| ObjectivePim-1 is a molecular marker that may identify at-risk population and have potential prognostic value in generalized cancer.Currently,its clinical significance,possible effects and related mechanisms in nasopharyngeal cancer have not been clarified.Radiation resistance and metastasis are the main obstacles affecting the prognosis of nasopharyngeal carcinoma.First,we planned to extract data from existing messy and limited studies on the relationship between Pim-1expression and clinicopathological features and prognosis in generalized cancer for meta-analyses the controversial results to clarify the research direction.Secondly,nasopharyngeal carcinoma tissue samples and clinical data are collected in order to make clear the expression of Pim-1 in nasopharyngeal carcinoma and analyze its relationship with clinical pathological characteristics and prognosis.And then,nasopharyngeal carcinoma cells and nude-mouse transplanted tumor model are constructed to understand whether Pim-1 may affect radiosensitivity of nasopharyngeal carcinoma in vitro and in vivo by use of lentivirus carrier.Furthermore,the possible role and regulatory mechanism of Pim-1 in nasopharyngeal carcinoma invasion and metastasis and epithelialmesenchymal transformation are discussed.Thus,providing theoretical support for revealing the potential value of PIM-1 in nasopharyngeal carcinoma and identifying possible therapeutic targets.Method1.Search Pub Med,Embase,Cochrane Library and Web of Science databases.Literature was screened according to inclusion and exclusion criteria.The extracted data included the year,type,location,sample size,subjects and other main characteristics of the study,as well as the number of samples with clinicopathological features under different levels of PIM-1 expression in cancer tissues,and the outcome indicators OR,HR and 95%CI values.If there is no direct data,Engauge Digitizer 11.1 software,Tierney table and Review Manager 5.4software are used for indirect extraction.Finally,Stata 15.0 and Review Manager5.4 software were used for statistical analysis.2.Fresh samples were collected by biopsy including 20 cases of NPC and 15 cases of nasopharyngeal mucositis who were pathologically confirmed from July to September 2020.Paraffin samples and clinical data of patients with NPC were retrospectively collected covering 80 cases of NPC and 30 cases of nasopharyngeal mucositis who were initially diagnosed by pathology from October 2017 to October 2018.The differential expression of Pim-1m RNA and protein in fresh frozen and paraffin tissues of carcinoma and mucositis were detected respectively by q PCR and immunohistochemistry.The relationship between Pim-1 and clinicopathological parameters and prognosis was further analyzed.3.The differential expression of Pim-1m RNA and protein between NPC cell lines(CNE2,C6661)and nasopharyngeal normal epithelial cell lines(NP69)were detected by q PCR and Western blot respectively to screen cells.According to the relative expression level of Pim-1,the CNE2 and C6661 cell lines with stable Pim-1 overexpression and silencing were constructed respectively.Radiosensitivity in vitro was measured by CCK-8,clone formation and flow cytometry.The nude-mouse transplanted tumor models were constructed to observe the radiosensitivity in vivo.The expression of Pim-1 in transplanted tumor tissues was verified in vivo by IHC.4.The migration and invasion abilities of Pim-1 overexpressed and silencing cell groups were detected by wound healing and Transwell assay,EMT-related and C-Myc-related protein expression was detected by WB.And the consistency of EMT-related protein expression in transplanted tumor tissues was verified by IHC in vivo.The endogenous expression of C-Myc and its phosphorylated protein p-C-Myc-S62 and the changes of C-Myc and p-C-Myc-S62 protein expression after exposure to 10058-F4 at different concentration(40u M and 80 u M)were detected by WB.WB and Transwell invasion assay were further used to investigate whether EMT-related protein expression and invasion ability might be reversed after exposure to 10058-F4.Result1.By October 31,2020,2538 literatures were retrieved,and 15 literatures were finally included for analysis,involving 10 cancer types.A total of 1651 patients were involved.The effect size of Meta-analysis showed that the high expression of Pim-1 was correlated with N stage,M stage and clinical stage.High pim-1indicates poor OS and DFS.Subgroup analysis suggested that the heterogeneity came from different detection methods of Pim-1 products,and two studies using q PCR to detect Pim-1 m RNA showed high homogeneity.The other 13 studies using IHC to detect Pim-1 protein showed significant heterogeneity.Further subgroup analysis showed that race,sample size and tissue origin were factors affecting heterogeneity,but the combined effect sizes of most factors in the subgroup were like the results of the overall study population.Sensitivity analysis showed that the effect size combination results were robust and reliable,and Begg’s test showed no significant publication bias.2.Pim-1 m RNA and protein showed relatively high expression in nasopharyngeal carcinoma,and undifferentiated non-keratin carcinoma was the main pathological type.Pim-1 protein was expressed in the nucleus,and the expression level of Pim-1 protein was correlated with T stage,N stage,pretreatment plasma EBV-DNA load and radiotherapy sensitivity,but not with gender,age and clinical stage.Up to May 31,2021,the median follow-up time was 1,120 days(range: 316-1323 days),including 38 cases of disease progression and 31 cases of death.The 3-year PFS and OS of high pim-1 were 39.5%(27/43)and 46.4%(23/43),respectively,while the 3-year PFS and OS of low pim-1 were73%(11/37)and 81.1%(8/37),respectively.High pim-1 had poor OS and PFS.The difference was statistically significant(p<0.05).Pim-1 was an independent prognostic factor for PFS and OS.3.The expression of Pim-1 m RNA and protein was relatively high in NPC cell lines(CNE2 and C6661).Cne2-OE-Pim-1 and C6661-sh RNA-Pim-1 cells and their corresponding lentivectors CNE2-OE-NC and C6661-sh RNA-NC cells were successfully constructed.Compared with NC cells in each group,CCK-8 and colony formation showed that the survival fraction of CNE2-OE-Pim-1 was relatively higher and that of C6661-sh RNA-Pim-1 was relatively lower at different X-ray doses.The sensitization enhancement ratio of CNE2-OE-Pim-1group was 0.88 < 1,which decreased the sensitivity to X-ray,while the SER of C6661-sh RNA-Pim-1 group was 1.12 > 1,which increased the sensitivity to Xray.In flow cytometry,before X-ray the apoptosis rate of CNE2-OE-Pim-1 was slightly lower,3.49±0.05% vs.2.78±0.06%,and that of C666-1-shr NA-Pim-1was increased,6.54±0.49% vs.9.22±1.11%;after X-ray the apoptosis rate of each group cells was significantly inhibited,the apoptosis rate of CNE2-OE-Pim-1was significantly decreased,18.23±0.31% vs.11.11±0.89%,while the apoptosis rate of C6661-sh RNA-Pim-1 cells was significantly increased,10.93±0.91% vs.23.15±2.67%,and the differences were statistically significant(p< 0.05).In flow cycle assay,G0/G1 decreased,S increased and G1/S activated in CNE2-OE-Pim-1 cells without irradiation,while G0/G1 increased,S and G2 remained unchanged and G1 was blocked in C666-1-sh RNA-Pim-1 cells.After X-ray,G0/G1 of C666-1-sh RNA-Pim-1 cells decreased and G2 block was induced.The growth curve of transplanted tumor in nude mice showed that the volume of tumor was larger in CNE2-OE-Pim-1 group and smaller in C666-1-sh RNA-Pim-1 group at all time points without X-ray.After X-ray,the growth of tumor was inhibited,the CNE2-OE-Pim-1-8Gy group was less affected,while the C666-1-sh RNA-Pim-1-8Gy group was significantly inhibited,the differences were statistically significant(p<0.05).IHC of transplanted tumor showed that pim-1 expression was upregulated in the CNE2-OE-Pim-1 group and down-regulated in the C666-1-sh RNA-Pim-1 group,and pim-1 protein was expressed in the nucleus.4.Compared with NC cells in each group,wound healing and Transwell assay showed that the wound healing rate of CNE2-OE-Pim-1 increased at 18 h and 36 h,and the cell number of migration and invasion increased,while the wound healing rate of C666-1-sh RNA-Pim-1 decreased,and the cell number of migration and invasion decreased,with statistical significance(p<0.05).The expression of Ecadherin was down-regulated in CNE2-OE-pim-1 and up-regulated in N-cadherin and Vimentin by WB,while the expression of E-cadherin was up-regulated in C666-1-shr NA-Pim-1 and the expression of N-cadherin and Vimentin was downregulated,and the differences were statistically significant(p<0.0001).IHC showed that the expression of EMT-related proteins in the transplanted tumor was consistent with that in cells.WB also showed that the expression of C-Myc was unchanged,and the phosphorylated p-C-My C-S62 was up-regulated and downregulated in CNE2-OE-Pim-1 and C6661-sh RNA-Pim-1 respectively.After exposure to 10058-F4 at different concentrations(0μM,40μM and 80μM),the expression of C-Myc and p-C-Myc-S62 was inhibited in CNE2,and the expression of C-Myc remained unchanged at the same concentration,while p-CMyc-S62 was inhibited in CNE2-OE-Pim-1.After exposure to 10058-F4 at 0μM and 40μM concentration,further WB and Transwell invasion assays showed that Inhibition of P-C-My C-S62 protein expression can effectively inhibit the invasion of EMT transformation induced by overexpression of Pim-1 in CNE2 cells.Conclusion1.In generalized carcinomas involving head and neck squamous cell carcinoma,Pim-1 overexpression may reflect poor prognosis and cancer phenotype.Pim-1may be a candidate molecular marker for cancer.2.In nasopharyngeal carcinoma,the expression of Pim-1 is relatively high,and associated with the invasion depth of cancer,lymph node metastasis,pretreatment plasma EBV-DNA load and radiosensitivity.Pim-1 overexpression shows unfavorable PFS and OS,which could be used as a potential predictive marker.3.In CNE-2,Pim-1 overexpression can promote proliferation and inhibit apoptosis after X-ray and promote G1/S,and more cells enter S phase to make radiosensitivity tends to decrease.In C666-1,Pim-1 silencing inhibit proliferation and promote apoptosis after X-ray and block the cells in G1 phase;In addition,Pim-1 silencing also can induced the cell to block in G2 phase after X-ray and further promote apoptosis and enhance the radiosensitivity.Pim-1 could be used as a candidate predictor of radiosensitivity.4.In CNE2,Pim-1 overexpression can promote migration and invasion and EMT;In C666-1,Pim-1 silencing can inhibited migration and invasion and EMT.In addition,Inhibition of p-C-My C-S62 protein expression can effectively inhibit the invasion of EMT transformation induced by Pim-1 overexpression in CNE2 cells.Pim-1 may be a therapeutic target that mediates EMT affecting invasion and migration by targeting C-Myc. |
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