Blank Plasmid Group Research Articles

Preparation of interleukin-1β-targeted nanobodies and their effects on apoptosis in hypoxic cardiomyocytes of mice

Objective: To prepare interleukin-1β-targeted nanoantibodies and observe their effects on apoptosis in hypoxic cardiomyocyte of mice. Methods: Using DNA recombination technology, the pET-16b and pHEN1 expression vectors were used to construct the prokaryotic expression plasmids of interleukin-1β-targeted nanobodies (pET-16b-4G6M-VHH, pET-16b-5BVP-VHH, pET-16b-5MVZ-VHH, pHEN1-4G6M-VHH, pHEN1-5BVP-VHH and pHEN1-5MVZ-VHH, where VHH is a variable domain of heavy chain antibody, 4G6M-VHH, 5BVP-VHH, 5MVZ-VHH were three interleukin-1β-targeted nanoantibodies respectively). The constructed plasmids were transferred into Escherichia coli Rosetta2 (DE3) for induction of expression and nickel column purification, respectively. The sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting were employed to identify the expression product and purified product, and the enzyme-linked immune sorbent assay (ELISA) was performed to determine their affinity. The cardiomyocyte hypoxia model was used with the highest affinity IL-1β-targeted nanobody (pHEN1-5MVZ-VHH), and cell survival and apoptosis rates were detected (the experiment was divided into normal control group, hypoxia model group, blank plasmid group and 12.5, 25.0, 50.0 μg/ml pHEN1-5MVZ-VHH treatment groups). Results: SDS-PAGE and Western blotting results showed that the anti-interleukin-1β (IL-1β) nanobodies with a relative molecular mass of about 15 000 were successfully obtained. Likewise, ELISA results found that the nanobodies expressed in pHEN1 vector group had higher affinity for IL-1β antigen compared with pET-16b vector group (4G6M-VHH group: 3.20±0.03 vs 1.20±0.03, P<0.001; 5BVP-VHH group: 3.18±0.06 vs 1.21±0.02, P<0.001; 5MVZ-VHH group: 3.38±0.05 vs 1.62±0.04, P<0.001). Additionally, the results of cell survival assay and apoptosis assay detected that compared with the hypoxia model group, HL-1 cell activity was significantly increased in the 25.0 μg/ml and 50.0 μg/ml pHEN1-5MVZ-VHH treatment groups [(75.55±2.23)% vs (46.90±2.51)%, P<0.001; (74.36±1.96)% vs (46.90±2.51)%, P<0.001], and apoptosis rate was significantly reduced [(6.83±0.27)% vs (10.24±0.76)%, P<0.001; (6.68±0.38)% vs (10.24±0.76)%, P<0.001]. Conclusions: 4G6M-VHH, 5BVP-VHH, and 5MVZ-VHH are expressed by both pET-16b and pHEN1 expression vectors and the nanobodies produced by the pHEN1 vector display enhanced antigen affinity. Furthermore, in hypoxic cardiomyocytes, pHEN1-5MVZ-VHH treatment reduces cell apoptosis.

Role and mechanism of miR-548-3p/DAG1 in the occurrence and malignant transformation of laryngeal carcinoma.

The AMC-HN-8 cell line and the primary human laryngeal epi-thelial cell lines were utilized in this work to explore the molecular mecha-nism of miR-548-3p regulating the gene DAG1 to induce the occurrence and malignant transformation of laryngeal carcinoma. Non-coding RNA miR-548-3p overexpression plasmid, interference plasmid and blank plasmid were con-structed, and the plasmids were transfected into AMC-HN-8 cells, respectively. Meanwhile, a non-transfected plasmid group and a human laryngeal epithelial primary cell group were set up. Five groups of cells were named as NC (Nor-mal control), Model, Ov-miR-548-3p, Sh-miR-548-3p and Blank-plasmid group. The luciferase reporter experiment was used to analyze the regulation charac-teristics of hsa-miR-548-3p on dystrophin-associated glycoprotein 1 (DAG1). Immunofluorescence was used to analyze the relative expression characteris-tics of the protein DAG1. The cell cloning experiment was used to analyze the proliferation characteristics of AMC-HN-8. The scratch healing test was used to analyze the migration ability of AMC-HN-8. The transwell test was used to analyze the invasion ability of AMC-HN-8. The RT-PCR was used to analyze the expression level of miR-548-3p. Western blot experiments were used to analyze the expression of protein DAG1, laminin α2 (LAMA2) and utrophin (UTRN). The luciferase report experiment and immunofluorescence test found that the expression of DAG1 and miR-548-3p are positively correlated. Cell cloning, scratching and migration experiments identified that the activity of laryngeal cancer cells was positively correlated with the expression of DAG1. The results of Western blot analysis further strengthened the above conclusions. Through carrying out research on the cellular levels, our work has demonstrated that miR-548-3p regulated the content of protein DAG1, and then further induced malignant transformation of laryngeal carcinoma.

The Regulatory Mechanism of EpCAM N-Glycosylation-Mediated MAPK and PI3K/Akt Pathways on Epithelial-Mesenchymal Transition in Breast Cancer Cells.

It was to investigate the regulation of motigen-activated protein kinase (MAPK) and phosphatidylinositol-3-kinase (PI3K)/protein kinase B (Akt) pathways mediated by epithelial cell adhesion molecules (EpCAM) N-glycosylation on epithelial-mesenchymal transition (EMT) in breast cancer cells. Breast cancer cells MCF-7 and MDA-MB-231 were taken in the control group; the blank vector plasmid transfection (blank plasmid group) and EpCAM N-glycosylation mutant plasmid transfection (Mutant-EpCAM group) were analyzed. The EpCAM in breast cancer cells was localized by immunofluorescence technique, the proliferation activity of cells in each group was detected by methyl thiazolyl tetrazolium (MTT) assay, and the clonality of cells was detected by plate cloning. and apoptosis-related proteins (Caspase-3, Bcl-2, and Bax), EMT-related molecular markers (E-cadherin, N-cadherin, and Vimentin), as well as MAPK and PI3K/Akt pathway-related proteins (p38, PI3K, and Akt) in cells were detected by western blotting (WB). EpCAM N-glycosylation mutations did not alter the expression localization of EpCAM in breast cancer cells. Compared with the control group and the blank plasmid group, the cell proliferation activity and the number of colonies formed were decreased in the Mutant-EpCAM group (P<0.05). The protein expressions of Caspase-3, Bax, and E-cadherin were up-regulated significantly, and the expressions of Bcl-2, N-cadherin, Vimentin, p-p38, p-PI3K, and p-Akt were greatly down-regulated (P<0.05). EpCAM N-glycosylation could regulate the EMT in breast cancer cells through MAPK and PI3K/Akt pathways.

Mechanism of ALDH2 improves the neuronal damage caused by hypoxia/reoxygenation.

To investigate the protective effect and mechanism of ALDH2 on PC12 cells and brain nerve tissue injury under hypoxia. The hypoxia model of PC12 cells with low ALDH2 expression was established and screened. The eukaryotic expression vector of wild type pEGFP-N1-ALDH2 and blank plasmid pEGFP-N1 were constructed and transfected into PC12 hypoxia cells respectively. After reoxygenation culture, the morphology, quantity, ALDH2 expression level and apoptosis rate of the two groups were observed, and the role of ALDH2 in cell hypoxia injury was analyzed. Eighty SD rats were randomly divided into model group (ischemia-reperfusion injury group), Alda-1 group (intraperitoneal injection of alda-1 12 hours before and after modeling), DMSO group (intraperitoneal injection of dimethyl sulfoxide) and sham operation group, with 20 rats in each group. The neurobehavioral score, apoptosis rate of nerve cells, the content and activity of ALDH2 in active cerebral cortex and hippocampal CA1 area were compared. The number of PC12 cells in hypoxia group was lower than that in control group. The expression level of ALDH2 protein in PC12 cells after 4 hours of hypoxia was lower than that in normal culture group. The number of PC12 cells transfected with wild-type recombinant plasmid was significantly more than that of blank plasmid group. Compared with the hypoxia group, the pre apoptotic and post apoptotic cells in wild type transfection group decreased after hypoxia treatment. Compared with sham operation group, nerve injury and apoptosis were increased in group M and DMSO, while ALDH2 activity and expression did not change significantly. Compared with M group and DMSO group, the nerve injury and apoptosis in Alda-1 group were improved, ALDH2 activity was increased, and ALDH2 expression was not significantly changed in Alda-1 group. Increasing the expression of ALDH2 or enhancing the activity of ALDH2 can improve the injury of neurons induced by hypoxia/reoxygenation.

Repair of osteoarthritis in animal model with exosomes derived from BMSCs transfected by the siRNA -Piezo1 through CT navigation

To investigate the effect of the exosomes from bone marrow mesenchymal stem cells (BMSCs) transfected with silence plasmid of Piezol small interference RNA (siRNA)on osteoarthritis (OA) animal model. Twenty male SD rats with specific pathogen free (SPF) were selected, ranging in age from 5.46 to 6.96 months, with a mean of (6.21± 0.75) months;and ranging in weight from 385.76 g to 428.66 g, with a mean of (407.21±21.45) g. BMSCs were extracted. The siRNA silencing plasmid of piezo1 was constructed by siRNA technology. After lentivirus was transfected into BMSCs, the exosomes were extracted. At the cellular level, BMSCs were divided into blank plasmid group and siRNA silencing plasmid group according to whether siRNA-Piezo1 was transfected or not. The osteogenic induction ability of siRNA-Piezo1 on BMSCs was detected by RT-PCR and Western blot. At the animal model level, the OA model was established by surgical resection of cruciate ligament of knee joint.According to different treatment schemes, SD rats were divided into 4 groups:blank control group, model group, BMSCs group and exosome group. SD rats in the blank control group were not treated. In the model group, the cruciate ligaments of rats were excised and OA animal model was established. In BMSCs group, BMSCs were injected into knee joint under CT guidance after OA model establishment, and the cell volume was 5×105/ml, loading amount of 2 ml, twice a week for 4 weeks. In the exosome group, 100 μg exosomes from siRNA BMSCs were added twice a week for 4 weeks after OA model establishment. The cartilage of the animal model was detected by hematoxylin eosin (HE) staining and safranin solid green staining, and quantified by the modified Minkin score and the score of the international society for osteoarthritis research (OARSI). Reverse transcription polymerase chain reaction (RT-PCR) was used to detect the relative mRNA expression level of aggrecan type II collagen in cartilage. The lentivirus transfection efficiency was(92.11±4.22)%. RT-PCR showed that the relative expression of Piezo1 mRNA in blank plasmid group was 1.07±0.06, which was significantly different from that of 0.31±0.01 in siRNA silencing plasmid group (t=2.907, P<0.05). The results of HE staining and safranine solid green staining showed that there was cartilage structure and smooth cartilage surface in the knee joint of SD rats in the blank control group. The knee joint structure in the model group had been completely destroyed, the knee joint cartilage structure in the BMSCs group was not clear, and there were subchondral bone components in the OA rats in the exosomes group. There was significant difference between the modified Minkin score of HE staining and the OARSI score of safranin solid green staining (F=15.903, 19.005;P<0.05). Among them, the repair effect of exosome group was significantly better than that of BMSCs group and model group (P<0.05). RT-PCR results showed that the relative expression of aggrecan mRNA in BMSCs group was significantly higher than that in model group (P< 0.05), and the relative expression of aggrecan mRNA in exosome group was higher than that in BMSCs group and model group (P<0.05). The relative expression of CollagenⅡmRNA in BMSCs group was higher than that in model group (P<0.05), and the relative expression of CollagenⅡmRNA in exosomes group was higher than that in BMSCs group and model group (P<0.05). Piezo1 siRNA silencing vector can promote the differentiation of BMSCs into chondrocytes and effectively inhibit the progression of OA, so as to delay the disease of OA.

Lentivirus mediated siRNA hsa-circ-0000885 transfection of BMSCs and osteoclast co-culture system on cell differentiation, proliferation and apoptosis

To explore the effects of siRNA hsa-circ-0000885 modified bone marrow mesenchymal stem cells (BMSCs) on osteogenic differentiation, cell proliferation and apoptosis in order to provide new ideas and methods for the clinical treatment of osteoporosis (OP). From September 2018 to February 2020, 13 patients with osteoporosis admitted to our hospital were selected as the research objects, including 11 females and 2 males, with an age of (65.45±10.77) years old. After obtaining the informed consent of patients, peripheral blood tissues were extracted. Then the expression level of cir-cRNA in peripheral blood mononuclear cells(PBMC) was detected by circ RNA chip. The expression of circ RNA was silenced by siRNA technology. The BMSCs were transfected with lentivirus. According to the siRNA interference plasmid hsa-circ-0000885, the cells were divided into the blank group, the empty vector group and the siRNA interference group. After 72 hours of treatment, the cell cycle was detected by flow cytometry, the apoptosis level was detected by AV-PI kit, and the osteogenic differentiation ability of BMSCs was detected by ALP staining. The expression of hsa-circ-0000885 in PBMC of patients with osteoporosis was significantly higher than that of healthy controls (t=2.119, P<0.05). ALP staining showed that siR-NA hsa-circ-0000885 could promote the osteogenic differentiation of BMSCs, which was obviously too much in the blank group and blank plasmid group (F=9.132, q=2.995, 2.897;P=0.009, 0.012<0.05). The results of CCK-8 showed that siRNA hsa-circ-0000885 could promote the proliferation of BMSCs, which was significantly higher than that of the blank group and blank plasmid group (F=9.881, q=2.457, 2.904;P=0.032, 0.016<0.05). The results of AV-PI showed that the apoptosis rate of siRNA interference group was significantly lower than that of blank group and blank plasmid group(F=10.208;q=2.885, 3.001; P=0.019, 0.011<0.05). The lentivirus mediated siRNA hsa-circ-0000885 plasmid transfected into BMSCs and osteoclast co culture system can promote cell proliferation, inhibit apoptosis and promote osteogenic differentiation of BMSCs, which can be used as a potential therapeutic target for OP patients.

Preparing PAMAM-NK4 nano complexes and examining their in vitro growth suppression effects in breast cancer.

This study sought to examine the suppression of the NK4 (which is a fragment that originates from the trypsin digestion of the hepatocyte growth factor) gene as mediated by new nano material polyamidoamine (PAMAM) dendrimers in the growth of breast cancer cells MDA-MB-231 and MCF-7, and the therapeutic effects in a nude mice model of transplanted tumor cell MDA-MB-231. We built PAMAM-NK4 nano particles and detected the in vitro transfection rate. Nano complexes and blank plasmid PAMAM dendrimers were transfected to MDA-MB-231 and MCF-7 cells, respectively. The western-blotting method, MTT experiment method, and bead method were used to detect the effects of the nano complexes on NK4 protein expression, cell proliferation, and cell apoptosis. The nude mice model of transplanted tumor cell MDA-MB-231 comprised 40 nude female mice who were subject to injections. The mice were randomly divided into four groups, comprising 10 mice per group. The control, blank plasmid and treatment groups were subcutaneously injected with 0.2 mL of 0.9% NaCl (Sodium chloride) solution, 0.2 mL of plasmid solution (including 100 µg PAMAM pcDNA3.1(-) blank plasmid nano complexes) and 0.2 mL of plasmid solution (including PAMAM-NK4 100 µg) beside the tumor inoculation spot, respectively. The positive control group was intraperitoneally injected with 0.2 mL of doxorubicin solution, including 100 µg doxorubicin. Western blotting was used to detect the NK4 protein expression of the transplanted tumor tissues of the various groups. NK4 protein was successfully expressed in MDA-MB-231 and MCF-7 cells transfected with PAMAM-NK4 nano particles, and cell proliferation was suppressed and cell apoptosis was induced. The tumor volumes and masses of the treatment and positive control groups were obviously smaller than those of the control group. The differences were statistically significant (P<0.05). The treatment group had an obviously higher mean value of NK4 protein expression than the control group. The differences were statistically significant (P<0.05). PAMAM-NK4 nano complexes suppress the growth of the breast cancer cells MDA-MB-231 and MCF-7, and had a treatment effect on this tumor nude mice model of breast cancer cells.

Expression of miR-495 and its effect on MHCC-97H hepatocellular carcinoma cells

Objective: To investigate the expression of miR-495 and its effect on MHCC-97H hepatocellular carcinoma cells. Methods: Fifty-six hepatocellular carcinoma tissue specimens (HCC group) and 40 normal liver tissue specimens (control group) preserved in our hospital from January 2017 to January 2018 were selected. Reverse transcription real-time fluorescent quantitative PCR (qRT-PCR) was used for miR-495 expression detection. MHCC-97H HCC cells were randomly selected and then divide into control group, blank plasmid group and transfection group. The blank plasmid group was transfected with blank plasmid, and the transfection group was transfected with miR-495 inhibitor. The expression of miR-495 in each group of cells were detected using qRT-PCR. CCK method was used to detect each group proliferation activity. Transwell cell migration assay was used to detect each group migration ability. Analysis of variance was used for comparison between multiple groups. Furthermore, LDS-t test was used for pairwise comparison, and t -test was used for comparison between the two groups. Results: The relative expression levels of miR-495 in the HCC group was (2.043 ± 0.382), which was higher than the control group, and the difference between the two groups was statistically significant (P < 0.05). The relative expressions levels of miR-495 in patients with stage III to IV and lymph node metastasis were 2.265 ± 0.284 and 2.290 ± 0.355, which were significantly higher than those of stage I to II and no lymph node metastasis (P < 0.05). The relative expression levels of miR-495 in transfection group was 0.653 ± 0.102, which were significantly lower than control group and blank plasmid group (P < 0.05). The A values of MHCC-97H cells cultured for 24 h and 48 h in transfection group were 0.404 ± 0.106 and 0.604 ± 0.136, which were significantly lower than control group and blank plasmid group (P < 0.05). MHCC-97H cells migration number in the transfection group was (6.10 0 ± 20), which was significantly lower than that of control group and blank plasmid group (P < 0.05). Conclusion: miR-495 high expression has certain relationship with clinicopathological characteristics of HCC tissues. In addition, miR-495 has a certain effect on the proliferation and migration ability of MHCC-97H HCC cells.

Effects of hnRNP E1 on expression of early genes E2, E6 of HPV16 and biological function in cervical cancer cells

Objective: To explore the effects of hnRNP E1 on the expression of early genes E2, E6 of HPV16 and the biological function in cervical cancer SiHa cell lines. Methods: The cell experiments in vitro were carried out in cervical cancer cell lines SiHa. The expression levels of E2, E6 mRNA and protein of HPV16 were detected by Real-time PCR and Western blot, respectively, before and after up-regulating hnRNP E1. Meanwhile, the cell proliferation, cycle and apoptosis were evaluated by CCK-8 and flow cytometry. Data analyses were performed using SPSS 22.0 and Graphpad Prism 7.0 software. Results: Compared with the blank and the blank plasmid group, the cells activity and proliferation decreased at 24, 48 and 72 h after up-regulating hnRNP E1 (P<0.05), while the percentage of cells in G0/G1 phase increased and the percentage in S and G2/M phase and proliferation index decreased (P<0.05). Moreover, the late apoptotic rate and the total apoptotic rate increased (P<0.05). The expression levels of E6 mRNA and protein of HPV16 in hnRNP E1 up-regulated group were significantly lower than that in both blank group and blank plasmid group, the differences were significant (P<0.05), showing the tendency of cells proliferation index decrease and total apoptotic rate increase with decreased HPV16 E6 expression. There were no significant differences in the expression of E2 mRNA of HPV16 among the three groups (P=0.427), and no E2 protein of HPV16 was detected. Conclusions: hnRNP E1 could inhibit the transcription and translation of E6 oncogene of HPV16 and further inhibit the proliferation and promote apoptosis of cervical cancer cells, suggesting that hnRNP E1 might be a potential target marker to prevent cervical lesions. But no association between hnRNP E1 and HPV16 E2 was found in SiHa cells.

Application of Taxol Nanomicelles with Lyp-1 Target in Targeted Therapy of Colon Cancer

As a new type of non-viral gene drug carrier, paclitaxel with Lyp-1 target has unique transmembrane ability due to its unique structure. In this paper, amino acids and surfactants are used to disperse SWCNTs in water, and non-covalent interactions are used to adsorb paclitaxel to the surface of SWCNTs. DSPE-PEG-Maleimide is then connected to NGR to achieve active targeting. To investigate the effect of NGR-SWCNTs-Paclitaxel on isolated cells, and to observe the antitumor effect of NGR-SWCNTs-Paclitaxel on S180 colon cancer mice in vivo, we provide theoretical and experimental basis for targeted cancer treatment. The luciferase activity test results showed that mi R-218 mimics had no significant effect on the intensity of the blank reporter plasmid group and p MIR-REPORT/UTR mutant luciferase activity, but in mi R-218 mimics and p MIR-REPORT/UTR Luciferase activity decreased after co-transfection of wild-type plasmids into cells. The validation results of the luciferase activity analysis system showed that mi R-218 was able to bind to Sp13’UTR. Overexpression of mi R-218 can significantly reduce the expression level of Sp1 protein but has no significant effect on Sp1 m RNA level, indicating that mi R-218 can target the regulation of Sp1 expression at the translation level.

Effects and potential mechanisms of bradykinin B2 receptor on growth and biological function of human extravillous trophoblast cells

Objective To investigate the effects and its mechanisms of bradykinin B2 receptor (B2R) on the growth and function of human extravillous trophoblast cells (HTR-8/SVneo cells). Methods B2R expression plasmid (pcDNA3.1-B2R) was constructed and B2R-specific small interfering RNA (siRNA) was synthesized. HTR-8/SVneo cells were divided into four groups and transfected with pcDNA-3.1 (blank plasmid group), pcDNA3.1-B2R (B2R expression plasmid group), siRNA negative control and B2R-specific siRNA, respectively. Quantitative real-time reverse transcription-polymerase chain reaction and Western blot were used to detect the changes in the expression of B2R, matrix metalloproteinase-2, matrix metalloproteinase-9, cyclin D1 and vascular endothelial growth factor-A at both mRNA and protein levels in HTR-8/SVneo cells. Cell counting kit-8 and flow cytometry were used to detect cell activity and cell cycle, respectively. Cell migration assay and cell invasion assay were used to detect cell migration and invasion, respectively. Tube formation assay was used to evaluate the tube formation abilities of HTR-8/SVneo cells. All data were analyzed with t test. Results (1) Compared with the blank plasmid group, expression of B2R in HTR-8/SVneo cells in the B2R expression plasmid group were significantly increased at both mRNA (5.06±0.49 vs 1.00±0.28, t=7.226, P=0.002) and protein levels (1.34±0.07 vs 1.00±0.05, t=3.727, P=0.006). And the expression of B2R in HTR-8/SVneo cells transfected with B2R-specific siRNA were significantly reduced at both mRNA (0.34±0.05 vs 1.00±0.17, t=3.667, P=0.021) and protein levels (0.74±0.03 vs 1.00±0.05, t=4.097, P=0.006) comparing with the siRNA negative control group. (2) Compared with the blank plasmid group, HTR-8/SVneo cells being transfected with B2R expression plasmid showed a higher proliferation activity (1.50±0.03 vs 1.34±0.04) promoting G0/G1 to S phase transition; compared with the siRNA negative control group, B2R-specific siRNA inhibited the proliferation of HTR-8/SVneo cells (1.06±0.04 vs 1.20±0.02) and arrested the cell cycle at G0/G1 phase (all P<0.05). (3) Compared with the blank plasmid group, B2R expression plasmid significantly increased the HTR-8/SVneo cell migration distance [(80.67±0.33) vs (41.33±5.24) μm], the number of cells penetrating matrigel gel (360.70±12.33 vs 268.70±14.45) and the number of cells having tube-like structures (28.20±2.47 vs 14.00±1.67), while significantly decrease was shown in these three parameters in B2R-specific siRNA group comparing with the siRNA negative control group [HTR-8/SVneo cell migration distance: (56.00±3.51) vs (87.00±1.53) μm, number of cells penetrating matrigel gel: 143.30±12.91 vs 252.30±17.07; number of tube-like structures: 6.25±1.49 vs 15.75±2.02; all P<0.05]. (4) Expression of matrix metalloproteinase-2 and matrix metalloproteinase-9 at mRNA level, and expression of cyclin D1 and vascular endothelial growth factor-A increased in the B2R expression plasmid group than in the blank plasmid group, and decreased in the B2R-specific siRNA group than in the siRNA negative control group at both mRNA and protein levels (all P<0.05). Conclusions B2R might enhance the activity, migration, invasion and tube formation ability of human extravillous trophoblast cells through promoting the expression of matrix metalloproteinase-2, matrix metalloproteinase-9, cyclin D1 and vascular endothelial growth factor-A. Key words: Receptor, bradykinin B2; Trophoblasts; Cell line; Cell proliferation

Differentiation of rat adipose-derived mesenchymal stem cells into corneal-like epithelial cells driven by PAX6.

Corneal integrity, transparency and vision acuity are maintained by corneal epithelial cells (CECs), which are continuously renewed by corneal limbal stem cells (LSCs). Deficiency of CECs and/or LSCs is associated with numerous ocular diseases. Paired box (PAX)6 is an eye development-associated transcription factor that is necessary for cell fate determination and differentiation of LSCs and CECs. In the present study, the PAX6 gene was introduced into adipose-derived rat mesenchymal stem cells (ADMSCs) to investigate whether PAX6-transfected cells were able to transdifferentiate into corneal-like epithelial cells and to further verify whether the cells were suitable as a cell source for corneal transplantation. The ADMSCs were isolated from the bilateral inguinal region of healthy Sprague Dawley rats. The characteristics of ADMSCs were identified using flow cytometric analysis. After subculture, ADMSCs underwent transfection with recombinant plasmid containing either PAX6-enhanced green fluorescent protein (EGFP) complementary (c)DNA or EGFP cDNA (blank plasmid group), followed by selection with G418 and determination of the transfection efficiency. Subsequently, the morphology of the ADMSCs and the expression profiles of corneal-specific markers CK3/12 and epithelial-specific adhesion protein were determined. E-cadherin was detected using immunofluorescence staining and western blot analysis at 21 days following transfection. An MTT cell proliferation and a colony formation assay were performed to assess the proliferative activity and clonogenicity of PAX6-transfected ADMSCs. Finally, the PAX6-expressing ADMSCs were transplanted onto the cornea of a rabbits with limbal stem cell deficiency (LSCD). At 21 days after transfection, the ADMSCs with PAX6 transfection exhibited a characteristic flagstone-like appearance with assembled corneal-like epithelial cells, and concomitant prominent expression of the corneal-specific markers cytokeratin 3/12 and E-cadherin. Furthermore, the proliferation and colony formation ability of PAX6-overexpressing ADMSCs was significantly retarded. The transplantation experiment indicated that PAX6-reprogramed ADMSCs attached to and replenished the damaged cornea via formation of stratified corneal epithelium. Taken together, these results suggested that conversion of ADMSCs into corneal-like epithelium may be driven by PAX6 transfection, which makes ADMSCs a promising cell candidate for the treatment of LSCD.

GSK3β to adjust the role of autophagy in Parkinson's disease nerve cells and mechanism research

Objective To screen stable express A30P a-synuclein PC12 cell as the research object, and to preliminarily investigate the activity of different glycogen systhesis kinase 3β (GSK3β) for the regulation of autophagy pathway causes a-synuclein autophagy level change and its effects on cell growth. Methods The vector of A30P a-synuclein was used to transfect to PC12 cells. G418 was used to screen stable expressed cells. Liposome method was used to transfect GSK3β-expressed plasmid, GSK3β silence plasmid, and blank plasmid into these cell lines, and stable expressed activity of GSK3β cell lines were screened. Western blot was used to detect the expression of GSK3β after transfection and verify the transfection efficiency. Western blot was used to test groups of cells in LC-3Ⅱ, Beclin1, and a-synuclein expressions. Methyl thiazolyl tetrazolium (MTT) method was used to detect the change of cell proliferation. Terminal dexynucleotidyl transferase (TdT) mediated dUTP nick end labeling (TUNEL) method was used to detect apoptosis. Results ⑴ When GSK3β expressed group was compared to GSK3β silence group, blank plasmidgroup, and blank control group, the expressions of LC-3Ⅱ and Beclin1 were significantly increased (P<0.05), the expression of a-synuclein was significantly reduced (P<0.05), the results of MTT and TUNEL showed that the cells were the decrease of apoptosis and increase of cell proliferation, with a statistically significant difference (P<0.05). ⑵ When GSK3β silence group was compared to GSK3β express group, blank plasmidgroup, and blank control group, the expressions of LC-3Ⅱ and Beclin1 were significantly reduced (P<0.05), the expression of a-synuclein was significantly increased (P<0.05), the results of MTT and TUNEL showed that the cells were the increase of apoptosis and decrease of cell proliferation, with a statistically significant difference (P<0.05). Conclusions ⑴ Activation of GSK3β activity can improve the level of cell autophagy, enhance the ability of alpha-synuclein degradation, and promote cell survival. ⑵ Inhibition of GSK3β activity can reduce the level of cell autophagy, weaken the ability of alpha-synucleindegradation, and reduce cell survival. ⑶ Autophagy is closely related to the pathogenesis of Parkinson's disease (PD). The improvement of the level of autophagy and enhancing of the degradation of a-synuclein is a new way of the future treatment of PD. Key words: Glycogen synthase kinase 3; Autophagy; alpha-synuclein; Parkinson disease

The proliferative inhibition and apoptosis promotion of Smac on human lens epithelial cells

Background Posterior capsular opacification (PCO) is a primary complication after extracapsular cataract extraction.The mechanism of PCO is associated with proliferation, migration and epithelial-mesenchymal transition (EMT) of human lens epithelial cells (LECs). To explore the target treatment of PCO is very important. Objective This study was to investigate the biological effects of second mitochondria-derived activator of caspases (Smac) on the proliferation and apoptosis of LECs. Methods Human LECs line (HLE-B3) and Smac-overexpressed LECs line were cultured, and the cells were transfected using small interfering RNA (siRNA)-Smac3 plasmid with green fluorescent protein (GFP) for 24 hours.Different concentration of transforming growth factor -β2 (TGF-β2) (5, 10, 20 and 50 μg/ml) or 200 μmol/L H2O2 were added respectively into the culture medium to establish PCO model and oxidative stress model.Cell counting kit-8 (CCK-8) assay was used to compare the cell proliferative activity among PBS group, TGF-β2 group and Smac-hyperexpression+ TGF-β2 group.Flow cytometry was used to evaluate the apoptotic rate of the PBS group, H2O2 group and siRNA-Smac+ H2O2 group.The expressions of Smac, caspase-3 and proliferating cell nuclear antigen (PCNA) mRNA and their proteins in the cells were detected by real-time quantitative PCR (RT-PCR) and Western blot. Results The GFP+ cells were≥ 80% 12 hours after siRNA-Smac3 transfection, with the optimal plasmid of siRNA-Smac3.GFP+ cell rate was (72.32±2.31)% in the siRNA-Smac3 transfection group, which was significantly higher than that in the blank plasmid group([4.91±0.24]%) (t=116.342, P<0.001). The relevant expression levels of Smac was 35.21±4.11 in the Smac-hyperexpression group, and that in the blank plasmid group was 15.24±2.48, with a significant difference between them (t=215.47, P<0.05). The cell viability of 20 ng/ml TGF-β2 affected PBS group, TGF-β2 group and Smac-hyperepression+ TGF-β2 group was (98.4±1.7)%, (98.9±0.1)% and (64.2±3.1)%, and the cell viability of Smac-hyperepression+ TGF-β2 group was significantly lower in the Smac-hyperepression+ TGF-β2 group than that in the TGF-β2 group (P<0.05). The apoptotic rate in the PBS group, H2O2 group and siRNA-Smac+ H2O2 group were (2.9±1.2) %, (45.1±4.5)% and (27.5±1.8)%, and the apoptotic rate was evidently lower in the siRNA-Smac+ H2O2 group than that in the H2O2 group (P<0.05). RT-PCR results showed that the expression levels of caspase-3 mRNA in PBS group, H2O2 group and siRNA-Smac+ H2O2 group were 0.321±0.103, 0.715±0.112 and 0.479±0.209, respectively.Compared with the H2O2 group, the relative expression level of caspase-3 mRNA in siRNA-Smac+ H2O2 group was significantly decreased, the difference was statistically significant (P<0.05). The PCNA mRNA expression levels in PBS group, TGF-β2 group and Smac-hyperepression+ TGF-β2 group were 0.299±0.013, 0.645±0.102 and 0.490±0.209, respectively.Western blot results showed that the relative expression of caspase-3 protein in siRNA-Smac+ H2O2 group and H2O2 group was 0.712±0.012 and 0.973±0.051, with significant difference between the two groups (t=132.52, P<0.05). The relative expression of PCNA protein in Smac-hyperepression+ TGF-β2 group was 0.782±0.212, which was lower than 1.126±0.251 in the TGF-β2 group (P<0.05). Conclusions Smac may prevent and treat PCO by inhibiting the proliferation and promoting apoptosis of human LECs. Key words: Smac; Apoptsis; Proliferation; After-cataract; Human; Lens epithlial cells; Small interfering RNA

Effects of PKM2 Gene Silencing on the Proliferation and Apoptosis of Colorectal Cancer LS-147T and SW620 Cells

Background/Aims: This paper aims to explore the effects of pyruvate kinase (PK) M2 gene silencing on the proliferation and apoptosis of colorectal cancer (CRC) LS-147T and SW620 cells. Methods: CRC LS-147T and SW620 cells highly expressing PKM2 were randomly selected by quantitative real-time polymerase chain reaction (qRT-PCR) and then assigned into the blank (no transfection), PKM2-shRNA (transfection with shRNA) and empty plasmid (transfection with empty plasmid) groups. Immunofluorescence was applied to detect PKM2 protein expression. qRT-PCR and Western blotting were conducted to assess mRNA and protein expression of PKM2, p53 and p21. The cell counting kit-8 (CCK-8) assay was used to assess cell proliferation. Flow cytometry was used to assess the cell cycle and apoptosis rate, and a senescence-associated β-galactosidase staining kit was used to assess cell senescence. Results: PKM2 exhibited high mRNA expression among CRC LS-147T and SW620 cells with remarkable protein expression noted in the cytoplasm and nucleus. The PKM2-shRNA group exhibited reduced PKM2 mRNA and protein expression, whereas p53 and p21 expression was increased compared with the blank and empty plasmid groups. Cell proliferation in PKM2-shRNA cells decreased significantly compared with the blank group and empty plasmid groups. The PKM2-shRNA group exhibited more cells in the G₁ phase and fewer cells in the G₂/M phase compared with the blank and empty plasmid groups. In addition, the PKM2-shRNA group exhibited significantly increased apoptosis rates and β-galactosidase activity compared with the blank and empty plasmid groups. Conclusion: Our study demonstrates that PKM2 gene silencing suppresses proliferation and promotes apoptosis in LS-147T and SW620 cells.

CCL22 and IL-37 inhibit the proliferation and epithelial-mesenchymal transition process of NSCLC A549 cells.

In the present study, we aimed to investigate the effects of CC chemokine ligand 22 (CCL22) and interleukin-37 (IL-37) on the proliferation and epithelial-mesenchymal transition (EMT) of non-small cell lung cancer (NSCLC) A549 cells. pDsRed-CCL22 and pEGFP-IL-37 plasmids were constructed. A549 cells were divided into six groups: the control, the pDsRed-N1 blank plasmid, the pEGFP-C1 blank plasmid, the pDsRed-CCL22 plasmid, the pEGFP‑IL-37 plasmid and the pDsRed-CCL22+pEGFP-IL-37 plasmid group. Expression levels and localization of CCL22 and IL-37 in cells were detected by confocal microscopy. Phase-contrast microscopy was applied for observing cellular morphology. Real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) was used for detecting the mRNA levels of vimentin, N-cadherin and E-cadherin, and their protein expression levels were tested using western blotting. Constructed plasmids expressed CCL22 and IL-37, both of which had a co-localization in the cell membrane. MTT assay and cell observation results revealed that CCL22 and IL-37 inhibited the proliferation and EMT process of the A549 cells. The results of RT-qPCR and western blotting revealed that decreased vimentin and N-cadherin mRNA and protein expression levels, and increased E-cadherin mRNA and protein expression levels were found in the pDsRed-CCL22 plasmid, pEGFP-IL-37 plasmid and pDsRed‑CCL22+pEGFP‑IL-37 plasmid groups when compared with the control, the pDsRed-N1 blank plasmid and the pEGFP-C1 blank plasmid groups (all P<0.05), and decreased vimentin and N-cadherin mRNA and protein expression levels and increased E-cadherin mRNA and protein expression levels were found in the pDsRed‑CCL22+pEGFP‑IL-37 plasmid group when compared with the pDsRed-CCL22 plasmid and the pEGFP‑IL-37 plasmid groups (all P<0.05). CCL22 and IL-37 with a co-localization in the A549 cells inhibited the proliferation and EMT process in A549 cells. The antitumor effects of CCL22 and IL37 provide a strategy for the treatment of NSCLC.

Expression of Jagged1 mRNA in human epithelial ovarian carcinoma tissues and effect of RNA interference of Jagged1 on growth of xenograft in nude mice

To investigate the expression of Jagged1 in human epithelial ovarian carcinoma tissues and the effect of Jagged1 on growth of xenograft in nude mice. (1) Forty-eight cases of ovarian cancer and 30 cases of patients with benign epithelial ovarian tumor in the Henan Province Xinxiang Central Hospital during Feb. 2011 to Mar. 2014 were enrolled in this study. The mRNA expression of Jagged1, Notch1 and the downstream target genes Hes1, Hey1 were analyzed by using realtime PCR method. (2) The ovarian cancer xenograft models in nude mice were constructed by injecting SKOV3 cells in axillary subcutaneouswere. The nude mice were randomly divided into Jagged1 interference group, blank plasmid group and control group. Each group had 10 mice. They were transfected with pcDNA3.1(+)-siRNA-Jagged1, blank plasmid pDC3.1 and phosphate buffer, respectively. The tumor volumes and tumor masses were measured 14 days after transfection and the inhibition rate was calculated. The relative mRNA expression of Jagged1, Notch1, Hes1 and Hey1 in xenograft tissues after transfection in each group was detected by using realtime PCR technique and the relative protein expression of Jagged1, Notch1, Hes1 and Hey1 in xenograft tissues was detected by utilizing western blot method. (1) The relative mRNA expression of Jagged1, Notch1, Hes1 and Hey1 in ovarian cancer tissues were higher than benign ovarian tumor tissues, the differences were statistically significant (P<0.01). (2) The tumor volume was (491± 68) mm(3) and tumor mass was (2.6±0.4) g in Jagged1 interference group, which were significantly lower than that in the blank plasmid group [(842±88) mm(3) and (4.4±0.8) g, respectively] and that in the control group [(851±90) mm(3) and (4.5±0.9) g, respectively; P<0.05], the tumor inhibition rate was 42.2% in Jagged1 interference group, which was significantly higher than that in the blank plasmid group and that in the control group (2.2% and 0, respectively), the differences were statistically significant (P<0.05). The relative mRNA and protein expression of Jagged1, Hes1 and Hey1 in xenograft tissues of nude micein Jagged1 interference group were lower than that in the other two groups, the differences were statistically significant (P<0.05). There were no differences of relative mRNA and protein expression of Notch1 in xenograft tissues of nude mice among the three groups (P>0.05). Jagged1 is highly expressed in epithelial ovarian carcinoma. Jagged1 gene interference in xenograft tumor can inhibit ovarian cancer cell growth and improve tumor suppressor rate, which probably play roles by inhibiting Notch1 signaling pathway.

Construction of Smac siRNA lentiviral vector and its silencing effect on Smac gene in human LECs

Background Research comfirmed that second mitochondrial activator of caspase (Smac) is a promoting tumor cell apoptosis protein.Our previous study showed that the expression level of Smac in LECs is obviously higher in cataract than that in normal eyes.We assumed that silencing Smac gene in LECs can inhibit the apoptosis of LECs.The way to transfect Smac siRNA into LECs is a key step. Objective This study was to construct siRNA lentiviral vector of Smac and identify its silencing efficiency in human lens epithelial B3 cell line(HLE-B3) and establish low-expressed Smac HLE-B3 line. Methods Based on the genebank and our previous study, siRNA sequence of Smac was designed and composed.The synthetic double-stranded DNA was linked to the lentiviral vector GV118 by T4 DNA ligase and then transformed DH5α competent cells.The plasmids were transformed into the DH5α competent cells.Recombinant colonies were screened by PCR and sequenced.Recombinant plasmids and two other auxiliary plasmids were used to infect 293T cells.Cell culture supernatant was collected for the measurement of viral titer.Recombinant lentiviral vector was used to infect HLE-B3 cells to calculate the viral multiplicity of infection (MOI) under the fluorescence microscope.Transfection efficiency was examined by calculating the GFP-positive cells.HLE-B3 cells were divided into negative control group, siRNA plasmid tranfected group and GV118-Smac-siRNA1 tranfected group.The relative expression levels of Smac mRNA in the cells were detected and compared among the three groups by real-time fluorescent quantitative PCR. Results GV118-Smac-siRNA was successfully constructed with the positive colonies 340 bp and blank vector colonies 299 bp, and viral titer was 3.0×108 TU/ml.At a MOI of 100, the infecting efficiency of the vector on HLE-B3 cells was about 82% and the cytotoxicity was low.The relative expression levels of Smac mRNA were (101.290±8.349)%, (92.330±6.320)% and (32.540±4.221)% in the negative control group, siRNA plasmid tranfected group and GV118-Smac-siRNA1 tranfected group, respectively, showing a significant difference among the three groups(F=32.871, P<0.01), and the relative expression level of Smac mRNA was significantly lower in the GV118-Smac-siRNA1 tranfected group than that in the negative control group (P=0.000). However, no significant difference was found in the Smac mRNA expression between the blank plasmid group and the negative control group (P=0.535). Conclusions GV118-Smac-siRNA lentiviral vector is successfully constructed.Smac-siRNA can effectively inhibit the expression of Smac mRNA in human LECs. Key words: Mitochondrial proteins; Apoptosis; RNA, small interfering; Genetic vectors; Lentivirus/genetics; Gene transfer techniques; Humans; Lens epithelial cells

Construction of murine model of TAO by immunification with recombinant extracellular domain of human thyrotropin receptor in cationic liposomes

Background Thyroid-associated ophthalmopathy (TAO) is a kind of clinically common and incurable ocular disease, and its incidence is at top place.The etiology and pathologic mechanism of TAO are still unknown because of shortness of replicative animal models and difficulty to acquire the ocular tissues in the early stage of the disease.To better understand the pathogenesis of TAO and investigate effective treatable measures, an appropriate animal model should be developed. Objective This study was to immunize female BALB/c mice with the recombinant plasmid of human thyroid-stimulating hormone receptor (TSHR) extracellular domain in cationic liposomes for the establishement of TAO models. Methods Thirty-two 6- to 8-week-old female BALB/c mice were randomly assigned to four groups according to computer random allocation.pcDNA3.1+ /hTSHR289 of 100 μg in an adjuvant cationic liposomes was injected via anterior tibialis muscle and peritoneal cavity separately in the recombinant plasmid injection group in 0, 3, 6 weeks, and pcDNA3.1 or cationic liposomes was injected in the liposomes injection group or the blank plasmid group in the same way, respectively, and normal saline solution was injected in the blank control group.Body weight of the mice was measued before and 1 month, 2, 3 and 4 months after initial injection.The manifestations were observed after modeling.The mice were sacrificed 17 weeks after initial injection, and the histopathology examination was carried out on the thyroid gland and orbital tissue.The heart blood was collected from the mice, and serum contents of total thyroxin 4 (TT4) and thyroid-stimulating hormone (TSH) were assayed by ELISA. Results Protrusion, eyelid swell and keratitis occurred in 12 eyes of 6 mice in the recombinant plasmid injection group after immunization.A significant difference in the body weight of the mice was found among the blank control group, blank plasmid group, liposomes injection group and recombinant plasmid injection group (Fgroup=3.425, P=0.028), and the body weight was considerably reduced in the recombinant plasmid injection group in comparison with the blank control group, blank plasmid group, liposomes injection group (Ftime=0.838, P=0.023). The serum levels of TT4 were (7.75±1.00), (7.96±0.76), (6.76±1.10) and (4.43±2.88)μg/dl in the blank control group, liposomes injection group, blank plasmid group, and recombinant plasmid injection group, and those of TSH were (6.36±2.58), (4.83±3.96), (6.63±1.71) and (1.60±1.76)ng/ml, showing significant differences among the groups (F=7.150, P<0.001; F=5.521, P<0.01), and the serum levels of TT4 and TSH were remarkably lower in the recombinant plasmid injection group than those of the blank control group, liposomes injection group and blank plasmid group (all at P<0.05). Histopathology revealed the lymphocyte infiltration of thyroid gland in 6 mice and proliferation of orbital adipose tissue, infiltration of lymphocytes and mastocytes, deposition of hyaluronic acid as well as swell, breakage and inflammatory cell infiltration of extraocular muscle in 15 eyes of the recombinant plasmid injection group. Conclusions A murine model of TAO can be successfully induced by immunization with recombination plasmid pcDNA3.1+ /hTSHR289 and cationic liposomes.The histopathology characteristics and ocular findings of the animal models are similar to human TAO. Key words: Graves disease; Disease models, animal; Immunization/methods; Mice, inbred BALB/c; Receptors, thyrotropin/blood; Thyroxine/blood; Recombinant plasmid; Thyroid-associated ophthalmopathy

Abnormal expression of WIF1 in hepatocellular carcinoma cells and its regulating effect on invasion and metastasis factors of TIMP-3 and caveolin-1 of hepatocellular carcinoma.

To discuss the abnormal expression of Wnt inhibitory factor (WIF1) in hepatocellular carcinoma cells and its regulating effect on the hepatocellular carcinoma invasion and metastasis factors of tissue inhibitor of matrix metalloproteinases-3 (TIMP-3) and caveolin-1. RT-PCR and Western blot were employed to detect the expression of WIF1 in six hepatocellular carcinoma cell lines of HepG2, Hep3B, Huh7, PLC/PRF/5, SMMC-7721 and MHCC97 and the immortalized human liver cell line THLE-3. Besides, Lipofectamine 2000 was employed to transfect the eukaryotic expression vector pcDNA3.1-WIF1 and blank plasmid pcDNA3.1 into hepatocellular carcinoma cell lines. Transwell assay was used to detect the effect of WIF1 on the invasion ability of hepatocellular carcinoma cells; Western blot was used to detect the effect of WIF1 on the expression of TIMP-3 and caveolin-1 in hepatocellular carcinoma cells, it also discussed the effect on the expression of β-catenin. The expression of WIF1 in hepatocellular carcinoma cell lines was lower than that in the normal liver cell lines (P<0.01); while there was basically no expression of WIF1 in the human highly metastatic cell line MHCC-97 and moderate expression in HepG2 and SMMC-7721. Therefore, HepG2 and SMMC-7721 were chosen as the further experimental cell lines. After transfecting the eukaryotic expression vector pcDNA3.1-WIF1 and blank plasmid pcDNA3.1 into hepatocellular carcinoma cell lines, compared with the blank plasmid group, the cell viability and invasion ability in the WIF1 group were all reduced (P<0.01), the expression of TIMP-3, caveolin-1 and mRNA were all down-regulated (P<0.01), and the expression of β-catenin was decreased (P<0.01). Because of down-regulation or missing of expression of WIF1 in hepatocellular carcinoma cell lines, the up-regulation of WIF1 expression can significantly inhibit the invasion and metastasis of HepG2 and SMMC-7721 of hepatocellular carcinoma cell lines, which are related to the up-regulated expression of TIMP-3 and down-regulated expression of caveolin-1 and may be realized through the Wnt/β-catenin signaling pathway.