MiR-4443 promotes migration and invasion of breast cancer cells by inhibiting PEBP1 expression / 南方医科大学学报

OBJECTIVE@#To investigate the effect of miR-4443 expression on migration and invasion of breast cancer.@*METHODS@#We examined the expression of miR-4443 in breast carcinoma in situ and paired adjacent tissues from 3 breast cancer patients with high-throughput sequencing and verified the results using TCGA database. We also detected miR-4443 expressions using real-time quantitative PCR (RT-qPCR) in low invasive and highly invasive breast cancer cells (MCF-7 and MDA-MB-231 cells, respectively). The changes in apoptosis, migration and invasion of MCF-7 and MDA-MB-231 cells after transfection with miR-4443 mimics, mimics-NC, miR-4443 inhibitor or inhibitor-NC were analyzed using flow cytometry, wound healing assay and Transwell invasion assay. The target gene of miR-4443 was predicted by bioinformatics software and validated by a dual luciferase reporter gene system. RT-qPCR and Western blotting were performed to detect the expression of recombinant human phosphatidyl ethanolamine binding protein 1 (PEBP1) in the transfected cells.@*RESULTS@#The expression of miR-4443 was significantly higher in the breast cancer tissues than in the adjacent tissues (@*CONCLUSIONS@#MiR-4443 promotes the migration and invasion of breast cancer cells by inhibiting the expression of PEBP1, suggesting the possibility of suppressing miR-4443 expression as a potential therapeutic strategy for breast cancer.

The biological complexity of RKIP signaling in human cancers

The Raf kinase inhibitory protein (RKIP) has been demonstrated to modulate different intracellular signaling pathways in cancers. Studies have shown that RKIP is frequently downregulated in cancers; therefore, attempts have been made to upregulate the expression of RKIP using natural and synthetic agents for the treatment of human malignancies. Moreover, various regulators such as specific proteins and microRNAs (miRNAs) that are involved in the regulation of RKIP expression have also been identified. RKIP mechanistically modulates the apoptotic regulators of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) signaling. Because of its critical role in human cancers, RKIP has drawn much research attention, and our understanding is expanding rapidly. Here, we summarize some of the biological complexities of RKIP regulation. However, we restrict our discussion to selected tumors by focusing on TRAIL, miRNAs and natural agents. Emerging evidence suggests a role for natural agents in RKIP regulation in cancer cells; therefore, naturally occurring agents may serve as cancer-targeting agents for cancer treatment. Although the literature suggests some advancement in our knowledge of RKIP biology, it is incomplete with regard to its preclinical and clinical efficacy; thus, further research is warranted. Furthermore, the mechanism by which chemotherapeutic drugs and novel compounds modulate RKIP and how nanotechnologically delivered RKIP can be therapeutically exploited remain to be determined.

Roles of phosphatidylethanolamine-binding protein in cell signaling and its biological functions / 生理学报

Phosphatidylethanolamine-binding protein (PEBP) is a cytoplasm soluble protein with a high conserved structure. It has been approved recently that PEBP is a multifunctional molecule regulating several important cellular signal pathways, including ERK cascade, NF-κB pathway, and signaling of G protein-coupled receptors. Furthermore, the role of PEBP in tumor metastasis also got a comprehensive attention in the field of clinical cancer research. Together, as a signal regulator at multiple paths in cell, PEBP is becoming a new focus in several research fields. This review is aimed to introduce the newest biological progress on PEBP.

Expression of Raf kinase inhibitor protein and its significance in invasion and metastasis of hepatocellular carcinoma / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To investigate the expression of RKIP, p65 and pERK in hepatocellular carcinoma (HCC) and theIr correlation with invasion and metastasis of HCC.</p><p><b>METHODS</b>Reverse transcription polymerase chain reaction (RT-PCR) was used to detect the expression of RKIP mRNA. The expression levels of RKIP, p65 and pERK proteins in HCC tumor and peritumoral tissues were determined by immunohistochemistry and Western blot analysis. Statistical analysis was performed to determine the relationship between their expression and clinicopathological parameters.</p><p><b>RESULTS</b>RKIP protein expression level (RKIP/actin) was 0.579 ± 0.380 in HCCs, 1.178 ± 0.659 in peritumoral tissues and 1.115 ± 0.442 in normal liver tissues. The pERK protein level was 1.023 ± 0.478, 0.605 ± 0.367 and 0.461 ± 0.293, p65 protein level was 0.83 ± 0.376, 0.63 ± 0.337 and 0.466 ± 0.345, respectively. Immunohistochemistry analysis showed that the RKIP positive rates in HCCs, peritumoral tissues and normal liver tissues, were 22.2%, 86.0%, and 93.8%, positive rates of p65 were 73.6%, 56.0% and 37.5%, positive rates of pERK were 65.3%, 38.0% and 31.3%, respectively. Statistical analysis revealed that there was a significant difference in RKIP protein expression levels (P < 0.05), but no significant difference in RKIP mRNA expression levels (P > 0.05) among HCC tumors, peritumoral tissues and normal liver tissues. The p65-positive and pERK-positive rates were higher in tumor tissues than that in peritumoral tissues and in normal liver tissues (P < 0.05), but RKIP-positive rates were lower in tumor tissues than that in paritumoral tissues and normal liver tissues (P < 0.05). RKIP protein expression levels were significantly lower in HCCs with intrahepatic or lymphatic metastasis than that in without. The RKIP positive rates in moderately and well differentiated HCCs were significantly higher than that in poorly differentiated HCCs. There was a relationship between RKIP and pERK expressions (P = 0.04), but RKIP expression was not correlated with p65 expression in HCCs (P = 0.143).</p><p><b>CONCLUSIONS</b>Our findings indicate that the down-regulation of RKIP expression may serve as a predictive marker for HCC development, progression and metastasis, which may contribute to the elevated ERK activity. The inhibiting effect of RKIP on invasion and metastasis of liver cancer cells may be due to the down-regulation of pERK expression rather than p65 expression.</p>

L-arginine-glycine amidinotransferase, betaine-homocysteine s-methyltransferase, and neuropolypeptide h3 are diminished in renal clear cell carcinoma of humans

To identify renal clear cell carcinoma-associated marker proteins. Twelve patients with renal cell carcinoma [RCC] were collected and processed in the Department of Urology, Renmin Hospital, Wuhan University, China, between January 2008 and September 2009. Two-dimensional polyacrylamide gel electrophoresis and matrix assisted laser desorption ionisation time-of-flight mass spectrometry [MALDI-TOF-MS] were employed to investigate differentially expressed protein spots between RCC tissues and adjacent normal tissues, then reverse transcription polymerase chain reaction and western blot were employed to confirm the proteomic results. One protein spot was up regulated, 13 were downregulated, and 22 were absent in RCC tissues. Four of the absent proteins were L-arginine-glycine amidinotransferase [AGAT], Betaine-homocysteine S-methyltransferase [BHMT], Ketohexokinase [KHK], and Neuropolypeptide h3 [NPh3]. The reverse transcriptase-polymerase chain reaction analysis demonstrated mRNA expression of AGAT, BHMT, and Nph3 was significantly decreased in 12 RCC tissues. In addition, Western blot analysis showed AGAT protein was absent in 11/12, BHMT in 9/12, and Nph3 in 5/12 RCC tissues. Absence of AGAT, BHMT, and Nph3 is common events in clear cell RCC; hence, it may be involved in the development of RCC; therefore, they have the potential to be tumor markers for diagnosis, treatment, and prognosis of RCC patients

Comparative proteomic analysis of human gallbladder carcinoma / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To find out potential molecular targets for gallbladder carcinoma diagnosis and treatment by analyzing and comparing the proteins expressed in human gallbladder carcinoma tissue and benign gallbladder tissue.</p><p><b>METHODS</b>Proteomic analysis of 6 human gallbladder carcinoma tissues and 6 benign gallbladder tissues was carried out. Total proteins of the carcinoma tissue and benign gallbladder tissue were separated by two-dimensional gel electrophoresis (2-DE). The differentially expressed proteins were analyzed and identified by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS). Immunohistochemistry was used to examine the expression of PEBP1 protein in an independent series of samples.</p><p><b>RESULTS</b>Protein extracts of individual samples in each type of tissues were separated on two-dimensional gels. There were forty six differentially expressed proteins in the gallbladder carcinom tissues. Seventeen proteins were successfully identified by MS, in which nine proteins were overexpressed in tumors while the other eight proteins were underexpressed. The increased level of PEBP1 protein in gallbladder carcinoma was further confirmed by immunohistochemical analysis.</p><p><b>CONCLUSION</b>Seventeen differentially expressed proteins were successfully characterized by comparative proteomic analysis. Those results may provide scientific foundation for screening the molecular biomarkers which can be used in diagnosis and treatment of gallbladder carcinoma, as well as to improve its prognosis and provide a new clue for carcinogenesis research of gallbladder carcinoma.</p>

Expression of Raf kinase inhibitor protein and E-cadherin in prostate cancer tissues / 中南大学学报(医学版)

OBJECTIVE@#To detect the expression of Raf kinase inhibitor protein (RKIP) and epithelial cadherin (E-cadherin) in human prostate cancer tissues, and their correlation.@*METHODS@#We discussed the relationship between RKIP and E-cadherin and the clinical stage and pathological classification of prostate cancer by immunofluorescence histochemistry staining in the test of expression of RKIP in 26 prostate cancer tissues and 14 BPH tissues, and analyzed the correlation between them.@*RESULTS@#The expression of RKIP and E-cadherin in prostate cancer tissues was obviously lower than that in the benign prostatic hypertrophy tissues. The expression of RKIP and E-cadherin in the dys-good differentiation group (Gleason 8-10) was significantly lower than that in the good differentiation group(Gleason

Decreased basic activity and induced activity of ERK1/2 pathway in hippocampal CA1/CA2 region of ovariectomized rats / 中国应用生理学杂志

<p><b>AIM</b>To investigate the relationship between the spatial learning and memory and hippocampal ERK1/2 pathway activity in ovariectomized rats.</p><p><b>METHODS</b>Female SD rats were randomly divided into sham operated group (Sham group) and ovariectomized group (OVX group), and fed 4 months. Then spatial learning and memory of rats were evaluated by the Morris water maze task. Rats in each group were randomly divided into training group and untraining group before the test. Induced activity of ERK 1/2 stimulated by learning and memory was detected in the training group, and basic activity of ERK 1/2 was detected in the untraining group. The protein expression of p-ERK 1/2 and Raf kinase inhibitor protein (RKIP) were assayed by Western blotting respectively.</p><p><b>RESULTS</b>(1) During the training session the OVX rats held longer escape latenci than the sham rats did (P < 0.05). (2) The relative level of pERK1/2 protein in training rats of the both groups was higher than that in untraining rats (P < 0.05). (3) The relative level of p-ERK1/2 protein both training and untraining rats in OVX group was lower than that in sham group correspondingly (P < 0.05). (4) Compared with sham group, the relative expression of RKIP in OVX group was significantly higher (P < 0.05).</p><p><b>CONCLUSION</b>Spatial learning and memory deficits in ovariectomized rats might be correlated with the decreased basic and induced activity of ERK1/2 pathway and increased expression of RKIP in the CA1/CA2 region of hippocampus.</p>

Influence of electromagnetic radiation on raf kinase inhibitor protein and its related proteins of hippocampus / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To study the development of changes for Raf kinase inhibitor protein (RKIP) and its mRNA in rats hippocampus after electromagnetic radiation.</p><p><b>METHODS</b>Rats were exposed to X-band high power microwave (X-HPM), S-band high power microwave (S-HPM) and electromagnetic pulse (EMP) radiation source respectively. The animal model of electromagnetic radiation was established. Western blot was used to detect the expression of RKIP, and RT-PCR was applied to detect the expression of RKIP mRNA. The interaction of RKIP and Raf-1 was measured with co-immunoprecipitation method, and the expression of cerebral choline acetyltransferase (CHAT) was measured by immunohistochemistry.</p><p><b>RESULTS</b>The expression of RKIP significantly down-regulated at 6 h after radiation, and recovered at 1 d in group EMP, but the down-regulation continued during 1 approximately 7 d after radiation in the two microwave groups. The expression of RKIP mRNA changed wavily during 6 h approximately 7 d after radiation, which showed down-regulation at 6 h, and up-regulation at 3 d. The interaction of RKIP and Raf-1 decreased during 6 h approximately 7 d after radiation, most significantly at 7 d, and the two microwave groups were more significant. The expression of CHAT decreased continuously during 6 h approximately 7 d after radiation, and generally recovered on 14 d.</p><p><b>CONCLUSION</b>The down-regulation of RKIP and its related proteins of hippocampus is induced by electromagnetic radiation.</p>

Expression and identification of PEBP-like gene from Saussurea involucrate Kar.er kir in Escherichia coli / 生物工程学报

This assay was designed to construct the prokaryotic expression vector, investigate the expression of PEBP-like in Escherichia coli and purify its product. The PEBP gene was inserted into the vector pET30a (+). The recombinant vector was transferred into E. coli BL21 (DE3)and induced the expression of protein by low concentration of IPTG and low temperature overnight. After purification, the supernatants were analyzed by SDS-PAGE and the results were identified by Western blotting. After IPTG induction, a new anticipating fusion protein of 28 kD appeared as an expected size, and its product was 26.8% in total protein, the fusion protein was positive by Western blotting. The prokaryotic expression system of PEBP-like is successfully constructed. It lays the foundation for the further application study on the antifreeze characters of the PEBP.

Differential analysis of proteomic profiles between cryptorchid and normal mouse testes / 生理学报

To screen factors related to spermatogonial stem cell (SSC) proliferation, and to investigate the mechanism of infertility caused by cryptorchidism, ten-day-old Kunming (KM) mice were used and experimental cryptorchidism was conducted. On the 35th day after cryptorchid operation, the left testes were fixed in Bouin's fluid and used for histological analysis. The testes of 45-day-old mice were subjected to the same histological analysis, and it was found that they contained germ cells at every stage of development, from SSCs to sperm, indicating that the animals were fully sexually mature at this age. While in experimental cryptorchid mice, the spermatogenesis was arrested at the stage of spermatocytes, and only spermatogonia and primary spermatocytes were present in cryptorchid testes. The proportion of spermatogonia to other types of germ cells was much higher than that in sexually mature mice. On the other hand, the right testes were used for proteomic analysis. The total protein in testes was extracted on the 35th day after cryptorchid operation. The differentially expressed proteins in cryptorchid mice and sexually mature mice were screened and compared by the proteomic techniques. Through the separation of two-dimensional gel electrophoresis (2-DE), 20 differential protein spots were found, and 9 of them were digested and identified by the matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrum. In cryptorchid mice, 6 out of 9 proteins were down-regulated, and 3 were up-regulated. Among these proteins, 4 proteins were identified, and they were Stathmin, phosphatidylethanolamine-binding protein1 (PEBP1), HES-related basic helix-loop-helix protein (HERP), and one unnamed protein (we temporarily named it Px). More Stathmin, PEBP1 and Px were expressed in sexually mature mice than in experimental cryptorchid mice. But HERP1 was the other way round. In the present study, we have screened 4 proteins related to cryptorchidism. It is helpful to study the mechanism of SSC proliferation and infertility caused by cryptorchidism.