Journal: Translational Oncology

Article Title: MicroRNA-376a-3p sensitizes CPT-11-resistant colorectal cancer by enhancing apoptosis and reversing the epithelial-to-mesenchymal transition (EMT) through the IGF1R/PI3K/AKT pathway

doi: 10.1016/j.tranon.2024.102125

Figure Lengend Snippet: IGF1R and its downstream PI3K/ AKT pathway are upregulated in CPT-11 resistant cells. Online bioinformatic tools, namely TargetScan, miRTarbase and miRTargetLink, were used to identify genes predicted to be targeted by miRNA-376a-3p (A). A total of sixteen genes were identified, overlapping across the three tools. Among, was IGF1R (B). The DAVID online bioinformatic platform was used to elucidate biological functions and pathways associated with these genes. Among them was PI3K/AKT pathway aligned with IGF1R (C). Before proceeding with the subsequent experiments, the expression of IGF1R in CPT-11R cells was evaluated by western blot analysis and immunofluorescence, comparing with that of LoVo cells as well as another CRC cell line Caco2 ( D -E). The expression of downstream survival regulated by IGF1R, the PI3K/AKT pathway, and its response to CPT-11 treatment in CPT-11R were evaluated in CPT-11R cells and compared to their LoVo cells (F). Results are presented as means ± standard deviation from three independent experiments. Statistical significance was determined with the following annotations: ns (not significant). ns: not significant p < 0.05 (* p < 0.05, ** <0.01, *** p < 0.001).

Article Snippet: These membranes were then blocked in 5 % skimmed milk in tris-buffered saline with tween 20 (TBST) at room temperature for 1 hour and incubated with primary antibodies in TBST at the following dilutions: PARP (Cell Signaling; #9542 anti-rabbit, 1:1000), procaspase 3 (Cell Signaling; anti-rabbit, #9662, 1:1000), Cleaved caspase 3 (Cell Signaling; #9664, anti-rabbit, 1:1000), Housekeeping control β-actin (Santa Cruz; sc-47,778, anti-mouse, 1:1000), IGF1R (Santa Cruz, sc-81,464, anti-mouse, 1:1000), PI3K p100 (Santa Cruz, Sc 365,404, anti-mouse, 1:1000), p-PI3K p85 (Cell Signaling, anti-rabbit, 1:1000), AKT1 (Santa Cruz; sc5298, anti-mouse, 1:1000), p-AKT (Cell Signaling; #9275, anti-rabbit, 1:1000), E-cadherin (Santa Cruz, sc-8426, anti-mouse, 1:1000), N-cadherin (Santa Cruz, sc-271,386, anti-mouse, 1:1000), vimentin ( Santa Cruz, sc-32,322, anti-mouse, 1:1000) and SNAI1(Santa Cruze, sc-10,433, anti-goat, 1:1000) at 4 °C overnight.

Techniques: Expressing, Western Blot, Immunofluorescence, Standard Deviation

Journal: Translational Oncology

Article Title: MicroRNA-376a-3p sensitizes CPT-11-resistant colorectal cancer by enhancing apoptosis and reversing the epithelial-to-mesenchymal transition (EMT) through the IGF1R/PI3K/AKT pathway

doi: 10.1016/j.tranon.2024.102125

Figure Lengend Snippet: Downregulation of IGF1R sensitizes resistant cells to CPT-11 . To explore the role of IGF1R in CPT-11R cells, IGF1R was knocked down using siRNA. Knockdown of IGF1R was confirmed by western blot (A). Subsequently, the impact of IGF1R knockdown on cell viability and EMT and sensitization to CPT-11 was assessed by MTT and western blot respectively ( B -C). Additionally, transwell and wound healing assay were performed to determine the effect of IGF1R downregulation on response to CPT-11(D). Furthermore, IGF1R downstream, PI3K/AKT signaling pathway was assessed for its response to IGF1R knockdown (E). Results are presented as means ± standard deviation from three independent experiments. Statistical significance was determined with the following annotations: ns (not significant). ns: not significant, p < 0.05 (* p < 0.05, ** <0.01, *** p < 0.001).

Article Snippet: These membranes were then blocked in 5 % skimmed milk in tris-buffered saline with tween 20 (TBST) at room temperature for 1 hour and incubated with primary antibodies in TBST at the following dilutions: PARP (Cell Signaling; #9542 anti-rabbit, 1:1000), procaspase 3 (Cell Signaling; anti-rabbit, #9662, 1:1000), Cleaved caspase 3 (Cell Signaling; #9664, anti-rabbit, 1:1000), Housekeeping control β-actin (Santa Cruz; sc-47,778, anti-mouse, 1:1000), IGF1R (Santa Cruz, sc-81,464, anti-mouse, 1:1000), PI3K p100 (Santa Cruz, Sc 365,404, anti-mouse, 1:1000), p-PI3K p85 (Cell Signaling, anti-rabbit, 1:1000), AKT1 (Santa Cruz; sc5298, anti-mouse, 1:1000), p-AKT (Cell Signaling; #9275, anti-rabbit, 1:1000), E-cadherin (Santa Cruz, sc-8426, anti-mouse, 1:1000), N-cadherin (Santa Cruz, sc-271,386, anti-mouse, 1:1000), vimentin ( Santa Cruz, sc-32,322, anti-mouse, 1:1000) and SNAI1(Santa Cruze, sc-10,433, anti-goat, 1:1000) at 4 °C overnight.

Techniques: Knockdown, Western Blot, Wound Healing Assay, Standard Deviation

Journal: Translational Oncology

Article Title: MicroRNA-376a-3p sensitizes CPT-11-resistant colorectal cancer by enhancing apoptosis and reversing the epithelial-to-mesenchymal transition (EMT) through the IGF1R/PI3K/AKT pathway

doi: 10.1016/j.tranon.2024.102125

Figure Lengend Snippet: MiRNA-376a-3p downregulates IGF1R in CPT-11 resistant LoVo cells. Cells were transfected to overexpress MicroRNA-376a-3p. Transfection efficiency was determined by RT-qPCR (A). The effect of miRNA-376a-3p mimic on IGF1R expression was assessed by western blot (B). To confirm the regulatory effect of miRNA-376a-3p on IGF1R, the TargetScan bioinformatics tool was utilized to identify the predicted target region of miRNA-376a-3p on the 3′ UTR of IGF1R (C). Subsequently, a luciferase activity assay was performed to validate this targeting (D). Results are presented as means ± standard deviation from three independent experiments. Statistical significance was determined with the following annotations: ns (not significant). ns: not significant, p < 0.05 (* p < 0.05, ** <0.01, *** p < 0.001). MNC: Mimic negative control, WT: Wild-type, MUT: Mutant-type.

Article Snippet: These membranes were then blocked in 5 % skimmed milk in tris-buffered saline with tween 20 (TBST) at room temperature for 1 hour and incubated with primary antibodies in TBST at the following dilutions: PARP (Cell Signaling; #9542 anti-rabbit, 1:1000), procaspase 3 (Cell Signaling; anti-rabbit, #9662, 1:1000), Cleaved caspase 3 (Cell Signaling; #9664, anti-rabbit, 1:1000), Housekeeping control β-actin (Santa Cruz; sc-47,778, anti-mouse, 1:1000), IGF1R (Santa Cruz, sc-81,464, anti-mouse, 1:1000), PI3K p100 (Santa Cruz, Sc 365,404, anti-mouse, 1:1000), p-PI3K p85 (Cell Signaling, anti-rabbit, 1:1000), AKT1 (Santa Cruz; sc5298, anti-mouse, 1:1000), p-AKT (Cell Signaling; #9275, anti-rabbit, 1:1000), E-cadherin (Santa Cruz, sc-8426, anti-mouse, 1:1000), N-cadherin (Santa Cruz, sc-271,386, anti-mouse, 1:1000), vimentin ( Santa Cruz, sc-32,322, anti-mouse, 1:1000) and SNAI1(Santa Cruze, sc-10,433, anti-goat, 1:1000) at 4 °C overnight.

Techniques: Transfection, Quantitative RT-PCR, Expressing, Western Blot, Luciferase, Activity Assay, Standard Deviation, Negative Control, Mutagenesis

Journal: Translational Oncology

Article Title: MicroRNA-376a-3p sensitizes CPT-11-resistant colorectal cancer by enhancing apoptosis and reversing the epithelial-to-mesenchymal transition (EMT) through the IGF1R/PI3K/AKT pathway

doi: 10.1016/j.tranon.2024.102125

Figure Lengend Snippet: MicroRNA-376a-3p sensitizes CPT-11 resistant cells to CPT-11 through PI3K/AKT pathway via IGF1R downregulation. To determine the mechanism by which miRNA-376a-3p sensitizes CPT-11R cells, we transfected cells with miRNA-376a-mimic, CPT-11, their combination and the respective controls, and assessed the expression of IGF1R using both immunocytochemistry and western blot ( A -B). Subsequently, we examined the expression of the downstream pathway of IGF1R, the PI3K/AKT pathway, through western blot analysis (C). Results are shown as means ± standard deviation from three independent experiments. ns: not significant, p < 0.05 (* p < 0.05, ** <0.01, *** p < 0.001). MNC: Mimic negative control.

Article Snippet: These membranes were then blocked in 5 % skimmed milk in tris-buffered saline with tween 20 (TBST) at room temperature for 1 hour and incubated with primary antibodies in TBST at the following dilutions: PARP (Cell Signaling; #9542 anti-rabbit, 1:1000), procaspase 3 (Cell Signaling; anti-rabbit, #9662, 1:1000), Cleaved caspase 3 (Cell Signaling; #9664, anti-rabbit, 1:1000), Housekeeping control β-actin (Santa Cruz; sc-47,778, anti-mouse, 1:1000), IGF1R (Santa Cruz, sc-81,464, anti-mouse, 1:1000), PI3K p100 (Santa Cruz, Sc 365,404, anti-mouse, 1:1000), p-PI3K p85 (Cell Signaling, anti-rabbit, 1:1000), AKT1 (Santa Cruz; sc5298, anti-mouse, 1:1000), p-AKT (Cell Signaling; #9275, anti-rabbit, 1:1000), E-cadherin (Santa Cruz, sc-8426, anti-mouse, 1:1000), N-cadherin (Santa Cruz, sc-271,386, anti-mouse, 1:1000), vimentin ( Santa Cruz, sc-32,322, anti-mouse, 1:1000) and SNAI1(Santa Cruze, sc-10,433, anti-goat, 1:1000) at 4 °C overnight.

Techniques: Transfection, Expressing, Immunocytochemistry, Western Blot, Standard Deviation, Negative Control

Journal: Translational Oncology

Article Title: MicroRNA-376a-3p sensitizes CPT-11-resistant colorectal cancer by enhancing apoptosis and reversing the epithelial-to-mesenchymal transition (EMT) through the IGF1R/PI3K/AKT pathway

doi: 10.1016/j.tranon.2024.102125

Figure Lengend Snippet: MiRNA-376a-3p improved antitumor efficacy against murine CPT-11 resistant colorectal cancer xenograft in BALB/c nude mice. CPT-11 resistant tumors were induced by intracutaneously injecting CPT-11 resistant cells into the flanks of mice. Tumor growth was closely monitored both before and throughout the six-week treatment period (A). At the conclusion of the study, mice were humanely euthanized, and tumors were excised. Tumor sizes and weights were then measured and compared against controls (B-D). Subsequently, tumors were embedded in paraffin sections, sliced, and mounted on microscope slides for staining. TUNEL and H & E staining techniques were employed (E). Proteins were extracted from the tumors, and Western blot analysis was conducted to evaluate apoptotic marker, caspase 3 (F), IGF1R (G), EMT markers (H), and the expression of the PI3K/AKT pathway (I). Additionally, paraffin-embedded slides were utilized for immunohistochemistry to assess the expression of IGF1R, E-cadherin, N-cadherin,vimentin and SNAI1 (J). The survival probability of the mice involved in the study was predicted using Kaplan Meier survival curves (K). Results are presented as means ± standard deviation from three independent experiments. "ns" denotes non-significance, with statistical significance set at p < 0.05 (* p < 0.05, ** <0.01, *** p < 0.001). MNC stands for Mimic Negative Control.

Article Snippet: These membranes were then blocked in 5 % skimmed milk in tris-buffered saline with tween 20 (TBST) at room temperature for 1 hour and incubated with primary antibodies in TBST at the following dilutions: PARP (Cell Signaling; #9542 anti-rabbit, 1:1000), procaspase 3 (Cell Signaling; anti-rabbit, #9662, 1:1000), Cleaved caspase 3 (Cell Signaling; #9664, anti-rabbit, 1:1000), Housekeeping control β-actin (Santa Cruz; sc-47,778, anti-mouse, 1:1000), IGF1R (Santa Cruz, sc-81,464, anti-mouse, 1:1000), PI3K p100 (Santa Cruz, Sc 365,404, anti-mouse, 1:1000), p-PI3K p85 (Cell Signaling, anti-rabbit, 1:1000), AKT1 (Santa Cruz; sc5298, anti-mouse, 1:1000), p-AKT (Cell Signaling; #9275, anti-rabbit, 1:1000), E-cadherin (Santa Cruz, sc-8426, anti-mouse, 1:1000), N-cadherin (Santa Cruz, sc-271,386, anti-mouse, 1:1000), vimentin ( Santa Cruz, sc-32,322, anti-mouse, 1:1000) and SNAI1(Santa Cruze, sc-10,433, anti-goat, 1:1000) at 4 °C overnight.

Techniques: Microscopy, Staining, TUNEL Assay, Western Blot, Marker, Expressing, Immunohistochemistry, Standard Deviation, Negative Control