Dataset Information

MicroRNA-30d target TIMP3 induces pituitary tumor cell growth and invasion.

ABSTRACT:

Background

As one of the most common intracranial tumors, pituitary adenomas, especially the Cushing's disease subtype, have been studied for many years. However, at present, effective methods for the early diagnosis of pituitary adenomas are very limited, especially for subtypes such as Cushing's disease. Therefore, it is of urgent importance to find effective molecular targets to develop new diagnostic and therapeutic methods for pituitary adenomas.

Methods

We showed the abnormally high expression of miR-30d in pituitary adenomas by analyzing data in the Gene Expression Omnibus (GEO) database and revealed a novel molecular mechanism of miR-30d in regulating the proliferation and invasion of a pituitary adenoma cell line (AtT-20). Cell culture and transfection, and RNA interference (RNAi) were used to treat AtT-20 cells to test the effects of miR-30d and TIMP3 on cells. Quantitative polymerase chain reaction (qPCR) was used to determine the messenger RNA (mRNA) expressions. We used 3-(4,5-diphenyltetrazolium bromide) (MTT) to determine cell viabilities. An invasion assay was performed using Transwell chambers. Luciferase activity was tested with a dual-luciferase assay.

Results

We found that the expression of miR-30d in pituitary adenoma was higher than that in normal pituitary tissues. It was revealed that miR-30d promoted the proliferation and invasion of AtT-20 cells by inhibiting the expression of TIMP3. In the above process, miR-30d could bind to the 3'-untranslated region (3'-UTR) of TIMP3 mRNA.

Conclusions

The mir-30d/TIMP3 signaling pathway plays an important regulatory role in pituitary adenomas. These new discoveries may reveal more functions of miR-30d and lay the foundation for future clinical development of new drug targets.

SUBMITTER: Sun B

PROVIDER: S-EPMC8749101 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The present study aimed to investigate the expression of miR-200b and protein kinase Cα (PKCα) in pituitary tumors and to determine whether miR-200b may inhibit proliferation and invasion of pituitary tumor cells. The regulation of PKCα expression was targeted in order to find novel targets for the treatment of pituitary tumors. In total, 53 pituitary tumor tissue samples were collected; these included 28 cases of invasive pituitary tumors and 25 cases of non-invasive tumors, in addition to 5 normal pituitaries. The expression level of miR-200b in the pituitary tumor tissue was detected by quantitative polymerase chain reaction (qPCR) and the expression of PKCα protein was detected by immunohistochemistry. A PKCα 3'untranslated region (UTR) luciferase vector was constructed and a dual luciferase reporter gene assay was employed in order to examine the effect of miR-200b on the PKCα 3'UTR luciferase activity. AtT-20 cells were transfected with miR-200b mimics, PKCα siRNA and miR-200b mimics + PKCα, and the changes in cellular proliferation, invasion and apoptosis were observed via MTT, Transwell assay and flow cytometric analysis. Furthermore, PKCα mRNA expression was determined by qPCR, and Western blotting was performed to detect the expression of PKCα protein. miR-200b revealed downregulation in invasive pituitary tumor tissue, and the expression level was significantly down-regulated compared with normal and non-invasive pituitary tumor tissue (P<0.01). In addition, the positive rate of PKCα protein expression in invasive pituitary tumor tissues was significantly higher than in normal and non-invasive tissues (P<0.01). PKCα protein levels are inversely correlated with miR-200b levels in invasive pituitary tumor tissues (r=-0.436, P=0.021). The dual luciferase reporter gene assay revealed that miR-200b could specifically bind to the 3'UTR of PKCα and significantly inhibit the luciferase activity by 39% (P<0.01). Upregulation of miR-200b or downregulation of PKCα could suppress cell proliferation and invasion, and increase apoptosis of AtT-20 cells. It was revealed that PKCα siRNA could suppress both proliferation and invasion of AtT-20 cells and partially simulate the function of miR-200b. Expression of PKCα mRNA and protein decreased significantly in AtT-20 cells overexpressing miR-200b. Additionally, miR-200b was significantly down-regulated in invasive pituitary tumor tissue and inversely correlated with PKCα protein levels. In conclusion, miR-200b inhibited proliferation and invasiveness and promoted the apoptosis of pituitary tumor cells by targeting PKCα. The observations of the present study indicate that miR-200b and PKCα may serve as promising therapeutic targets for invasive pituitary tumors.