The low efficiency of clustered, regularly interspaced, palindromic repeats-associated Cas (CRISPR/Cas) system editing genes in vivo limits the application. A components of the extracellular matrix (ECM), the extra domain A positive fibronectin (EDA+FN), may be a target for CRISPR/Cas system for the pro-oncogenic effects. The exclusion of EDA exon would alter the microenvironment and inhibit tumor progression, even the frequency of gene editing is still limited.The pro-oncogenic effects were confirmed by the exclusion of EDA exon from the fibronectin gene, as illustrated by the down-regulated proliferation, migration and invasion of CNE-2Z or SW480 cells (P<0.05). Furthermore, although the efficacy of EDA exon knockout through CRISPR/Cas system was shown to be low in vivo, the EDA+FN protein levels decrease obviously, inhibiting the tumor growth rate significantly (P<0.05), which was accompanied by a decrease in Ki-67 expression and microvessel numbers, and increased E-cadherin or decreased Vimentin expression (P<0.05).Human nasopharyngeal carcinoma cell line CNE-2Z, and the colorectal carcinoma cell line SW480 were transfected with CRISPR/Cas9 plasmids targeting EDA exon. The effects of the exclusion of EDA on the cell proliferation, motility and epithelial-mesenchymal transition (EMT) were investigated, and the western blot and real-time PCR were performed to analyze the underlying mechanisms. Furthermore, CRISPR/Cas9 plasmids were injected into xenograft tumors to knockout EDA exon in vivo, and tumor growth, cell proliferation, EMT rate, or vascularization were investigated using western blot, PCR and immunohistochemistry.CRISPR/Cas system targeting ECM components was shown to be an effective method for the inhibition of tumor progression, as these paracrine or autocrine molecules are necessary for various tumor cells. This may represent a novel strategy for overcoming the drug evasion or resistance, in addition, circumventing the low efficiency of CRISPR/Cas system in vivo.
Abstract Purpose Anlotinib is a novel oral multi-targeted receptor tyrosine kinase inhibitor, which selectively inhibits VEGFR2/3, FGFR1-4, PDGFR α/β, c-kit, and Ret. It shows antitumor effect in patients with advanced refractory solid tumors. The detailed absorption, metabolism, and excretion pathways of anlotinib have not yet been fully investigated. Methods Six male patients were enrolled and divided into two groups. Group A (containing two patients) received 14.15 mg/80 µCi/subject [ 14 C]-anlotinib hydrochloride. Group B (containing four patients) received 14.15 mg/120 µCi/subject [ 14 C]-anlotinib hydrochloride. The blood, urine, and feces of all the six patients after orally administration of [ 14 C]-anlotinib were collected. The absorption, metabolism, and excretion of [ 14 C]-anlotinib were investigated, and the efficacy and safety of anlotinib were evaluated. Results In plasma, the average time to peak concentration ( T max ) of total radioactivity was 4.42 h and the average peak concentration ( C max ) of total radioactivity was 18.80 ng Eq./g. The average values of AUC 0-last , AUC 0-∞ , and MRT 0-t were 4071 h.ng Eq./g, 13,555 h.ng Eq./g, and 125 h, respectively. The average recovery of total radioactivity (TRA) in urine and feces was 62.03%, accounting for 48.52% and 13.51% in feces and urine of the total dosage, respectively. The parent drug, a carboxylic metabolite (M30), and mono-oxidation products (M46/M66) were major drug-related components in human plasma. Oxidative metabolism played the major role in drug clearance in human. The major metabolic pathways include oxidative deamination to M2, mono-oxidation to M1, and the formation of M30. Adverse events occurred in five patients and severe adverse events (SAE) occurred in one. Tumor response were evaluated as stable disease (SD) in three, partial response (PR) in one, and progressive disease (PD) in one of the patients, respectively. Conclusions Anlotinib had a good pharmacokinetic profile with rapid absorption, long half-life, and extensive hepatic metabolism. The adverse events and efficacy were as expected.
The purpose of this study is to establish in vivo and in vitro models for studying lymphatic metastasis of squamous cell carcinoma (SCC). Three cell lines CAL-27, Tca-83, and HeLa were injected into the tongue of nude mice. Forty days after injection, we could isolate cells of 2 homologous cell lines LN-CAL-27 and LN-HeLa from lymph node metastasis lesions. Then, the homologous cell pairs were compared by the CCK-8 assay, wound healing assay, real-time PCR, western blot, and animal experiments. The results showed that all the three cell lines could be used to establish lymphatic metastasis animal models, and the lymphatic metastasis process was observed clearly. In addition, the homologous cell pairs performed differently from parent lines with respect to biological behavior and lymphatic metastasis-related gene and protein expression. In conclusion, CAL-27, Tca-83, and HeLa cells could be used to simulate the lymphatic metastasis process of oral cancer in vivo. Furthermore, the homologous cell pairs (CAL-27 and LN-CAL-27; HeLa and LN-HeLa) are potential tools for in vitro investigation of the mechanisms underlying metastasis.
To study the clinical efficacy of digital design combined with three-dimensional (3D) printing model minimally invasive extraction of complex impacted mandibular third molars.
MicroRNAs (miRNAs) are small noncoding RNAs approximately with 22 nucleotides.Accumulating evidence indicates that they are involved in carcinogenesis and tumor progression and recent investigations have also reported that several microRNAs can act as biomarkers in cancer diagnosis and prognosis.MicroRNA-589-5p (miR-589-5p) is a less studied microRNA, and herein we examined its roles in hepatocellular carcinoma (HCC).We analyzed miR-589-5p expression in HCC tissues by sequencing data and proved its expression in liver cancer cell lines by quantitative real-time PCR (qRT-PCR).We then studied its effect on liver cancer cell growth by MTT assay, colony formation and flow cytometry, and identified its target gene by luciferase reporter assay.We found that miR-589-5p was commonly over-expressed in HCC specimens.High expression of miR-589-5p was a risk factor for HCC patients (Hazard ratio [HR]=1.434;95% confidence intervals [CI]=1.006-2.044;p=0.046).We also found miR-589-5p had higher expression in the HepG2 and HuH-7 hepatocarcinoma cell lines than in the normal Lo-2 hepatocyte.We established that suppression of miR-589-5p inhibited cell proliferation and cell cycle progression by loss-of-function studies.Furthermore, we found mitogen-inducible gene 6 (MIG-6) is a target for miR-589-5p.Our study demonstrated that miR-589-5p facilitated the growth of liver cancer cells by targeting MIG-6 and that it could be a prognosis biomarker for HCC.Suppression of miR-589-5p may be a feasible approach for inhibiting HCC progress.
Ovarian cancer (OC) is one of the deadliest gynecological malignancy. Epithelial ovarian cancer (EOC) is its most common form. OC has both, a poor prognosis and a high mortality rate due to the difficulties of early diagnosis, limitation of current treatment and resistance to chemotherapy. Extracellular vesicles (EVs) is a heterogeneous group of cell-derived submicron vesicles, which can be detected in body fluids, and it can be classified into three main types including exosomes, micro-vesicles, and apoptotic bodies. Cancer cells can produce more EVs than healthy cells. Moreover, the contents of these EVs have been found distinctive from each other. It has been considered that EVs shedding from tumor cells may be implicated in clinical applications, such as a tool for tumor diagnosis, prognosis and potential treatment of certain cancers. In this review, we provide a brief description of EVs. in diagnosis, prognosis, treatment, and drug-resistantance of OC. Cancer-related EVs show powerful influences on tumors by various biological mechanisms. However, the contents mentioned above remain in the laboratory stage and there is a lack of large-scale clinical trials, and the maturity of the purification and detection methods is a constraint. In addition, amplification of oncogenes on ecDNA is remarkably prevalent in cancer. It may be possible that ecDNA can be encapsulated in EVs and thus detected by us. In summary, much more research on EVs needs to be performed to reveal breakthroughs in OC and to accelerate the process of its application in clinic.
We performed a study to investigate the role of ERCC1 (rs11615, rs2298881, and rs3212986) and ERCC2 (rs13181, rs238406, and rs1799793) polymorphisms in the prognosis of gastric cancer.A total of 346 patients with gastric cancer were recruited between May 2009 and May 2012.Single nucleotide polymorphism genotyping was performed using the Sequenom MassARRAY platform.The GA+AA genotype of ERCC2 rs1799793 showed significant and favorable response to chemotherapy than the wide-type GG genotype in multivariate analysis (OR = 1.78, 95%CI = 1.13-2.81).In a Cox proportional hazard model, carriers of ERCC2 rs1799793 GA+AA genotype exhibited longer duration of survival than did those with the GG genotype (hazards ratio = 0.57, 95%CI = 0.35-0.92).In conclusion, our study suggests that ERCC2 rs1799793 polymorphic variation could be used as a predictor for the prognosis of gastric cancer.
Abstract Background Given the difficulties or incapacity of teeth movement in orthodontic treatment, the ways to speed tooth movement must be investigated. Besides, nonsteroidal anti-inflammatory drugs (NSAIDs) were utilized to treat pain caused by tooth movement during orthodontic treatment. The purpose of this study is to examine the impact of aspirin and low-frequency high-intensity ultrasound (LFHIU) on rat orthodontic tooth movement in rats. Methods Thirty-six male Sprague-Dawley rats were divided into three groups: orthodontic (O), ultrasound-treated orthodontic (OU), and ultrasound-treated orthodontic with aspirin gavage (OUA) group. In the OU and OUA group, LFHIU (44 W/cm2, 28 kHz) was applied to the buccal side of the maxillary first molar alveolar bone for 10 s every day. In the OUA group, aspirin was given by gavage every day. The rats were sacrificed on days 1, 3, 7, and 14. Results After ultrasonic treatment, the speed of tooth movement was increased by about 1.5 times. And the number of osteoclasts considerably increased by about 2 times. However, they decreased slightly after aspirin gavage. By Applying ultrasound therapy, Receptor Activator for Nuclear Factor-κ B Ligand (RANKL) levels in periodontal tissue were elevated. Aspirin was able to reduce these increases. Results from Micro Computed Tomography (Micro-CT) revealed that bone mineral density decreased by about 1/5 after ultrasound treatment on the compression side. The rate of bone mineral apposition indicated that bone was forming under tension, and that of the OU group increased by about 1.3 times that O group. Conclusions Although aspirin slowed this trend, LFHIU still enhanced overall tooth mobility in orthodontic treatment.
Early-stage temporomandibular joint osteoarthritis (TMJOA) is characterized by excessive subchondral bone loss. Emerging evidence suggests that TMJ disc displacement is involved, but the pathogenic mechanism remains unclear. Here, we established a rat model of TMJOA that simulated disc displacement with a capacitance-based force-sensing system to directly measure articular surface pressure in vivo. Micro-CT, histological staining, immunofluorescence staining, IHC staining, and Western blot were used to assess pathological changes and underlying mechanisms of TMJOA in the rat model in vivo as well as in RAW264.7 cells in vitro. We found that disc displacement led to significantly higher pressure on the articular surface, which caused rapid subchondral bone loss via activation of the RANTES-chemokine receptors-Akt2 (RANTES-CCRs-Akt2) axis. Inhibition of RANTES or Akt2 attenuated subchondral bone loss and resulted in improved subchondral bone microstructure. Cytological studies substantiated that RANTES regulated osteoclast formation by binding to its receptor CCRs and activating the Akt2 pathway. The clinical evidence further supported that RANTES was a potential biomarker for predicting subchondral bone loss in early-stage TMJOA. Taken together, this study demonstrates important functions of the RANTES-CCRs-Akt2 axis in the regulation of subchondral bone remodeling and provides further knowledge of how disc displacement causes TMJOA.
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