Journal: Cell Death & Disease

Article Title: MEX3A promotes the malignant progression of ovarian cancer by regulating intron retention in TIMELESS

doi: 10.1038/s41419-022-05000-7

Figure Lengend Snippet: A Heatmap showing the expression of 46 differential key RNA binding proteins in ovarian cancer ( n = 426), normal fallopian tube (FT, n = 5) and ovary tissues ( n = 88) from TCGA-GTEX database. B Kaplan–Meier analysis showed the effect of MEX3A expression on the overall survival (High expression group, n = 325; Low expression group, n = 330) and progression-free survival (High expression group, n = 307; Low expression group, n = 307) of ovarian cancer patients from Kaplan–Meier Plotter ( http://kmplot.com/ ). C Kaplan–Meier analysis showed the effect of MEX3A expression on the overall survival based the Tissue Microarray of high-grade serous ovarian cancer from our tissue bank. D Immunohistochemical staining images of MEX3A in fallopian tube and ovarian cancer based the Tissue Microarray of high-grade serous ovarian cancer from our tissue bank. E The relative expression of MEX3A in ovarian cancer, according to the current molecular typing of ovarian cancer from TCGA-OV. (Fallopian, n = 134; Immunoreactive, n = 108; Mesenchymal, n = 106; Proliferative, n = 135). F qRT-PCR analysis of MEX3A mRNA expression between ovarian cancer ( n = 24) and fallopian tube ( n = 12) tissues ( n = 4 biologically independent samples). G Western blotting showed the differences in the MEX3A protein levels between ovarian cancer ( n = 10) and fallopian tube ( n = 6) tissues. H Genetic alterations of MEX3A in ovarian cancer from TCGA cohort (Firehose Legacy, n = 579). I Relative MEX3A mRNA expression of samples with different copy number variation status from TCGA. (Non-amplified group, n = 190; amplified group, n = 55). P -value was obtained by Log-rank test ( B , C ) or unpaired t -test ( E , F , and I ). * P < 0.05, ** P < 0.01.

Article Snippet: The MEX3A overexpression lentiviral plasmid was purchased from OriGene (RC215359L3, USA).

Techniques: Expressing, RNA Binding Assay, Microarray, Immunohistochemical staining, Staining, Quantitative RT-PCR, Western Blot, Amplification

Journal: Cell Death & Disease

Article Title: MEX3A promotes the malignant progression of ovarian cancer by regulating intron retention in TIMELESS

doi: 10.1038/s41419-022-05000-7

Figure Lengend Snippet: A , B MEX3A knockdown and overexpression efficiency were confirmed by qRT-PCR ( n = 4 biologically independent samples) and western blotting. C Growth curve showing the growth of ovarian cancer cells upon MEX3A knockdown or overexpression ( n = 5 biologically independent samples). D EdU assay showed the proliferation ability changes of ovarian cancer cells after MEX3A knockdown ( n = 5 biologically independent samples). E The clonogenic assay showed the clonogenic capacity changes of ovarian cancer cells after MEX3A knockdown and overexpression ( n = 5 biologically independent samples). F Images of tumors isolated from subcutaneous implantation of nude mice with MEX3A knockdown and control cells ( n = 6 mice per group). G Tumor size and weight statistics. P -value was obtained by unpaired t -test. Results represent the mean ± SD of three independent experiments. * P < 0.05, ** P < 0.01.

Article Snippet: The MEX3A overexpression lentiviral plasmid was purchased from OriGene (RC215359L3, USA).

Techniques: Over Expression, Quantitative RT-PCR, Western Blot, EdU Assay, Clonogenic Assay, Isolation

Journal: Cell Death & Disease

Article Title: MEX3A promotes the malignant progression of ovarian cancer by regulating intron retention in TIMELESS

doi: 10.1038/s41419-022-05000-7

Figure Lengend Snippet: A Transwell assays were used to evaluate the effect of MEX3A expression on the migration of ovarian cancer cells ( n = 5 biologically independent samples). B Transwell assays were used to evaluate the effect of MEX3A expression on the invasion of ovarian cancer cells ( n = 5 biologically independent samples). P -value was obtained by unpaired t -test. Results represent the mean ± SD of three independent experiments. * P < 0.05, ** P < 0.01.

Article Snippet: The MEX3A overexpression lentiviral plasmid was purchased from OriGene (RC215359L3, USA).

Techniques: Expressing, Migration

Journal: Cell Death & Disease

Article Title: MEX3A promotes the malignant progression of ovarian cancer by regulating intron retention in TIMELESS

doi: 10.1038/s41419-022-05000-7

Figure Lengend Snippet: A Heatmap of differentially expressed genes from RNA-seq analysis of MEX3A knockdown in A2780 cells. B Bubble diagram showing the results of GO analysis of differentially expressed genes described in ( A ). C Venn diagram showing the key DEGs involved in critical alternative splicing events. D Heatmap of differential expression of selected genes ( n = 7) from RNA-seq analysis after MEX3A knockdown. E The differential expression of selected genes ( n = 7) in ovarian cancer ( n = 426) compared to normal ovaries ( n = 88) and fallopian tubes ( n = 5) through TCGA-GTEX database. F , G Relative mRNA and protein expression of TIMELESS from TCGA ovarian cancer (Fallopian tubes, n = 5; Ovary, n = 88; HGSOC, n = 426) and CPTAC databases (Ovary, n = 17; HGSOC, n = 77). H Correlation analysis between MEX3A and TIMELESS mRNA in ovarian cancer tissues from TCGA ovarian cancer ( n = 489). I qRT-PCR analysis of the relative mRNA expression of TIMELESS in ovarian cancer after MEX3A knockdown ( n = 4 biologically independent samples). J western blotting showed the MEX3A and TIMELESS protein levels after MEX3A downregulation. P -value was obtained by unpaired t -test ( F , G , and I ). Results represent the mean ± SD of three independent experiments. * P < 0.05, ** P < 0.01.

Article Snippet: The MEX3A overexpression lentiviral plasmid was purchased from OriGene (RC215359L3, USA).

Techniques: RNA Sequencing Assay, Expressing, Quantitative RT-PCR, Western Blot

Journal: Cell Death & Disease

Article Title: MEX3A promotes the malignant progression of ovarian cancer by regulating intron retention in TIMELESS

doi: 10.1038/s41419-022-05000-7

Figure Lengend Snippet: A TIMELESS knockdown reduced the colony formation abilities of MEX3A-overexpressing HEY cells ( n = 5 biologically independent samples). B Xenograft experiments showed TIMELESS knockdown partially reversed the effect of MEX3A overexpression on the growth of ovarian cancer cell in vivo ( n = 6 mice per group). C , D TIMELESS inhibition partially reversed the effect of MEX3A overexpression on cell migration and invasion ( n = 5 biologically independent samples). P -value was obtained by unpaired t -test. Results represent the mean ± SD of three independent experiments. * P < 0.05, ** P < 0.01.

Article Snippet: The MEX3A overexpression lentiviral plasmid was purchased from OriGene (RC215359L3, USA).

Techniques: Over Expression, In Vivo, Inhibition, Migration

Journal: Cell Death & Disease

Article Title: MEX3A promotes the malignant progression of ovarian cancer by regulating intron retention in TIMELESS

doi: 10.1038/s41419-022-05000-7

Figure Lengend Snippet: A Sashimi plot visualization of RNA-seq reads mapped to TIMELESS in A2780 cells after MEX3A knockdown. B Schematic showing three splicing variants of the TIMELESS mRNA transcript, as identified from the Ensemble genome browser. C Schematic diagram showing the position of intron retention and primers used for RT-PCR. D – F Relative mRNA expression of TIMELESS and its splicing variants described from TCGA, GTEX and CCLE databases. (CCLE, n = 47; Fallopian tubes, n = 5; Ovary, n = 88; HGSOC, n = 426) G Kaplan–Meier analysis of the effect of TIMELESS-205 expression on the overall survival of ovarian cancer patients from GEPIA (High expression, n = 212; Low expression, n = 209). H , I RT-PCR was used to analyze the splicing isoforms of TIMELESS in ovarian cancer cells with MEX3A knockdown ( n = 3 biologically independent samples). J qRT-PCR analysis of the proportion of TIMELESS intron retained transcripts after downregulation of MEX3A in ovarian cancer cell lines using specific intron 23 primers ( n = 4 biologically independent samples). K HEY cells were cotransfected with specific MEX3A siRNA or control and UPF1 siRNA. TIMELESS expression was examined by qRT-PCR ( n = 4 biologically independent samples). L RIP-qPCR was applied to determine the interaction between MEX3A protein and TIMELESS mRNA in MEX3A overexpressed HEY cells using Flag antibody ( n = 5 biologically independent samples). P -value was obtained by unpaired t -test. Results represent the mean ± SD of three independent experiments. * P < 0.05, ** P < 0.01.

Article Snippet: The MEX3A overexpression lentiviral plasmid was purchased from OriGene (RC215359L3, USA).

Techniques: RNA Sequencing Assay, Reverse Transcription Polymerase Chain Reaction, Expressing, Quantitative RT-PCR

Journal: Cell Death & Disease

Article Title: MEX3A promotes the malignant progression of ovarian cancer by regulating intron retention in TIMELESS

doi: 10.1038/s41419-022-05000-7

Figure Lengend Snippet: Abundant MEX3A regulates TIMELESS expression and performs oncogenic functions through maintaining the normal splicing of TIMELESS. At the deficiency of MEX3A, TIMELESS splicing goes wrong which leads to reduction of TIMELESS and decreases the oncogenic ability of MEX3A.

Article Snippet: The MEX3A overexpression lentiviral plasmid was purchased from OriGene (RC215359L3, USA).

Techniques: Expressing