Fig 1: let-7i-5p and miR-181a-2-3p modulates cervical CSCs via EGF/PI3K/SOX2 pathway. (a) Forced expression of let-7i and miR-181a-2: The clones expressing let-7i-5p and miR-181a-2-3p were purchased from Origene and transfected by Lipofectamine 2000 in CaSki cell lines and the efficacy of the clones was examined by real time PCR. The transfection of the respective clones resulted in an increase of 1.53 log2 fold change in let-7i-5p and an increase of 5.21 log2 fold change in miR-181a-2-3p. (b) Clonogenic assay: The number of colonies formed by CaSki cells transfected with let-7i-5p or miR-181a-2-3p reduced significantly in comparison to the untransfected CaSki cells. The quantitative change was calculated by dissolving the stained colonies in 30% glacial acetic acid and recording their absorbance at 570 nm. The decrease was about 66.5% and 65% in let-7i-5p and miR-181a-2-3p overexpressed cells, respectively. (c) Sphere formation assay: The number of spheres reduced from 28 in untransfected CaSki cells to 9 in cells transfected with let-7i-5p (67% decrease) and 8 in cells transfected with miR-181a-2-3p (71% decrease). (d) let-7i-5p indirectly targets SOX2 through HMGA2: The western blotting data shows that the forced expression of let-7i-5p reduces the expression of HMGA2 and SOX2 to −0.14 and −1.64 log2 fold change, respectively. This is in significant contrast to the increase in HMGA2 and SOX2 expression in cervical CSCs. The blots were cropped according to the molecular weight of the proteins and then probed with specific antibodies. Where molecular weight of the proteins was similar, for instance SOX2 (34 kDa) and GAPDH (37 kDa), the blot was first probed with one antibody, stripped and then reprobed with another antibody. (e) miR-181a-2-3p targets EGF/PI3K/SOX2 pathway: The western blotting data confirms that EGF/PI3K/SOX2 pathway is activated in the CSCs of CaSki cells. The exogenous expression of miR-181a-2-3p reverses the expression of EGF pathway genes in CaSki cells. miR-181a-2-3p inhibits the expression of PI3K55γ (a validated target of miR-181a-2-3p), SOX2 and the phosphorylated AKT. The reverse pattern is clearly depicted in the densitometric analysis of the western blot data. The blots were cropped according to the molecular weight of the proteins and then probed with specific antibodies. Where molecular weight of the proteins was similar, for instance in case of SOX2 (34 kDa) and GAPDH (37 kDa) or AKT and pAKT (~55 kDa), the blot was first probed with one antibody, stripped and then reprobed with another antibody. *p value < 0.05, **p value < 0.01, ***p value < 0.001.
Fig 2: Schematic diagram explaining the mechanism of CSC maintenance in cervical cancer via let-7i-5p, miR-181a-2-3p and EGF/PI3K/SOX2 axis. In this study, it was observed that exogenous EGF added to the media used for CSC enrichment induces the expression of PI3K and phosphorylates AKT, leading to the enhanced expression of SOX2. SOX2 silencing drastically reduced the CSC population in CaSki cells. The inhibition of EGFR phosphorylation and PI3K reduced the expression of SOX2 and subsequently suppressed the CSC population, thereby establishing the role of EGF pathway in the maintenance of cervical CSCs. While EGF pathway promotes CSC maintenance, let-7i-5p and miR-181a-2-3p counteracts by indirectly targeting SOX2 and thereby suppressing the cervical CSCs. let-7i-5p inhibits HMGA2 (an inducer of SOX2 expression) and miR-181a-2-3p inhibits PI3kinase p55γ (PI3K activator). Hence, activation of EGF pathway and inhibition of let-7i-5p and miR-181a-2-3p are vital for cervical CSC maintenance. The reported literature suggests that HPV infection may cause activation of EGF pathway. If proven, this would perhaps explain the formation of cervical CSCs and hence the origin of cervical cancer.
Fig 3: miRNA profiling in CaSki CSCs and validation of differentially expressed miRNAs. (a) The small RNA sequencing was carried out for CaSki cells and CSCs enriched from CaSki cell line. This experiment identified 42 differentially expressed miRNAs (12 downregulated and 30 upregulated) in CSCs with a p value < 0.05. As per the miRNA nomenclature guidelines, the mature miRNA sequences are mentioned as miR- and precursor sequences as mir-. (b) miRNAs (let-7i-5p, miR-181a-2-3p, miR-615-3p, and miR-663a) were selected from the list of differentially expressed miRNAs for further validation. The expression of these miRNAs were analyzed by real time PCR and found to be in concordance with the data obtained from small RNA sequencing experiment. *p value < 0.05.
Fig 4: (A) Biodistribution of hpMSC exosomes (MExo, 1 × 108 particles per mouse) and engineered exosomes (EExo, 1 × 109 particles per mouse) conjugated with Cy7 mono NHS ester in the heart, lungs, liver, kidney, spleen, and bladder of mice, using ex vivo bioluminescence imaging assay, measured at 0, 2, 24, and 48 h after intraperitoneal administration. Data were obtained from 3 mice sacrificed at each time point in both groups. * p < 0.05, versus the 0-h mark. (B) Pharmacokinetic analysis of MExo and EExo. The plasma concentrations of MExo and EExo were measured through the assay of Cy7 mono NHS ester signal intensities. The MExo and EExo concentrations were measured at 0.5 h after intraperitoneal administration and were used as the baseline. Data were obtained from 3 mice in each group. hpMSC exosomes: exosomes from human placenta-derived mesenchymal stem cells. Engineered exosomes: engineered exosomes from RAW264.7 cells overexpressing hsa-let-7i-5p. The dashed line indicates a 50% fold change compared to the 4-h mark.
Fig 5: Characteristics of isolated exosomes. (A) Representative transmission electron microscopic images of isolated exosomes. (B) Representative gel photography of markers ALIX and CD63 in isolated exosomes using the Simple Western method. (C) Particle sizing assay of isolated exosomes. (D) The microRNA concentrations of hsa-let-7i-5p in isolated exosomes. Data were obtained from 3 exosomes in each group and presented as mean ± standard deviations. * p < 0.05, versus the RExo group. EExo: engineered exosomes from RAW264.7 cells overexpressing hsa-let-7i-5p. MExo: exosomes from human placenta-derived mesenchymal stem cells. RExo: exosomes from RAW264.7 cells without genetic modification.
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