Rela re rela program

Learn more. Why does the linker generate seemingly useless relocations in. Ask Question. Asked 4 years, 11 months ago. Active 4 years, 11 months ago. Viewed 2k times. First, the toy program I'm playing with: prog. But then Value Sym. Update 1: To be clear, this is about PIC code and 64bit code. Thanks yugr. Improve this question. Rahm Urwin. Rahm Urwin Rahm Urwin 73 4 4 bronze badges. This is a known issue. See ewontfix. In particular, it discusses the relocations for the function call in the code in the.

Add a comment. Active Oldest Votes. Which makes sense, since this address is known at link time Is it? Improve this answer. RahmUrwin "Snarky" - never meant to, sorry. RahmUrwin 1 Hm, there must be some misunderstanding as I fail see how my posts above disagree with your statements. Laporkan Komentar. Terima kasih. Kami sudah menerima laporan Anda. Terkini Lainnya.

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The single miR can simultaneously modulate hundreds of gene expression, vice versa, the single gene can be cooperatively regulated by multiple miRs.

The specificity of target gene of miRs is usually determined by the seed region of mature sequences, association of which down-regulated gene expression by diverse mechanisms including mRNA degradation, translation suppression and de-adenylation. Notably, accumulative evidence suggests that miRs are involved in tumor biology of a variety of human malignancies [11]. MiRb-3p has been unraveled involving in several human cancers. For example, Xie et al. Wang et al. Liu et al.

Cai et al. In respect to prostate cancer, three studies so far focused on miRb with contradictory conclusions. Here we identified miRb-3p as differentially expressed gene in prostate cancer patients and sought to elucidate its roles in prostate cancer.

Blood samples were collected from 10 patients with prostate cancer and 10 health individual donors in Longhua Hospital Shanghai University of Traditional Chinese Medicine. Written informed consents were provided by all enrolled subjects. The human related study was approved by the Ethics Committee of Longhua Hospital Shanghai University of Traditional Chinese Medicine and in strict compliance with the reviewed protocol. Read count was calculated as relative expression value and differential expression genes were identified.

The cell identities and mycoplasma-free states were authenticated by Abace Beijing, China. The sequence of miRb-3p precursor was constructed and sub-cloned into the pMXs-miR plasmid. Mutagenesis PCR was adopted for generating mutation in the putative recognizing sites. The indicated cells were collected and subjected to lysis in RIPA buffer 30 min on ice with allcell debris removed.

Scanned images were processed and analyzed using ImageJ software. Briefly, the indicated single-cell suspension was prepared in serum-free medium. One hour later, the unattached cells were washed off and the attached ones were counted after trypsinization. For detachment assay, after being cultured for 24 h, the cells were partially digested and detached with 0. After removal the detached cells, the remaining cells were harvested and counted.

Mice were sacrificed at post-injection day 15 and tumor was weighed. The Prism GraphPad software was employed for processing and analyzing the data. The P value was generated and P less than 0. Here we set out to apply RNA-seq for the patient blood samples to discover the novel microRNAs with differential expression and association with prostate cancer.

Hence, we collected serum samples from 10 prostate cancer patients and 10 health donors. As shown in Figure 1A, our RNA-seq data unambiguously identified that miRb-3p was markedly and consistently reduced in cancer group in comparison with control across three independent repeats, which suggested the potential tumor suppressing function of miRb-3p in prostate cancer.

Therefore, our results demonstrated the downregulated miRb-3p in prostate cancer both in vivo and in vitro. Mature microRNA assay for miRb-3p was conducted in the same prostate cancer patients and healthy control blood samples. Next, we aimed to identify the molecular events downstream aberrant suppressed miRb-3p in prostate cancer. Aided by a well-recognized bioinformatic online tool, we predicted RELA as the potential candidate gene to be targeted by miRb-3p.

The putative recognizing site was disrupted via mutagenesis PCR in our mutation construct in comparison with wild type one Figure 2B.

Co-transfection with miRb-3p significantly inhibited the relative activities of luciferase, which was abolished by the introduction of mutation in the seed region Figure 2C.

Our foregoing results suggested potential anti-tumor properties of miRb-3p in prostate cancer, which prompted us to investigate this probability in vitro. To address this, we employed a stable cell line in PC-3 overexpressing miRb-3p. High levels of miRb-3p significantly suppressed cell migrative and invasive capacity in our system Figure 3A, 3B. The EMT phenotype was evaluated by cell attachment and detachment as well. As demonstrated in Figure 3C and 3D, miRb-3p over-expression greatly inhibited cell attachment and detachment in PC-3 cells.

Consistent with compromised migrative, invasive, attachment and detachment capacities, we observed remarkable down-regulation of both N-cadherin and Vimentin, and up-regulation of E-cadherin upon miRb-3p over-expression in PC-3 cells Figure 3E.

Therefore, we presented evidence supporting the suppressive effect of miRb-3p on cell migration, invasion and EMT-related behaviors. MiRb-3p inhibits migration, invasion and suppresses EMT phenotypes of prostate cancer cells in vitro. PC-3 cells stably expressing miRb-3p mimic miRb or negative control miR miR-NC were subjected to in vitro assays to analyze their A migration, B invasion, C attachment and D detachment abilities.

E Protein expressions of epithelial marker E-cadherin, and mesenchymal markers N-cadherin and vimentin, were assessed using Western blot in PC-3 cells stably expressing negative control miR miR-NC or miRb-3p mimic miRb. To exclude the possibility that in vitro cell cultures may generate artifacts, we further evaluated the anti-tumor activity of miRb-3p in vivo using a xenograft mouse model. The miRv-3p-proficient PC-3 cells were inoculated subcutaneously into immunodeficient mice, after which tumor development was regularly checked and recorded.

As shown in Figure 4A, ectopic expression of miRb-3p significantly delayed the xenograft tumor progression in comparison with scramble control. Consistently, the average weight of the xenograft tumors resected from sacrificed miRb-3p mice was remarkably less than the control group Figure 4B.

Consequently, the favorable prognosis was noticed in the miRb-3p-proficient mice Figure 4C , which further consolidated our preliminary observations in respect to the anti-tumor activity of miRb-3p in vivo. MiRb-3p inhibits growth of xenograft tumor from inoculated prostate cancer cells, and promotes survival of xenograft mice.

On day 15, mice were sacrificed to extract the xenograft and the tumor were resected and weighed, with representative tumor images shown on the left. Next, we sought to evaluate the importance of RELA for the anti-tumor properties in prostate cancer.

Similarly, supplementation with RELA evidently reversed the decrease in cell attachment and detachment elicted by miRb-3p over-expression Figure 6C, 6D. The up-regulated epithelial marker E-cadherin was significantly decreased and down-regulated mesenchymal markers including N-cadherin and Vimentin were prominently increased by RELA in the miRb-3p-expressing PC-3 cells Figure 6E. E Protein expressions of epithelial marker E-cadherin, and mesenchymal markers N-cadherin and vimentin, were analyzed using Western blot in the above-mentioned PC-3 cells.

To confirm our observations that RELA predominantly mediated the anti-tumor features of miRb-3p in vivo, here we further established stable cell line in miRb-3p-proficient PC-3 cells with over-expression of RELA. Consistent with our in vitro conclusion, we noticed significant increase of tumor growth in RELA-mice in comparison with control group Figure 7A.

More importantly, the overall survival was remarkably compromised by RELA re-introduction as well Figure 7C , which unambiguously underlined the oncogenic properties of RELA in prostate cancer as well as its predominance in mediating the anti-tumor activities of miRb-3p.

B On day 15, mice from all three groups were sacrificed to extract the xenograft and weigh the tumor, with representative tumor images shown on the left. The next-generation sequencing has been increasingly adopted for diagnostic and prognostic exploitations in various human malignancies [16]. Here we aimed to analyze the blood samples from both prostate cancer patients and health individual donors using RNA-seq technology and notably identified the miRb-3p as among the most significantly differentially expressed genes in this disease.

The aberrant miRb-3p reduction was further confirmed by quantitative rt-PCR. Our data supported potential anti-tumor capacities of miRb-3p in prostate cancer. Noting worthily, the previous investigation by Mathieu et al. On the contrary, Poliseno et al. Recently, Guo et al. In sharp contrast to the archived oncogenic roles, here we uncovered novel anti-tumor properties of miRb-3p in prostate cancer both in vivo and in vitro.

Along this direction, we used bioinformatics to predict and experimentally validated that RELA was directly targeted by miRb-3p in prostate cancer in this scenario. Consistently, the endogenous expression of RELA was greatly compromised at both transcript and protein levels by ectopic introduction of miRb-3p in PC-3 cells as well. In addition, we examined the potential influences of miRb-3p on tumor metastatis-related behaviors in prostate cancer cells. The remarkable decreases in respect to migration, invasion, attachment and detachment were observed in our system.

Similarly, E-cadherin, anepithelial molecular marker, was greatly induced while N-cadherin and Vimentin, the mesenchymal markers, were tremendously repressed by miRb-3p in PC-3 cells. In vivo study using xenograft model mice further consolidated the anti-tumor activities of miRb-3p in prostate cancer, leading to more favorable overall survival in our animal experiment.