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RAD54B

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Last Updated: 02 July 2021

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General | Latest Info

RAD54B

Identifiers
AliasesRAD54B , RDH54, RAD54 homolog B (S. cerevisiae), RAD54 homolog B
External IDsOMIM: 604289 MGI: 3605986 HomoloGene: 8240 GeneCards: RAD54B
Orthologs
Wikidata
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Every item is shipped based on the best shipping method assess for temperature requirements of that specific item. Items are grouped and shipped together whenever possible, and separate shipping charges will be included for each shipping method require. Shipping charges list below are from our US warehouse to Contiguous US, Alaska, Hawaii, Canada and Puerto Rico. Shipping charges for countries outside the US and Canada will be determined once the order has been received. The RAD54B Antibody is a high quality monoclonal RAD54B Antibody suitable for detection of RAD54B protein of human origin. The RAD54B Antibody is available as a Non-conjugate anti-RAD54B Antibody. RAD52 family members mediate DNA double-strand break repair for DNA damage that otherwise could cause cell death, mutation or neoplastic transformation. Rad51 interacts with BRCA1 and BRCA2 to influence subcellular localization and cellular response to DNA damage. BRCA2 inactivation may be a key event leading to genomic instability and tumorigenesis from deregulation of Rad51. RAD52 forms heptameric ring that bind single-strand DNA ends and catalyze DNA-DNA interaction necessary for annealing of complementary strands. RAD52 can interact with Rad51. Rad54A of the DEAD-like helicase superfamily bind to double-strand DNA and induces DNA topological change, which is thought to facilitate homologous DNA pairing and stimulate DNA recombination. RAD54B of DEAD-like helicase superfamily bind to double-strand DNA and displays ATPase activity in the presence of DNA. RAD54B is abundant in testis and spleen, and mutations of this gene occur in primary lymphoma and colon cancer. MRE11 is a nuclear 3-5 exonuclease / endonuclease that Associate with RAD50 and influences homologous recombination, telomere length maintenance, and DNA double-strand break repair. MRE11 is the most abundant in proliferating tissues. For Research Use Only. Not intended for Diagnostic or Therapeutic Use.

* Please keep in mind that all text is machine-generated, we do not bear any responsibility, and you should always get advice from professionals before taking any actions.

* Please keep in mind that all text is machine-generated, we do not bear any responsibility, and you should always get advice from professionals before taking any actions

Cancer

IHC staining was performed to determine the relationship between RAD54B expression and prognosis of liver cancer patients. RAD54B protein expression was investigated in 83 samples of liver cancer tissue. Overall survival data was obtained for all 83 patients. Results reveal that 52 / 83 samples of liver cancer were positive for nuclear staining and RAD54B protein expression, while the other 31 samples were negative. Using Kaplan-Meier survival analysis, we find that the median OS time was 18. 3 months for patients with positive expression, whereas OS was 22. 0 months for patients with RAD54B negative expression. Liver cancer patients with positive RAD54B expression were associated with reduced OS. Collectively, these results indicate that expression of RAD54B could be a prognostic marker for liver cancer.

* Please keep in mind that all text is machine-generated, we do not bear any responsibility, and you should always get advice from professionals before taking any actions.

* Please keep in mind that all text is machine-generated, we do not bear any responsibility, and you should always get advice from professionals before taking any actions

Introduction

Primary liver cancer is a common aggressive malignant disease, and the third leading cause of cancer-related deaths worldwide. An estimated 40 710 new cases of disease and 28 920 cancer-associate deaths were reported in 2017 in the USA. More than 90 % of primary liver cancers are hepatoma, which is mainly caused by infection with hepatitis B or C. Incidence of liver cancer has been increasing in Asia and ~50 % of reported mortalities are Chinese individuals. As early Detection is difficult, ~70 % of all hepatoma patients cannot be treated by surgical resection at time of diagnosis, which results in poor prognosis and median overall survival time of few months. Molecular pathogenesis of liver cancer has been extensively investigated in recent years; However, no effective biomarker for early diagnosis and treatment of hepatoma has been establish. Emerging evidence has demonstrated that abnormal homologous recombination repair is closely related to the development and progression of human cancers. The function of proteins that participate in homologous recombination is primarily to maintain genomic stability and suppress tumor development by repairing DNA double strand breaks and damaging replication forks. RAD51 is a central member of the HRR signaling pathway, which is expressed in number of cancer cell lines and is associated with cell sensitivity to DNA-damaging agents. RAD51-associate Protein 1 mRNA expression is increased in liver cancer tissues compared with corresponding normal liver tissues. Obama et al reveal that RAD51AP1 expression was increased in intrahepatic cholangiocarcinoma, and downregulation of RAD51AP1 by shRNA could effectively suppress proliferation of cholangiocarcinoma cells. Recent findings have revealed that RAD52 may be a potential therapeutic target for BRCA1 and BRCA2-deficient familial breast and ovarian cancer. It has also been demonstrated that disease-free survival is correlated with RAD50 expression in tissues from patients with non-small Cell Lung cancer; RAD50 knockdown increases Cell sensitivity to radiation whereas RAD50 upregulation induces radioresistence in NSCLC cells. These previous studies indicate that many HR-associate proteins are involved in the development of cancers, including liver cancer; However, there are still many HRR-associated proteins that regulate growth and function of cancer cells and need to be further elucidate. Recently, our group investigated RAD54B, which is a vital motor protein of HR. RAD54B belongs to the SNF2 / SWI2 superfamily and plays an important role in the DNA repair system. It has been revealed that distant metastasis was significantly increased in colorectal cancer patients with high RAD54B expression compared with low expression group, which may be associated with degradation of p53 Protein in clinical samples. In addition, high expression of RAD54B may act as an independent prognostic factor for Lung adenocarcinoma. To the best of our knowledge, few studies have reported the effect of RAD54B on the development of cancer and the mechanism by which it function. Furthermore, there have been no previous reports regarding expression and biological function of RAD54B in liver cancer.

* Please keep in mind that all text is machine-generated, we do not bear any responsibility, and you should always get advice from professionals before taking any actions.

* Please keep in mind that all text is machine-generated, we do not bear any responsibility, and you should always get advice from professionals before taking any actions

Results

Yeast rdh54 and RAD54 have each previously been shown to exhibit SL / SGD interactions with rad27. To determine if similar genetic interaction is conserved in human cells, RAD54B proficient and deficient cells were transiently transfected with siRNA duplexes specifically targeting FEN1, homolog of yeast rad27. All FEN1 duplexes specifically target unique non-overlapping regions within the FEN1 coding region and cause reduce FEN1 expressions 24 h to at least 7 days post-transfection. 2 most effective independent siRNA duplexes were used to demonstrate specificity of phenotype, while FEN1-Pool was used to decrease the number of off-target effects that are potentially observed when using a single duplex. Mitotic kinase, PLK1, was included as positive control as it is an essential mitotic kinase known to decrease cellular proliferation through increased cytotoxicity that is independent of any known SL interaction. High-content digital imaging microscopy was performed on fixed cells and total numbers of Hoechst positive cells imaged were calculate. Percentages of cells relative to GAPD were determined for each of the conditions, and are present in Fig. 4. As anticipated, PLK1 silencing diminish relative total number of cells significantly, irrespective of RAD54B status. However, visually striking decreases in relative cell numbers were also apparent in RAD54B-deficient cells in which FEN1 expression had been diminish, that were not apparent in RAD54B-proficient cells. Moreover, large difference in relative cell numbers observed in RAD54B-deficient cells for various FEN1 conditions appear to reflect the efficiency of FEN1 knock-down in general. For example, FEN1-Pool was visually the most effective down-regulator and exhibited the greatest decrease in relative cell numbers. FEN1-3 however, was visually less efficient at silencing and exhibited less profound, but still significant, effect on relative cell numbers, while FEN1-2 knockdown efficiency was intermediate, as was its effect on relative cell numbers. Student's T tests comparing mean relative cell numbers identify highly statistically significant differences for PLK1 in both RAD54B-proficient and deficient cells, while highly significant differences were only observed after FEN1 silencing in RAD54B deficient cells. Because HCT116 cells are MLH1-deficient, there is possibility that second-site gene mutation was clonally fixed in the background of RAD54 knockout cell line that is actually responsible for SL interaction with FEN1 knockdown. Accordingly, we perform dual RNAi against RAD54B and FEN1 in the parental HCT116 cell line. In analogous fashion to that described above, diminish RAD54B and FEN1 expression in concert result in a highly statistically significant decrease in total cell numbers relative to GAPD-silence control that is not observed for either of independent knockdowns. The Extent of decrease in total cell numbers for dual RNAi system was not as great as with the RAD54B knockout cell line, and is likely due to residual RAD54B and FEN1 expression levels. To further assess the specificity of RAD54B / FEN1 synthetic interaction, 12 randomly selected human gene targets were subject to silencing in RAD54B-deficient background.

* Please keep in mind that all text is machine-generated, we do not bear any responsibility, and you should always get advice from professionals before taking any actions.

* Please keep in mind that all text is machine-generated, we do not bear any responsibility, and you should always get advice from professionals before taking any actions

Discussion

Present work demonstrates that RAD54B plays a critical role in targeted integration in human cells without affecting cell growth, cell survival to DNAdamaging agents or SCE. Since RAD54B shares structural similarity with Scerevisiae TID1 / RDH54 not only in ATPase domains but also in the Nterminal region, it is probable that RAD54B is Human homolog of TID1 / RDH54. Properties of RDH54 single mutants and RAD54 TID1 / RDH54 double mutants in mitosis and meiosis have already been characterize. Yeast mutants do not show hypersensitivity to MMS. The TID1 / RDH54 gene was required for interchromosomal recombination but not for intrachromosomal gene conversion in mitosis. This phenotype was observed in TID1 / RDH54 single mutant, whereas elsewhere it was reported that TID1 / RDH54 single mutant do not show deficiency in mitotic recombination. Interchromosomal recombination at HIS4 locus was reduced in RAD54 TID1 / RDH54 double mutant but not in TID1 / RDH54 single mutant, suggesting that TID1 / RDH54 and RAD54 act in recombinational repair pathway with partially overlapping roles. In meiosis, tid / RDH54 mutant exhibit significant defects in sporulation, spore viability and recombination. Although few comparative experiments have been performed between RAD54B deficient Human cells and TID1 / RDH54 mutants, there are a couple of similarities between these mutants. First, in contrast to RAD54 mutants in Scerevisiae, chickens and mice, neither of these mutants show hypersensitivity to MMS. Secondly, both mutants were defective in recombination, although the respective experiments were not comparable. The effect of TID1 / RDH54 on targeted integration, which is reduced in RAD54 mutants in mice, chickens, schizosaccharomyces pombe and Scerevisiae as well as in RAD54B mutants, has not been report. Interchromosomal recombination has been studied in tid / RDH54 mutants. Although the relationship between target integration and interchromosomal recombination has not been establish, they may share some similarity, since both use homologous sequences that are not present on the same chromosome. Since the roles of RAD54B in meiosis and overlapping roles of RAD54B and RAD54 in humans remain to be demonstrate, we are not able to conclude that RAD54B is a functional homolog of yeast TID1 / RDH54 gene. However, our findings indicate that some, if not all, roles in recombinational repair pathway in mitosis might be conserved between RAD54B and TID1 / RDH54. In the mitotic cell cycle of yeast, RAD54 is essential for repair by sister chromatid, whereas TID1 / RDH54 is not required for this pathway. The role of RAD54 in sisterchromatidbased repair may be conserve from yeast to higher eukaryotes. RAD54 deficient DT40 cells have been shown to be extremely sensitive to ionizing radiation in G 2 as well as G 1, while wildtype DT40 cells were resistant to irradiation in G 2. Sister chromatids serve as the main template for recombination in G 2. Therefore, hypersensitivity to irradiation of RAD54 mutant in G 2 implies that RAD54 is required for DNA repair mediated by sister chromatids. Consistent with this idea, DNA damageinduced SCE was reduced in RAD54 deficient DT40 and mouse ES cells.

* Please keep in mind that all text is machine-generated, we do not bear any responsibility, and you should always get advice from professionals before taking any actions.

* Please keep in mind that all text is machine-generated, we do not bear any responsibility, and you should always get advice from professionals before taking any actions

Immunohistochemistry (IHC)

Harvest hepatoma cells were washed with phosphate-buffer saline and lysed using RIPA lysis buffer. The concentrations of protein were detected using Bio-Rad protein assay kit. Equal amounts of total protein were separated by 10 % sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. Membranes were blocked with 5 % skim milk-TBST and incubate with primary antibodies against RAD54B at 4C overnight. Membranes were washed three times using TBST before being incubate with HRP-conjugate secondary antibodies at room temperature for 1 H. Membranes were then washed three times with TBST and proteins were visualized using electrochemiluminescence assay. Images were taken by fusion-capture software.

* Please keep in mind that all text is machine-generated, we do not bear any responsibility, and you should always get advice from professionals before taking any actions.

* Please keep in mind that all text is machine-generated, we do not bear any responsibility, and you should always get advice from professionals before taking any actions

Western blot analysis

To further investigate the effect of RAD54B on proliferation of hepatoma cells, we inhibit expression of RAD54B in BEL-7404 and SMMC-7721 cell lines. Cells were infected with lentivirus-shRAD54B and changes to cell proliferation were observe. Lentivirus-shCtrl infected cells were used as control. We observe that > 80 % of cells were infected by shRAD54B or shCtrl, which was confirmed by fluorescence microscopy. MRNA and protein expression of RAD54B were obviously suppressed in cells infected by shRAD54B compared with those cells infected with shCtrl, as determined by RT-qPCR and western blot analysis. In addition, Celigo calculations and MTT assay were used to determine the effect of RAD54B inhibition on proliferation of infected cells. Results of Celigo calculation reveal that fluorescence intensities of shRAD54B groups were reduced compared with shCtrl groups from days 2 to 5 in both BEL-7404 and SMMC-7721 cell lines. In addition, cell counts of shRAD54B groups were significantly lower than shCtrl groups from days 2 to 5. Similarly, MTT assay reveal that cell growth in shRAD54B groups was significantly suppressed compared with shCtrl groups from days 2 to 5. These results reveal that downregulation of RAD54B inhibits cell proliferation in BEL-7404 and SMMC-7721 cell lines.

* Please keep in mind that all text is machine-generated, we do not bear any responsibility, and you should always get advice from professionals before taking any actions.

* Please keep in mind that all text is machine-generated, we do not bear any responsibility, and you should always get advice from professionals before taking any actions

Lentivirus infection assay

A Lung adenocarcinoma tissue microarray containing 15 lung adenocarcinoma tissues and match healthy lung tissues was used for IHC staining. Slides were deparaffinized with xylene and then rehydrated in ethanol series. Endogenous peroxidases were inactivated by incubation with 3 % H 2 O 2 for 10 minutes at room temperature. Antigen retrieval was then conducted by microwaving in 0. 01 M citrate buffer for 20 minutes. Non-specific antigens were blocked by incubating with 10 % normal goat serum for 30 minutes. Subsequently, slides were incubated with rabbit anti-RAD54B primary antibody at 1: 500 dilution overnight at 4C, then with horseradish peroxidase-conjugate anti-rabbit IgG at 1: 100 dilution for 1 hour at room temperature. Immunoreactivity was developed with 3 3-diaminobenzidine substrate and counterstaining was performed with hematoxylin. Intensity of immunoreactivity score was assessed by two investigators independently as IRS = staining intensity percentage of positive cells. SI was graded as 0 1 2, and 3; PP was scored as 0 1 2 3, and 4. IRS 6 was considered to represent high RAD54B expression. Write informed consent was obtained from all patients, and study protocol was approved by the ethics committee of Guizhou Provincial Peoples Hospital. Total RNA was extracted from cells using TRIzol according to manufacturers ' protocol. A total of 2 g RNA was Reverse-transcribed into cDNA with oligo primer using M-MLV Reverse Transcriptase. CDNA was then used for quantitative PCR with SYBR Master Mixture according to manufacturers ' instructions. GAPDH was used for normalization. Primers were: RAD54B forward, 5-GCCAAACACTGATGATTTGTGG-3 andRAD54Breverse, 5-CCTGAGAAGAATGCGAGATAGC-3; GAPDH forward, 5-TGACTTCAACAGCGACACCCA-3and GAPDH Reverse, 5-CACCCTGTTGCTGTAGCCAAA-3. Real-time PCR was carried out in Agilent MX3000p qPCR System using an initial denaturation step at 95C for 30 seconds, followed by 45 cycles of amplification with denaturation at 95C for 5 seconds, and annealing and extension at 60C for 30 seconds. The RAD54B expression was calculated in triplicate using the 2 Ct method. Cell extracts were separate on 10 % sodium dodecyl sulfate polyacrylamide gel and proteins were transferred to nitrocellulose membranes. These were incubate overnight at 4C with rabbit anti-RAD54B primary antibody at 1: 100 dilution and anti-actin antibody at 1: 200 dilution, then with HRP-conjugate anti-rabbit IgG at 1: 1000 dilution for 2 hours. Western blotting signals were developed by the BCA Protein Assay Kit. After color development, image was analyzed using ChemiScope Mini chemiluminescence meter to calculate optical density of target and internal control band for protein expression as follow: Protein expression = integral optical density value of target Protein / integral optical density value of-actin. Lentiviral vector GV115, containing hU6-MCS-CMV-EGFP, was used for shRNA-mediate RNA interference. EGFP was used as an indicator of infection efficiency. ShRNAs targeting human RAD54B and control shRNA were separately synthesize and inserted between Age I and Eco RI sites of multiple cloning sites of GV115, and confirmed by sequencing.

* Please keep in mind that all text is machine-generated, we do not bear any responsibility, and you should always get advice from professionals before taking any actions.

* Please keep in mind that all text is machine-generated, we do not bear any responsibility, and you should always get advice from professionals before taking any actions

Sources

* Please keep in mind that all text is machine-generated, we do not bear any responsibility, and you should always get advice from professionals before taking any actions.

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