Non-transfected Group Research Articles

#2222 Suppression of miR-199a-3p ameliorates TGF-β-induced EMT in renal tubular cells

Abstract Background and Aims Various kidney conditions, such as acute kidney injury and fibrosis, cause dysregulation of miRNAs. TGF-β is one of the most potent inducer of epithelial-to-mesenchymal transition (EMT) and fibrosis. We recently reported increased miR-199 expression in TGF-β-induced kidney fibrosis mouse model (doi: 10.3390/ijms242115520). However, the exact role of miR-199 in fibrosis and EMT development is still unknown. Method Primary tubular cells were isolated from a 4-week-old C57BL6 mouse and kept in DMEM/F12 medium containing 2% serum, supplemented with insulin/transferrin/selenium. Cells were treated for 24 hours with 30 nM miR-199-3p miRNA inhibitor (anti-miR), with or without TGF-β (10 ng/ml). The expression of profibrotic genes, as well as miR-199a-3p, was assessed by qPCR. Western blot was used to evaluate the protein expression. Results are presented as mean fold expression relative to calibrator. Statistical significance was assessed using the Kruskal-Wallis test, and the level of significance was set to p < 0.05. Results The miR-199a-3p expression in the anti-miR transfected control and TGF-β groups decreased by 93.5% and 85.5%, respectively, as compared to non-transfected groups (p < 0.05). Morphologically, cells transfected with miR-199a-3p and TGF-β1 showed no difference from cells exposed to only TGF-β1 but appeared more elongated compared to the control groups. However, the suppression of miR-199a-3p alleviated TGF-β1-induced fibrotic cell responses as shown by downregulated fibronectin mRNA (Fn1) and protein expression by 30.2% (p < 0.05) and by 4.3%, respectively. This was accompanied by reduced Acta2 (α-SMA) and Tgfb1 mRNA expressions by 30.2% and 17.5%, respectively (p < 0.05). Conclusion Our findings suggest that in murine renal tubular epithelial cells, miR-199a-3p suppresses TGF-β1-induced EMT by inhibiting FN1, Acta2, and Tgfb1 both at mRNA and protein levels. Further studies are needed to elucidate the underlying molecular interactions.

Experimental Feasibility Study of TRAIL Gene Transfected into Neural Stem Cells.

To investigate the feasibility of transfecting the TNF-related apoptosis-inducing ligand (TRAIL) gene into neural stem cells (NSCs) in vitro, and explore whether NSCs retain their proliferative and differentiated activities after transfection. NSCs were obtained from fetal mouse brains, cultured in serum-free medium and identified by immunofluorescence staining. Lentivirus vector solution containing green fluorescent protein (GFP) gene was added to the NSCs based on the multiplicity of infection (MOI). The transfection efficiency of GFP was observed using a fluorescence microscope and detected by flow cytometry. NSCs were transfected with GFP-TRAIL fusion genes mediated by the optimized MOI lentivirus solution. The expression of TRAIL proteins in NSCs was detected by immunofluorescence and Western blot analysis. The differentiation of NSCs were induced and identified by immunofluorescence staining. The optimal MOI value of virus transfection was 10, resulting in a transfection rate was higher than 90%. GFP fluorescence could be observed at 24 hours after transfecting GFP-TRAIL genes into NSCs with an MOI of 10, and reached the maximum value at 72 hours. Immunofluorescence and Western-blot assays confirmed that GFP-TRAIL fusion proteins could be continuously expressed stably. Transfected NSCs could differentiate into neurons and glial cells without any statistically significant difference compared to the non-transfected group. Neural stem cells retained their proliferative and differentiated potential after being transfected with the TRAIL gene while sustainably expressing TRAIL protein.

LncRNA model predicts liver cancer drug resistance and validate in vitro experiments.

Introduction: Hepatocellular carcinoma (HCC) patients may benefit from chemotherapy, but drug resistance is an important obstacle to favorable prognoses. Overcoming drug resistance is an urgent problem to be solved. Methods: Differential expression analysis was used to identify long non-coding RNAs (LncRNAs) that differed in chemotherapy-sensitive and chemotherapy-resistant patients. Machine learning algorithms including random forest (RF), lasso regression (LR), and support vector machines (SVMs) were used to identify important chemotherapy-related LncRNAs. A back propagation (BP) network was then used to validate the predictive capacity of important LncRNAs. The molecular functions of hub LncRNAs were investigated via qRT-PCR and cell proliferation assay. Molecular-docking technique was used to explore candidate drug of targets of hub LncRNA in the model. Results: A total of 125 differentially expressed LncRNAs between sensitive and resistant patients. Seventeen important LncRNAs were identified via RF, and seven factors were identified via LR. With respect to SVM, the top 15 LncRNAs of AvgRank were selected. Five merge chemotherapy-related LncRNAs were used to predict chemotherapy resistance with high accuracy. CAHM was a hub LncRNA of model and expression high in sorafenib resistance cell lines. In addition, the results of CCK8 showed that the sensitivity of HepG2-sorafenib cells to sorafenib was significantly lower than that of HepG2; and the sensitivity of HepG2-sorafenib cells transfected with sh-CAHM was significantly higher than that of Sorafenib. In the non-transfection group, the results of clone formation experiments showed that the number of clones formed by HepG2-sorafenib cells treated with sorafenib was significantly more than that of HepG2; after HepG2-sorafenib cells were transfected with sh-CAHM, the number of clones formed by Sorafenib treatment was significantly higher than that of HepG2 cells. The number was significantly less than that of HepG2-s + sh-NC group. Molecular Docking results indicate that Moschus was candidate drug for target protein of CAHM. Conclusion: Five chemotherapy-related LncRNAs could predict drug resistance in HCC with high accuracy, and the hub LncRNA CAHM has potential as a new biomarker for HCC chemotherapy resistance.

Inhibition of Wnt7b reduces the proliferation, invasion, and migration of colorectal cancer cells.

Colorectal cancer is one of the most common gastrointestinal tumors. The role of Wnt7b as a ligand of the Wnt signaling pathway in colorectal cancer remains to be studied. Through bioinformatics online analysis, we found that Wnt7b is abnormally highly expressed in a variety of gastrointestinal tumors. This study mainly explored the effects of Wnt7b regulating the Wnt/β-catenin signaling pathway on the proliferation, migration, and invasion of SW480 cells in colorectal cancer. Applying the TCGA data set, Wnt7b was found to be highly expressed in most gastrointestinal tumor samples. Real-time quantitative PCR(q-PCR), Western blotting(WB) results showed that Wnt7b was significantly higher expressed in colorectal cancer cell lines compared with normal intestinal epithelial cells. SW480 cells transfected with the sh-Wnt7b showed successful knockdown of Wnt7b. MTT colorimetry showed the proliferation ability of sh-Wnt7b group decreased significantly compared with the non-transfected group. The results of double staining flow cytometry showed that the sh-Wnt7b group had more apoptosis. Cell scratch test showed that the cell migration rate of sh-wnt7b group considerably reduced. The Transwell invasion experiment demonstrated that the number of cell invasions in the sh-Wnt7b group decreased significantly. After SW480cells was transfected with sh-Wnt7b, the protein levels of β-catenin, CCND1, and CD44 in this group of cells were detected to be reduced by WB, and the same results were obtained by q-PCR detection of mRNA. Wnt7b is highly expressed in colorectal cancer cells, which may affect the proliferation, migration, and invasion of colorectal cancer cells by activating the Wnt/β-catenin signaling pathway.

Biological function and regulation mechanism of MTA2 expression in bladder cancer

Bladder cancer that occurs on the bladder mucosa is the most common malignant tumor in the urinary system. Exposure to aromatic amine chemicals and cigarette smoking are the two risk factors which causes bladder cancer. The mechanism of complex molecular signaling during its progress is still not understood clearly. High expression of metastasis associated gene 2 (MTA2) in malignant tumors such as ovarian cancer and hepatocellular carcinoma indicates its possible relationship with migration and invasion. However, expression level and biological function of MTA2 in bladder cancer tissues have not been studied. Here, we investigated the biological function, regulation mechanism and clinical application of metastasisrelated genes 2 (MTA2). The expression level of MTA2 in various bladder tumor cells was determined by Western Blot. T24 and EJ cells were selected for in vitro study. T24 cells and EJ cells were transfected with viral vector and SiMTA2, respectively, to induce MTA2 over- or low- expression. MTS colorimetric assay was used to explore the proliferative ability of bladder cancer cells in vitro under MTA2 over- or low-expression. Matrigel invasion assay and Transwell migration assay were used to detect the invasion and migration of cancer cells in vitro under different MTA2 expression conditions. The results of Western Blot assay on bladder cancer cell lines have shown that the MTA2expression level to be significantly lower in T24 cells compared to that of EJ and J82 cells. MTA2 level was significantly higher in MTA2 overexpressed T24 cells than those in the non-transfected and CD511B transfected bladder cancer cells, while the level of MTA2 was much lower in the MTA2-knockdown EJ cells than that in the non-transfected and si-NC transfected. The MTS colorimetric assay significant increase in proliferation of T24 cells with MTA2 overexpression, while the proliferation of EJ cells with MTA2 knockdown was decreased. Results of Transwell migration assay showed that MTA2 overexpression could significantly increase the migration ability of T24 cells, and MTA2 knockdown could significantly reduce the migration ability of EJ cells. Matrigel invasion assay showed that MTA2 overexpression could enhance the invasive ability of T24 cells significantly, while MTA2 knockdown weaken the invasive ability of EJ cells. The expression level of E-cadherin was lower in the T24 cells with MTA2 overexpression than that in the non-transfected group and CD511B transfected group, but the expression level of N-cadherin protein was significantly higher in the T24 cells with MTA2 overexpression compared to the other two groups. The expression level of E-cadherin protein was significantly higher in the EJ cells with MTA2 knockdown than that of the non-transfected group and si-NC transfected group, while the expression level of N-cadherin protein was significantly lower than that in the non-transfected group and si-NC transfected group.

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.

Abstract P1138: Combination Of Pluripotency And Cardiac Transcription Factors Reprogram Skin Fibroblasts Into Cardiomyocytes

Direct reprogramming of autologous somatic cells into cardiomyocytes is a novel approach which is being employed for cardiac regeneration. Multiple studies have combined different cardiac-specific factors that could directly reprogram cardiac fibroblasts into induced cardiomyocyte-like cells. However, this approach needs heart biopsy for the isolation of autologous fibroblasts which involves extensive surgical procedures that require precision. In this study, we aimed to develop an approach which can directly reprogram somatic cells into cardiomyocyte-like cells using a combination of pluripotency and cardiac transcription factors. Skin fibroblasts were isolated from rat neonatal pups and were induced into cardiomyocyte-like cells using non-viral integration of cardiac transcription factors (GATA4, Mef2c and Nkx2.5) and OKSIM plasmid carrying the iPSC factors, Sox2, Oct4, Klf4 and cMyc. After 72 h, cells were analyzed for the expression of cardiac markers by qPCR and immunocytochemistry. Gene expression analysis showed significantly higher expression of cardiac markers GATA4, cMHC, Mef2c, cTnT, cTnI, and Nkx2.5 Immunostaining confirmed the expression of cardiac proteins GATA4, cMHC, cTnT, cTnI, and Nkx2.5. These results imply that the transfected cells started differentiating towards cardiac lineage. Transfected cells were also transplanted in rat myocardial infarction (MI) model immediately after ligation of left ventricle descending (LAD) artery. After 2 and 4 weeks of cell transplantation, the animals were assessed for cardiac function via echocardiography. They showed significant improvement in the cardiac function as compared to MI and non-transfected groups. After 4 weeks, the hearts were harvested, and histological analysis was performed for the assessment of fibrosis and left ventricular wall thickness. Rats transplanted with transfected cells showed significantly reduced fibrosis and increased wall thickness as compared to MI and non-transfected groups. In conclusion, transient and combined expression of cardiac transcription and iPSC factors in neonatal somatic cells leads to the transdifferentiation of skin cells into myogenic lineage. This approach can be useful for future therapeutic application for cardiovascular diseases.

Expression of POT1-AS1 in GC Tissue, Its Effect on Biological Behavior of Gastric Cancer, and Its Significance on Prognosis of Gastric Cancer.

Objective To study the correlation between gold in GC and biological indicators of gastric cancer (GC) and its effect on prognosis and correlation of POT1-AS1 with GC cellular growth, and to explore its impact in the processes of GC, to supply histological basis for medical treatment of GC. Methods From September 2019 to December 2021, 80 pairs of GAC specimens and healthy para-carcinoma tissue were immediately stored in paraformaldehyde solution. POT1-AS1 levels in 77 postoperative patients with GC were detected by immunohistochemical method. The correlation of the above indexes and the relationship between the above indexes and the biological behavior and prognosis of GC were analyzed. Results POT1-AS1 was strongly displayed in GAC specimens, and the difference between groups was statistically significant (P < 0.05). After sh-POT1-AS1 plasmid transfection, the relative expression of POT1-AS1 mRNA in SGC-7901 cells was remarkably lower compared to nontransfection group, and the difference between groups was statistically significant (P < 0.05). After POT1-AS1 knockdown, the SGC-7901 proliferation ability and the number of clones of SGC-7901 decreased remarkably. The relative level of cyclin D1 and cyclin-dependent kinase 4 (CDK4) in SGC-7901 reduced remarkably, while relative expression of cyclin-dependent kinase inhibitor 1A (CDKI1A) increased remarkably, and the difference between groups was statistically significant (P < 0.05). The positive expression of POT1-AS1 was found in GC and stromal cells. TIMP-1 in tumor stromal cells was related to the maximum diameter of tumor (P = 0.027), invasion depth (P = 0.001), lymph node metastasis (P = 0.006), and clinical stages (P = 0.006). TIMP-1 had an effect on the prognosis, while the strong positive group had a poor prognosis. The expression of TIMP-1 in GC cells was not related to clinical biological behavior and prognosis of GC. The VEGF level in GC was correlated to tumor maximum diameter (P < 0.05), invasive depth (P < 0.05), and lymph node metastasis (P < 0.05) that was linked to clinical phases, and the difference between groups was statistically significant (P < 0.05), which was positively correlated with Ki67-LI; the correlation coefficient was 0.254 and P = 0.026, which was not related to the positive expression of TIMP-1 in GC cells and stromal cells. VECF has an effect on the prognosis, and the outcomes of the positive group are worse. Conclusion The correlation between TIMP-1 of GASTRIC cancer mesenchymal cells of POT1-AS1 and VEGF and Ki-67-Li suggests that TIMP-1 produced by mesenchymal cells can facilitate tumor progression and lead to poor prognosis by promoting tumor cell proliferation. VEGF can strengthen tumor angiogenesis and then promote tumor cell proliferation, which has an adverse effect on the prognosis. Ki-67-LI is correlated to the medical biological behavior and prognosis of the tumor, reflecting the malignant process of the tumor.

The Expression of MDM2 Gene Promoted Chondrocyte Proliferation in Rats with Osteoarthritis via the Wnt/β-Catenin Pathway.

This study aimed to investigate the regulatory mechanism of MDM2 gene expression on cartilage cell proliferation in Osteoarthritis (OA) rats. For this purpose, 22 SD rats were randomly divided into normal control (10 cases) and treated (12 cases) groups. Treated group was used for OA modelling with the modified Hulth method. After a week, RT-PCR was used to detect MDM2 in cartilage tissue of rats, Wnt 1, Wnt 3 a, Wnt 10 b and β-catenin genes mRNA expression. Rat chondrocytes were isolated and cultured, and the recombinant eukaryotic expression vector pcDNA3.1 myc-siRNA-MDM2-β-catenin and co-expression plasmid pcDNA3.1 myc-siRNA-MDM2-β-catenin was used to transfect chondrocytes and the proliferation and related gene expression levels of the transfected chondrocytes were detected by MTT method and RT-PCR. The results showed that compared with the control group, MDM2, Wnt 1, Wnt 3 a, Wnt 10b and β-catenin genes in OA rat cartilage constructed by Hulth method were increased (p<0.05). The pcDNA3.1 myc-beta-catenin transfection slowed down the proliferation of OA chondrocytes, different from the non-transfected OA group (p<0.001), and increased Wnt 1, Wnt 3a, Wnt 10b and β-catenin genes expression compared with the Control group (p<0.05), but did not affect the expression of MDM2. The transfection of siRNA-MDM2 was opposite to pcDNA3.1 myc-β-catenin. The co-expression plasmid pcDNA3.1 myc-siRNA-MDM2-beta-catenin transfection did not affect the proliferation of OA chondrocytes. In general, the high expression of MDM2 in OA rats restricts the proliferation of chondrocytes, which may be related to the main pathogenesis of the occurrence and development of OA in vivo, and the regulation of MDM2 on the proliferation of chondrocytes may be achieved through the Wnt/ β-catenin pathway.

Radiosynthesis of a novel antisense imaging probe targeting LncRNA HOTAIR in malignant glioma

BackgroundLong non-coding RNA (LncRNA) HOTAIR was amplified and overexpressed in many human carcinomas, which could serve as a useful target for cancer early detection and treatment. The 99mTc radiolabeled antisense oligonucleotides (ASON) could visualize the expression of HOTAIR and provide a diagnostic value for malignant tumors. The aim of this study was to evaluate whether liposome-coated antisense oligonucleotide probe 99mTc-HYNIC-ASON targeting HOTAIR can be used in in vivo imaging of HOTAIR in malignant glioma xenografts.MethodsThe ASON targeting LncRNA HOTAIR as well as mismatched ASON (ASONM) were designed and modified. The radiolabeling of 99mTc with two probes were via the conjugation of bifunctional chelator HYNIC. Then probes were purified by Sephadex G25 and tested for their radiolabeling efficiency and purity, as well as stability by ITLC (Instant thin-layer chromatography) and gel electrophoresis. Then the radiolabeled probes were transfected with lipofectamine 2000 for cellular uptake test and the next experimental use. Furthermore, biodistribution study and SPECT imaging were performed at different times after liposome-coated 99mTc-HYNIC-ASON/ASONM were intravenously injected in glioma tumor-bearing mice models. All data were analyzed by statistical software.ResultsThe labeling efficiencies of 99mTc-HYNIC-ASON and 99mTc-HYNIC-ASONM measured by ITLC were (91 ± 1.5) % and (90 ± 0.6) %, respectively, and both radiochemical purities were more than 89%. Two probes showed good stability within 12 h. Gel electrophoresis confirmed that the oligomers were successfully radiolabeled no significant degradation were found. Biodistribution study demonstrated that liposome-coated antisense probes were excreted mainly through the kidney and bladder and has higher uptake in the tumor. Meanwhile, the tumor was clearly shown after injection of liposome coated 99mTc-HYNIC-ASON, and its T/M ratio was higher than that in the non-transfection group and mismatched group. No tumor was seen in mismatched and blocking group.ConclusionThe liposome encapsulated 99mTc-HYNIC-ASON probe can be used in the in vivo, real-time imaging of LncRNA HOTAIR expression in malignant glioma.

Osteogenic and Angiogenic Potency of VEGF165-Transfected Canine Bone Marrow Mesenchymal Cells Combined with Coral Hydroxyapatite in Vitro.

To explore the osteogenic and angiogenic potential of human vascular endothelial growth factor 165 (hVEGF165) gene-transfected canine bone marrow mesenchymal stem cells (BMSCs) combined with coral hydroxyapatite (CHA) scaffold. We constructed a lentiviral vector and transfected canine BMSCs with the best multiplicity of infection. Osteogenesis was induced in the transfected groups (GFP-BMSCs group and hVEGF-BMSCs group) and non-transfected group (BMSCs group), followed by the evaluation of alkaline phosphatase (ALP) activity and alizarin red S staining. Cells from the three groups were co-cultured with CHA granules, respectively to obtain the tissue-engineered bone. MTT assay and fluorescence microscopy were employed to assess cell proliferation and adhesion. The expression of osteogenic and angiogenic related genes and proteins were evaluated at 7, 14, 21, and 28days post osteoinduction in cell culture alone and cell co-culture with CHA, respectively using RT-PCR and ELISA. The hVEGF165 gene was transfected into BMSCs successfully. Higher ALP activity and more calcified nodules were found in the hVEGF-BMSCs group than in the control groups (p < 0.001). Cells attached and proliferated in CHA particles. Both cells cultured alone and cells co-culture with CHA expressed more osteogenic and angiogenic related genes and proteins in the hVEGF-BMSCs group compared to the GFP-BMSCs and BMSCs groups (p < 0.05). High expression of hVEGF165 in BMSCs potentially promote the osteogenic potential of BMSCs, and synergically drive the expression of other osteogenic and angiogenic factors. hVEGF-BMSCs co-cultured with CHA expressed more osteogenic and angiogenic related factors, creating a favorable microenvironment for osteogenesis and angiogenesis. Also, the findings have allowed for the construction of a CHA-hVEGF-BMSCs tissue-engineered bone.

Effect and Mechanism of Transthyretin over-Expression on Proliferation and Cell Cycle of Lung Cancer A549 Cells.

Background:The effects of transthyretin (TTR) over-expression on the proliferation and cell cycle of non-small cell lung cancer (NSCLC) A549 cells and its possible mechanism were verified.Methods:A total of 196 LC patients and 20 healthy controls were enrolled at Tianjin Hospital, Tianjin, China between Apr 2017 and Oct 2017. The serum TTR content was detected by ELISA. Through lentiviral transfection method, NSCLC cells were divided into non-transfected group (group A), negative control group (group B) transfected with empty vector and experimental group (group C) transfected with TTR over-expression. Cell proliferation was detected by CCK-8 method, TTR mRNA expression was detected by real-time quantitative polymerase chain reaction (RT-qPCR), and TTR protein expression was tested by Western blot (WB). Cell cycle was detected by flow cytometry, Wnt3a/β-catenin protein expression was detected by WB, and mRNA expression was detected by RT-qPCR.Results:The serum TTR content in early, middle and late LC group was remarkably lower than that in healthy group (P<0.05). Compared with late stage, TTR content in early and middle stages of LC group was higher, and the difference was statistically marked (P < 0.05). The absorbance value of group C was lower than that of groups A and B, indicating that the cell proliferation activity dramatically decreased, with statistically marked difference (P<0.05). LC A549 cells in group C were obviously blocked in G2M, with statistical significance (P<0.05).Conclusion:TTR over-expression can inhibit the proliferation of NSCLC A549 cells, and the expression is related to Wnt3a/β-catenin pathway. TTR in serum of patients was helpful for diagnosing LC and has certain clinical value.

DCLK1 Inhibition Sensitizes Colorectal Cancer Cells to Radiation Treatment.

Colorectal cancer (CRC) is one of the most prevalent diagnosed cancers and a common cause of cancer-related mortality. Despite effective clinical responses, a large proportion of patients undergo resistance to radiation therapy. Therefore, the identification of efficient targeted therapy strategies would be beneficial to overcome cancer radioresistance. Doublecortin-like kinase 1 (DCLK1) is an intestinal and pancreatic stem cell marker that showed overexpression in a variety of cancers. The transfection of DCLK1 siRNA to ‎normal HCT-116 cells was performed, and then cells were irradiated with X-rays. The effects of DCLK1 inhibition on cell survival, apoptosis, cell cycle, DNA damage response (ATM and γH2AX proteins), epithelial-mesenchymal transition (EMT) related genes (vimentin, N‐cadherin, and E-cadherin), cancer stem cells markers (CD44, CD133, ALDH1, and BMI1), and β‐catenin signaling pathway (β‐catenin) were evaluated. DCLK1 siRNA downregulated DCLK1 expression in HCT-116 cells at both mRNA and protein levels (P <0.01). Colony formation assay showed a significantly reduced cell survival in the DCLK1 siRNA transfected group in comparison with the control group following exposure to 4 and 6 Gy doses of irradiation (P <0.01). Moreover, the expression of cancer stem cells markers (P <0.01), EMT related genes (P <0.01), and DNA repair proteins including pATM (P <0.01) and γH2AX (P <0.001) were significantly decreased in the transfected cells in comparison with the nontransfected group after radiation. Finally, the cell apoptosis rate (P <0.01) and the number of cells in the G0/G1 phase in the silencing DCLK1 group was increased (P <0.01). These findings suggest that DCLK1 can be considered a promising therapeutic target for the treatment of radioresistant human CRC.

Expressions of miR-122a and miR-3195 in laryngeal cancer and their effects on the proliferation and apoptosis of laryngeal cancer cell Hep-2.

Laryngeal cancer (LC) is one of the common malignant tumors in the head and neck area, and the survival rate for patients is low. To investigate miR-122a and miR-3195 expressions in LC tissue, their correlations with clinicopathological features, and their impacts on Hep-2 proliferation and apoptosis. Thirty LC and 20 peritumoral tissue specimens were analyzed. miR-122a, miR-122a-negative control sequence, miR-3195, and miR-3195-NG sequence were transfected into Hep-2 in the miR-122a-mimics, miR-122a-NG, miR-3195-mimics, and miR-3195-NG groups, respectively. The miR-122a-mimics-non-transfected and miR-3195-mimics-non-transfected groups used non-transfected Hep-2. There were lower miR-122a, miR-3195 and occludin protein, and higher TBX1 protein expressions in LC than in the peritumoral tissue; the miR-122a level was associated with clinical stage (all p < 0.001). Positive correlations between miR-122a and miR-3195, and miR-122a and occludin expressions, and a negative correlation between miR-3195 and TBX1 expressions were observed (r = 0.418, r = 0.541, r = -0.428, all p < 0.001). The miR-122a and miR-3195 levels in the 2 mimics groups increased respectively compared to their NG and the non-transfected groups. At different time points after 24 h of transfection, the optical density in the 2 mimics groups was lower than in their NG groups. The miR-122a-mimics group had an increased occludin level and the miR-3195-mimics group had a decreased TBX1 level, and both groups had greater apoptosis rates than their NG groups and in the non-transfected groups (all p < 0.001). miR-122a is associated with clinical stage. miR-122a and miR-3195 may act as tumor suppressors and play a role in LC pathogenesis. They can suppress Hep-2 proliferation and promote its apoptosis, probably owing to the upregulation of occludin by miR-122a and suppression of TBX1 by miR-3195.

Lentivirus-mediated BMP-2 overexpression plasmid transfection into bone marrow mesenchymal stem cells combined with silk fibroin scaffold for osteoblast transformation

To explore the effect of lentivirus-mediated BMP-2 overexpression plasmid transfection into bone marrow mesenchymal stem cells and silk fibroin scaffold on osteoblast transformation. The lentivirus BMP-2 overexpression vector was constructed, bone marrow mesenchymal stem cells were cultured, and the combined culture system of nuclear scaffolds was constructed. Alizarin red staining and alkaline phosphatase staining were used to detect the osteogenic transformation of bone marrow mesenchymal stem cells in vitro. Ten New Zealand white rabbits, weighing 3.2 to 4.5 kg(averaging 3.9 kg), aged (2.89±0.45) years old, were selected to construct the rabbit tibial defect model by drilling a conical tibial defect (5 mm in length, 2 mm in width and 3 mm in depth) with an oral drill. The repair of the tibial defect in the animal model was observed by HE staining. The experimental group was implanted with silk fibroin scaffold + BMP-2 overexpression vector bone marrow mesenchymal stem cell complex, while the negative control group was implanted with silk fibroin scaffold+non-transfected bone marrow mesenchymal stem cell complex. Compared with the control group(silk fibroin scaffold+non-transfected bone marrow mesenchymal stem cells), the number of adherent cells on the surface of the scaffold in the experimental group(silk fibroin scaffold+transfected BMP-2 overexpression vector BMP-2 complex) increased significantly. Compared with the control group, the ECM secretion in the experimental group increased significantly. EDX analysis showed that the content of calcium ion was 0.22% in the control group and 0.86% in the experimental group, which showed that the ability of inducing calcium ion formation in the experimental group was stronger than that in the control group. Alizarin red staining of calcium nodules showed that there was no obvious change in the naked eye of the control group, and a small amount of calcium nodules could be seen under the microscope. In the experimental group, obvious red area staining was observed by naked eye, and a large number of calcium nodules were observed by microscopy. The results of alkaline phosphatase staining showed that there was no obvious change in the naked eye of the control group, and no obvious change in the microscopic observation. In the experimental group, purple area staining was observed by naked eyes, and ALP staining was strongly positive by microscopy. The combined culture system of silk fibroin scaffold and bone marrow mesenchymal stem cells can repair cartilage defects. The repair effect of BMP-2 bone marrow mesenchymal stem cells after transfection is obviously better than that of non-transfection group. HE staining showed that inflammatory cells decreased and scaffolds disappeared slightly in the control group. In the experimental group, inflammatory cells were significantly reduced, scaffolds disappeared and angiogenesis was observed. Lentivirus-mediated BMP-2 overexpression plasmid can promote BMSC to differentiate into osteocytes and secrete more extracellular matrix containing Ca²⁺ to promote bone defect repair.

Effects of bone morphogenetic protein 2 on ossification of posterior longitudinal ligament and possible mechanism

Objective To investigate the effect of bone morphogenetic protein 2(BMP2)on ossification of the posterior longitudinal ligament (OPLL) and its relationship with transforming growth factor-β (TGF-β)/Smad signaling pathway. Methods The expression vectors of wild type pcDNA3.1-BMP2 (WT), mutant pcDNA3.1-BMP2 (37G), mutant pcDNA3.1-BMP2 (190T) and mutant pcDNA3.1-BMP2 (37G/190T) were constructed and identified by agarose gel electrophoresis. The constructed vector was transfected into mouse embryonic fibroblasts C3H10T1/2 mediated by liposome to detect the expression of BMP2. Six groups were divided according to the transfection situation: (1) the non-transfection group; (2) empty vector pcDNA3.1 transfection group; (3) pcDNA3.1-bmp2 (WT) transfection group; (4) pcDNA3.1-bmp2 (37G) transfection group; (5) pcDNA3.1-bmp2 (190T) transfection group; (6) pcDNA3.1-bmp2 (37G/190T) transfection group. The experimental and control group were defined according to whether BMP2 polymorphism was included. Therefore, the non-transfection group and empty vector pcDNA3.1 transfection group were control groups, and the other groups were experimental groups. The expression of phosphorylated Smad1/5/8 and Smad4 in positive cell clones were detected by western blotting, and the alkaline phosphatase (ALP) was detected by quantitative detection kits. The protein expressions were compared among the experimental groups. Results Two fragments digested from pcDNA3.1-BMP2 represented 1.2 kb and 5.4 kb by agarose electrophoresis. The direct sequencing results were in accordance with target gene sequence. BMP2 gene was successfully transfected and stably expressed in C3H10T1/2 cells. Western blotting showed that the expression of phosphorylated Smad1/5/8 protein in the experimental groups was increased significantly after transfection, with significant difference between the experimental groups and the control groups (P 0.05). The expressions of Smad4 protein transfected by wild or mutation type pcDNA3.1-BMP2 were significantly higher than those in the control groups (P 0.05). However, there were significant differences between the two groups and other experimental groups (P<0.05). The Ser37Ala (T/G) polymorphism in exon 2 of BMP2 gene was positively correlated with ALP activity in stably transfected C3H10T1/2 cells. Conclusion The Ser37Ala (T/G) polymorphism in exon 2 of BMP2 gene promotes OPLL ossification through TGF-β/Smad signaling pathway, the possible mechanism for which is to up-regulate the protein expressions of Smad4 and ALP. Key words: Ossification, posterior longitudinal ligament; Bone morphogenetic protein 2; Signal transduction; Single nucleotide polymorphisms

Effect of adenosine kinase small interfering RNA modified corneal endothelial cells on the proliferation and secretion of regulatory T cells

Objective To investigate the role of adenosine kinase (ADK)small interfering RNA(siRNA) modified corneal endothelial cells on the proliferation and secretion of regulatory T cells (Treg cells). Methods The experiment was divided into transfected group and non-transfected group, FAM-ADK siRNA was transfected into corneal endothelial cells with Lipofectamine 3000 in transfected group, and the average mass concentration of adenosine was detected by high performance liquid chromatography in both groups.The experiment was divided into control group (transfected with control siRNA), rapamycin group (added 10 ng/ml rapamycin to culture supernatant after transfected with control siRNA) and ADK siRNA group (added 10 ng/ml rapamycin to culture supernatant after transfected with ADK siRNA). TUNEL staining was used to detect the proportion of apoptosis.Flow cytometry was used to detect the expressions of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and E-selectin in corneal endothelial cells.The cells were divided into single cultured group (lymphocytes were cultured separately), co-culture group (lymphocytes were co-cultured with corneal endothelial cells) and ADK siRNA group (lymphocytes were co-cultured with ADK siRNA transfected corneal endothelial cells), and the proportion of CD4+ CD25+ Foxp3+ Treg cells was detected by flow cytometry, and the content of interleukin-10 (IL-10) and transforming growth factor-β (TGF-β) in the supernatant was detected by ELISA in the three groups.The access and use of clinical specimens was approved by Ethic Committee of Tianjing Taida Hospital (TD201705140901). Results Flow cytometry showed that 95.1% of the endothelial cells expressed siRNA.The average mass concentration of adenosine in the supernatant of the transfection group was significantly higher than that in the control group ([38.020±6.658]ng/ml vs.[1.663±0.581]ng/ml)(t=5.437, P=0.006). TUNEL staining showed that the average apoptotic cell proportion of corneal endothelial cells in the rapamycin group was significantly higher than that in the control group, and the average apoptotic cell proportion of corneal endothelial cells in the ADK siRNA group was significantly lower than that in the rapamycin group, with significant differences between them (t=3.763, P=0.020; t=4.405, P=0.012). Flow cytometry showed that the average fluorescence intensities of ICAM-1, VCAM-1 and E-selectin in the ADK siRNA group was weaker than that in the control group (4.060±1.179 vs.11.600±2.427, 3.600±1.234 vs.11.030±2.291, 5.223±1.734 vs.24.270±4.332), with significant differences between them (t=2.794, P=0.049; t=2.857, P=0.046; t=4.081, P=0.015). The proportions of Treg cells in the single cultured group, co-culture group and ADK siRNA group was significantly different (F=12.890, P=0.007), and the proportion of Treg cells in the ADK siRNA group was significantly lower than that in the co-culture group (t=3.650, P=0.022). ELISA assay showed that, the contents of IL-10 and TGF-β in the supernatant in the single cultured group, co-culture group and ADK siRNA group were significantly different (F=20.960, P=0.003; F=27.320, P=0.001), and the content of IL-10 and TGF-β in the supernatant in the ADK siRNA group was significantly higher than that in the co-culture group, respectively (t=4.492, P=0.011; t=5.280, P=0.006). Conclusions ADK siRNA modified corneal endothelial cells can induce Treg cells to proliferate and secrete IL-10 and TGF-β, which provides a new method for induction of corneal allograft immune tolerance. Key words: Adenosine kinase; Corneal transplantation; Endothelial cells; Regulatory T cells

Matrix Production in Chondrocytes Transfected with Sex Determining Region Y-Box 9 and Telomerase Reverse Transcriptase Genes: An In Vitro Evaluation from Monolayer Culture to Three-Dimensional Culture.

This study aimed to observe the cartilaginous matrix production in SRY (sex determining region Y)-box 9 (SOX9)- and/or telomerase reverse transcriptase (TERT)-transfected chondrocytes from monolayer to three-dimensional (3D) culture. The genes were transferred into chondrocytes at passage-1 (P1) via lipofection. The post-transfected chondrocytes (SOX9-, TERT- and SOX9/TERT) were analysed at P1, P2 and P3. The non-transfected group was used as control. The 3D culture was established using the chondrocytes seeded in a disc-shaped PLGA/fibrin and PLGA scaffolds. The resulting 3D "cells-scaffolds" constructs were analysed at week-1, -2 and -3. The histoarchitecture was evaluated using haematoxylin and eosin, alcian blue and safranin o stains. The quantitative sulphated glycosaminoglycan (sGAG) content was measured using biochemical assay. The cartilage-specific markers expression were analysed via real-time polymerase chain reaction. All monolayer cultured chondrocytes showed flattened, fibroblast-like appearance throughout passages. Proteoglycan and sGAG were not detected at the pericellular matrix region of the chondrocytes. The sGAG content assay indicated the matrix production depletion in the culture. The cartilage-specific markers, COL2A1 and ACAN, were downregulated. However, the dedifferentiation marker, COL1A1 was upregulated. In 3D "cells-scaffolds" constructs, regardless of transfection groups, chondrocytes seeded in PLGA/fibrin showed a more uniform distribution and produced denser matrix than the PLGA group especially at week-3. Both sGAG and proteoglycan were clearly visualised in the constructs, supported by the increment of sGAG content, quantitatively. Both COL2A1 and ACAN were upregulated in SOX9/TERT-PLGA and SOX9/TERT-PLGA/fibrin respectively. While, COL1A1 was downregulated in SOX9/TERT-PLGA. These findings indicated that the SOX9/TERT-transfected chondrocytes incorporation into 3D scaffolds facilitates the cartilage regeneration which is viable structurally and functionally.

Apollon silencing enhanced the sensitivity to cisplatin-induced cytotoxicity of chemoresistant breast cancer cell line MCF-1/cisplatin

Objective To explore the effect of Apollon on cisplatin-induced chemoresistance of breast cancer cell line MCF-1/cisplatin (DDP). Methods Apollon silencing was performed by transient transfection of small interfering RNA (siRNA). The inhibitory effect of different concentrations of cisplatin (10, 20, 40, 80, and 100 μmol/L) on cell viability was measured by methyl thiazol tetrazolium (MTT) assays. The mRNA and protein expression levels were detected by real-time quantitative polymerase chain reaction (Real-time PCR) and Western blotting, respectively. Results As compared with MCF-7 cells, Apollon mRNA and protein levels were 1.70±0.21 folds and 2.23±0.33 folds enhanced in cisplatin-resistant breast cancer cell line MCF-1/DDP, respectively (P=0.007, 0.003). Knockdown of Apollon increased the sensitivity to cisplatin-induced cytotoxicity of MCF-1/DDP. As compared with non-transfected control group, the half maximal inhibitory concentration (IC50) value of cisplatin was significantly decreased from (81.39±3.67) μmol/L to (33.24±2.01) μmol/L (P=0.002). Meanwhile, the mRNA and protein expression levels of Apollon and β-catenin were remarkably declined to 0.43±0.16 (P=0.005), 0.54±0.10 (P=0.006) and 0.23±0.08 (P=0.003), 0.44±0.20 (P=0.005) after Apollon silencing, respectively. In addition, the mRNA and protein expression levels of β-catenin were significantly 1.91±0.23 folds (P=0.0044) and 2.21±0.31 (P=0.0023) folds enhanced with little influence on Apollon expression (P=0.890, 0.730) upon the treatment of WAY-262611 in siRNA-transfected cells. Conclusion Apollon silencing enhanced the sensitivity to cisplatin-induced cytotoxicity of chemoresistant breast cancer cell line MCF-1/DDP, which might be partially dependent on the reduced β-catenin expression. Key words: Breast cancer; Apollon; Cisplatin-resistant

MiR-122 exerts anti-proliferative and apoptotic effects on nasopharyngeal carcinoma cells via the PI3K/AKT signaling pathway

To investigate the effects of microRNA-122 (miR-122) on the proliferation and apoptosis of nasopharyngeal carcinoma (NPC) HONE-1 cells, and its correlation with the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway. Human NPC cell line (HONE-1) was transfected with miR-122 inhibitor (anti-miR-122 group), negative controls (vector control group) via lipofectamines, and HONE-1 cell lines undergoing no transfection were selected (non-transfection group). The expression of miR-122, cell proliferation, apoptosis, and expressions of PI3K/AKT pathway and downstream target proteins in the three groups were determined using fluorescence quantitative polymerase chain reaction (qPCR), cell counting kit-8 (CCK8), immunofluorescence (IF) and Western blotting, respectively. The expression of miR-122 in the anti-miR-122 group was significantly lower than corresponding expressions in the non-transfection and vector control groups after 48h of transfection (p &lt;0.05). The proliferation of cells in the anti-miR-122 group was significantly reduced with time after transfection (p &lt;0.05). After 48h of transfection, the extent of apoptosis in the anti-miR-122 group (47.11 ± 1.95%) was significantly higher than that in normal control (7.37 ± 0.82%) and vector control group (8.54 ± 0.96%; p &lt;0.05). There were no significant differences in the expressions of PI3K, AKT, mTOR protein, and the downstream signal proteins (p70S6K and 4E-BP1) in the three groups (p &gt;0.05). However, the expressions of phosphorylated forms of these proteins were significantly lower in the anti-miR-122 group than in the non-transfection and vector control groups (p &lt;0.05). IF results revealed that there were no significant differences in the fluorescence intensity value of PI3K and Akt among the three groups of patients (p&gt;0.05). Inhibition of the expression of miR-122 in NPC suppresses the proliferation, and promotes their apoptosis through the PI3K/AKT signal transduction pathway.