| Backgroud Prostate cancer(PCa) is the most common solid-organ malignancy and the second leading cause of cancer-related death in males. This heterogeneous neoplasm has been reported to be primarily regulated by androgenic hormones and influenced by dietary habits. Chronic inflammation causes 20% of human cancers and increases the risk of cancer, including gastric, pancreatic, colon and lung cancer. Increasing epidemiological and histopathological evidence also suggests that the incidence of PCa is correlated with inflammation, yet the concrete mechanism involved, particularly in human studies, is still not fully understood. Basically, chronic inflammation creates a milieu rich in pro-inflammatory cytokines and growth factors that may lead to an uncontrolled proliferative response and genetic mutations of rapidly dividing cells. Thus, PCa is closely associated with inflammation, particularly with the role of inflammatory factors. Interleukin-6(IL-6) is a multifunctional pro-inflammatory cytokine whose expression and function are altered in a variety of human cancers. IL-6 has also been found to be widely expressed in clinical specimens obtained from PCa patients and in several PCa cell lines(2). G protein-coupled receptor(GPCR) 48, also known as leucine-rich repeat-containing GPCR(LGR) 4, is a glycoprotein hormone receptor belonging to the GPCR superfamily that plays an important role in the development of multiple organs. In general, LGR4 encodes one of the GPCRs for R-spondins. A recent study found that IL-6 enhanced the expression of LGR4 protein in osteosarcoma cells, suggesting that LGR4 may be a novel responsive gene of IL-6 in cancer progression. Furthermore, previous preclinical and clinical studies have confirmed that GPCR is overexpressed in PCa tissues. LGR4 contributes greatly to the formation of various types of cancers, including lung, breast, prostate and gastric cancer, as well as hepatoma. The effect of LGR4 on cancer progression has been recognized, yet the regulatory mechanism on LGR4 expression is still unclear. micro RNAs(mi RNAs) are a set of endogenous small non-coding RNAs(19-22 bases in length) and are used to regulate gene expression by inhibiting translation or cleaving RNA transcripts in a sequence-specific manner. Cumulative evidence indicates that mi RNAs regulate diverse biological processes, including cell proliferation, invasion, migration and apoptosis. mi RNAs downregulate multiple target genes, including oncogenes and tumor-suppressor genes, while some mi RNAs function as tumor suppressors and others act as oncogenes. In particular, mi R-218, a tumor-suppressing mi RNA, has been extensively studied in several types of cancers and is highly downregulated in PCa. In the present study, we demonstrated that LGR4 is a downstream target of mi R-218 and that mi R-218 inhibited PCa cell proliferation and invasion through suppressing LGR4 expression.Objective(1) By real-time fluorescent quantitative PCR method and ELISA method to detect the expression of mi R-218 and IL-6 in the adjacent normal tissue(NP), benign prostatic hyperplasia(BPH), prostate cancer.(2) established a IL-6 of long-term incubation with low concentrations of LNCa P-IL-6 subline, using real-time fluorescent quantitative PCR method for detection of mi R-218 in LNCa P and the expression of LNCa P-IL-6 in cell subculture trend.(3) using Brd U and Transwell method to IL-6 and mi R-218 in the statistical analysisof the effect of LNCa P-IL-6 cell line.(4) detected by Western blot analysis IL-6 and mi R-218 effects of LGR4 protein expression in LNCa P-IL-6+ cells.Materials and Methods 1. Cases selection: From July 2012 July 2015 in my hospital for treatment of 58 patients with diagnosed cancer tissue samples from patients with prostate cancer(Pca), 51 cases of prostate cancer in patients with normal tissue(NP), and 56 patients with benign prostatic hyperplasia(BPH) in patients with prostate tissue while saving both at-80 c freezer. All patients have been received into a set of standard screening for confirmed cases. 2. Exclusion criteria: The patients with endocrine or others have been excluded. 3. The experimental methods: Using real-time fluorescent quantitative PCR method and ELISA method for detection of mi R-218 and IL-6 in the adjacent normal tissue(NP), benign prostatic hyperplasia(BPH), the expression levels of prostate cancer was established in IL-6 of long-term incubation with low concentrations of LNCa P-IL-6 subline, using real-time fluorescent quantitative PCR method for detection of LNCa P-IL-6 and mi R-218 in LNCa P cells were cultured in the expression of trends Using Brd U and Transwell method to IL-6 and mi R-218 in the statistical analysis of the effect of LNCa P-IL-6 cell line using Western blot analysis detected IL-6 and mi R-218 effects of LGR4 protein expression in LNCa P-IL-6+ cells. 4. Statistical methods: Using SPSS20.0 statistical software for statistical analysis, the determination results showed as the mean ±standard deviation. Using one-way analysis of variance for the data of the RT-PCR, With P<0.05 for the difference of statistically significant.Results(?) The level of expression of mi R-218 and IL-61. mi R-218 in adjacent normal tissue(NP), benign prostatic hyperplasia tissue(BPH) and prostate cancer(Pca) levels decreased, respectively(1.49 ± 0.07),(0.89 ± 0.02),(0.51 ± 0.02), a significant difference(P=0.012),(P=0.024),((P=0.001) was statistically significant. 2.IL-6 in adjacent normal tissue(NP), benign prostatic hyperplasia tissue(BPH) and prostate cancer(Pca) levels increased, respectively(1.09 ± 0.05),(1.89 ± 0.01),(2.52 ± 0.02), a significant difference(P=0.002),(P=0.004),((P=0.020) was statistically significant.(?) In the LNCa P-IL-6 subline, IL-6 for a long time after the decline in levels of mi R-218 expression in LNCa P cells, results indicate that mi R-218 into LNCa P-IL-6+ the expression in LNCa P cells in the process of gradually declining.(?) mi R-218 IL-6-induced LNCa P-IL-6 cell proliferation and invasion. Brd U analysis shows IL-6 for LNCa P-IL-6 cell proliferation, while mi R-218 transfection inhibited the effect. And IL-6 led to a reduction period of the cell cycle in G0/G1 cells, instead G0/G1 the increase in the number of cells transfected by mi-R218. Transwell shows, invasion of LNCa P-IL-6 cells increased significantly after the use IL-6, however this role was significantly inhibited by mi R-218 transfection.(?) Detected by Western blot analysis, respectively IL-6 and mi R-218 LNCa P-IL-6+ LGR4 protein expression in cells of the effect: LGR4 in IL-6 in LNCa P-IL-6 cells induced by the relative expression levels(2.20±0.30); LGR4 in mi R-218 in LNCa P-IL-6 cells induced by the relative expression levels(0.40±0.01) LGR4 relative expression in LNCa P-IL-6 cells induced by IL-6+mi R-218(1.30±0.21) comparison between the three, the differences were statistically significant.(?) The expression of mi R-218 detected by RT-PCR in the LNCa P-IL-6+ cell line transfected by mi R-218: the relative expression of mi R-218(1.20 ± 0.20), was significantly increased in comparison with the control group, the differences were statistically significant.(?) The expression of LGR4 detected by Western blot analysis in the LNCa P-IL-6+ cell line transfected by mi R-218: the relative expression of LGR4(0.30 ± 0.03), was significantly reduced in comparison with the control group, the differences were statistically significant.Conclusions mi R-218 expression is downregulated and IL-6 expression is upregulated in the progression of prostate cancer. h&E stainings for human histological specimens were examined, and representative images of NP, BPh and PCa tissue samples are shown, in which the probable pathological progression of prostate cancer can be observed. q RT-PCR assay showed that mi R-218 expression in the BPh tissues was lower than that in the NP tissues and mi R-218 expression in the PCa tissues was lower when compared with that in the BPh tissues. however, ELISA assay showed that IL-6 levels in the NP, BPh and PCa tissues exhibited an opposite trend. The expression levels of mi R-218 in the longterm IL-6-stimulated LNCa P cells decreased with increasing passage. These results revealed that mi R-218 expression was gradually decreased and IL-6 expression was gradually increased in the process of prostate cancer progression from NP, BPh to PCa and from LNCa P to LNCa P-IL-6+ cells. mi R-218 impedes IL- 6-induced LNCa P-IL- 6+ cell proliferation and invasion. Brdu assay revealed that IL-6 promoted LNCa P-IL-6+ cell proliferation, whereas mi R-218 transfection inhibited the enhanced cell growth. The data from FACS analysis showed that IL-6 led to a reduction in the percentage of cells in the G0/G1 phase of the cell cycle as compared to the control group; conversely, mi R-218 transfection promoted an accumulation of cells in the G0/G1 phase. Transwell assay showed that the invasive ability of the LNCa P-IL-6+ cells was significantly promoted by IL-6 and markedly inhibited by mi R-218 transfection. In addition, mi R-218 transfection suppressed the increased expression of cyclin A1 and MMP-9 proteins induced by IL-6. Taken together, these findings indicated that mi R-218 impeded IL-6-induced LNCa P-IL-6+ cell proliferation and invasion. mi R-218 inhibits IL-6-induced LGR4 expression in LNCa P-IL-6+cells.Western blotting showed that LGR4 expression was upregulated by IL-6 pretreatment while mi R-218 transfection abolished the enhanced expression of LGR4 protein induced by IL-6 incubation in the LNCa P-IL-6+ cells. These data suggest that mi R-218 may be a promising candidate for directly targeting LGR4 in LNCa P-IL-6+ cells. mi R-218 targets LGR4 by binding to its 3'-UTR. Bioinformatic prediction showed that there was one putative binding site between mi R-218 and the 3'-UTR of LGR4. To confirm the binding, a luciferase reporter assay was performed by evaluating the luciferase activity of h EK293 T cells transfected with the p MIR-LGR4 3'-u TR plasmids and comparing this activity with that transfected with control plasmids. The results showed that mi R-218 significantly suppressed luciferase expression of LGR4-wt, whereas LGR4-mut induced no suppressive effect. In addition, q RT-PCR analysis confirmed that mi R-218 transfection resulted in an increase in mature mi R-218 in the LNCa P-IL-6+ cells. In addition, the LGR4 protein level was suppressed following mi R-218 transfection in the LNCa P-IL-6+ cells. In summary, these results indicated that mi R-218 directly targeted LGR4 in the LNCa P-IL-6+ cells. |
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