In addition, silencing or elevating PTEN protein could partially reverse the effect of miR-21-specific shRNA or pre-miR-21 on apoptosis, cell cycle distribution, and invasion of CRC cells. Furthermore, the transfection of CRC cells with pre-miR-21 could inhibit apoptosis and promote cellular proliferation, invasion, cell cycle progression and growth of xenografts in nude mice, whereas the transfection of miR-21-specific shRNA resulted in the opposite phenomena. The PTEN protein level in CRC tissues and cells was inversely correlated with miR-21 expression. Patients with advanced Tumor-Node-Metastasis (TNM) stage, lymph node metastasis, local invasion and higher serum carcinoembryonic antigen (CEA) levels displayed significantly high expression of miR-21. In this study, we showed that miR-21 expression was significantly up-regulated in CRC compared to that in normal tissues. Tumorigenesis was also evaluated by xenografting HCT-116 cells into nude mice. Furthermore, we transfected CRC cells with different combinations of ectopic-expression vector or shRNA expression vector of miR-21 and phosphatase and tensin homolog (PTEN) to modulate the expression levels of miR-21 and PTEN respectively, and then analyzed the phenotypic alterations of CRC cells. The protein and mRNA expression levels of PTEN and miR-21 were examined using western blot analysis and real-time reverse transcription-PCR (qRT-PCR).
The aim of the present study was to explore the oncogenic function of miR-21 and its molecular mechanism in CRC.Ī total of 105 paired tumor and tumor-adjacent normal tissue specimens from CRC patients and two CRC cell lines (HCT-116 and SW480) were studied. MicroRNA-21 (miR-21) has been demonstrated to play an important role in carcinogenesis however, its mechanism of action in colorectal cancer (CRC) has not been fully elucidated. Among them, the PTEN protein was reported to be frequently silenced in CRC, and it has been shown to suppress tumor formation by inhibiting the PI3K/AKT pathway. According to recent reports, several significant targets of miR-21 associated with malignancy have been experimentally validated, such as PTEN, programmed cell death 4 (PDCD4), reversion-inducing cysteine-rich protein with Kazal motifs (RECK), forkhead box O1 (FOXO1), RhoB and Cdc25a, and these targets may exert different effects on tumorigenesis. miR-21, a putative oncogene, is most frequently over-expressed in various malignancies, and it has been implicated in multiple malignancy-related processes including uncontrolled cell proliferation, inhibition of apoptosis and promotion of invasion and metastasis. Furthermore, it has been shown that miRNAs can function as tumor suppressors or oncogenes and are involved in various biological processes such as tumor initiation, tumor progression and drug resistance in various types of cancer, including CRC. Targeting of microRNA-21 is sufficient to limit tumor cell proliferation and invasion in a manner that is likely to involve associated changes in multiple targets, suggesting that suppression of microRNA-21 may be a novel approach for the treatment of hepatocellular carcinoma. MicroRNA-21 simultaneously regulates multiple programs that enhance cell proliferation, apoptosis or tumor invasiveness by targeting PTEN, PDCD4, and RECK in hepatocellular carcinomas. Moreover, knockdown of microRNA-21 resulted in alterations of the Akt signaling pathway, the expression of p21 and MMP families, which are associated with apoptosis, and the cell cycle or invasiveness of cancer cells.
Silencing of PTEN and PDCD4 to prevent their induction by anti-microRNA-21 treatment led to decreased apoptosis and increased invasion, while silencing of RECK only led to increased invasion. We demonstrate that hepatocellular carcinoma is characterized by elevated levels of microRNA-21 and marked reductions of PTEN, PDCD4, and RECK expression. MicroRNA-21 was suppressed using anti-microRNA-21 to further uncover its effect on several critical signaling pathways. We designed a set of rescue experiments using different combinations of anti-microRNA-21, siRNA, and a negative control to modulate the protein level of microRNA-21 targets and resulting phenotypic alterations. In this study, we investigate the function of microRNA-21 as a potent oncomir and probe the relationship between microRNA-21, its targets, and phenotypic alterations. However, its mechanism of action in human hepatocellular carcinoma is poorly understood, and no direct evidence has shown a correlation between microRNA-21 function and phenotype. MicroRNA-21 negatively regulates several targets, thereby affecting tumorigenesis.
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