The mechanism and clinical significance of long non-coding RNA plasmacytoma variant translocation gene 1 in regulating the differentiation, proliferation, invasion and metastasis of breast cancer cells through target genes / 中国医师进修杂志

Objective:To analyze the mechanism and significance of long non-coding RNA (LncRNA) plasmacytoma variant translocation gene 1 (PVT1) in regulating the proliferation, differentiation, invasion and metastasis of breast cancer cells through the target gene.Methods:From January 2018 to December 2019, the expression levels of LncRNA PVT1 and microRNA (miR)-1207-5p in breast cancer cell line MCF-7 and normal breast epithelial cell line MCF-10A were detected by real-time fluorescence quantitative polymerase chain reaction (qRT-PCR). The human breast cancer cell line MCF-7 was transfected with PVT1 overexpression vector plasmid, PVT1 silencing lentivirus plasmid and negative control plasmid, and the expression levels of PVT1 and miR-1207-5p after transfection were detected. The activities of proliferation differentiation, metastasis and invasion in transfected breast cancer cells were detected by CCK-8 method, cell scratch test and Transwell invasion experiment. The expression levels of signal transducer and activator of transcription 6 (STAT6) and P21 mRNA/protein after transfection miR-1207-5p were detected by qRT-PCR and Western blotting method.Results:The expression levels of PVT1 and miR-1207-5p in breast cancer cells were significantly higher than those in normal breast epithelial cells (1.271 ± 0.305 vs. 0.023 ± 0.006 and 1.679 ± 0.347 vs. 0.031 ± 0.009), and there were statistical differences ( P<0.01). The expression levels of PVT1 and miR-1207-5p in breast cancer cells after transfection PVT1 overexpression vector plasmid were significantly higher than those in breast cancer cells after transfection negative control plasmid and PVT1 silencing lentivirus plasmid (2.357 ± 0.271 vs. 1.000 ± 0.000 and 0.103 ± 0.021, 3.265±0.375 vs. 1.000 ± 0.000 and 0.265 ± 0.024), the indexes in breast cancer cells after transfection negative control plasmid were significantly higher than those in breast cancer cells after transfection PVT1 silencing lentivirus plasmid, and there were statistical differences ( P<0.05). The proliferation activity in breast cancer cells 48, 72, 96 and 168 h after transfected with PVT1 overexpression vector plasmid was significantly higher than that in breast cancer cells transfected with negative control plasmid and PVT1 silencing lentivirus plasmid, proliferation activity in breast cancer cells transfected with negative control plasmid was significantly higher than that in breast cancer cells transfected with PVT1 silencing lentivirus plasmid, and there were statistical differences ( P<0.05). The metastasis activity and invasion activity in breast cancer cells 24 h after transfected with PVT1 overexpression vector plasmid were significantly higher than those in breast cancer cells transfected with negative control plasmid and PVT1 silencing lentivirus plasmid, the metastasis activity in breast cancer cells transfected negative control plasmid was significantly higher than that in breast cancer cells transfected PVT1 silencing lentivirus plasmid, and there were statistical differences ( P<0.05). The STAT6 and P21 mRNA in transfection miR-1207-5p overexpression group were significantly lower than those in transfection mimic group (0.476 ± 0.102 vs. 1.000 ± 0.000 and 0.429 ± 0.097 vs. 1.132 ± 0.236), and there were statistical differences ( P<0.01); the STAT6 and P21 protein in miR-1207-5p overexpression group was significantly lower than that in mimic group (0.396 ± 0.104 vs. 1.062 ± 0.002 and 0.434 ± 0.067 vs. 1.141 ± 0.218), and there were statistical differences ( P<0.01). Conclusions:LncRNA PVT1 may be a regulated host gene of miR-1207-5p, which synergistically affects the proliferation, differentiation, invasion and metastasis of breast cancer cells through the regulation of target gene STAT6. Inhibiting the transcription of this gene may be a new research direction for breast cancer treatment.

The change of platelet mitochondrial ATPase-6 gene in patients with benzene induced chronic myeloid leukemia / 中国职业医学

OBJECTIVE: To investigate the changes of platelet mitochondrial adenosine triphosphate synthase 6( ATPase-6)gene in patients with occupational chronic myeloid leukemia( CML) caused by benzene. METHODS: Five occupational benzene induced CML patients were selected as the case group and 21 healthy workers without benzene exposure were selected as the control group,venous blood were drew from these study subjects. The number of platelet was counted. The level of mitochondrial ATPase-6 gene was examined by quantitative polymerase chain reaction. RESULTS: The platelet count in case group was lower than that of control group [( 135. 2 ± 32. 6) ×10~9/ L vs( 239. 7 ± 77. 5) ×10~9/ L,P < 0. 05]. The ratio of ATPase-6 gene / platelet was higher than that of control group( median: 3. 43 ×10~(-4)copies vs 1. 87 ×10~(-4)copies,P < 0. 05). The ATPase-6 gene level in case group showed no significant difference when compared with that of control group( median: 4. 83 ×10~7 copies / L vs 3. 24 ×10~7 copies / L,P > 0. 05). CONCLUSION: The benzene induced CML patients shows more sensitive change in ratio of ATPase-6 gene / platelet than in ATPase-6 gene level.

Effects of Rapamycin and Rapamycin-loaded Poly(lactic-co-glycolic)Acid Nanoparticles on Apoptosis and Expression of bcl-2 and p27(kip1) Proteins of Human Umbilical Arterial Vascular Smooth Muscle Cell / 中国医学科学院学报

<p><b>OBJECTIVE</b>To investgate the effects of rapamycin(RPM)and RPM-loaded poly(lactic-co-glycolic)acid(PLGA)nanoparticles(NPs)on the apoptosis of human umbilical arterial vascular smooth muscle cells(HUASMCs)in vitro and expression of bcl-2 and p27(kip1) protein.</p><p><b>METHODS</b>HUASMCs were cultured in vitro and divided to RPM and RPM-PLGA-NPs groups treated at 3 different concentration by 12 and 24 hours,with M231-smooth muscle growth supplements medium and null-PLGA-NPs treated groups as controlled. The apoptosis of HUASMCs was determined by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling staining and flow cytometry. The expressions of bcl-2 and p27(kip1) were detected by streptacidin/peroxidase immunohistochemical method. The effect on cellular proliferation was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromidecolorimetry.</p><p><b>RESULTS</b>The proliferation of HUASMCs was inhibited by RPM and RPM-PLGA-NPs in a dose-dependent manner. DNA electrophoresis showed DNA ladder in RPM and RPM-PLGA-NPs groups and classical scalar strips in control groups. The apoptotic indexes of RPM 100 ng/ml group and RPM-PLGA-NPs 500 ng/ml group detected by flow cytometry were(45.45<2.36)% and(35.04<5.64)%,respectively,which were significantly higher than that of M231-smooth muscle growth supplements control group [(2.60<0.95)%,all P<0.01]. The apoptotic indexes of groups incubated with RPM and RPM-PLGA-NPs for 24 hours were significantly higher than those of groups which incubated for 12 hours(P<0.05,P<0.01). The positive expression indexes(PEI)of p27(kip1) and bcl-2 protein were higher in RPM and RPM-PLGA-NPs groups than that of control groups. The Spearman's rank correlation coefficient test showed that there was no significant correlation between the PEI of p27(kip1) and the apoptotic indexes in the RPM group and RPM-PLGA-NPs group(P>0.05).</p><p><b>CONCLUSIONS</b>Rapamycin-loaded PLGA nanoparticles and rapamycin have similar effects in inhibiting proliferation and inducing apoptosis;meanwhile,they upregulate the expression of p27(kip1) protein without downregulating the expression of bcl-2 protein in HUASMCs in vitro. RPM-PLGA-NPs has more potent pro-apoptotic effect than equivalent dose of RPM but is not linearly correlated with the p27(kip1) expression level.</p>

Facial paralysis induced by herpes simplex virus type 1 and the study of facial neurons apoptosis in mice / 中华耳鼻咽喉头颈外科杂志

<p><b>OBJECTIVE</b>To study the apoptosis of facial motor neurons and the expression of apoptosis-related genes, Bcl-2 and Bax, in the animal model of viral facial paralysis.</p><p><b>METHODS</b>Total of 84 Balb/c mice were divided into viral inoculation group and nerve transaction group. The animals were executed 1, 3, 7, 10, 15, 20 and 30 days after being operated respectively. The histopathological features of facial neurons in brain stem were observed by HE and Nissl stain. The changes of facial neuronal apoptosis were observed by TUNEL. The changes of expression of Bcl-2 and Bax genes in facial neurons were observed by immunohistochemistry staining.</p><p><b>RESULTS</b>After nerve transection, increased apoptotic cells were found in homolateral facial motor nucleus and the peak appeared at 10 and 15 days. The level of Bcl-2 expression in neurons declined while the expression of Bax increased gradually. Correspondingly, the ratio of Bcl-2/Bax declined. In the viral inoculation group, no visible change of apoptosis and Bax expression, but the level of Bcl-2 and the ratio of Bcl-2/Bax increased gradually.</p><p><b>CONCLUSIONS</b>Comparing to axotomy, facial motor nucleus in HSV-1 infective animal model are free of apoptosis. Both the mild form of lesion and the ability to block apoptosis of HSV-1 are likely to be involved into the phenomenon. Bcl-2 and Bax might interfere with the apoptotic response.</p>

Dynamic change study of dermatitis medicamentosa-like of trichloroethylene patients with liver damage / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>Observing the dynamic change characteristics of serum liver function indexes in occupational dermatitis medicamentosa-like of trichloroethylene patients with liver damage, we can underlie for guiding therapy, prognosis and mechanism of dermatitis medicamentosa-like of trichloroethylene patients with liver damage.</p><p><b>METHODS</b>We collected serum of 10 cases of occupational dermatitis medicamentosa-like of trichloro-ethylene patients with liver damage from different time points since they were hospitalized, using automatic biochemistry analyzer to detect total protein (TP), albumin (ALB), total bilirubin (TBIL), direct bilirubin (DBIL), indirect bilirubin (IBIL), alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma-glutamyl transpeptidase (GGT), alkaline phosphatase (ALP), albumin/globulin ratio etc 11 liver function biochemical indicators. We used Excel to establish database, professional drawing software gnuplot to draw dynamic variation diagram of each index.</p><p><b>RESULTS</b>The variation range of 11 liver function indexes of 10 cases was TP 43.2-74.2 g/L, ALB 24.6-44.6 g/L, A/G 0.77-2.10, TBIL 3.7-268.2 umol/L, DBIL 1.0-166.0 umol/L, IBIL 2.4 -167.5 umol/L, ALT 11-5985 U/L, AST 14-5586 U/L, GGT 15-1500 U/L, ALP 35-309 U/L, S/L 0.07-1.94, respectively. TBIL, DBIL, ALT, AST, GGT, ALP concentration significantly increased, especially ALT, AST, GGT, ALT topped 5985 U/L, AST topped 5586 U/L, GGT topped 1500 U/L. But TP, ALB and S/L significantly decreased, TP lowest to 43.2 g/L, S/L lowest to 0.07. A/G basically remained unchanged, but IBIL didn't change regularly.</p><p><b>CONCLUSION</b>The early liver damage in dermatitis medicamentosa-like of trichloroethylene patients was serious, and repeatedly attacked, so we should lead to enough attention to the clinical work and prevention. This also provided the basis for studying the mechanism of trichloroethylene poisoning.</p>

Construction and identification of Mdr-1-shRNA eukaryotic expression vector / 中国实验血液学杂志

This study was purposed to construct and identify the short hairpin RNA (shRNA) eukaryotic expression vector for targeting gene mdr-1 which may play an important role in K562/A02. Short hairpin RNA (shRNA) aiming at the target sequence was to synthesized, the 3491-3509, 1539-1557and 3103-3121 nucleotide of mdr-1 mRNA were selected as targets. The selected nucleotides were cloned in the plasmid pGCSilencer-U6-neo-GFP respectively, and the resultant recombinant plasmids were named as pGY1-1, pGY1-2 and pGY1-3. The sequences of the recombinant plasmids were identified by DNA sequencing and PCR electrophoresis. The recombinant plasmids were transfected into the cell line K562/A02 by lipofection. After being transfected for 48 hours, the inhibition of mdr-1 mRNA was detected by real time-PCR, and P-gp expression was detected by Western blot. The results showed that the specific oligonucleotide was cloned into the vector successfully, and the expression of mdr-1 mRNA and P-gp in K562/A02 cells was reduced after transfecting the recombinant plasmid, as compared to the control group. It is concluded that the shRNA eukaryotic expression vector has been successfully established which can inhibit the expression of mdr-1 mRNA, setting up the basis to futher explore the effects of mdr-1 on cell line of K562/A02.

Effects of rapamycin-loaded poly(lactic-co-glycolic) acid nanoparticles on distribution of cell cycle, expression of p27 protein, and proliferation of human umbilical arterial vascular smooth muscle cell in vitro / 中国医学科学院学报

<p><b>OBJECTIVE</b>To evaluate the effects of rapamycin (RPM)-loaded poly (lactic-co- glycolic) acid (PLGA) nanoparticles (NPs) on the proliferation, distribution of cell cycle, and expression of p27 protein in human umbilical arterial vascular smooth muscle cell (HUASMC) in vitro.</p><p><b>METHODS</b>The primarily culture model of HUASMC was successfully established by explant-attached method in vitro. The cells were administrated with different doses of RPM, and RPM-PLGA NPs were observed as treat groups compared with PLGA NPs and M231-SMGs medium cultured group. The effect of RPM-PLGA NPs on proliferation of HUASMC was assessed using 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) colorimetry method. The influences of RPM-PLGA NPs on the cell cycle and cellular growth kinetics of HUASMCs were tested by flow cytometry. The effect of RPM-PLGA NPs on the expression of p27 protein of HUASMCs was assessed through an immunohistochemical method.</p><p><b>RESULTS</b>Compared with the control group, the proliferation of HUASMCs was inhibited by 50 microg/L and higher concentration of RPM-PLGA NPs in a dose-dependent manner (P < 0.05). The numbers of cells entering cell cycle of S/G2/M phases were significantly lower in RPM-PLGA NPs and RPM treated groups. Histologically, the expression of p27 were up-regulated in 500 microg/L RPM-PLGA NPs and 100 microg/L RPM treated group (all P < 0.01 ) when compared with the control group.</p><p><b>CONCLUSIONS</b>RPM-PLGA NPs has a similar effects as RPM in inhibiting the growth of in vitro cultured HUASMC. It can remarkably suppress the expression of in vitro cultured HUASMC p27 protein, arrest its cell cycle at G1/S phase, and inhibit its proliferation.</p>

The effect of serum high-density lipoprotein on the growth rate of human endothelial progenitor cells / 中华病理学杂志

<p><b>OBJECTIVE</b>To study the effect of high-density lipoprotein (HDL) on the proliferation of endothelial progenitor cells (EPC) isolated from human umbilical cord blood; to further explore its effect on prevention and development of atherogenesis.</p><p><b>METHODS</b>EPC isolated by density gradient centrifugation were cultured in a M200 medium. Immunofluorescence staining for CD133, CD34, KDR and Factor VIII were adopted respectively as the specific markers for identification. The effect of HDL on EPC proliferation was estimated on the 7th day of cell cultivation using MTT assay, confocal microscopy and fluorescence activated cell sorting.</p><p><b>RESULTS</b>HDL, when incubated with EPC, was able to promote remarkably the proliferation rate of EPC, dose- and time-dependent. HDL participated in the transcriptional regulation of cell cycle by affecting the regulatory proteins such as cyclin D1.</p><p><b>CONCLUSIONS</b>A subtype of progenitor cells was isolated from human cord blood with a potential of differentiating into mature endothelial cells (known as endothelial progenitor cells). HDL plays an important role on EPC fluorescence activated cell sorting differentiation and proliferation. Further studies are required to identify the signal pathway and the molecular mechanism of HDL effect on EPC proliferation.</p>

Effect of oxidized-LDL on NF-kappaB nuclear translocation in aortic smooth muscle cells originated from rats of different ages / 中国医学科学杂志(英文版)

<p><b>OBJECTIVE</b>To investigate the molecular mechanism of atherosclerosis that related to age.</p><p><b>METHODS</b>Immunohistochemistry staining and Western blot were adopted to determine the nuclear translocation of nuclear factor-kappa B (NF-kappaB) and expression of platelet-derived growth factor B (PDGF-B) in smooth muscle cells (SMCs) co-cultured with low density lipoprotein (LDL), oxidized LDL (ox-LDL), and ox-LDL+high density lipoprotein (HDL) originated from rats of 2 and 10 months old respectively. Fat stain was used to identify the lipid intake in SMCs.</p><p><b>RESULTS</b>The optimal stimulation time of ox-LDL to SMCs was 12 hours. NF-kappaB intensity increased in most nuclei of SMCs that originated from rats of either 2 or 10 months old co-cultured with ox-LDL. The intensity of NF-KB and the amount of intracellular lipid taken in SMCs were more obvious in cells from 10-month-old rats than from the younger ones. Change of PDGF-B expression in SMCs was not remarkable in each group of rats.</p><p><b>CONCLUSIONS</b>The 10-month-old rats are more susceptive to ox-LDL than 2-month-old rats in activating nuclear translocation of NF-kappaB. Maybe this is one of the important reasons contributing to the difference between the older and younger rats on the initiation and development of atherosclerosis lesion. Expression of PDGF-B is not associated with the activity of nuclear translocation of NF-kappaB.</p>