Dataset Information

Hsa-miR-195-5p inhibits cell proliferation of human thyroid carcinoma cells via modulation of p21/cyclin D1 axis

ABSTRACT:

Background

Based on existing evidence, microRNAs (miRs) are gene regulators that undertake key functions in the oncogenesis and tumor progression of every single human malignant disease, such as thyroid carcinoma (TC). Previous clinical findings showed that expression of miR-195 is down-regulated in TC, which implies that miR-195 may be practically involved in TC pathogenesis. Nevertheless, the function of hsa-miR-195-5p in TC is still largely unclear. Herein, we detected the conceivable involvement of hsa-miR-195-5p in TC cell proliferation.

Methods

Real time PCR examination was performed to assess the expression level of hsa-miR-195-5p in TC cell lines TPC-1 and B-CPAP. TPC-1 cells were transfected with either hsa-miR-195-5p mimics or hsa-miR-195-5p inhibitor. After confirmation of transfection efficiency, the effect of hsa-miR-195-5p on proliferation and cell cycle of TPC-1 cells was assessed. The expression of cyclin D1 and p21 was simultaneously detected by western blotting. Moreover, targetScan 6.2 was used to predict hsa-miR-195-5p target genes. Subsequently, luciferase reporter was performed to examine whether there is a possible binding of hsa-miR-195-5p to 3’-UTR of cyclin D1 mRNA. Furthermore, cyclin D1 mRNA and protein levels were measured to check whether hsa-miR-195-5p exerts its function at the post-transcriptional level. In addition, to explore the function of cyclin D1 in TPC-1 cells overexpressing hsa-miR-195-5p, cyclin D1 siRNA was used to silence the expression of cyclin D1 in TPC-1 cells overexpressing hsa-miR-195-5p.

Results

We quantified the expression of hsa-miR-195-5p in TC cells and normal thyroid cells and found a remarkable decrease in hsa-miR-195-5p expression in TC cells. Over-expression of hsa-miR-195-5p obviously resulted in downgraded proliferation of TC cells. Moreover, hsa-miR-195-5p caused cell arrest at the GO/G1 phase. Further in silico analyses and the dual-luciferase reporter assay confirmed that 3’-UTR of cyclin D1 is a direct target of hsa-miR-195-5p. Western blot analysis uncovered that hsa-miR-195-5p over-expression led to decreased levels of cyclin D1 and p21. In mechanistic analyses, we found that silencing of cyclin D1 reversed the inhibitory effect of hsa-miR-195-5p on the proliferation of TC cells, which indicates that hsa-miR-195-5p suppresses TC cell proliferation by adversely regulating cyclin D1.

Conclusions

We concluded that hsa-miR-195-5p is a candidate tumor-suppressor miRNA in TC and that the hsa-miR-195-5p/p21/cyclin D1 pathway could be a potential therapeutic target for TC.

SUBMITTER: Liu D

PROVIDER: S-EPMC8799136 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Heparanase (HPSE) is an endo-β-D-glucuronidase overexpressed in different types of human cancer, and a predicted target of microRNA (miRNA/miR)-219a-2-3p in thyroid cancer. The present study aimed to investigate the potential role of HPSE and miR-219a-2-3p in thyroid cancer, and the molecular mechanism of miR-219a-2-3p regulating the proliferation of thyroid cancer cells via HPSE was confirmed. Immunohistochemistry analysis was performed to detect HPSE expression in thyroid cancer sections. In addition, reverse transcription-quantitative PCR analysis was performed to detect mRNA and miR-219a-2-3p expression levels in thyroid cancer samples and cell lines. miR-219-2-3p mimic or HPSE plasmid were transfected into B-CPAP and TPC-1 thyroid cancer cells. Furthermore, western blot analysis was performed to detect the protein expression levels of HPSE and cyclin D1. Cell cycle analysis was performed using propidium iodide staining and flow cytometry, and EdU incorporation was performed to detect cell proliferation. The results demonstrated that high HPSE expression was significantly associated with tumor size, extracapsular invasion and lymph node metastasis. Notably, a statistically negative correlation was observed between HPSE mRNA expression and miR-219a-2-3p expression in thyroid cancer tumors, as well as in thyroid cancer cell lines. When exogenously expressed in B-CPAP and TPC-1 cells, miR-219a-2-3p induced cell cycle arrest at the G0/G1 phase and decreased the percentage of proliferating cells. Furthermore, HPSE and cyclin D1 protein expression decreased following transfection with miR-219a-2-3p. Notably, when HPSE was ectopically expressed in miR-219a-2-3p transfected cells, cyclin D1 expression and the number of proliferative cells increased. Taken together, these results suggest that HPSE contributes to the proliferation of thyroid cancer cells. In addition, miR-219a-2-3p was confirmed to target HPSE and inhibit cell proliferation, which was associated with cyclin D1 suppression-mediated cell cycle arrest.