[Overexpression of miR-382-5p Mediated Downregulation of PTEN on Malignant Biological Behavior of Human Glioma U251 Cells].

OBJECTIVE: To investigate the effect of overexpression of miR-382-5p overexpression on malignant biological behavior of human glioma U251 cells. METHODS: U251 cells were transfected with miR-382-5pmimic. Then miR-382-5p and PTEN mRNA levels were detected by reverse transcription-polymerase chain reaction (RT-PCR) after transfection. Used bioinformatics to predicted the presence of base binding sites between miR-382-5p and PTEN, and constructed PTEN pcDNA vector overexpression plasmid was constructed. Luciluciase reporting experiment was used to detect the targeting relationship between miR-382-5p and PTEN. Cells were randomly divided into four groups: control group, mimics group, pc-PTEN group and mimics+pc-PTEN group for follow-up experiments. RT-PCR was carried out to detect the level of PTEN mRNA in each group. Cell proliferation was detected by clone formation method. The mRNA levels of Ki67, Survivin and c-Myc were detected by RT-PCR. Transwell experiment was used to assayed cell invasion ability. The expression levels of E-cadherin, N-cadherin and Vimentin were determined by Western blot. RESULTS: Results showed that miR-382-5p directly targeted PTEN. Compared with the control group, miR-382-5p and c-Myc mRNA levels and E-cadherin protein level were increased (P<0.05),PTEN, Ki67 and Survivin mRNA levels were decreased (P<0.05), cell clonal formation rate and cell invasion number were decreased (P<0.05), N-cadherin and Vimentin protein levels were decreased (P<0.05) in the mimics group; In pc-PTEN group, miR-382-5p mRNA and c-Myc mRNA levels and E-cadherin protein level were decreased (P<0.05),PTEN, Ki67 and Survivin mRNA levels were increased (P<0.05), cell clonal formation rate and cell invasion number were increased (P<0.05), N-cadherin and Vimentin protein levels were increased (P<0.05). Compared with pc-PTEN group, PTEN, Ki67 and Survivin mRNA levels, the cell clone formation rate, the number of invasion cells and the N-cadherin and Vimentin levels of mimics+PC-PTEN group decreased significantly (P<0.05), while the c-Myc mRNA level and E-cadherin protein level increased significantly (P<0.05). CONCLUSION: Overexpression of miR-382-5p mediates the downregulation of PTEN expression, causing the inhibition of the proliferation, invasion, growth and EMT of U251 glioma cells.

Hypermethylation Of ADHFE1 Promotes The Proliferation Of Colorectal Cancer Cell Via Modulating Cell Cycle Progression.

BACKGROUND: Colorectal cancer (CRC) is one of the most common malignancies worldwide. Studies have demonstrated that epigenetic modifications play essential roles in the development of CRC. ADHFE1 is a differentially expressed gene that has been reported to be hypermethylated in CRC. However, the role and mechanism of ADHFE1 in the proliferation of CRC remain unclear. MATERIALS AND METHODS: ADHFE1 expression was analyzed in CRC tissues by IHC and qRT-PCR, and the relationship between ADHFE1 expression and the clinicopathological parameters was analyzed. Cell proliferation were assessed by the in vitro and in vivo experimental models. GSEA assay was performed to explore the mechanism of ADHFE1 in the proliferation of CRC. Flow cytometry and Western blot were used to detect the activation of the cell cycle signaling. Bisulfite genomic sequence (BSP) assay was used to test the methylation degree of ADHFE1 gene promoter in CRC tissues. RESULTS: Here, we verified that ADHFE1 was down-regulated and hypermethylated in CRC tissues. The down-regulation of ADHFE1 was correlated with poor differentiation and advanced TNM stage of CRC patients. And ADHFE1 expression restored when the CRC cell line SW620 was treated with the demethylating agent 5-Aza-CdR. Overexpression of ADHFE1 inhibited the proliferation of CRC, while ADHFE1 knockdown promoted the proliferation of CRC cells in vitro and in vivo. Moreover, ADHFE1 overexpression could induce a significant G1-S cell cycle arrest in CRC cells and vice versa. CONCLUSION: Hypermethylation of ADHFE1 might promote cell proliferation by modulating cell cycle progression in CRC, potentially providing a new therapeutic target for CRC patients.

Downregulation of microRNA-382 is associated with poor outcome of esophageal squamous cell carcinoma.

AIM: To study the potential prognostic role of microRNA-382 (miR-382) in esophageal squamous cell carcinoma (ESCC). METHODS: Forty six patients were divided into 2 groups according to postoperative survival time: the poor outcome group (28 patients), who showed early metastasis but no recurrence, and died within 1 year after surgery, 12 patients of the group received postoperative chemotherapy treatment that was given after early metastasis happening; the good outcome group (18 patients), who had no clinical metastasis and recurrence, and survived 5 years or more after surgery, all patients did not receive any postoperative treatment. Total RNA was extracted from the patients' formalin-fixed and paraffin-embedded esophageal cancer tissues. miR-382 level was evaluated using high-throughput real-time quantitative polymerase chain reaction analysis. The correlation between miR-382 level and clinicopathologic features was analyzed through COX regression model, and Kaplan-Meier analysis was used to analyze the relationship between miR-382 level and patient survival time. RESULTS: miR-382 was differentially expressed in the two groups. Overall the average miR-382 level in the ESCC patients with good outcome was 9.8 ± 3.8, while miR-382 level in the ESCC patients with poor outcome was 3.0 ± 0.8. The differences of miR-382 levels between two groups were significant (P < 0.05). Kaplan-Meier analysis results showed that miR-382 expression level generally had a significant reverse-correlation with ESCC patient survival time (P < 0.001), in which the patients with higher expressions of miR-382 had a longer survival time either among individuals with the same tumor stage or among the overall patients. CONCLUSION: miR-382 levels are reverse-correlated with ESCC poor outcomes, suggesting that miR-382 could be a potential predictive biomarker for both prognosis and treatment of ESCC.

Screening of microRNA in patients with esophageal cancer at same tumor node metastasis stage with different prognoses.

Patients at the same pathological stage of esophageal cancer (EC) that received the same surgical therapy by the same surgeon may have distinct prognoses. The current study aimed to explore the possibility of differentially- expressed microRNAs (miRNAs) underlying this phenomenon. Samples were collected from EC patients at the same tumor node metastasis (TNM) stage but with different prognoses. Paracancerous normal tissues were taken as controls. The specimens were histopathologically analyzed. Differentially-expressed miRNAs were analyzed using real-time quantitative reverse transcription polymerase chain reaction. Compared with patients with poor prognosis, those with good prognosis exhibited 88 two-fold or more than two-fold increased miRNA fragments and 4 half-decreased miRNAs. The most noticeably up-regulated miRNAs included hsa-miR-31, hsa-miR- 196b, hsa-miR-652, hsa-miR-125a-5p, hsa-miR-146b, hsa-miR-200c, hsa-miR-23b, hsa-miR-29a, hsa-miR-186, hsa-miR-205, hsa-miR-376a, hsa-miR-410, hsa-miR-532-3p, and hsa-miR-598, whereas the most significantly- downregulated miRNAs were hsa-let-7e, hsa-miR-130b, and hsa-miR-103. EC patients at same TNM stage but with different prognoses show differentially-expressed miRNAs.

Whole-exome sequencing identifies mutated PCK2 and HUWE1 associated with carcinoma cell proliferation in a hepatocellular carcinoma patient.

Hepatocellular carcinoma (HCC) is diagnosed in more than half a million individuals worldwide every year. It is often invasive and metastatic, resulting in a poor prognosis. Our knowledge of the genomic alterations implicated in HCC initiation and progression is fragmentary, and few molecular alterations unique to HCC are known. We performed whole-exome sequencing for a pleomorphic cell-type HCC tissue and matched normal tissue, and uncovered seven non-synonymous somatic variants in SPATA21, PPCS, CDH12, OR1L3, PCK2, HUWE1 and PHF16. These variants were validated by PCR and sequencing, with the exception of that in PPCS. We further performed a bioinformatics analysis of the six validated variants. The results suggested that the function of the proteins of the three mutated genes, PCK2, HUWE1 and PHF16, may be changed significantly. Among these genes, PCK2, within the insulin signaling pathway, and HUWE1, within the ubiquitin-mediated proteolysis pathway, may be essential for cell proliferation. These pathways are known to be important for hepatocarcinogenesis. Hence, we suggest that PCK2 and HUWE1 are associated with carcinoma cell proliferation in HCC.