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Objective:To investigate the effect of miRNA-34b/c-5p on the expression of neurokinin 1 receptor-truncated(NK1R-Tr)in breast cancer and the effect of miRNA-34b/c-5p and NK1R-Tr on the migration and invasion abilities of breast cancer cells.Methods:Real-time fluorescence quantitative PCR(RT-PCR)was used to detect the expression of miRNA-34b/c-5p and NK1R-Tr in 50 breast can-cer specimens that were collected at Tianjin Medical University Cancer Institute&Hospital from February to May 2013.Western blot analysis was performed to detect the expression of miRNA-34b/c-5p and NK1R-Tr in breast cancer cell lines MDA-MB-231 and MCF-7. Scratch and Transwell assays were carried out to explore the effects of miRNA-34b/c-5p and NK1R-Tr on the migration and invasion abilities of MDA-MB-231 cells.Results:The expression of miRNA-34b/c-5p and NK1R-Tr in breast cancer tissues and cells were signifi-cantly negatively correlated.The relative expression of NK1R-Tr in breast cancer patients with lymph node metastasis was 5.75,and the relative expression of NK1R-Tr in patients with non-lymph node metastasis was 4.29.The relative expression was significantly dif-ferent between the two groups(P=0.026).Overexpression of miRNA-34b/c-5p and knockdown of NK1R-Tr could significantly inhibit the migration and invasion abilities of MDA-MB-231 cells(all P<0.001).Conclusions:The expression of miRNA-34b/c-5p and NK1R-Tr was significantly negatively correlated.MiRNA-34b/c-5p and NK1R-Tr might be potential therapeutic targets for inhibiting the invasion of breast cancer cells.
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Aim To observe the optimal temperature and optimal pH of uricase-catalase liposomes(UCALP) and free uricase(UAE), and study the abilities of UCALP to reduce uric acid and hydrogen peroxide in mice with hyperuricemia.Methods UCALP were prepared by reverse phase evaporation, optimal temperature and optimal pH of UCALP and UAE were determined, respectively.Mouse model of hyperuricemia was established by intraperitoneally injection of uric acid, and the model mice were intravenously injected UCALP and UAE, respectively, then the serum concentration of uric acid and hydrogen peroxide in mice at different time points were measured by the assay kits, respectively.Results Optimal temperature of UCALP and UAE was 40℃, and optimal pH was 8.0 and 8.5, respectively.UCALP could more significantly lower uric acid level of hyperuricemia mice than that of UAE, and the concentration of hydrogen peroxide in UCALP group was lower than in UAE group.Conclusion UCALP can effectively decrease the level of uric acid and control the level of hydrogen peroxide in mice with hyperuricemia.
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Objective To analyze the association of staphylococcal nuclease domain-containing protein 1(SND1) and T-cell intracellular antigen 1(TIA-1) on stress granules, and the regulation of SND1 on stress granules under stress stimuli. Methods The immunofluorescence assay and laser scanning confocal microscopy were used to observe the co-localization of SND1 protein and TIA-1 protein under stress stimuli, and the over-expression plasmids of pEGFP vector were transfected into HeLa cells and to verify which domain of SND1 co-localized with TIA-1 under stress stimuli. RNA interference-mediated knockdown of the expression of SND1 protein in HeLa cells was measured by Western Blotting assay. Then whether the knockdown of SND1 affected the recruitment of TIA-1 on stress granules was observed. Heat shocks under different times were used to identify whether there were dynamic changes in transportation of SND1 and TIA-1 on stress granules. Results SND1 co-localized with TIA-1 on stress granules under stress stimuli, and the associated domain of SND1 were SN domain. TIA-1 still can be recruited on stress granules but a large amount of stress granules were reduced even though the expression of SND1 protein was decreased. And the transportation of SND1 on stress granules was laged behind TIA-1 under different-times of heat shocks. Conclusion SND1 protein co-localizes with TIA-1 on stress granules, and which co-regulates the cellular stress response under stress stimuli.
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Objective To analyze the regulation of estrogen receptor α (ERα) on truncated neurokinin-1 receptor (NK1R-Tr), and the influence of this regulation on cell proliferation in estrogen receptor-positive breast cancer cell lines. Methods The chromatin immune coprecipitation (CHIP) was used to observe the transcriptional regulation function of ERαon NK1R-Tr in breast cancer cells. Luciferase reporter gene assay was used to verify whether ERα played a positive regulatory role in the expression of NK1R-Tr. Western blot assay and real-time-PCR were used to detect the expression of ERα and NK1R-Tr in breast cancer cells, MCF-7 and T47D, as well as the expression of NK1R-Tr protein and mRNA level. NK1R-Tr levels were also detected after using estradiol (E2, ERα agonist) and small interfering RNA (knock out ERα). CCK-8 and clone formation experimen were used to detect the proliferation ability of breast cancer cells after knocking out NK1R-Tr with small interfering RNAs. Results CHIP test and Luciferase reporter gene assay proved that ERα can positively regulate the expression of NK1R-Tr via the ERα sequences in the upstream of the NK1R-Tr gene promoter. The expression of NK1R-Tr at both protein level and mRNA level dropped in the estrogen receptor-positive breast cancer cell line MCF-7 upon knocking out ERα. After knocking out NK1R-Tr, the proliferation ability of estrogen receptor-positive breast cancer cells was lower than that of the control group. Conclusion The ERα positively regulates the expression of NK1R-Tr, resulting in the increased cell proliferation in estrogen positive breast cancer cells.
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Objective:To determine the expression of the full-length (NK1R-FL) and truncated (NK1R-Tr) neurokinin-1 receptor (NK1R) and the neurokinin-2 receptor (NK2R) in breast cancer tissues and cell lines, as well as to study the effects of the NK1R and NK2R antagonists on the growth of breast cancer cells. Methods:Immunohistochemistry and Western blot assays were used to detect NK1R, NK1R-FL, and NK2R expression in clinical samples of primary breast cancer tissue, benign lesions, and normal breast tissue, as well as in different breast cancer cell lines. Cell proliferation and soft agar growth tests were performed on cells treated with the NK1R and NK2R antagonists to study the ectopic overexpression of NK1R-FL and NK1R-Tr in breast cancer cell lines. Results:Total NK1R expression was detected in the breast cancer tissues, benign lesions, and normal breast tissues. Compared with the normal breast epithe-lia and benign breast lesions, the expression levels of NK1R-FL and NK2R decreased in the carcinoma. These changes were also relat-ed to the carcinoma type, histological grade, lymph node metastasis, HER2 and Ki-67 expression, and estrogen and progesterone recep-tors in breast cancer. The expression levels of NK1R-FL and NK2R were high in the HBL-100 breast cell lines of para-neoplastic tis-sues, but NK1R-Tr expression was low. The MDA-MB-231, T-47D, and MCF-7 cells only expressed NK1R-Tr. NK1R-Tr or NK1R-FL overexpression caused the decreased inhibition rate or increased levels of the NK1R and NK2R antagonists in the breast cancer cells. Conclusion:NK1R-FL and NK2R are co-expressed in normal cells. NK1R-Tr is highly expressed in breast cancer cells and exerts nega-tive feedback to regulate NK1R-FL and NK2R expression in all cells, especially cancer cells.
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<p><b>OBJECTIVE</b>To characterize the property of uricase loaded in uricase-catalase liposomes (BUCLPs) prepared using borate buffer.</p><p><b>METHODS</b>BUCLPs were prepared using reverse-phase evaporation, and the physicochemical properties of uricase in the prepared BUCLPs were examined.</p><p><b>RESULTS</b>The optimal temperature of BUCLP and URI was 40 degrees celsius, their optimal pH values were 8.0 and 8.5, and their Michaelis-Menten constants were 14.207 µmol/L and 13.623 µmol/L, respectively. Fluorescence intensity of nanoliposome-loaded uricase-catalase that bound to FITC was higher than that of uricase-catalase binding directly with FITC; the fluorescence intensity of BUCLP was higher than that of free uricase-catalase at 280 nm.</p><p><b>CONCLUSION</b>Uricase activity is enhanced after loading in uricase and catalase liposomes.</p>
Subject(s)
Borates , Catalase , Liposomes , Nanoparticles , Chemistry , Temperature , Urate Oxidase , ChemistryABSTRACT
Objective To investigate the expression of substance P ( SP) and neurokinin-1 recep-tor (NK1R) in patients with breast cancer and to further understand the correlations of them with the clinico-pathological features and the prognosis of breast cancer.Methods SP levels in serum samples and superna-tants of breast cell culture were measured by ELISA.The expression of total NK1R, full-length NK1R (NK1R-FL) and truncted NK1R (NK1R-Tr) in 82 patients with breast cancer and 30 patients with breast hyperplasia were detected by using immunohistochemistry and Western blot.Results The levels of SP in patients with breast cancer were higher than those in patients with breast hyperplasia and healthy subjects ( P0.05).Conclusion The invasion and metastasis of breast cancer showed a negative cor-relation with the expression of NK1R-FL, but a positive correlation with the expression of NK1R-Tr and SP.
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Objective:To investigate the clinical value of serological test indicators in the identification of primary and metastatic liver cancers. Methods:We detected the serum levels of ALT, AST, ALP, TBIL, DBIL, GGT, CHE, 5'-NT, CA199, CEA, and AFP in primary hepatic carcinoma (PHC;120 cases), metastatic hepatic carcinoma (MHC;135 cases), and no liver metastatic control (135 cas-es) groups. The methods used were variance analysis and Scheffe test. ROC curve analysis was used to determine the diagnostic value of CA199 , CEA, and AFP in PHC and MHC. Results:The difference between the serum levels of AST, ALP, GGT, 5'-NT, AFP, and CEA of the PHC and MHC groups was statistically significant (P<0.05). The AFP, CEA, and CA199 areas under the ROC curve of the PHC and MHC groups revealed that AFP diagnosis of primary liver cancer had certain accuracy, whereas CEA and CA199 have some diagnostic value in differentiating primary and metastatic liver cancers. Conclusion: The detection of serum levels of AST, GGT, 5'-NT, AFP, CA199, and CEA in malignant tumor was a preliminary diagnosis of liver metastasis and can provide evidence for the dif-ferential diagnosis of PHC and MHC.
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Objective To investigate how SDF-1α and c-MYC protein regulates TACRl-Tr expression. Methods c-myc shRNA vector was constructed, small interfering RNA was employed for silencing c-myc gene in MCF-7 breast cancer cell. SDF-1α neutralized antibody was used in c-myc+ cell group and c-myc- cell group, while other c-myc+ cell group and c-myc- cells group were cultured under normal condition. The mRNA level of TACRl-Tr was determined by real-time PCR. Results c-myc shRNA vector was constructed successfully, in the normal presence of SDF-la, the level of TACRl-Tr mRNA in c-myc- cell group were lower than that in c-myc+ cell group( P < 0.05). But in the presence of SDF-la neutralized antibody, TACRl-Tr mRNA level of c-myc- cell group was higher than that of c-myc+ cell group(P < 0.05). Conclusion In the normal culture condition, c-MYC protein may transactivate TACRl-Tr transcription in MCF-7 cell, in the presence of SDF-1α neutral antibody, c-MYC protein lost the activity of transactivating for TACRl-Tr transciption.