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@# Objective: To explore the molecular mechanism of miR-17-5p regulating the proliferation and invasion of nasopharyngeal carcinoma cells by regulating the expression of breast cancer metastasis suppressor 1 like (BRMS1-like or BRMS1L) gene. Methods:A total of 40 cases of nasopharyngeal carcinoma tissues and corresponding paracancerous tissues resected from nasopharyngeal carcinoma patients, who were admitted to the General Hospital of Pingdingshan Shenma Medical Group during January 2014 to December 2017, were included in this study; in addition, nasopharyngeal carcinoma cell lines CNE 2, HONE 1, C666-1 and nasopharyngeal immortalized epithelial cell line NP69 were also collected for this study. The expression of miR-17-5p in nasopharyngeal carcinoma tissues and cell lines was detected by qPCR. The targeted relationship between BRMS1L and miR-17-5p was predicted by the StarBase and verified by the Dual luciferase reporter gene assay. Effects of transfection of miR-17-5p mimics and inhibitors on the expression of BRMS1Lin CNE2 cells were detected by WB assay. CCK-8, Transwell and Flow cytometry were used to detect the effects of miR-17-5p/BRMS1L axis on the proliferation, migration, invasion and apoptosis of CNE 2 cells. Results: miR-17-5p was highly expressed in nasopharyngeal carcinoma tissues and cell lines (P<0.05 or P<0.01). Knockdown of miR-17-5p significantly inhibited proliferation, invasion and migration of CNE2 cells but promoted apoptosis (P<0.05 or P<0.01); miR-17-5p targeted BRMS1Land down-regulated its expression. Over-expression of BRMS1Lsignificantly inhibited the proliferation, invasion and migration of CNE2 cells but promoted apoptosis (all P<0.01); while simultaneous over-expression of miR-17-5p and BRMS1L reversed the above effects (all P<0.01). Conclusion: miR-17-5p promoted proliferation, invasion, migration and inhibited apoptosis of CNE 2 cells by down-regulating the expression of BRMS1L.
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Objective To investigate the clinical significance of peripheral blood metastasis in breast cancer and its relationship with the metastasis suppressor gene BRMS1.Methods Reverse transcription polymerase chain reaction (RT-PCR) was used to detect the expression of micrometastatic marker hMAM-RNA in the peripheral blood of 149 cases of invasive breast carcinoma.Immunohistochemical method was used to detect the expression of BRMS1 protein in breast cancer tissues after surgery.The recurrence was followed up.SPSS19.0 statistics software was used to analyze the data.Results Among the 149 cases of invasive breast carcinoma patients with preoperative peripheral blood,expression of hMAM-RNA was found in 71 cases,and the micrometastasis rate was 47.65％.Peripheral blood micrometastasis rate in breast cancer was closely related to tumor TMN stage,lymph node metastasis and postoperative recurrence (P＜0.05);while it had nothing to do with patients' age,tumor size,pathological types or tumor tissue typing (P＞0.05).The expression of BRMS1 in postoperative breast cancer tissue was detected in 56 cases,and the positive rate was 37.58％.For BRMS1 positive cases,16 cases had peripheral blood micrometastasis (the positive rate was 28.57％);For BRMS1 negative cases,55 cases had peripheral blood micrometastasis (the positive rate was 51.93％).The difference had statistical significance and the two showed a significant negative correlation(r=-0.296,P＜0.01).With the gradual increase of positive staining intensity of BRMS1 protein,the micrometastasis rate of peripheral blood of breast cancer showed a significant decrease (P＜0.05).At the same time,among patients with positive peripheral blood micrometastasis,the recurrence rate of patients with positive BRMS1 (12.5％) was significantly lower than that of patients with negative BRMS1 (43.64％),and the difference was statistically significant(P＜0.05).Conclusions BRMS 1 expression and breast cancer micrometastasis in peripheral blood is closely related.BRMS1 can also be used as an important molecular marker for determining micrometastasis in peripheral blood of breast cancer.Routine detection of BRMS1 expression in breast cancer tissue is helpful for clinical understanding of breast cancer patients,peripheral blood micrometastasis and postoperative recurrence,thus guiding clinical individualized treatment and prognosis.
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OBJECTIVE@#To discuss the effect of BRMS1 on the proliferation, migration and adhesion of mouse forestomach carcinoma.@*METHODS@#The constructed pCMV-myc-BRMS1 recombinant plasmid and blank plasmid were transfected into mouse forestomach carcinoma. MTT method was employed to measure the activity of gastric cancer cell; the scratch assay and Transwell assay to measure the migration and invasion of gastric cancer cell; the adhesion assay to measure the adhesion of gastric cancer cell; while the Western blot assay to measure the expression of The NF-κB signal pathway, downstream matrix metalloproteinase (MMP)-2, MMP-9 and osteopontin and E-cadherin in the gastric cancer cell. Besides, the transplanted animal model of gastric cancer in mice was constructed to measure the size of tumor xenograft.@*RESULTS@#Results of MTT assay showed that, compared with the empty vector control group, the activity of gastric cancer cell was not affected in the BRMS1 transfection group. The improved expression of BRMS1 could inhibit the adhesion, migration and invasion of gastric cancer cell (P < 0.01). Besides, compared with the empty vector control group, the phosphorylation of NF-κB p65 and IκBα was reduced in the BRMS1 transfection group, with the decreased expression of MMP-2, MMP-9 and osteopontin and the increased expression of E-cadherin (P < 0.01). Results of animal experiment also showed that the expression of BRMS1 did not affect the transplanted tumor.@*CONCLUSIONS@#The expression of BRMS1 can significantly inhibit the adhesion, migration, invasion and metastasis of mouse forestomach carcinoma gastric cancer cell, which is related to The NF-κB signal pathway.
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Objective: To discuss the effect of BRMS1 on the proliferation, migration and adhesion of mouse forestomach carcinoma. Methods: The constructed pCMV-myc- BRMS1 recombinant plasmid and blank plasmid were transfected into mouse forestomach carcinoma. MTT method was employed to measure the activity of gastric cancer cell; the scratch assay and Transwell assay to measure the migration and invasion of gastric cancer cell; the adhesion assay to measure the adhesion of gastric cancer cell; while the Western blot assay to measure the expression of The NF-κB signal pathway, downstream matrix metalloproteinase (MMP)-2, MMP-9 and osteopontin and E-cadherin in the gastric cancer cell. Besides, the transplanted animal model of gastric cancer in mice was constructed to measure the size of tumor xenograft. Results: Results of MTT assay showed that, compared with the empty vector control group, the activity of gastric cancer cell was not affected in the BRMS1 transfection group. The improved expression of BRMS1 could inhibit the adhesion, migration and invasion of gastric cancer cell (P < 0.01). Besides, compared with the empty vector control group, the phosphorylation of NF-κB p65 and IκBα was reduced in the BRMS1 transfection group, with the decreased expression of MMP-2, MMP-9 and osteopontin and the increased expression of E-cadherin (P < 0.01). Results of animal experiment also showed that the expression of BRMS1 did not affect the transplanted tumor. Conclusions: The expression of BRMS1 can significantly inhibit the adhesion, migration, invasion and metastasis of mouse forestomach carcinoma gastric cancer cell, which is related to The NF-κB signal pathway.
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Breast cancer metastasis suppressor 1(BRMS1)can suppress the tumor cells metastasis. and significantly reduces the metastasis without affecting tumor growth. The relevant mechanism(s)may be related to intercellular communication, phosphoinositide signal transduction and interaction with NF-KB, and so on. Therefore, exploring the mechanisms of inhibition of tumor metastasis might be helpful for use of BRMS1 as tumor gene therapy.
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Objective To construct and identify the recombinant vector pcDNA3. 1 (-) B/myc-BRMS 1 carrying breast-cancer metastasis suppressor 1 (BRMS 1) which can express in eukaryote cells and which will provide the basis for further researching the mechanisms of metastasis suppression and working on cancer metastasis gene ther-apy. Methods To isolate total RNA from MCF - 7 cells and design a pair of primers, and coding sequence of aRMS 1 cDNA were amplified from human breast cancer cells MCF -7 by reverse transcription-polymerase chain reaction (RT-PCR). Then the product was inserted to the PcDNA3. 1/myc-His (-) B plasmid. The recombined pcDNA3. 1 (-)B/myc-BRMS1 was identified by gene sequence analysis,then recombinants was transfected into HEK-293 cells and was identified by Western blot. Results The recombinant of pcDNA3.1 (-) B/myc-BRMS1 was structurally confirmed by analysis of sequencing. The inserted fragment in the vector was in the right direction and its sequence was structurally confirmed to be consistent with CDS sequence of human BRMSI cDNA that of the published data. GenBank, [AF159141]. The recombinants was transfected into HEK-293 cells ,then the cells expressed protein tagged c-myc identified by Western blot indicated it can express in eukaryote cells. Conclusion cDNA of human BRMS1 can be successfully cloned and inserted into Eukaryote-expression vector. The newly constructed vector may serve as the potential tool to conduct further comprehensive experiments in future on BRMS1 function and on gene therapy.
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Objective To investigate the effect of 5-Aza-CdR on experimental lung metastasis of breast cancer and the possible mechanisms.Methods MDA-MB-231 cells were divided into control group and 5-Aza-CdR-treated group.The mRNA expressions and promotor methylation status of BRMS1 and CXCR4 of the MDA-MB-231 cells were evaluated by SqRT-PCR and MSP respectively.Then,the MDA-MB-231 cells of control group and 5-Aza-CdR-treated group were injected into BALB/c nude mice through lateral tail veins,respectively.five weeks later,the mRNA abundance of the target gene HPRT and internal control gene GAPDH of the lung tissues from the mice were evaluated by FqRT-PCR.Results 5-Aza-CdR upgraded the BRMS1mRNA expression significantly than that in control group(0 versus 0.39?0.001,P0.05) and the status of unmethylated CXCR4 CpG island 1 in promotor were not changed significantly.The Ct values of HPRT and GAPDH in control and 5-Aza-CdR-treated groups were 24.75?1.55,16.19?0.69 versus 27.61?1.67,17.48?0.96 respectively,2-??Ct=0.34.The mRNA abundance of the HPRT was lower and there were fewer metastases in the lungs of 5-Aza-CdR-treated group compared with control group.Conclusions 5-Aza-CdR can activate tumor metastasis suppressor gene BRMS1 by demethylation mechanism,and thus,decreased the ability of breast cancer MDA-MB-231 cells for experimental metastasis to lungs.
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Objective To investigate the expression of BRMS1 in different metastatic breast cancer cells and its relation to HDAC activity.Methods The gene expression of BRMS1 in 3 types breast cancer cell lines(non-metastasis MCF-7(A cell line),low metastasis MDA-MB-453(B cell line)and high metastasis MDA-MB-231(C cell line)) was determied by RT-PCR technique;the protein expression of BRMS1 was measured with Western blot technique and HDAC activity by enzyme linked immunosorbent assay.Results(1) Gene expressive ratio of BRMS1 was 3.1∶2.0∶1.0 in the A、B、C cell lines,respectively.The gene expression of BRMS1 in the C cell line decreased 210% compared to A cell line,and expression of BRMS1 was markedly reduced as the degree of metastasis increased(P