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Gain-of function and loss-of function studies further indicated that PVT1 promoted the proliferation and invasion of SCCHN both in vitro and in vivo , which was accompanied by epithelial-mesenchymal transition and enhanced cancer stem cell-like properties.
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Despite considerable improvements in diagnosis and therapies have been achieved for decades, life quality and survival rate of SCCHN patients are far from satisfactory. Unlimited proliferation and metastasis are major challenges in the treatment of SCCHN patients [ 1 , 2 ]. Molecular mechanisms underlying the tumorigenesis and progression of SCCHN are complicated, which are intensively studied in diverse gene types including protein coding genes and non-coding genes [ 3 , 4 ]. Hence, a comprehensive and profound understanding of the molecular mechanisms underlying cancer development and progression is critical for the discovery of novel and promising treatment strategies for the management of SCCHN patients.
It is well established that lnc RNAs play critical roles in the regulation of various biological and pathological processes, such as proliferation, apoptosis, cell cycle progression, migration and invasion etc [ 5 ]. Emerging studies have shown that lncRNAs are frequently deregulated in a wide type of malignancies including SCCHN, and are involved in the development and progression of cancer [ 6 ].
Plasmacytoma variant translocation 1 PVT1 , as one lncRNA with a length of nucleotides, is located adjacent to oncogene c-Myc on chromosome 8q Aberrant PVT1 expression has been detected in a variety of malignancies, including non-small-cell lung cancer [ 10 - 12 ], breast cancer [ 13 ], hepatocellular carcinoma [ 14 , 15 ], clear cell renal cell carcinoma [ 16 ] and prostate cancer [ 17 ], etc. Available evidence indicates that PVT1 has broad functions in cancer cell proliferation, apoptosis, cell cycle arrest, invasion, radioresistance and chemoresistance [ 18 , 19 ]. Taken together, PVT1 is a potential oncogene and a valuable biomarker in human cancers.
Importantly, PVT1 overexpression predicted a poor prognosis. Zhuo G. Cells in the logarithmic growth phase were used in all the subsequent experiments.
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SCCHN patients enrolled in the study had to follow these inclusion criteria: i primary squamous cell carcinoma without other malignancies; ii no history of previous radiotherapy or chemotherapy. The main clinicopathological features of patients were listed in Table 1. Pathological tumor-node-metastasis TNM stage was determined based on the criteria of the 7th American Joint Committee on Cancer staging system in The follow-up range was from 13 to 60 months.
Clinical physical examination, imaging evaluation, and pathological examination were employed to confirm the recurrence or metastasis. Overall survival OS was calculated from the day of surgery to the date of death. The study was approved by Research Ethics Committee of Xiangya Hospital, Central South University, Changsha, China, and written informed consent was obtained from each patient before surgery. Empty vector was used as a control.
Lentiviral particles were harvested 48 hours after transfection and freshly used for cell transfection.
Stable cells were screened by puromycin for 2 weeks and efficiency of gene modulation was assayed by qPCR. FaDu cells or Tu cells after PVT1 regulation were seeded into 6-well plate for cell counting at different time points, or into well plate for proliferation assay via Cell Counting Kit-8 Beyotime Institute of Biotechnology [ 25 ]. Transwell cell migration and invasion assays were performed as we previously described [ 25 - 27 ]. In brief, 4. Stained cells were visualized under a microscope and counted from 5 random fields.
The western blotting analysis was performed as we previously described [ 25 , 27 ]. FaDu cells were cultured as a monolayer before being harvested as single cell suspension. Culture medium was changed every 3 days. Changsha, Hunan. Mice were housed in specific pathogen-free laboratory. Mice at the age of weeks were subcutaneously injected into the position of hind flank with 2. Basic condition and individual mouse weight were monitored during the whole period. Thirty days after cancer cell injection, all mice were euthanized and sacrificed.
Xenograft tumors in all groups were removed and final weight of each tumor was measured. Each tumor sample was spliced into 2 parts and stored in nitrogen for the following qPCR and western blotting assays, respectively. Two-tailed Student's t -test was employed to determine the significant difference. The Cox proportional hazards regression model was used to identify factors that were associated with overall survival.
Survival curves were plotted with the Kaplan-Meier method and compared by the log-rank test. Collectively, these clinical data suggest that PVT1 overexpression is associated with a progressive behavior and indicates a poor prognosis in SCCHN patients. ANCE, adjacent non-cancerous epithelial samples.
B Cells in each group were counted. D Quantitative results of transwell migration and invasion assays. NOTE: All the clinicopathological variables listed in the table were included in the univariate and multivariate analyses. Subsequently, alterations including growth capacity, migration and invasion were examined. EMT is a critical molecular reprogram in cancer cells that enhances the malignancy in numerous cancer types [ 29 ].
Moreover, recent evidence has shown that EMT reprogram enhanced cancer stemness [ 28 ], we also studied the effect of PVT1 on the stem cell-like phenotype by tumorsphere formation assay. C Tumorsphere formation assays were used to determine cancer stem cell-like properties after PVT1 overexpression.
B Cell proliferation was assayed by CCK8. C Transwell migration and D invasion assays were used to examine the changes of migration and invasion. In the end, we extended to the in vivo environment to further confirm the above in vitro results. Consequently, 2. Tumor size was monitored every days and mice were euthanized at day 30 post cell injection.volunteerparks.org/wp-content/caboxor/156.php
Final weight of xenograft tumors were declined from 0. Available evidence indicates that PVT1 plays an important role in carcinogenesis and cancer progression. In the region of head and neck, PVT1 is remarkably upregulated in thyroid cancer, and PVT1 silencing significantly inhibited proliferation and cell cycle progression of cancer cells [ 34 ]. More recently, PVT1 knockdown induces apoptosis of nasopharyngeal carcinoma via preventing the DNA damage repair pathway after radiotherapy [ 19 ].
Final tumor images A , tumor size B , and tumor weight C were shown. One major finding of our study is that high PVT1 expression is tightly associated with several clinical parameters and a poor prognosis in SCCHN patients, which is consistent with reports in other solid tumors including non-small-cell lung cancer [ 10 , 12 ], hepatocellular carcinoma [ 15 ], clear renal cell carcinoma [ 35 ], prostate cancer [ 17 ], glioma [ 36 , 37 ], gastric [ 23 ] and cervical cancer [ 38 ], etc. We have to mention that our current clinical analysis is based on a relative small sample size, therefore, verification in a larger patient cohort will further strengthen the clinical significance of PVT1 in SCCHN patients.