Journal: Molecular Oncology
Article Title: Replenishing co‐downregulated mi R ‐100‐5p and mi R ‐125b‐5p in malignant germ cell tumors causes growth inhibition through cell cycle disruption
doi: 10.1002/1878-0261.13757
Figure Lengend Snippet: Combination miR‐100‐5p/miR‐125b‐5p replenishment results in LIN28A depletion and late concomitant increases in let‐7 tumor suppressor microRNA (miRNA) levels in malignant germ cell tumor (GCT) cells. (A) LIN28A quantitative RT‐PCR (qRT‐PCR) levels in TCam2 (left) and 1411H (right) cells at day (d) 1, d2, and d3 following microRNA (miRNA) replenishment. For this work, cells were treated as described in Fig. , namely total miRNA/mimic‐negative‐control (MNC) concentrations were used at 16.7 n m ; and for combination miRNA mimics, this was an 8.33 n m equimolar concentration of each, as per the color‐code below the qRT‐PCR plots. (B) Representative LIN28A protein levels by western blot on d2 and d3 in TCam2 cells. (C) Let‐7b‐5p miRNA expression levels as quantified by qRT‐PCR at d1, d2, d3, and d6, and (D) representative LIN28A protein levels by western blot at d4 and d7; both following mimic replenishment in TCam2 cells as detailed in (A) above. Note that the experiments in (A) and (C) were performed in biological triplicate (i.e., n = 3) for individual treatment conditions, each with technical replicates; the data shown in (A) and (C) represent the mean and error bars = SEM of these biological triplicate values. For (A) and (C), statistical significance using Student's t ‐test is shown with the appropriate comparisons: * P ≤ 0.05, ** P < 0.01, *** P < 0.005, **** P < 0.001. Only significant comparisons are shown. For representative western blots shown in (B) and (D), these were from experiments performed in biological triplicate (i.e., n = 3), with tubulin protein used as a loading control. The experimental condition used is shown above each blot. The quantification number under each band on the blot represents densitometry analysis of protein expression normalized to tubulin using imagej software and referenced to expression in mimic‐negative‐control (MNC)‐treated cells, which are given an arbitrary value of 1.00. The size of the specific protein is listed in kilodaltons (kDa) on the right‐hand side of the blots. (E) Co‐expression analysis of let‐7b‐5p levels ( x ‐axis) with LIN28A mRNA levels ( y ‐axis) in malignant GCT tissue samples ( n = 156). Data obtained from the independent ENCORI Pan‐Cancer Analysis Platform. For the co‐expression analysis in (E), statistical significance is determined using linear regression analysis; the red line indicates the best fit line based on this analysis. (F) Graphical abstract summarizing potential miRNA effects on the cell cycle in malignant GCTs derived from data from this manuscript and published studies. Reduced levels (blue arrows) of tumor suppressor miR‐100‐5p/miR‐125b‐5p miRNAs result in increased levels (red arrows) of, for example, TRIM71 , with concomitant reductions in cyclin‐dependent kinase (CDK)‐interacting protein/kinase inhibitory protein (CIP/KIP) family members such as CDKN1A (blue arrow) , coding for p21 protein. In turn, this allows cell cycle progression through reduced physical inhibition (i.e., increased activity; black arrows) of CDK2 and CDK4 . As demonstrated in this study, replenishment of these downregulated miRNAs, or TRIM71 kd, reduces cell cycle proliferation through converse effects on the pathway and thus G0/G1 and G1/S transition. Further contributions to malignant GCT pathogenesis are likely from, for example, reduced miRNA‐mediated control of LIN28A levels. High LIN28A levels cause increased degradation of let‐7 tumor suppressor miRNA family members, reducing its own inhibition via the LIN28A / let‐7 feedback loop, resulting in cellular proliferation . To add to the complexity, TRIM71 itself is also a direct mRNA target of the let‐7 family . Finally, the effects of overexpressed oncogenic miR‐371~373 and miR‐302/367 miRNAs also converge on the cell cycle through CIP/KIP family members . 3′UTR, 3′ untranslated region. Schematic created with BioRender.com .
Article Snippet: Next, gDNA was isolated using phenol:chloroform from malignant GCT cells, control gDNA from normal adult testis tissue (Catalogue: R1234260‐50; BioChain Institute, Newark, CA, USA), control samples from three primary cultures of normal cervical squamous epithelium (95:14, 95:15, and NCX6) [ ] available in our laboratory, the normal fibroblast cell line HFFF2 (RRID: CVCL_2489), and the retinal pigment epithelium cell line 340‐RPE‐11tv (CVCL_W078) (both gifts from within the Department of Pathology, Cambridge, UK).
Techniques: Quantitative RT-PCR, Negative Control, Concentration Assay, Western Blot, Expressing, Control, Software, Derivative Assay, Inhibition, Activity Assay
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