINC01619 up-regulation reduced miR-129-5p expression in SPCA1 cells, whereas LINC01619 down-regulation elevated miR-129-5p expression in A549 cells. G. miR-129-5p expression was reduced in NSCLC tissues than that in normal tissues. H. In NSCLC tissues, prominently negative correlation between LINC01619 and miR-129-5p expression level was found. **P < 0.01.

Article Snippet: Luciferase reporter gene assay Fragments of LINC01619 wild type (WT), LINC01619 mutant type (MUT), PAX6 WT and PAX6 MUT containing the miR-129-5p binding sites were designed by GenePharma Co., Ltd, (Shanghai, China).

Techniques: Binding Assay, Luciferase, Reporter Assay, Expressing

Journal: American Journal of Translational Research

Article Title: LINC01619 promotes non-small cell lung cancer development via regulating PAX6 by suppressing microRNA-129-5p

doi:

Figure Lengend Snippet: PAX6 was directly suppressed by miR-129-5p. A. Luciferase reporter assay indicated that PAX6 was directly suppressed by miR-129-5p. B. LINC01619 enhanced PAX6 mRNA expression and miR-129-5p inhibited PAX6 mRNA expression in NSCLC cells. C and D. LINC01619 enhanced PAX6 protein expression and miR-129-5p inhibited PAX6 protein expression in NSCLC cells. E. PAX6 mRNA expression was increased in NSCLC tissues than that in normal tissues. F and G. In NSCLC tissues, PAX6 expression was positively correlated with LINC01619 and negatively correlated with miR-129-5p. H. PAX6 protein expression was increased in 5 pairs of NSCLC tumor tissues (T1, T2, T3, T4 and T5) than that in normal tissues (N1, N2, N3, N4 and N5). I. qRT-PCR and IHC indicated that, patients with advanced TNM stage had higher PAX6 mRNA expression level in their tumor tissues, and more PAX6 positive expression signals were found in tumor tissues (sample I, II and III) than that in normal tissues. **P < 0.01.

Article Snippet: Luciferase reporter gene assay Fragments of LINC01619 wild type (WT), LINC01619 mutant type (MUT), PAX6 WT and PAX6 MUT containing the miR-129-5p binding sites were designed by GenePharma Co., Ltd, (Shanghai, China).

Techniques: Luciferase, Reporter Assay, Expressing, Quantitative RT-PCR

Journal: International Journal of Oncology

Article Title: m 6 A-mediated LINC02038 inhibits colorectal cancer progression via regulation of the FAM172A/PI3K/AKT pathway via competitive binding with miR-552-5p

doi: 10.3892/ijo.2023.5529

Figure Lengend Snippet: Relationship between LINC02038 expression and clinical characteristics in patients with colorectal cancer (n=68).

Article Snippet: Cells were co-transfected with vector, pmirGLO LINC02038 wild type (WT) or LINC02038 mutant (MUT), FAM172A WT or FAM172A MUT (Promega Corporation) as well as miR-552-5p mimic using Lipofectamine ® 3000 (Invitrogen; Thermo Fisher Scientific, Inc.).

Techniques: Expressing

Journal: International Journal of Oncology

Article Title: m 6 A-mediated LINC02038 inhibits colorectal cancer progression via regulation of the FAM172A/PI3K/AKT pathway via competitive binding with miR-552-5p

doi: 10.3892/ijo.2023.5529

Figure Lengend Snippet: LINC02038 is downregulated in CRC. (A) Heatmap of lncRNAs. lncRNAs with an FC ≥5 and P<0.05 were considered down- or upregulated. Downregulation is represented by a blue scale, while upregulation is represented by a red scale. (B) lncRNA volcano diagram of CRC vs. non-tumorous tissues. Upregulated genes are indicated in red and downregulated genes are indicated in green (defined by FC ≥1 and P<0.05). Genes in black were differentially expressed by FC <1. (C) Expression of LINC02038 based on pan-cancer analysis utilizing the TCGA and GTEx databases. LINC02038 expression was lower in COAD tissues than in normal colorectal tissues. (D and E) The expression of LINC02038 in CRC and normal colorectal tissues based on data obtained from GTEx and TCGA datasets. (F) The relationship between the LINC02038 and OS in the obtained datasets. (G) Bubble plots showing the top 21 dysregulated KEGG pathways in terms of genes associated with LINC02038 in CRC. * P<0.05, *** P<0.001. CRC, colorectal cancer; lncRNA, long non-coding RNA; TCGA, The Cancer Genome Atlas; GTEx, Genotype-Tissue Expression project; KEGG, Kyoto Encyclopedia of Genes and Genomes; COAD, colon adenocarcinoma; OS, overall survival; ns, no significance.

Article Snippet: Cells were co-transfected with vector, pmirGLO LINC02038 wild type (WT) or LINC02038 mutant (MUT), FAM172A WT or FAM172A MUT (Promega Corporation) as well as miR-552-5p mimic using Lipofectamine ® 3000 (Invitrogen; Thermo Fisher Scientific, Inc.).

Techniques: Expressing

Journal: International Journal of Oncology

Article Title: m 6 A-mediated LINC02038 inhibits colorectal cancer progression via regulation of the FAM172A/PI3K/AKT pathway via competitive binding with miR-552-5p

doi: 10.3892/ijo.2023.5529

Figure Lengend Snippet: LINC02038 suppresses CRC cell growth, motility and invasion while inducing apoptosis. (A) LINC02038 expression in 68 paired CRC and normal colorectal tissue. (B and C) LINC02038 expression in several different CRC cell lines. (D) LINC02038 was successfully overexpressed in CRC cells transfected with pcDNA3.1-LINC02038. (E) CCK-8 assay results showed the proliferative effects of LINC02038 overexpression on CRC cells. (F) Overexpression of LINC02038 affected CRC cell cycle regulation. (G) CRC proliferation was detected using colony formation assays following LINC02038 overexpression. (H) Overexpression of LINC02038 promoted apoptosis in a fraction of the cells as measured by flow cytometry. Transwell assays showing CRC (I) migration and (J) invasion following LINC02038 overexpression. Magnification, ×200. * P<0.05, ** P<0.01 vs. normal tissues, NCM460, or empty vector; CRC, colorectal cancer.

Article Snippet: Cells were co-transfected with vector, pmirGLO LINC02038 wild type (WT) or LINC02038 mutant (MUT), FAM172A WT or FAM172A MUT (Promega Corporation) as well as miR-552-5p mimic using Lipofectamine ® 3000 (Invitrogen; Thermo Fisher Scientific, Inc.).

Techniques: Expressing, Transfection, CCK-8 Assay, Over Expression, Flow Cytometry, Migration, Plasmid Preparation

Journal: International Journal of Oncology

Article Title: m 6 A-mediated LINC02038 inhibits colorectal cancer progression via regulation of the FAM172A/PI3K/AKT pathway via competitive binding with miR-552-5p

doi: 10.3892/ijo.2023.5529

Figure Lengend Snippet: LINC02038 sponges miR-552-5p in the CRC cells. (A) LINC08038 localized to the cytoplasm (red) and nucleus (blue) in LoVo and SW620 cells using FISH test. (B) The predicted binding site between LINC02038 and miR-552-5p. (C) Using data obtained from TCGA-COAD dataset, it was shown there was a negative association between LINC02038 and miR-552-5p expression in CRC tissues. (D) TCGA-COAD database analysis confirmed that miR-552-5p expression was higher in CRC tissues than in matched and unpaired normal tissues. (E) RT-qPCR analysis of miR-552-5p expression in CRC and normal tissues. (F) RT-qPCR detection of miR-552-5p expression in CRC cells. (G) miR-552-5p expression in LoVo and SW620 cells transfected with LINC02038 overexpression or an empty vector was determined using RT-qPCR. (H) The relative luciferase activity in LoVo and SW620 cells co-transfected with LINC02038 WT or MUT and miR-552-5p mimics. (I) RIP assays were used to examine LINC02038 enrichment in LoVo and SW620 cells co-transfected with LINC0203 and miR-552-5p mimic. (J) The relationship between miR-552-5p and overall survival in CRC patients in TCGA-COAD dataset. ** P<0.01, *** P<0.001. miR, microRNA; CRC, colorectal cancer; FISH, Fluorescence in situ hybridization; TCGA, The Cancer Genome Atlas; COAD, colon adenocarcinoma; RT-qPCR, reverse transcription quantitative PCR; MUT, mutant; WT, wild-type; Ago2, argonaute 2; RIP, RNA immunoprecipitation.

Article Snippet: Cells were co-transfected with vector, pmirGLO LINC02038 wild type (WT) or LINC02038 mutant (MUT), FAM172A WT or FAM172A MUT (Promega Corporation) as well as miR-552-5p mimic using Lipofectamine ® 3000 (Invitrogen; Thermo Fisher Scientific, Inc.).

Techniques: Binding Assay, Expressing, Quantitative RT-PCR, Transfection, Over Expression, Plasmid Preparation, Luciferase, Activity Assay, Fluorescence, In Situ Hybridization, Reverse Transcription, Real-time Polymerase Chain Reaction, Mutagenesis, RNA Immunoprecipitation

Journal: International Journal of Oncology

Article Title: m 6 A-mediated LINC02038 inhibits colorectal cancer progression via regulation of the FAM172A/PI3K/AKT pathway via competitive binding with miR-552-5p

doi: 10.3892/ijo.2023.5529

Figure Lengend Snippet: LINC02038 exerts its function by inhibiting miR-552-5p in CRC cells. (A) miR-552-5p expression was determined using RT-qPCR after transfection with miR-552-5p mimic, LINC02038 overexpression + miR-552-5p mimic or the control. (B) CCK-8 and (C) colony formation assays were used to assess the proliferation of transfected cells. (D and E) Flow cytometry was used to identify the cell cycle distribution and apoptosis of SW620 and LoVo cells transfected with miR-552-5p mimic, LINC02038 overexpression + miR-552-5p mimic or the control. LINC02038 overexpression resulted in G 1 phase cell cycle arrest and increased rates of apoptosis and partially reversed the effects of miR-552-5p mimic transfection in CRC cells. (F) Migration and (G) invasion rates were determined using Transwell assays in CRC cells co-transfected with LINC02038 overexpression vector and miR-552-5p mimics as well as the corresponding controls. * P<0.05, ** P<0.01 vs. NC mimic; miR, microRNA; CRC, colorectal cancer; RT-qPCR, reverse transcription quantitative PCR; NC, negative control.

Article Snippet: Cells were co-transfected with vector, pmirGLO LINC02038 wild type (WT) or LINC02038 mutant (MUT), FAM172A WT or FAM172A MUT (Promega Corporation) as well as miR-552-5p mimic using Lipofectamine ® 3000 (Invitrogen; Thermo Fisher Scientific, Inc.).

Techniques: Expressing, Quantitative RT-PCR, Transfection, Over Expression, Control, CCK-8 Assay, Flow Cytometry, Migration, Plasmid Preparation, Reverse Transcription, Real-time Polymerase Chain Reaction, Negative Control

Journal: International Journal of Oncology

Article Title: m 6 A-mediated LINC02038 inhibits colorectal cancer progression via regulation of the FAM172A/PI3K/AKT pathway via competitive binding with miR-552-5p

doi: 10.3892/ijo.2023.5529

Figure Lengend Snippet: miR-552-5p targets and regulates FAM172A. (A) Bioinformatics analysis revealed that miR-552-5p directly targets the 3′-UTR of FAM172A. (B and C) Differential FAM172A expression in CRC and normal tissues. FAM172A expression in (D) several CRC cell lines was determined using RT-qPCR and (E) in CRC tissues by western blotting. (F) The luciferase activity of the reporter including miR-552-5p mimic and FAM172A WT or MUT was evaluated. (G) RIP assay analysis showed FAM172A or miR-552-5p expression pulled down from the lysates of SW620 and LoVo cells with the anti-Ago2 antibody. Data obtained from TCGA was utilized to investigate the association between FAM172A expression and the (H) PFS and (I) overall survival in CRC patients. Data are presented as the mean ± SD of three repeats. ** P<0.01, *** P<0.001. miR, microRNA; UTR, untranslated region; CRC, colorectal cancer; RT-qPCR, reverse transcription quantitative PCR; WT, wild-type; MUT, mutant; RIP, RNA immunoprecipitation; TCGA, The Cancer Genome Atlas; PFS, progression free survival; NC, negative control.

Article Snippet: Cells were co-transfected with vector, pmirGLO LINC02038 wild type (WT) or LINC02038 mutant (MUT), FAM172A WT or FAM172A MUT (Promega Corporation) as well as miR-552-5p mimic using Lipofectamine ® 3000 (Invitrogen; Thermo Fisher Scientific, Inc.).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Luciferase, Activity Assay, Reverse Transcription, Real-time Polymerase Chain Reaction, Mutagenesis, RNA Immunoprecipitation, Negative Control

Journal: International Journal of Oncology

Article Title: m 6 A-mediated LINC02038 inhibits colorectal cancer progression via regulation of the FAM172A/PI3K/AKT pathway via competitive binding with miR-552-5p

doi: 10.3892/ijo.2023.5529

Figure Lengend Snippet: LINC02038 inhibits CRC progression by regulating FAM172A. (A) The knockdown efficiency of siRNAs targeting FAM172A was validated using RT-qPCR. (B) FAM172A expression was examined using western blotting in the FAM172A knockdown cells and/or LINC02038 overexpressing cells. (C) FAM172A mRNA levels are shown in SW620 and LoVo cells co-transfected with siFAM172A and LINC02038 overexpression vector. (D and E) CCK-8 and colony formation assays were used to evaluate cell viability. (F) Flow cytometry was used to examine the cell cycle distribution. (G) Apoptosis was detected in SW620 and LoVo cells using Annexin V/PI double-staining by fluorescence activated cell sorting. Transwell assays were used to evaluate (H) migration and (I) invasion of SW620 and LoVo cells. Scale bars, 100 µ m. (J) The protein expression levels of AKT, p-AKT and PI3K were analyzed by western blotting in SW620 and LoVo cells. Data are presented as the mean ± SD of three repeats. * P<0.05, ** P<0.01. CRC, colorectal cancer; siRNA, small interfering RNA; RT-qPCR, reverse transcription quantitative PCR; p-, phosphorylated.

Article Snippet: Cells were co-transfected with vector, pmirGLO LINC02038 wild type (WT) or LINC02038 mutant (MUT), FAM172A WT or FAM172A MUT (Promega Corporation) as well as miR-552-5p mimic using Lipofectamine ® 3000 (Invitrogen; Thermo Fisher Scientific, Inc.).

Techniques: Knockdown, Quantitative RT-PCR, Expressing, Western Blot, Transfection, Over Expression, Plasmid Preparation, CCK-8 Assay, Flow Cytometry, Double Staining, Fluorescence, FACS, Migration, Small Interfering RNA, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: International Journal of Oncology

Article Title: m 6 A-mediated LINC02038 inhibits colorectal cancer progression via regulation of the FAM172A/PI3K/AKT pathway via competitive binding with miR-552-5p

doi: 10.3892/ijo.2023.5529

Figure Lengend Snippet: In vivo , LINC02038 inhibits CRC tumorigenesis. (A) Images of the CRC tissues following subcutaneous injection of LoVo cells with LINC02038 overexpression or pcDNA3.1/Control empty vector. The (B) volume and (C) weight of xenograft tumors were measured. (D) Representative images of IHC-stained CRC tissues. Magnification, ×100 and ×200. Data are presented as the mean ± SD of three repeats. ** P<0.01. CRC, colorectal cancer; IHC, immunohistochemistry.

Article Snippet: Cells were co-transfected with vector, pmirGLO LINC02038 wild type (WT) or LINC02038 mutant (MUT), FAM172A WT or FAM172A MUT (Promega Corporation) as well as miR-552-5p mimic using Lipofectamine ® 3000 (Invitrogen; Thermo Fisher Scientific, Inc.).

Techniques: In Vivo, Injection, Over Expression, Control, Plasmid Preparation, Staining, Immunohistochemistry

Journal: International Journal of Oncology

Article Title: m 6 A-mediated LINC02038 inhibits colorectal cancer progression via regulation of the FAM172A/PI3K/AKT pathway via competitive binding with miR-552-5p

doi: 10.3892/ijo.2023.5529

Figure Lengend Snippet: METTL3 knockdown reduces the stability of LINC02038 via an m 6 A-YTHDF2-dependent mechanism. (A) SRAMP was used to predict the possible m 6 A modification locations of LINC02038. (B) The enrichment of m 6 A-modified LINC02038 was measured using a MeRIP-qPCR assay. (C) Using RT-qPCR and western blotting, METTL3 expression was determined in CRC cells following METTL3 knockdown. (D) Correlation analysis of METTL3 and LINC02038 mRNA on data obtained from TCGA. (E) LINC02038 expression in METTL3-deficient LoVo and SW620 cells was detected using RT-qPCR. (F) shMETTL3-mediated LINC02038 m 6 A modifications were demonstrated in LoVo and SW620 cells. (G) The interaction between METTL3, METTL14, FTO and YTHDF2. (H) YTHDF2 expression of LoVo and SW620 cells transfected with shYTHDF2 were detected using RT-qPCR and western blotting. (I) LINC02038 expression was determined using RT-qPCR in YTHDF2-deficient LoVo and SW620 cells. (J) The expression of LINC02038 was detected following treatment with Actinomycin D (5 mg/ml) in SW620 cells using RT-qPCR at the indicated time points. Data are presented as the mean ± SD of three repeats. * P<0.05, ** P<0.01. m 6 A, N6-methyladenosine; MeRIP, methylated RNA immunoprecipitation; qPCR, quantitative PCR; RT-qPCR, reverse transcription qPCR; TCGA, The Cancer Genome Atlas; shRNA, short hairpin RNA.

Article Snippet: Cells were co-transfected with vector, pmirGLO LINC02038 wild type (WT) or LINC02038 mutant (MUT), FAM172A WT or FAM172A MUT (Promega Corporation) as well as miR-552-5p mimic using Lipofectamine ® 3000 (Invitrogen; Thermo Fisher Scientific, Inc.).

Techniques: Knockdown, Modification, Quantitative RT-PCR, Western Blot, Expressing, Transfection, Methylation, RNA Immunoprecipitation, Real-time Polymerase Chain Reaction, Reverse Transcription, shRNA

Journal: International Journal of Oncology

Article Title: m 6 A-mediated LINC02038 inhibits colorectal cancer progression via regulation of the FAM172A/PI3K/AKT pathway via competitive binding with miR-552-5p

doi: 10.3892/ijo.2023.5529

Figure Lengend Snippet: Graphic illustrating the m 6 A-dependent effects of the LINC02038/miR-552-5p/FAM172A/PI3K/AKT regulatory loop on CRC growth. m 6 A, N6-methyladenosine; CRC, colorectal cancer; miR, microRNA.

Article Snippet: Cells were co-transfected with vector, pmirGLO LINC02038 wild type (WT) or LINC02038 mutant (MUT), FAM172A WT or FAM172A MUT (Promega Corporation) as well as miR-552-5p mimic using Lipofectamine ® 3000 (Invitrogen; Thermo Fisher Scientific, Inc.).

Techniques:

Journal: Oncology Letters

Article Title: miR-148b-3p inhibits malignant biological behaviors of human glioma cells induced by high HOTAIR expression

doi: 10.3892/ol.2016.4743

Figure Lengend Snippet: Reciprocal negative regulation of miR-148b-3p and HOTAIR . (A) miR-148b-3p expression in HA1800 and A172 cell lines. (B and C) Effect of miR-148b-3p on HOTAIR expression. A172 cells were transfected with si-NC, (B) miR-148b-3p mimic or (C) miR-148b-3p inhibitor. (D) Upregulation of miR-148b-3p induced by ectopic expression of HOTAIR . A172 cells were transfected with si-NC or si- HOTAIR . At 24, 48 and 72 h post-transfection, mRNA expression was analyzed by reverse transcription-quantitative polymerase chain reaction. (E) miR-148b-3p binding sequence in HOTAIR . The red letters in the HOTAIR sequence show mutations. (F) A172 cells were co-transfected with the pmirGLO luciferase construct (pmirGLO-mutant or pmirGLO-wild-type) and miR-148b-3p mimic. After 24 h of incubation, cells were harvested and assayed using a luciferase reporter assay system. *P<0.05, **P<0.01 vs. si-NC. Data are presented as the mean ± standard deviation of three independent experiments. miR, microRNA. si-NC; siRNA-negative control; si- HOTAIR, siRNA HOTAIR .

Article Snippet: The human HOTAIR fragment containing the putative binding sites for miR-148b-3p was synthesized, annealed and inserted into the Not I and Xba I restriction sites of the pmirGLO luciferase reporter vector (Promega, Madison, WI, USA), downstream of the luciferase gene, to generate the recombinant vectors pmirGLO-wild-type (WT) and pmirGLO-mutant (MUT).

Techniques: Expressing, Transfection, Real-time Polymerase Chain Reaction, Binding Assay, Sequencing, Luciferase, Construct, Mutagenesis, Incubation, Reporter Assay, Standard Deviation, Negative Control