Fig 2: Effect of miR-195-5p upregulation on HUVECs. (A) After transfection with mimic control or miR-195-5p mimic for 48 h, the level of miR-195-5p in HUVECs was detected using RT-qPCR. After transfection with control-plasmid or Bcl-2-plasmid for 48 h, the mRNA and protein level of Bcl-2 in HUVECs was detected using (B) RT-qPCR and (C) western blotting. (D) After transfection with mimic control, miR-195-5p mimic or miR-195-5p mimic + Bcl-2-plasmid for 48 h, the cell viability of HUVECs was detected using MTT assay. (E) After transfection with mimic control, miR-195-5p mimic or miR-195-5p mimic + Bcl-2-plasmid for 48 h, the cell apoptosis of HUVECs was detected using flow cytometry, and the cell apoptosis rate (Q2 + Q4) was calculated and presented. (F) After transfection with mimic control, miR-195-5p mimic or miR-195-5p mimic + Bcl-2-plasmid for 48 h, the protein level of Bcl-2 and Bax in HUVECs was detected using western blotting. After transfection with mimic control, miR-195-5p mimic or miR-195-5p mimic+Bcl-2-plasmid for 48 h, the mRNA level of (G) Bcl-2 and (H) Bax in HUVECs was detected using RT-qPCR. Control, HUVECs without any treatment; mimic control, HUVECs transfected with mimic control for 48 h; miR-195-5p mimic, HUVECs transfected with miR-195-5p mimic for 48 h; control-plasmid, HUVECs transfected with control-plasmid for 48 h; Bcl-2-plasmid, HUVECs transfected with Bcl-2-plasmid for 48 h; miR-195-5p mimic+Bcl-2-plasmid, HUVECs co-transfected with miR-195-5p mimic + Bcl-2-plasmid for 48 h. Data are presented as the mean ± SD. **P<0.01 vs. mimic control or control-plasmid; ##P<0.01 vs. miR-195-5p mimic. HUVECs, human umbilical vein endothelial cells; miR, microRNA; PI, propidium iodide; Q, quadrant; RT-qPCR, reverse transcription-quantitative PCR.

Fig 3: SREBP1 induces progesterone resistance by activating the NF-KB pathway.A The Top 10 signaling pathways in IshMR cell lines compared with Ish cell lines in KEGG pathway analysis; in GO-BP pathway analysis; GO-CC; GO-MF. B Representative images of IHC staining of NF-?B expression in endometrium before and after progesterone conservative treatment in CR, PR, and PD/SD group. The intensities of IHC staining were quantitated by Image-Pro Plus 6.0. C Effect of NF-?B pathway after overexpression of SREBP1 on MPA in Ish cells. D Effect of NF-?B pathway of nuclear expression after overexpression of SREBP1 on MPA in Ish cells. E Expression of NF-?B in PCMV-SREBP1 Ish cells were determined by western blotting assay after transfected with siNF-?B or siNC for 72 h. F Growth curves in PCMV-SREBP1 Ish cells and PCMV-Ctrl Ish cells with 30 µM MPA for 48 h were determined by MTT assay after transfected with siNF-?B or siNC. G Apoptosis in PCMV-SREBP1 Ish cells and PCMV-Ctrl Ish cells with 30 µM MPA for 48 h were determined by flow cytometry assay after transfected with siNF-?B or siNC. H PCMV-SREBP1 Ish cells and PCMV-Ctrl Ish cells with siNF-?B or siNC were treated with 30 µM MPA for 48 h respectively. Expression of SREBP1, NF-?B, p-NF-?B, CylinD1, CDK4, Bcl-2, PARP, and cleaved PARP were determined by western blotting assay. All experiments were repeated three times at least. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 for statistical analysis of the indicated groups. Quantitation of western blotting assay bands shown in using Image J. Statistical analysis was performed using Student’s t test.

Fig 4: Bioengineered miR-34a-5p and miR-124-3p molecules are effective to modulate target gene expression and control human carcinoma cell proliferation. (A) Levels of miR-34a-5p and miR-124-3p were sharply higher in cells treated with htRNALeu/miR-34a-5p and htRNALeu/miR-124-3p, respectively, as compared with vehicle or htRNALeu control. MiRNA levels were determined by selective stem-loop RT-qPCR assays. Values are the mean ± SD (N = 6/group). ***P < 0.001, 1-way ANOVA with Bonferroni post-tests. (B) Humanized miR-34a and -124 prodrugs greatly reduced the proliferation of carcinoma cells. Values are the mean ± SD (N = 5/group). ***P < 0.001, 2-way ANOVA with Bonferroni post-tests). (C) and (D) Protein levels of miR-34a (BCL2, SIRT1, and c-MET) and miR-124 (VAMP3, MCT1, and STAT3) targets were significantly reduced in cells treated with miR-34a-5p and miR-124-3p prodrugs, respectively, compared to control treatments. Band intensity was normalized to corresponding ß-actin level. aP < 0.05 compared to vehicle treatment, and bP < 0.05 compared to htRNALeu; 1-way ANOVA with Bonferroni post-tests.

Fig 5: Western blot for Cyclin D1, cleaved caspase-3, and BCl2, with a ß-actin-loading control, in SW620 and HCT116 cells 72 h after treated with Arylquin 1; quantitative data are expressed as mean ± SEM. * p < 0.05, ** p < 0.01, and *** p < 0.001 compared to the control group.