Intelligent nanotherapeutic strategies for the delivery of CRISPR system

CRISPR, as an emerging gene editing technology, has been widely used in multiple fields due to its convenient operation, less cost, high efficiency and precision. This robust and effective device has revolutionized the development of biomedical research at an unexpected speed in recent years. The development of intelligent and precise CRISPR delivery strategies in a controllable and safe manner is the prerequisite for translational clinical medicine in gene therapy field. In this review, the therapeutic application of CRISPR delivery and the translational potential of gene editing was firstly discussed. Critical obstacles for the delivery of CRISPR system in vivo and shortcomings of CRISPR system itself were also analyzed. Given that intelligent nanoparticles have demonstrated great potential on the delivery of CRISPR system, here we mainly focused on stimuli-responsive nanocarriers. We also summarized various strategies for CIRSPR-Cas9 system delivered by intelligent nanocarriers which would respond to different endogenous and exogenous signal stimulus. Moreover, new genome editors mediated by nanotherapeutic vectors for gene therapy were also discussed. Finally, we discussed future prospects of genome editing for existing nanocarriers in clinical settings.

Effect of microRNA-29b on proliferation and migration of breast cancer cells and its molecular mechanism / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To investigate the effects of microRNA(miRNA)-29b on the proliferation and migration of breast cancer cells and its molecular mechanism.</p><p><b>METHODS</b>The recombinant lentiviral expression vector (lenti-miRNA-29b) was constructed and transfected into 293T cells to obtain lentivirus particles that were used to infect breast cancer MCF-7 cells. Transfection efficiency of lenti-miRNA-29b in MCF-7 cells was identified by the expression of green fluorescent protein (GFP). The expression of miRNA-29b was detected by real-time PCR. The cell proliferation and migration were detected by CCK8 assay and Transwell assay, respectively. The bioinformatics softwares were used to predict and screen the downstream target genes regulated by miRNA-29b, which were verified by double luciferase reporter gene assay, RT-PCR and Western blot. The effects of screened target gene RTKN on the growth and migration of MCF-7 cells were verified by RTKN siRNA.</p><p><b>RESULTS</b>Recombinant lentiviral expression vector of miRNA-29b were successfully constructed. About 90% and 60% of the breast cancer cells showed green fluorescence in lenti-miRNA-29b and lenti-miRNA-NC groups, respectively. The expression of miRNA-29b in lenti-miRNA-29b group increased significantly compared with the lenti-miRNA-NC group and blank control group (all<0.05); the proliferation and migration ability of MCF-7 cells significantly reduced compared with the control group (all<0.05). The screening with bioinformatics softwares found that the 3'UTR coding region RTKN had the binding site to miRNA-29b; the dual luciferase reporter gene assay showed that the luciferase activity decreased significantly after the MCF-7 cells were co-transfected with wild type RTKN-WT-3'UTR and miRNA-29b mimics report gene vector (<0.05). The RTKN proteins in MCF-7 cells were significantly decreased after transfection with siRNA-RTKN, and the proliferation and migration ability of MCF-7 cells were significantly reduced (all<0.05).</p><p><b>CONCLUSIONS</b>MiRNA-29b can inhibit the proliferation, invasion and metastasis of breast cancer cells by inhibiting the expression of RTKN.</p>

A survey of nurses' knowledge and attitude towards cancer-related fatigue / 医学研究生学报

Objective Cancer-related fatigue (CRF) is a key in the management of cancer patients' clinic syptoms.This article investigated the status quo of nurses' knowledge and attitude towards CFR.Methods THe method of cross-sectional survey and questionnaire was used to investigate the knowledge and attitude towards CRF among nurses from related departments of three Grade III hospitals in Nanjing.Results 142 nurses answered the questionnaire.The average correct rate was 76.25%, among which nurses from the oncology department had better congition rate than nurses from other medical and surgical departments (84.3%, 75.98%, 79.57%) , representing significant difference (P<0.05).64.79% of the nurses found the relatives of cancer patients and nurses often fail to understand cancer patients;complaint of fatigue, 76.76% of nurses assumed there is lack of communication in fatigue between patients and medical staff.94.36% of nurses agreed medical institutions should strengthen the management of CRF.Conclusion At present, the clinical nurses have inadequate knowledge about CRF, which should be enhanced in future work.

Androgen receptor inhibits the prolfieration of estrogen receptor-positive bre ast cancer cells / 医学研究生学报

Objective Androgen receptor ( AR) is extensively expressed in breast cancer and influences the proliferation of the malignant cells.Our study aimed to investigate the effect of AR on estrogen receptor (ER)-positive breast cancer. Methods ER-positive MCF-7 breast cells were exposed to various concentrations of agonist dihydrotestosterone ( DHT) or antagonist bicalutamide or left untreated .Then the proliferation and apoptosis of the cells were determined by MTT assay , cell counting , and flow cytometry , and the expressions of the proteins related to cell cycle regulation were detected by Western blot . Results The relative gray value of AR was significantly increased in the DHT group (1.055 ±0.020) but decreased in the bicalutamide group (0.705 ±0.010) as com-pared with the blank control (0.795 ±0.020) (both P<0.05).Flow cytometry showed that the early apoptosis rate of the breast cancer cells was markedly higher in the DHT group ([51.20 ±0.312]%) but lower in the bicalutamide group ([2.410 ±0.367]%) than in the blank control ([3.540 ±0.375]%) (both P<0 .01). In comparison with the control group , the expressions of the p53, p73 and p21 proteins in the MCF-7 cells were remarkably up-regulated in the DHT group but down-regulated in the bicalutamide group ( both P<0.05). Conclusion AR inhibits the proliferation of ER-posi-tive breast cancer cells , which suggests that it may be a potential ther-apeutic target for ER-positive breast cancer .

Serum-free culture of the MKN-45 gastric cancer cell line and preparation of stem cells / 医学研究生学报

[Abstract ] Objective Difficulties with the preparation of gastric cancer stem cells (CSCs) have been a main obstacle to the studies of gastric cancer .This article addresses the technology of the serum-free medium suspension cultivation of the MKN-45 gastric cancer cell line and screening of stem cells from the cell line based on the biomarkers of gastric CSCs . Methods MKN-45 cells were cultured in serum-free medium for 8 weeks and those in the logarithmic phase cultivated with hoechst 33342 followed by detection of the side cells by flow cytometry .When the side population cells reached 25%, all the cell microspheres were collected , hatched with CD133 and CD44, and subjected to fluorescence-activated cell sorting.The CDl33 +and CD44 +cells were selected as gastric CSCs . Results About 40%of the MKN-45 gastric CSCs were alive , prolif-erated, and formed floating cell balls .Side population cells constitu-ted 3.4% of the MKN-45 cells and 26.9% of the cell balls.The CDl33 +and CD44 +cells made up 11.2% of the MKN-45 cells and 90.3%of the cell balls. Conclusion Cell balls rich in CSCs can be successfully obtained by serum-free medium suspension culti-vation and CSCs can be screened out with hoechst 33342 and surface markers , which may serve as an experimental ground for the stud-ies of gastric CSCs .

MicroRNA-495 induces breast cancer cell migration by targeting JAM-A

MicroRNAs (miRNAs) are small, non-coding RNAs that function as post-transcriptional regulators of gene expression. The deregulated expression of miRNAs is associated with a variety of diseases, including breast cancer. In the present study, we found that miR-495 was markedly up-regulated in clinical breast cancer samples by quantitative real time-PCR (qRT-PCR). Junctional adhesion molecule A (JAM-A) was predicted to be a potential target of miR-495 by bioinformatics analysis and was subsequently verified by luciferase assay and Western blotting. JAM-A was found to be negatively correlated with the migration of breast cancer cells through loss-of-function and gain-of-function assays, and the inhibition of JAM-A by miR-495 promoted the migration of MCF-7 and MDA-MB-231 cells. Furthermore, overexpression of JAM-A could restore miR-495-induced breast cancer cell migration. Taken together, our findings suggest that miR-495 could facilitate breast cancer progression through the repression of JAM-A, making this miRNA a potential therapeutic target.

Bioinformatic analysis of the polymorphism and function of IGF2R gene 3′untranslated region / 医学研究生学报

Objective Genetic variants in microRNA (miRNA) binding regions of the gene 3′untranslated region (3′UTR) can affect the regulation of gene expression .The aim of this study was to predict insulin like growth factor 2 receptor (IGF2R) 3′UTR variants and to test their effects on IGF2R gene expression by bioinformatic analysis . Methods Single nucleotide polymorphisms (SNPs) with minor allele frequency (MAF) in the IGF2R gene 3′UTR were obtained from online databases .The frequency distribution of all the selected IGF2R 3′UTR variants genotypes among the different populations and the linkage disequilibrium ( LD) values of all SNPs were calculated .Additionally , the potential miRNA binding sites were also predicted with the help of online bioinformatic tool . Finally, correlation analysis of the mRNA expression of IGF 2R genotype and different variant genotypes in the lymphoblastoid cell lines was performed. Results In total, 33 SNPs were reported in the 3′UTR, of which only five SNPs (rs8191959, rs200237825, rs3832385, rs201568808, rs1050015) had available minor allele frequency (MAF) values ( >0.05).And only the effect of rs1050015 variant on IGF2R mRNA expression level had significant difference (P=0.010). Conclusion The expression of IGF2R gene can be up-regulated by rs10500105 variant in the 3′UTR, which might support its use as markers of cancer risk and individualized treatment.

Expression of p53 in breast cancer and regulatory effects of 5-aza-2′-deoxycytidine on p53 / 医学研究生学报

Objective Tumor suppressor gene p53 can inhibit tumor cell growth, arrest cell cycle, and promote apoptosis.Howev-er, the effects of p53 on the pathogenesis of breast cancer have not been fully elucidated.The aim of this study was to explore the expression of p53 protein and the correlation with clinical pathologic features in breast cancer.Furthermore, the regulatory effects of 5-aza-2′-deoxycyti-dine on p53 in breast cancer cell line were also studied. Methods The expression of p53 protein in 80 cases of breast cancer and normal and adjacent tissue were determined by the immunohistochemical staining .The expressions of p53 mRNA and p53 protein in breast cancer cell line were determined by RT-PCR and Western blotting. Results The positive rate of p53 in breast cancer (41.25%) was higher than that in the normal and adjacent tissue (22.5%) (P0.05).The low expression of p53 both in mRNA and in protein levels were found in breast cancer cell line of MCF-7.The expres-sion of p53 increased after 5-aza-2′-deoxycytidine administration . Conclusion p53 is highly expressed in breast cancer , which may play an im-portant role in the development and progression of breast cancer. 5-aza-2′-deoxycytidine, up-regulating the p53 expression in breast cancer cell line, which provides the evidents for the development of therapeutic drugs for the patients with low expression of p53 breast cancer.

Study on diallyi disulfide inhibiting the proliferation of human small cell lung cancer H446 cells / 中国医师杂志

Objective To investigate the effects of diallyl disulfide (DADS) on ceil proliferation in human small cell lung cancer H446 cells in vitro. Methods MTT assay was used to observe inhibitory effect of DADS on proliferation of H446 cells. Cell Proliferation in-hibition was measured by growth curve analysis, average doubling time, vitality detection and MT]" assay. Cell morphology was observed by inversion microscope and optics microscope. Cell apoptosis was analyzed by cell morphology observed under light microscope, flow cytometry (FCM). Results MTT assay showed that DADS from 4 to 60 μg/ml significantly inhibited t446 ceils and exhibited a dose-dependent and time-dependent model. After exposure to DADS, H446 cell average doubling time retarded from 25. 40 hours to 145. 64 hours( P＜0.05).Flow cytometry analysis revealed that the cell content of C0-phase declined, however, hypodiplod peak was increased, which means cell ap-optosis were induced by DADS. Conclusion DADS could significantly inhibit the proliferation of H446 cells and induce the apoptosis of H446 cell.

The Cross-talk Between p27~(kip1) and Its Interacting Molecules in Breast Cancer Cells / 生物化学与生物物理进展

p27kip1 is an important negatively regulator of cell cycle progression and plays a central role in the pathogenesis of a member of tumors including breast cancer. In breast cancer cells, the level of p27kip1 expression usually decreases during tumor development and progression, in addition, cytoplasm mislocalization of p27kip1 has been reported, but less is known about the exact molecular mechanisms. Studies have indicated that phosphorylation is the key regulation way, several signal transduction pathways are involved in the regulation of the expression and distribution of p27kip1. To further understand the mechanism, the disparity of the interacting protein profiling between tumor cells and normal cells must be identified first. Including cyclins, cyclin-depend kinases, CRM1, jab1, SKP2, p27kip1 has various interacting molecules. There are also several interacting molecules especially for breast cancer cells. It seems that different protein profiling cause the different expression and intracellular distribution in different cell cycle phase. So, disparity of the p27kip1 protein profiling may be the main mechanism of its down-expression and mislocalization in breast cancer cells.

Expressions of survivin and caspase-3 in human non-small cell lung cancer and their relationship with cell apoptosis / 中国肺癌杂志

<p><b>BACKGROUND</b>Survivin, a member of inhibitor of apoptosis protein (IAP) family, can directly inhibit caspase-3 and caspase-7 activity and plays an important role in oncogenesis. The aim of this study is to investigate the expressions of survivin and caspase-3 in human non-small cell lung cancer (NSCLC), and to evaluate their relationship with cell apoptosis.</p><p><b>METHODS</b>The expressions of survivin and caspase-3 in 88 patients with NSCLC were examined by using immunohistochemical SP methods, and TNUEL method was used to detect the cell apoptosis simultaneously.</p><p><b>RESULTS</b>The positive expression rates of survivin in NSCLC tissues and normal lung tissues were 61.4% (54/88) and 13.8% (4/29) respectively, there was a significant difference between them (P < 0.01). Survivin expression in NSCLC was not related to the histologic type, pathological grade and lymph node metastasis (P > 0.05), but correlated with TNM stage (P < 0.05). The positive expression rate of caspase-3 was 89.7% (26/29) in normal lung tissues, which was higher than that in NSCLC tissues (73.9%, 65/88), but there was no significant difference (P > 0.05). Caspase-3 expression in NSCLC was associated with pathological grade (P < 0.05). The average apoptosis index (AI) of survivin-positive cases was significantly higher than that of surviving-negative ones (1.63±0.58 vs 3.29±0.76)(P < 0.05). The average AI of the caspase-3 positive cases was significantly higher than that of the caspase23 negative cases (2.42±0.59vs1.28±0.65)(P < 0.05). Expression of survivin was negatively correlated with caspase-3 (P < 0.05).</p><p><b>CONCLUSIONS</b>Survivin may play an important role in the process of carcinogenesis of NSCLC by inhibiting cell apoptosis. Moreover, survivin may prevent cell apoptosis by inhibiting caspase-3 activation.</p>

Influence of radiosensitivity by mild hyperthermia in a pulmonary adenocarcinoma cell line / 医学研究生学报

Objective:To investigate the radiosensitization and the cell-cycle of mild hyperthermia(≤42℃)on human pulmonary adenocarcinoma cell line SPC-A-1 in vitro. Methods: The human pulmonary adenocarcinoma cell line SPC-A were treated with radiation and the combination of radiation with mild hyperthermia. Radiosensitivity was determined by clonogenic assay and quantified by calculating the thermal enhancement ratio (TER). Flow cytometry was used to observe the cell-cycle. Results: Do, Dq calculated from the dose-response curve for radiation combined with 41.5℃ were 1.390 Gy, 1.426 Gy, whereas 1.693 Gy, 2.453 Gy for radiation alone, respectively. TER was 1.218. The proportion of cells in S phase was found to be 14.81% in the radiation group. The values, after 48 hours and 72 hours, with 6Gy radiation combined immediate 41.5℃ one hour mild hyperthermia, were 5.89% and 9.08%, respectively, versus 18.8% and 31.91% with 6 Gy radiation alone. Conclusion:Radiosensitization of mild hyperthermia in SPC-A-1 cells associated with the hyper-radiosensitization of the cells in S phase.

Analysis of low density lipoprotein receptor function and gene mutation in familial hypercholesterolemic patients / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To investigate low density lipoprotein receptor (LDLR) function and gene mutation in Chinese patients with familial hypercholesterolemia(FH).</p><p><b>METHODS</b>Lymphocytes were isolated from 10 ml anticoagulated peripheral blood of the patients, then a flow-cytometric method (FCM) with 1,1'-dioctadecyl-3,3,3', 3-tetramethylindocarbocyanine perchlorate labelled low density lipoproetin (DiI-LDL) was used to identify the function of LDLR on the surface of lymphocytes. Genomic DNA was isolated from whole blood of FH patients and analyzed by PCR-single strand conformation polymorphism (SSCP) and nucleotide sequencing methods.</p><p><b>RESULTS</b>Defects of binding and uptaking of LDLR were identified by FCM in 2 FH patients in one family, and their parents were examined in the present study. Then they were analyzed genetically. The detected mutation was a deletion of A, which caused a frame shift in codon 297 of exon 6 and introduced a beforehand stop codon in codon 369.</p><p><b>CONCLUSION</b>A novel mutation of LDL receptor gene was detected by the combination of FCM and PCR-SSCP methods.</p>

Clinical significance of p27~(Kip1) expression in breast cancer at clinical stage Ⅰ and Ⅱ / 医学研究生学报

Objective:Tumor size,lymphnode metastasis,hormone level and so on have been the prognosis predictors of breast cancer,but they are not ideal. This study is to investigate the influence of the expression of p27Kip1 in early breast cancer prognosis and its relationship with clinical feature. Methods:Archival early breast carcinomas (n=106),were analyzed for p27Kip1 expression by immunohistochemistry EnVision system using specific mono-clonal antibody. Results:The p27Kip1 protein nucleus expression was low in 61 (57.55%) of the 106 cases in early breast carcinomas,which has statistic difference from the high expression in survival rate (P0.05),but correlated with lymph-node metastasis and histological grade (P

Preliminary application of positron emission tomography/computed tomography on the staging of malignant lymphoma / 医学研究生学报

Objective:Position emission tomography/computed tomography(PET/CT) is a new bio-imaging system which is combined metabolic with anatomic imaging.This study was to compare the accuracy of conventional staging methods(including computed tomography,ultrasound,magnetic resonance imaging,and detection of bone marrow) with that of PET/CT for lymphoma staging and re-staging. Methods:A total of 42 patients with lymphoma diagnosed by operation or biopsy,received conventional and PET/CT staging.The accuracy of these two methods and their impact on lymphoma staging were compared.Results:The accuracy of PET/CT scan was 95.2%(40/42),and that of conventional staging was 78.6%(33/42).The detection rates of internal lymph node were 97.1%(66/68) and 88.2%(60/68),respectively.The detection rates of outer lymph node were 91.7%(22/24) and 58.3%(14/24),respectively.Compared with conventional staging methods,7 cases were up-staged and 2 cases were down-staged by PET/CT,which led to the change of therapy in 8 cases.Conclusion:PET/CT scan is more sensitive and accurate than conventional staging methods in staging and restaging of lymphoma.

Clinical research of malignant hydrothorax or hydroperitoneum treated with intracavitary chemotherapy and local endogenetic thermotherapy / 医学研究生学报

Objective:To observe the recent effects and toxicity of thermochemotherapy on malignant hydrothorax or hydroperitoneum,to evaluate the changes of the immunological functions,and to investigate the mechanism of thermochemotherapy.Methods:Fifty-two patients were treated with weekly intracavitary chemotherapy,and then combined with local endogenetic thermotherapy twice a week.As the control,another 50 patients received weekly intracavitary chemotherapy.The treatment lasted for two weeks and was followed by one-week rest,and then the recent effects and toxicity were observed.The T cell subset,NK cells and VEGF levels in serum,hydrothorax or hydroperitoneum were tested.Results:Overall response rates of the malignant hydrothorax were 86.9% vs 60.0%(P

Progresses on the study of microRNA and cancer / 医学研究生学报

microRNA with 22 nucleotides in length are a growing family of small non-coding RNA that function as post-transcriptional regulators of target genes in eukaryote.Recent study indicates that microRNA are closely related to the oncogenesis additional to its biological function in the regulation of cell proliferation,differentiation and death.This review proposes the progresses on the study of(microRNA,) including the biological character,biogenesis and function,and its relationship with cancer.