Downregulation of PDCD4 by miR-21 suppresses tumor transformation and proliferation in a nude mouse renal cancer model

Abstract

Programmed cell death 4 (PDCD4) is known to suppress neoplastic transformation, cell proliferation and metastasis, and to be downregulated by microRNA-21 (miR-21) in renal cell carcinoma (RCC) cell lines and tissues. The aim of the present study was to investigate the roles of and association between PDCD4 and miR-21 in a nude mouse renal cancer model. A total of 24 BALB/c male nude mice were randomly assigned into the following three groups: Negative control (NC; n=8), miR-21 inhibitor (n=8) and miR-21 mimic (n=8). Subsequently, renal cell adenocarcinoma 786-O cells were subcutaneously transplanted into the armpits of the mice, which were then injected daily with NC small interfering (si)RNA, precursor-miR-21 (mimic) or anti-miR-21 (inhibitor). Tumors were removed from the mice and weighed 16 days following 786-O cell transplantation. In addition, the expression of miR-21 and PDCD4 mRNA in cancer tissues was analyzed using reverse transcription-quantitative PCR. The expression of PDCD4 protein in cancer tissues was also examined using immunohistochemistry and western blotting. Furthermore, 786-O cells were transfected with PDCD4 siRNA or NC siRNA, and the effects of silencing PDCD4 on tumor cell growth, proliferation and invasion were investigated using soft agar colony formation, EdU cell proliferation assay and Transwell migration and invasion assays. Another 16 BALB/c male nude mice were randomly assigned into two groups as follows: NC (n=8) and PDCD4 siRNA (n=8). The 786-O cells were subcutaneously transplanted into the armpits of the mice, which were subsequently injected daily with NC siRNA or PDCD4 siRNA. The tumors were removed and weighed 16 days following transplantation. Compared with the NC group, tumor weight in the miR-21 mimic group was significantly increased. By contrast, tumor weight in the miR-21 inhibitor group was significantly decreased. Similar to the results observed in human renal cancer tissue and cell lines, miR-21 expression in the nude mouse renal cancer models was significantly upregulated in the miR-21 mimic group compared with the NC group, while it was significantly lower in the miR-21 inhibitor group. Furthermore, there was a significant reduction in PDCD4 protein levels in the miR-21 mimic group and a significant increase in the miR-21 inhibitor group compared with the NC, whereas PDCD4 mRNA expression was not significantly altered. In the EdU proliferation assay, the mean percentage of new cells that incorporated EdU was 28.6% in the NC siRNA group and significantly increased to 44.7% in PDCD4 siRNA transfected cells. In the soft agar colony formation assay, Transwell and migration and invasion assays, a significant increase in colony formation, migration and invasion capacity in PDCD4 siRNA-transfected cells was observed compared with the NC. Furthermore, compared with the NC group, tumor weight in the PDCD4 siRNA group was significantly increased. Similar to the results observed in human renal cancer tissue and cell lines, miR-21 promoted cancer cell hyperplasia and prolifer...

The let-7 family of microRNAs suppresses immune evasion in head and neck squamous cell carcinoma by promoting PD-L1 degradation

“…Two μg of total cDNA was used as a templet, while U6 was regarded as the internal control. The relative expression of the targeted genes was calculated using the 2- ΔΔCt method [20], in which ΔΔCt = ΔCt (experiment group) – ΔCt (control group) and ΔCt = Ct (target genes) – Ct (U6). The experiment was performed in triplicate to obtain the mean value (Table 1).…”

Epidermal growth factor stimulates exosomal microRNA‐21 derived from mesenchymal stem cells to ameliorate aGVHD by modulating regulatory T cells

“…The primers used in the present study were synthesized by Takara Biotechnology Ltd. (Dalian, Liaoning, China) (Table 1). With U6 serving as an internal reference, the relative expression of miR‐21 was determined by relative quantitative method (2 −ΔΔCt method) 15 . The experiment was repeated for five times.…”

Construction and analysis of circular RNA molecular regulatory networks in clear cell renal cell carcinoma

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