MicroRNA 421 induces the formation of high-invasive cell subsets of ovarian cancer from low-invasive cell subsets mediated by exosomes by activating the PI3K/AKT pathway.

Intratumoral heterogeneity (ITH) results in treatment failure in ovarian cancer (OC). Exosomes are related to the formation of a heterogeneous tumor microenvironment, and microRNAs play a crucial role in the progression of OC. Therefore, we aimed to explore the effect of exosomes and microRNA 421 (miR-421), which is mediated by exosomes, on ITH and the diagnosis of OC. Exosomes derived from A2780 cells with the highest (AHC) or lowest (ALC) invasive/migratory capacity cells (AHE/ALE) were extracted by differential centrifugation. We conducted a series of experiments to verify the role of AHE and miR-421 in promoting the transformation of low-invasive cells to high-invasive cells by regulating the PI3K/AKT pathway, and we also measured the levels of CA125 in serum exosomes. The results of assays showed that the AHE and miR-421, mediated by exosomes, significantly increased the malignancy of ALC cells by activating the PI3K/AKT pathway. The expression of miR-421 was significantly increased in the serum exosomes derived from high-grade serous ovarian cancer (HGSOC) patients. Our findings indicate that MiR-421, mediated by exosomes, could induce the transformation of highly invasive cell subpopulations from subpopulations of OC cells with low invasive potential by activating the PI3K/AKT signaling pathway.

DUSP7 inhibits cervical cancer progression by inactivating the RAS pathway.

To determine the differentially expressed proteins (DEPs) between paired samples of cervical cancer (CC) and paracancerous tissue by quantitative proteomics and to examine the effects of DUSP7 expression on the tumorigenesis and progression of CC. Proteomic profiles of three paired samples of CC and paracancerous tissue were quantitatively analysed to identify DEPs. The relationship between DEP expression and patient clinicopathological characteristics and prognosis was evaluated. The effects of the selected DEPs on CC progression were examined in SIHA cells. A total of 129 DEPs were found. Western blot and immunohistochemistry (IHC) staining analyses confirmed the results from quantitative proteomic analysis showing that the selected DEP, HRAS, P-ERK1/2, and PLD1 levels were increased, whereas the DUSP7 level was decreased in CC tissue compared with the paired normal paracancerous tissues. The IHC results from the CC TMA analysis showed that the decreased expression of DUSP7 (p = 0.045 and 0.044) was significantly associated with a tumour size >2 cm and parametrial infiltration. In addition, the decreased expression of DUSP7 and increased expression of p-ERK1/2 were adversely related to patient relapse (p = 0.003 and 0.001) and survival (p = 0.034 and 0.006). The expression of HRAS and p-ERK1/2 was decreased in DUSP7-SIHA cells compared with NC-SIHA cells (p = 0.0003 and 0.0026). Biological functions in vitro, including invasion, migration and proliferation and tumour formation in vivo were decreased in DUSP7-SIHA cells (all p < 0.05) but increased in shDUSP7-SIHA cells (all p < 0.05). DUSP7 inhibits cervical cancer progression by inactivating the RAS pathway.

miR­34c­5p targets Notch1 and suppresses the metastasis and invasion of cervical cancer.

Micro (mi)RNAs are crucial participants in the progression of cervical cancer (CC). Growing evidence indicates that miRNA (miR)­34c­5p is a pivotal tumor suppressor in numerous types of cancer and its functions in CC require further investigating. The present study demonstrated that there was a decreased level of miR­34c­5p in CC­associated cell lines compared with healthy control samples. It also demonstrated that miR­34c­5p targeted Notch1 and suppressed CC progression. Dual­luciferase reporter assays verified the targeted relationship of miR­34c­5p and Notch1. The expression of Notch1 in HeLa cells was markedly reduced following miR­34c­5p overexpression and the proliferation, migration and invasion of HeLa cells were reduced although apoptosis was accelerated. However, overexpression of miR­34c­5p was reversed following the addition of Notch1, which supported the finding of the targeted relationship between miR­34c­5p and Notch1. Flow cytometry demonstrated that miR­34c­5p inhibited the proliferation of HeLa cells while accelerating apoptosis. The present study concluded that miR­34c­5p was a tumor suppressor in CC and may be a novel measure for the future treatment of CC.

A Polyethylene Glycol-Based Method for Enrichment of Extracellular Vesicles from Culture Supernatant of Human Ovarian Cancer Cell Line A2780 and Body Fluids of High-Grade Serous Carcinoma Patients.

OBJECTIVE: This study tried to evaluate whether 8% polyethylene glycol (PEG) 6000 precipitation combined with differential ultracentrifugation (PPDU) was an efficient and practical method for the enrichment and purification of extracellular vesicles (EVs) derived from the culture supernatant of human ovarian cancer cell line A2780 and from body fluids of patients with high-grade serous carcinoma (HGSC). METHODS: PPDU was used to enrich and purify the EVs derived from body fluids of patients with HSGC and cell culture supernatant of subclones of human ovarian cancer cell line A2780 with high/low invasive capacity (named as A-H/A-L, respectively). Transmission electron microscope (TEM) and nanoparticle tracking analysis (NTA) were used to identificate the EVs size and distribution. Western blots (WB) were used to detect the expression of CD9, CD63, Alix and Calnexin. The high-purity EVs derived from the cell culture supernatant of A-H/A-L were detected by the protein profile. Expression of integrins (ITGs) αV, ß1 and ß3 in the EVs derived from body fluids of HGSC patients was also evaluated. RESULTS: The diameter of EVs was about 30-260 nm observed under the TEM. Under the NTA identification, the peak size of EVs was ranged from 70 to 159nm. EVs derived from different specimens did not significantly differ in mean size and peak size. Presence of CD9, CD63 and Alix and absence of Calnexin were confirmed in the EVs. The protein concentrations of EVs' sample extracted from A-H/A-L cell culture supernatant were 0.36µg/µL and 0.20µg/µL, respectively. The total amount of protein obtained from 300ul EVs was 108.02ug and 61.44ug, respectively. Totally, 2397 peptides and 952 proteins were identified by isobaric tags for relative and absolute quantitation (ITRAQ). The expression of ITGαV, ß1, and ß3 in the EVs from plasma and ascites of HGSC patients was significantly higher than the control group (plasma: all P<0.0001; ascites: P=0.036, 0.001 and 0.004, respectively). The expression level of ITGαV and ß1 in EVs of HGSC's ascites was significantly higher than that in plasma (P= 0.004, 0.001, respectively). The expression of ITGß3 was also slightly elevated in EVs-derived HGSC patients' ascites (P=0.492). CONCLUSION: PPDU was an efficient and practical method to enrich EVs from body fluids and cell culture supernatant. The characteristic expression of ITGαV, ß1 and ß3 in ascites and plasma EVs of patients with HGSC provided useful information on the development of EVs in HGSC.