HIF-1α Induces HECTD2 Up-Regulation and Aggravates the Malignant Progression of Renal Cell Cancer via Repressing miR-320a

Abstract

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.

Keywords: HECTD2; HIF-1α; MiR-320a; progression; renal cell carcinoma.

GRAPHICAL ABSTRACT

FIGURE 1

Expression and prognosis of HIF-1α and HECTD2 in RCC (A,B). The number of HIF-1α and HECTD2 positive cells in RCC tissue and normal paracancerous tissues was analyzed in the Human Protein Atlas database (https://www.proteinatlas.org/). (C,D): HIF-1α and HECTD2 protein profiles in 7 cases of RCC tissues (T) and normal paracancerous tissues (N) were compared by WB. (E) The online database GEPIA was used for analyzing the expression relationship in KIRC (http://gepia.cancer-pku.cn/). (F,G): Prognostic analysis of HIF-1α and HECTD2 in RCC was performed on Kaplan-Meier Plotter (http://kmplot.com/analysis/).

FIGURE 2

Effect of HECTD2 overexpression on the malignant phenotype of RCC. Overexpression models of HECTD2 were constructed in 786-O and A-498 cell lines. (A): RT-qPCR was implemented to testify the HECTD2 expression. (B–D): RCC cell proliferation and apoptosis were determined by the colony formation experiment, BrdU assay and flow cytometry, respectively. (E) RCC cell migration and invasion were assessed by Transwell assay. (F,G): WB was conducted to examine the expression of P53, Bax, p21, c-Caspase3, E-cadherin, Vimentin, and N-cadherin. **p < 0.01, ***p < 0.001 vs. NC group. N = 3. Scale bar = 50 μm.

FIGURE 3

Influences of overexpressing HECTD2 on the malignant phenotypes of RCC in vivo. The HECTD2 overexpression model was established in 786-O and A-498 cells, which were then used for constructing xenografted tumor model in nude mice. The mice were sacrificed at the 35th day after cell transplantation. (A–C): Tumor volume and weight. (D): The number of Ki67-positive cells in 786-O and A-498 cells was evaluated by IHC. (E): The profile of HECTD2 in the formed tumor tissues was tested by WB. (F): The fluorescence intensity of HECTD2 was tested by tissue immunofluorescence. ns p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001. N = 5. Scale bar = 50 μm.

FIGURE 4

HIF-1α heightened the expression of HECTD2 and its malignant phenotype. HECTD2 was knocked down in 786-O cells overexpressing HIF-1α. (A): RT-qPCR was carried out to monitor the levels of HIF-1α and HECTD2. (B–D): RCC cell proliferation and apoptosis were checked using the colony formation experiment, BrdU assay and flow cytometry, respectively. (E): Transwell assay was conducted to measure RCC cell migration and invasion. (F,G): The expression of P53, Bax, p21, c-Caspase3, E-cadherin, Vimentin, and N-cadherin was compared by WB. ***p < 0.001 (vs. vector group). ns p > 0.05, && p < 0.01 (vs. HIF-1α group). N = 3. Scale bar = 50 μm.

FIGURE 5

Impacts of the HIF-1α/HECTD2 axis on RCC cell growth in vivo. HIF-1α overexpression and/or HECTD2 knockdown cell model was used for constructing xenografted tumor model in nude mice. The mice were sacrificed at the 35th day after cell transplantation. (A–C): Tumor volume and weight. (D): IHC was adopted to count the number of Ki67-positive cells in 786-O and A-498. (E): The profiles of HIF-1α and HECTD2 in 786-O and HECTD2 cells were monitored by WB. (F,G): The fluorescence intensity of HIF-1α and HECTD2 was checked by tissue immunofluorescence. ns p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001. N = 5. Scale bar = 50 μm.

FIGURE 6

miR-320a targeted HECTD2. (A) The positive or negative related genes of HECTD2 in TGCA database was analyzed via LinkedOmics, and those genes were subjected to GSEA enrichment analysis. HECTD2 is potentially involved in a KEGG pathway of MicroRNAs in Cancer. (B): Targetscan, miRmap, microT, PicTar and miRanda databases were utilized to search the miRNA target of HECTD2. Venn diagram was utilized to analyze the common target miRNAs of HECTD2. Seven common miRNAs were identified, including has-miR-221-3p, has-miR-30a-5p, has-miR-320a, has-miR-493-5p, has-miR-218-5p, has-miR-124-3p and has-miR-222-3p. (C): RT-PCR was used for determining the seven miRNAs in 786-O and A-498 cells transfected with HIF-1α. (D): HECTD2 contained the binding sites with miR-320a. (E,F). Dual-luciferase reporter assay and RIP assay were applied to probe the binding of miR-320a to HECTD2. G: HECTD2 level in 786-O cells transfected with miR-320a mimics or miR-320a inhibitors was tested by WB. NS p > 0.05, **p < 0.01, ***p < 0.001. N = 3.

FIGURE 7

Influences of miR-320a on the malignant phenotype in RCC. miR-320a mimics were transfected into A-498 cells. (A): The miR-320 profile was determined by RT-qPCR. (B–D): RCC cell proliferation and apoptosis were checked by the colony formation assay, BrdU assay and flow cytometry, respectively. (E): Transwell assay was implemented to verify RCC cell migration and invasion. (F–H): WB was conducted to evaluate the expression of P53, Bax, p21, c-Caspase3, E-cadherin, Vimentin, and N-cadherin. **p < 0.01, ***p < 0.001. N = 3.

FIGURE 8

miR-320a affected RCC growth and EMT via HECTD2. Transfection of miR-320a mimics was made in 786-O cells with overexpressed HECTD2. (A). RT-qPCR was implemented to examine miR-320 expression. (B–D): The colony formation assay, BrdU assay, and flow cytometry were applied to assess RCC cell proliferation and apoptosis. RCC cell migration and invasion were tested by Transwell assay. (F,G): The profiles of P53, Bax, p21, c-Caspase3, E-cadherin, Vimentin, and N-cadherin were compared by Transwell assay. ***p < 0.001, & p < 0.05, && p < 0.01, &&& p < 0.001. N = 3.