Adaptor protein Crk and CrkL were thought to be closely related because both consist of one SH2 and two SH3 domains and share 60% homology with the highest identity within their functional domains. Their functions were most presumed to be in part, if not all, redundant. And both were suggested to be implicated in carcinogenesis. In this study, both Crk and CrkL presented with much higher expression in ovarian cancer tissues than those in normal and benign ovarian tissues. However, in contrast with CrkL, high Crk expression displayed close association with advanced stages and high-grade diseases. Furthermore, the differential binding selectivity of Crk and CrkL to their downstream partners Dock 180 and C3G was demonstrated in ovarian cancer cell line SKOV3 through coimmunoprecipitation. Additionally, Crk-knockdown cells presented with changed morphology, reduced growth, and cell invasion but remained viable. In contrast, all CrkL-knockdown cells could not survive over time, gradually detaching from the bottom of plastic dish. In conclusion, these two highly homologous proteins hold features that allow for the differential association with each binding molecules, thereby activating different signaling pathways and being involved in diverse roles in ovarian cancer.

Renal cell carcinoma (RCC) is a frequent malignancy of the urinary system. It has been found that hypoxia mediates the malignant evolvement of RCC. Here, we probe the impact and potential mechanism of HECT domain E3 ubiquitin-protein ligase 2 (HECTD2) and HIF-1α on regulating RCC evolvement. RCC tissues and adjacent normal tissues were collected, and the association between the expression profiles of HECTD2 and HIF-1α and the clinicopathological features was analyzed. Additionally, we constructed HECTD2/HIF-1α overexpression and knockdown models in RCC cell lines to ascertain the impacts of HECTD2 and HIF-1α on RCC cell proliferation, apoptosis, migration, and growth in vivo. We applied bioinformatics to predict the upstream miRNA targets of HECTD2. Meanwhile, RNA immunoprecipitation (RIP), and the dual-luciferase reporter assays were employed to clarify the targeting association between HECTD2 and miR-320a. The effect of miR-320a on HECTD2-mediated RCC progression was investigated. The results suggested that both HIF-1α and HECTD2 were up-regulated in RCC (compared with adjacent non-tumor tissues), and they had positive relationship. Moreover, higher level of HECTD2 and HIF-1α is associated with poorer overall survival of RCC patients. HECTD2 overexpression heightened RCC cell proliferation and migration, and weakened cell apoptosis. On the other hand, the malignant phenotypes of RCC cells were signally impeded by HECTD2 or HIF-1α knockdown. Moreover, miR-320a targeted the 3′-untranslated region of HECTD2 and suppressed HECTD2 expression. The rescue experiments showed that miR-320a restrained HECTD2-mediated malignant progression in RCC, while up-regulation of HIF-1α hampered miR-320a expression. Collectively, HIF-1α mediated HECTD2 up-regulation and aggravated RCC progression by attenuating miR-320a.

Background Multiple studies have found that microRNAs contribute to the malignant progression and chemoresistance of renal cell carcinoma (RCC). This study intends to probe the effect of miR-19b-3p shuttled by exosomes derived from RCC cells on RCC development and its resistance to Sunitinib. Methods Sunitinib-resistant cell lines (OSRC-2R and Caki-1R) were constructed from OSRC-2 and Caki-1 RCC cells. Exosomes in the RCC cell supernatant were isolated, and the miR-19b-3p profile in cells and exosomes was measured by reverse transcription-polymerase chain reaction (RT-PCR). Subsequently, the TGFβR2/SMAD2/3 pathway was activated by TGFβ, and the KLF10 overexpression and miR-19b-3p overexpression/knockdown models were constructed. The cell counting kit-8 (CCK-8) assay, colony formation assay and flow cytometry were implemented to verify RCC cell proliferation, Sunitinib chemosensitivity, and apoptosis. The expression of apoptosis-related proteins and the TGFβR2-SMAD2/3-KLF10 pathway was monitored by Western blot. MiR-19b-3p was overexpressed in sunitinib-resistant RCC cell lines (OSRC-2R and Caki-1R) and their exosomes (vs. normal OSRC-2 and Caki-1 cell lines). Results In-vitro experiments showed that knocking down cellular and exosomal miR-19b-3p levels reduced the proliferation and colony-forming ability of OSRC-2 and Caki-1 cells and strengthened their apoptosis and sensitivity to Sunitinib. Bioinformatics analysis illustrated that miR-19b-3p targeted TGFβR2 and inhibited TGFβR2/SMAD2/3. Activation of the TGFβR2-SMAD2/3 pathway via TGFβ dampened ORSC-2 and Caki-1 cell proliferation, induced apoptosis, and enhanced their chemosensitivity to Sunitinib. Conclusion Moreover, TGFβ heightened KLF10 expression, and overexpressing KLF10 attenuated miR-19b-3p-mediated carcinogenic effects and resistance to Sunitinib by increasing SMAD2/3 phosphorylation. RCC cell-derived exosomal miR-19b-3p enhances RCC progression and Sunitinib chemoresistance by inactivating TGFβR2-SMAD2/3-KLF10.