Functional Study Of TTTY15-USP9Y And Mirna-182-5P Genes Related To Prostate Cancer

BackgroundProstate cancer is the most common cancer in men worldwide, no matter in the United States or China. In recent years, it showed a clear upward trend in China. Prostate cancer studies has two significant dilemma: one is effective therapeutic target. On the basis of growing surgery, hormonal therapy, biological therapy and chemotherapy treatment, localized prostate cancer cure rate has been improving. But the total mortality rate is still high, rooting in recurrent cancer and progressing into metastatic prostate cancer with hormone-refractory. Thus, exploring the molecular mechanisms in invasion and metastasis of prostate cancer, finding new therapeutic target for prostate cancer become the focus of prostate cancer research. Second is diagnostic markers. The current treatments are refered to the level of serum PSA. PSA detect has the flaw of false positives, leading to unnecessary biopsy and treatment burden. For years, researchers and clinicians dedicated to find more specific diagnostic modality, diagnostic markers. The study included studies related to prostate cancer miRNA, Lnc RNA, PSA isomers, common diagnostic PSA joint UN-length coding RNA and RNA ring of serum prostate cancer exosomes and exosomes gene related miRNA, proteins, fusion. Researchers are trying to find an excellent prostate cancer diagnostic markers and therapeutic targets.Fusion gene in blood disease first became famous because of the Philadelphia chromosome, which opened the field of research in human tumor fusion gene. The fusion gene has a good diagnostic efficiency in blood cancers, and provides support and guidance for the treatment. After this classical fusion gene, other tumor areas have started to explore the tissue-specific fusion gene. Because of the limited scientific research and technolog, the fusion genes were not found. With the development of sequencing technology and constantly improve cancer databases, some of the fusion gene was detected later. The earliest and most classic fusion gene in prostate cancer is ETS fusion gene family. The fusion gene contains a variety of subtypes. The fusion genes founded are fused on the classic DNA level in this period, which brings excellent diagnostic and prognostic guidance efficiency. After that, with the improvement in sequencing technology, we found some chimera RNA. Comparing to the level of integration of DNA, chimera RNA have greater flexibility, richer editing mode, which greatly expands the limited human protein-coding Category gene, makes the body could have a richer protein-coding genes or regulatory manner under limited circumstances. The chimera RNA in this period were almost all trans-splicing and trans-splicing. And the classic trans-splicing was found in trypanosomes and nematodes, latter found in humans. Only this step laid a good foundation for the fusion gene's richness. In recent years, research on chimeras found SLC45A3-ELK4, which is not the behavior of the chimeric DNA level but RNA level behavior after some experiments. And its cis or trans-splicing splicing behavior had been demonstrated. Studies tend to think that the chimera RNA cis-splicing is after read through, a new and more flexible form.Purpose:Identifying whether fusion gene exsits in prostate cancer and prostate cancer cell lines, the subtype of fusion gene and whether the fusion occurred in the DNA level. Study the correlation function in prostate cancer cells. Exploring the fusion gene's mechanism; screening miRNA significantly different expressing in prostate cancer and adjacent cancer tissues were, discuss MiR-182-5P fuction in prostate cancer proliferation, apoptosis, invasion area.Methods:1, DU145, PC3, LNCa P, 22RV1, C42-related prostate cell lines using PCR-based method to detect monoclonal TTTY15-USP9 Y is real, it contains identification of different subtypes2, DU145, PC3, LNCa P, RWPE, 22RV1, C42-related prostate cell lines using RT-PCR method to detect TTTY15-USP9 Y expression.3, Between the colon cancer cells, kidney cells, and umbilical cord mesenchymal stem cells, liver cells and DU145 detected fusion gene were TTTY15-USP9Y4, Northern blot identification of the TTTY15-USP9Y6,Construct expressing cell line DU145 advantage of clonal cell lines of monoclonal7, Walking PCR-based pure monoclonal cell lines DNA and RNA level TTTY15-USP9 Y levels were identified.8, Use crispr / cas9 technology advantages of cell lines expressing the DNA double-level targeting, and the cells of monoclonal pick stitching accurate monoclonal cell lines artificially constructed having TTTY15-USP9 Y fusion gene cell lines.9, After the artificial construct cell lines to identify DNA and RNA level, level, level further help to confirm the fusion gene occurs.10, New TTTY15-USP9 Y sequence to predict functional domains11, Using immunofluorescence Western Blot detection of protein-coding12, The original parent gene TTTY15, and USP9 Y before and after the fusion site interference, interference detection efficiency13, Using the scratch test, EDU test cycle testing, apoptosis detection methods to explore the fusion gene function14, The original fusion USP9 Y 5'UTR region after comparing the new 5'UTR region.15, The original promoter of USP9 Y with the new fusion gene promoter FeatureComparisonResults:1, Conducting in prostate cancer cell lines to identify relevant expression of the fusion gene, confirming the advantages of subtypes TTTY15-USP9Y-480, and confirm the superiority of cells expressing DU145.2, Among the cancer cells, kidney cells, and umbilical cord mesenchymal stem cells, liver cells and DU145 carried the fusion gene TTTY15-USP9 Y detected, TTTY15-USP9Y-480 the highest expression in prostate cancer cells.3, Successfully established prostate cancer cell line DU145 monoclonal cell line, built by the short-term line of monoclonal cell lines were identified DNA and RNA level level, confirm TTTY15-USP9 Y fusion DNA level is not the behavior of RNA levels chimeric4, The use crispr / cas9 technology prostate cancer cells DNA double-level targeting, successfully constructed, and pick out TTTY15 USP9 Y accurate docking with monoclonal cell lines artificially constructed TTTY15-USP9 Y fusion gene clonal cell lines.5, By artificially constructed fusion gene cell lines were identified RNA and DNA levels, further confirms the occurrence of the fusion gene does have RNA level accurate splicing and preferences, and further suggests that the fusion of the gene is RNA level along- splicing.6, According to the new sequence of the gene fusion of functional areas predicted results display area has not changed, the original protein coding region is not destroyed.7, Immunofluorescence and Western Blot showed that, USP9 Y protein is the highest expression in the DU145, and concentrated in the cytoplasm.8, The interference difference between the original parent gene fusion site before and after the relevant functional experiments confirmed that the fusion gene can promote the value of prostate cancer, prostate cancer and to promote greater access to G2 phase. Promote prostate cancer cell migration and invasion.9, The original fusion USP9 Y 5'UTR region after comparing the new 5'UTR region, there are some differences, but did not reach a statistically significant gap.10, The original promoter and USP9 Y new fusion gene promoter function, the difference was significant, statistically significant. TTTY15-USP9 Y promoter region of fusion is stronger than the original USP9 Y promoter.Conclusions:Chinese human prostate cancer tissue fusion TTTY15-USP9 Y does exist, which is also present in prostate cancer cell lines. TTTY15-USP9 Y gene fusion has four subtypes, the prostate cell lines built in Chinese human prostate cancer tissue or in the West are based on the dominant subtype, TTTY15-USP9Y-480. According to confirmed relevant experiments, TTTY15-USP9 Y fusion gene behaviors on the RNA level. This topic through a series of experiments confirmed that the fusion gene strongly upregulated the expression of USP9 Y to further promote the appreciation and the related tumor characteristics of prostate cancer. And further exploring the fusion gene promoter region in this new process had played an important role. According to the sequencing information of prostate cancer, followed by design primers pcr in prostate cancer cell lines of monoclonal, identified TTTY15-USP9 Y fusion does exist, and contains four subtypes. BackgroundmiRNA involved in regulating cell growth, proliferation, apoptosis and stress response, playing an important role cell homeostasis and development. The abnormal expression of miRNA is an important characteristic of malignant tumors. Compared to other areas, the study of prostate related miRNA started later, with smaller scale and fewer repeated verifications. And an ideal diagnostic marker needs to show stability in large scale sample screening.Purpose:Identifying whether fusion gene exsits in prostate cancer and prostate cancer cell lines, the subtype of fusion gene and whether the fusion occurred in the DNA level. Study the correlation function in prostate cancer cells. Exploring the fusion gene's mechanism; screening miRNA significantly different expressing in prostate cancer and adjacent cancer tissues were, discuss MiR-182-5P fuction in prostate cancer proliferation, apoptosis, invasion area.Methods:1, Using RT-PCR method to detect the expression of MiR-182-5P in DU145, PC3, LNCa P, RWPE, 22RV1, C42-related prostate cell lines. Function Synthesis MiR-182-5P of agomir, antagomir, agomir NC, antagomir NC for performing miRNA's. By clontech of miRNA specific transfection reagent Xfect ? siRNA Transfection Reagent, transfection prostate carcinoma DU145, LNCa P cell lines, to detect changes MiR-182-5P expression. Application Edu kit before and after transfection of cell proliferation; using flow cytometry cell cycle and apoptosis of change; change scratch assay cell migration; cell lines to detect changes in Transwell invasion ability. Investigate all aspects of the development of the role of MiR-182-5P occur in prostate cancer.2, Using bioinformatics methods to predict the presence or absence of the promoter region of ar-binding sites, using chip-related experiments to predict whether the binding sites of the AR. Mdv3100 use of DHT and stimulate androgen receptor-positive cells was observed in the expression levels of MiR-182-5P is affected AR change. Using RT-PCR method to detect relations between MiR-182-5P and AR downstream target.3, Using Bioinformatics prediction method with miRbase, star Base, Tarbase, target Scan etc., predicting possible target genes of MiR-182-5P,intersecting with the down-regulated genes in 65 pairs of the sequencing results. Then screened target genes were ordered according to predictions. six genes were chosen which was closely related to tumor development, and down-regulated in tumor tissue high-throughput sequencing. After the RT-PCR method, Western Blot and dual luciferase reporter assay experimental were used to validate target genes and further explore the mechanism MiR-182-5P relevant role in prostate cancer.4, Using Western Blot, periodic testing, detecting the level of apoptosis and scratch assay to detect compensation effect of 182, and the compensation of ARRDC3, ITGb4 for MiR-182-5P.5, Culturing cells, overexpressing MiR-182-5P, and selecting 10 5-week-old male nude mice bearing subcutaneous tumor experiments. Observe the change of generation and growth of tumors, and tumor tissues of Ki67 immunohistochemistry.Results:1, Through the organization RNA sequence sequenced and found a number of differences between the groups exist miRNA, after statistical analysis, found MiR-182-5P fold difference of more than 5 times, more significant differences between the groups. After realtime pcr experiments, which were significantly higher than non-cancerous tissue in the expression of prostate cancer.2, MiR-182-5P expression du145 lower amount, suitable for over-expression detection. Overexpression of MiR-182-5P treatment groups invasiveness stronger suppression group had decreased invasiveness.3,Changes MiR-182-5P expression levels may affect AR downstream target. And it is present on the path between the target and downstream ar.4, The experimental results show that the transfection MiR-182-5P agomir significantly inhibited p MIR-REPORT- ARRDC3- 3'UTR-Wt luciferase activity.5, Overexpression MiR-182-5P cell cycle, apoptosis and migration caused can be compensated by inhibiting ITGb4, inhibition of MiR-182-5P prostate cancer may be caused by interference balance ARRDC3.6, Tumor-bearing nude mice experiments showed that overexpression of MiR-182-5P could promote tumor formation and development.Conclusions:MiR-182-5P in adjacent cancer tissues with significant differences. MiR-182-5P can promote the proliferation of prostate cancer cells, inhibition of early apoptosis of prostate cancer cells, the prostate cancer cell invasion. miRNA ability plays an important role. MiR-182-5P associated with AR is regulated by the AR. MiR-182-5P by binding directly to ARRDC3 3'UTR region, downregulated ARRDC3, and thus play a proliferation of prostate cancer cells, invasion effect. Stable performance MiR-182-5P expected as a new target for clinical diagnostic markers and therapeutic intervention. MiR-182-5P act directly on ARRDC3, MiR-182-5P can affect ARRDC3 related binding proteins. MiR-182-5P in vivo also could promote tumor formation and development.