Dual targeting of Polo-like kinase 1 and baculoviral inhibitor of apoptosis repeat-containing 5 in TP53-mutated hepatocellular carcinoma

BACKGROUND Hepatocellular carcinoma(HCC),often diagnosed at advanced stages without curative therapies,is the fifth most common malignant cancer and the second leading cause of cancer-related mortality.Polo-like kinase 1(PLK1)is activated in the late G2 phase of the cell cycle and is required for entry to mitosis.Interestingly,PLK1 is overexpressed in many HCC patients and is highly associated with poor clinical outcome.Baculoviral inhibitor of apoptosis repeatcontaining 5(BIRC5)is also highly overexpressed in HCC and plays key roles in this malignancy.AIM To determine the expression patterns of PLK1 and BIRC5,as well as their correlation with p53 mutation status and patient clinical outcome.METHODS The expression patterns of PLK1 and BIRC5,and their correlation with p53 mutation status or patient clinical outcome were analyzed using a TCGA HCC dataset.Cell viability,cell apoptosis,and cell cycle arrest assays were conducted to investigate the efficacy of the PLK1 inhibitors volasertib and GSK461364 and the BIRC5 inhibitor YM155,alone or in combination.The in vivo efficacy of volasertib and YM155,alone or in combination,was assessed in p53-mutated Huh7-derived xenograft models in immune-deficient NSIG mice.RESULTS Our bioinformatics analysis using a TCGA HCC dataset revealed that PLK1 and BIRC5 were overexpressed in the same patient subset and their expression was highly correlated.The overexpression of both PLK1 and BIRC5 was more frequently detected in HCC with p53 mutations.High PLK1 or BIRC5 expression significantly correlated with poor clinical outcome.PLK1 inhibitors(volasertib and GSK461364)or a BIRC5 inhibitor(YM155)selectively targeted Huh7 cells with mutated p53,but not HepG2 cells with wild-type p53.The combination treatment of volasertib and YM155 synergistically inhibited the viability of Huh7 cells via apoptotic pathway.The efficacy of volasertib and YM155,alone or in combination,was validated in vivo in a Huh7-derived xenograft model.CONCLUSION PLK1 and BIRC5 are highly co-expressed in p53-mutated HCC and inhibition of both PLK1 and BIRC5 synergistically compromises the viability of p53-mutated HCC cells in vitro and in vivo.

SMAC exhibits anti-tumor effects in ECA109 cells by regulating expression of inhibitor of apoptosis protein family

BACKGROUND The poor prognosis and rising incidence of esophageal cancer highlight the need for improved therapeutics that are essential prior to treatment.LCL161 is an SMAC(second mitochondrial activator of caspases)mimic and inhibitor of apoptosis protein(IAP)antagonist which exhibits anti-tumor effects and improves the chemical sensitivity of many cancers.AIM To ascertain the effects and mechanisms of the SMAC analog LCL161 on esophageal cancer cells.METHODS MTT assay and TUNEL assay were used to detect cell proliferation and apoptosis,respectively.Western blot analysis was used to study the molecular mechanisms of LCL161-induced death of ECA109 cells.RESULTS LCL161 decreased ECA109 cell proliferation in dose-and time-dependent manner and induced apoptosis of ECA109 cells in a dose-dependent manner.Also,LCL161 induced a significant decrease in the expression of the XIAP and significant increase in the expression of Caspase-3.In addition,Bax increased significantly with increasing concentrations of LCL161,and the relative expression of Bax was significantly different between groups.CONCLUSION These findings support the hypothesis that LCL161 can inhibit proliferation and induce apoptosis in esophageal cancer cells by regulating the expression of IAP family members,suggesting that it has potential to be an effective treatment for esophageal squamous cell carcinoma.

Regulatory Effects of X-linked Inhibitor of Apoptosis Protein and Pro-apoptotic Protein Smac on Apoptosis Resistance to Chemotherapy in Pancreatic Cancer Cells~*

Objective： To investigate the relation of X-linked inhibitor of apoptosis （XIAP） and second mitochondria-derived activator of caspase （Smac） signaling pathway to chemoresistance in human pancreatic cancer Panc-1 and BXPC-3 cells. Methods： Apoptosis and the changes of XIAP expression in permeabilized cells induced by cisplatin and 5-fluorouracil （FU） were measured by flow cytometry. The cytosolic expression of XIAP, Smac and caspase-3 was detected by Western blot. A recombinant plasmid vector pEGFP-N1/Smac was constructed and transfected into of Pancol cells. The effect of cytosolic overexpression of Smac on apoptosis of Panc-1 cells was evaluated by flow cytometry. Results： Panc-1 was more resistant to cisplatin or 5-FU induced apoptosis than BXPC-3. Western blot revealed that chemoresistant Panc-1 highly expressed XIAP, and increased cytosolic expression of Smac might be responsible for the marked down-regulation of XIAP in chemo-sensitive BXPC-3 cells after exposure to cisplatin or 5-FU. Furthermore, cytosolic overexpression of Smac could significantly down-regulate the levels of XIAP and promote the activity of caspase-3, as well as sensitize Panc-1 cells to anticancer drug-induced apoptosis. Conclusion： Anticancer drug-induced apoptosis requires mitochondrial release of Smac and downregulation of XIAP, which may be an important determinant of chemo-sensitivity in pancreatic cancer cells. Up-regulation of cytosolic expression of Smac may act as an effective modifying signal to overcome apoptosis resistance to chemotherapy in pancreatic cancer cells.

Targeting X-linked inhibitor of apoptosis protein inhibits pancreatic cancer cell growth through p-Akt depletion

AIM: To determine whether lentivirus-mediated shRNA targeting the X-linked inhibitor of apoptosis protein (XIAP) gene could be exploited in the treatment of pancreatic cancer. METHODS: Human pancreatic cancer cells Panc-1, Mia-paca2, Bxpc-3 and SW1990, infected with lentivirus, were analyzed by real-time polymerase chain reaction (PCR). Western blotting was used to examine XIAP protein levels, survivin and p-Akt to confirm the result of real-time PCR and determine the possible mechanism. The 3-(4,5-cimethylthiazol-2-yl)-2,5- diphenyl tetrazolium bromide (MTT) assay was used to measure IC50 to determine chemosensitivity to the chemotherapeutic drugs 5-fluorouracil (5-FU) and gemcitabine. A colony assay, MTT assay and a tumorigenicity experiment were used to study cell proliferation in vitro and in vivo . Caspase-3/7 activity, 4',6-diamidino-2-phenylindole-staining and flow cytometric measurements were used to study apoptosis in SW1990 cells. RESULTS: XIAP proteins were found to be differen- tially expressed among pancreatic cancer cell lines Panc-1, Mia-paca2, Bxpc-3 and SW1990. Data of real-time PCR and Western blotting showed that XIAP was reduced persistently and markedly by lentivirus-mediated shRNA. Downregulation of XIAP by transfection with XIAP shRNA resulted in decreased p-Akt expression. XIAP shRNA also inhibited the growth of pancreatic cancer cells in vitro and in vivo , enhanced drug-induced apoptosis and increased chemosensitivity to 5-FU and gemcitabine. Results also suggest that inhibition of XIAP and subsequent p-Akt depletion may have an anti-tumor effect through attenuating the ability of cancer cells to survive. CONCLUSION: Lentivirus-mediated gene therapy is an attractive strategy in the treatment of pancreatic cancer and justifies the use of lentivirus in pancreatic cancer gene therapy studies.

Baculoviral inhibitor of apoptosis protein repeatcontaining protein 3 delays early Wallerian degeneration after sciatic nerve injury

Wallerian degeneration is a complex biological process that occurs after nerve injury,and involves nerve degeneration and regeneration.Schwann cells play a crucial role in the cellular and molecular events of Wallerian degeneration of the peripheral nervous system.However,Wallerian degeneration regulating nerve injury and repair remains largely unknown,especially the early response.We have previously reported some key regulators of Wallerian degeneration after sciatic nerve injury.Baculoviral inhibitor of apoptosis protein repeat-containing protein 3(BIRC3)is an important factor that regulates apoptosis-inhibiting protein.In this study,we established rat models of right sciatic nerve injury.In vitro Schwann cell models were also established and subjected to gene transfection to inhibit and overexpress BIRC3.The data indicated that BIRC3 expression was significantly up-regulated after sciatic nerve injury.Both BIRC3 upregulation and downregulation affected the migration,proliferation and apoptosis of Schwan cells and affected the expression of related factors through activating c-fos and ERK signal pathway.Inhibition of BIRC3 delayed early Wallerian degeneration through inhibiting the apoptosis of Schwann cells after sciatic nerve injury.These findings suggest that BIRC3 plays an important role in peripheral nerve injury repair and regeneration.The study was approved by the Institutional Animal Care and Use Committee of Nantong University,China(approval No.2019-nsfc004)on March 1,2019.

Expression of second mitochondria-derived activator of caspases, X-linked inhibitor of apoptosis protein, and caspase-3 in pituitary adenomas

Studies concerning correlations between pituitary adenomas and cell apoptosis have mainly focused on upstream apoptosis signaling, but seldom on downstream mediators. In the present study, second mitochondria-derived activator of caspases （Smac）, X-linked inhibitor of apoptosis protein （XIAP）, and caspase-3 protein were qualitatively analyzed using immunohistochemistry, and quantified by western blot. Smac, XIAP, and caspase-3 mRNA expressions were detected by reverse transcription-PCR. Results showed that XIAP protein and mRNA expressions were greater in the invasive pituitary adenoma group compared with the noninvasive pituitary adenoma group. However, Smac and caspase-3 protein and mRNA expressions were lower in the invasive pituitary adenoma group compared with the noninvasive pituitary adenoma group. In the invasive pituitary adenomas, Smac expression was positively correlated with caspase-3 protein and mRNA expression （Protein： r = 0.55, P 0.01; mRNA： r = 0.50, P 0.01）. Smac and caspase-3 expressions were negatively correlated with XIAP protein and mRNA expression （Protein： r = -0.56, -0.64, P 0.01; mRNA： r = -0.69, -0.67, P 0.01）. However, no significant differences in correlation among Smac, XIAP, and caspase-3 were detectable in noninvasive pituitary adenomas. These data indicated that high expression of XIAP and low expression of Smac and caspase-3 suppressed cell apoptosis and led to enhanced invasiveness of pituitary adenomas. Thus, Smac, XIAP, and caspase-3 may be useful markers in determining the invasive behavior of pituitary adenomas.

Expression of X-linked Inhibitor of Apoptosis Protein and Its Effect on Chemotherapeutic Sensitivity of Bladder Carcinoma

The expression of X-linked inhibitor of apoptosis protein (XIAP) gene and its effect on chemotherapeutic sensitivity in bladder carcinoma was explored. By using immunohistochemistry, the expression of XIAP was detected in 47 bladder carcinomas and 5 normal bladder tissues. The XIAP gene was transfected into bladder cancer cell line T24 by liposome and the positive clone was screened by G418. Cellular XIAP mRNA level was detected by RT-PCR. Low-dose mitocycin C was administered to induce the apoptosis of T24 cells. The in vitro growth of bladder carcinoma cells was analyzed by MTT colorimetry, and the apoptosis rate was assayed by TUNEL methods. It was found XIAP was moderately expressed in bladder carcinomas with the the positive rate being 78.73% (37/47), but the positive rate was not correlated with carcinoma stages and grades (P<0.05). XIAP mRNA level in transfected T24 cells was significantly increased by 3.8 times as compared with that in the cells not transfected with XIAP. After treatment with low-dose mitomycin C (0.005 and 0.05 mg/mL), the growth rate in XIAP no-transfected control group was increased by (11.60±0.25)% and (16.51±0.87)% (P<0.05), and the apoptosis rate was decreased by (10.1±0.2)% and (11.9±0.2%) (P<0.05) respectively as compared with XIAP transfected group. It was concluded that XIAP was expressed in most of bladder carcimoma samples. Overexpression of XIAP in T24 could significantly reduce the MMC-induced apoptosis of bladder carcinoma, suggesting its effect on the chemothera- peutic sensitivity of T24 cells.

Evolutionary and functional analyses of the interaction between the Bombyx mori inhibitor of apoptosis(IAP)and nucleopolyhedrovirus IAPs

As an important insect immune response,apoptosis plays a critical role in the interaction between baculoviruses and insect hosts.Previous reports have identified inhibitor of apoptosis(IAP)proteins in both insects and baculoviruses,but the relationship between these proteins is still not clearly understood.Here,we found that insect IAP proteins were clustered with baculovirus IAP3,suggesting that the baculovirus iap3 gene might be derived from the Lepidoptera or Diptera.We demonstrated that Bombyx mori inhibitor of apoptosis(Bmiap)gene had an inhibitory effect on apoptosis in silkworm cells.Further analysis of the effects of Bmiap genes on the proliferation of B.mori nucleopolyhedrovirus(BmNPV)showed that both the Bmiap and BmNPV iap genes increased BmNPV proliferation after BmNPV infected silkworm cells.Our results also indicated that BmNPV IAP1 and IAP2 directly interacted with BmIAP in silkworm cells,implying that the Bmiap gene might be hijacked by BmNPV iap genes during BmNPV infection.Taken together,our results provide important insights into the functional relationships of iap genes,and improve our knowledge of apoptosis in baculoviruses and insect hosts.

Expression of Livin in tissues of lung cancer and its correlation with the expression of caspase-3

Objective： To study the expressions of two isoforms of Livin in tissues of lung cancer and their relations to histological types and chemotherapy, and to study their correlations to the expression of caspase-3 as well. Methods： Expressions of Livin isoforms a, 13 and caspase-3 were detected by reverse transcription polymerase chain reaction （RT-PCR） assay in lung cancer tissues as well as in controls. Results： Livin isoforms a and ~ were expressed in 12 of 27, and 19 of 27 lung cancer tissues respectively, much more than those in lung para-cancereus [both were （0/6）] or benign disease lung tissues （0/12, 1/12; P 〈 0.01 and P 〈 0.01 ）. Moreover, they were detected in 7/14, 9/14 lung adenocarcinomas and 4/12, 9/12 squamocallular and large call carcinomas, respectively, and both showed expressions in one small cell carcinoma. The levels of these two isoforms in lung cancer were significantly higher than those in controls by Gel imaging system （P 〈 0.05 and P 〈 0.05）, the former was higher in adenocarcinoma than that in squamocellular carcinoma （P 〈 0.05）, while the latter was the same in both （P 〉 0.05）. Meanwhile, the levels of caspase-3 in lung cancer were significantly lower than those in controls, and it was suggested to be negatively associated with either each of two isoforms or their sum （P 〈 0.05, P 〈 0.01 and P 〈 0.01）. Two isoforms of Livin expression seemed to increase＇after chemotherapy but not related to clinical stages （P 〉 0.05）. Conclusion： Two isoforms of Livin are differently expressed in different histological types of lung cancer and may contribute to corresponding cancerous development; the levels of Livin are negatively associated with those of caspase-3, this may be due to the fact that Livin could resist against apoptosis; high expression of Livin seems to be related to chemotherapy but not clinical stages.

Transfection of gene Livin α/β into A549 cells and separate effect on the cell growth

Objective:To express two Livin isoforms (Livin α & β genes) with transfection techniques in A549 cell line respectively in order to observe their effect on growth of cell line. Methods:Two eukaryotic expression vectors of Livin, pcDNA3.1-Livin α & β, were transfected into A549 cell line by electroporation. Then G418-resistant clones were screened. RT-PCR, Northern blot and immunofluorescence cytochemistry were used to detect Livin α & β expression level in the transfected cells. Finally, observation of cell morphology, growth curve assay and colony formation analysis were performed to explore the effect of Livin on growth of the cells. Results:Livin α & β were expressed in transfected A549 cells, and induced a faster cell growth, shorter doubling time and stronger cell colony forming ability, yet had no morphology change.Conclusion:Both isoforms can accelerate the growth of A549 cells, indicating a close relationship between Livin expression and the genesis and development of lung cancer. The expression of Livin α & β in A549 cells provides basis for further study of their different biological functions of anti-apoptosis and of their role in lung cancer cell resistance to radiotherapy and chemotherapy.

The prediction of recurrence and survival of patients with Dukes’ B colorectal cancer after curative resection with immunohistochemical detection of Survivin and Livin expressions

Objective: To detect the expressions of Survivin and Livin in Dukes’ B colorectal cancer tissues and analyze the prognosis after curative resection. Methods: The expressions of Survivin and Livin were evaluated immunohistochemically in Dukes’ B colorectal cancer specimens from 81 patients after curative resection of the tumor. Their correlations to clinical characters and survival were also explored. Results: The positive rates of Survivin and Livin in colorectal cancer tissues were significantly higher than those in normal colorectal tissues (58.0% vs. 16.7% and 45.7% vs. 8.3% respectively, P < 0.05). The expressions of Survivin and Livin were not related to gender, tumor site, primary size, T stage, pathologic category, and degree of differentiation (P > 0.05), and no relationship was found between the expressions of Survivin and Livin (P > 0.05). The expression rate of Survivin in patients older than 50 years was higher than that in patients younger than 50 years (70.6% vs. 36.7%, P < 0.05). Both Survivin and Livin were related to recurrence and/or metastasis (P = 0.02 and P = 0.001, respectively), and shorter survival (P = 0.039 and P = 0.001, respectively). Cox multivariate analysis showed T4 and positive Livin expression were independent prognostic factors (P = 0.002 and P = 0.047, respectively). Conclusion: Survivin and Livin are over-expressed in Dukes’ B colorectal cancer tissues and are positively related to recurrence and/or metastasis and poor prognosis after curative resection of the tumor.

XIAP expression and its predictive significance of prognosis in stage IIB osteosarcomas

Objective： To evaluate X-linked inhibitor of apoptosis protein （XIAP） expression in biopsy specimens from the patients with stage lib osteosarcomas before chemotherapy to study XIAP expression and its predictive significance of progosis in stage lib osteosarcomas. Methods： The expression of XIAP was retrospectively detected by SP immunohistochem- istry in 31 cases biopsy specimens from stage lib osteosarcomas before chemotherapy, and the relationship between XIAP expression and clinicopathologic features of the patients with stage lib osteosarcomas was analyzed. Results： The osteo- sarcoma specimens showed strong cell nucleus immunoreaction of XIAP while normal bone specimens showed weak. High XIAP positive expression independently predicted the patient＇s poor outcome and had no relationship with clinicopathologic variables such as age, gender, location, tumor size, rate of tumor necrosis to preoperative chemotherapy, local recurrence （P 〉 0.05）. Significant correlations were found between high XIAP expression and metastasis （P -- 0.022）, and also between high XIAP expression and final survival （P = 0.011）. Conclusion： XIAP plays an important role in the development of stage liB osteosarcomas and XIAP expression of biopsy specimens before chemotherapy can be seen as a promising prognostic marker in early predicting the outcome of patients suffering from stage lib osteosarcomas,

siRNA targeting Livin decreases tumor in a xenograft model for colon cancer

AIM： To evaluate the effect of silencing Livin gene expression with siRNA to apoptosis and proliferation in a colon cancer cell line. METHODS： To investigate the anticancer effect of silencing Livin gene expression, we established an siRNA transfected cell line using the HCT116 colon cancer cell line. After confirming the successful transfection, MTT assay, flow cytometry and annexin V staining were em- ployed to evaluate the antiapoptotic effect. To confirm the in vivo effect of Livin-siRNA, different doses of LivinsiRNA were injected into xenografted tumors in BALB/c nude mice model. RESULTS： Livin expression was dramatically decreased after siRNA transfection, especially at 25 umol/L of siRNA, but this suppression was not dose-dependent. The cell count at 18 h after transfection was significantly reduced as compared with controls （P 〈 0.01）, but tended not to decrease proportionally depending on transfected dose or time. MTT assay revealed that silencing the Livin gene suppressed cellular proliferation at 18 h after transfection （P = 0.04）; however, the inhibitory effect disappeared thereafter. Also, there was no significant difference in cellular proliferation depending on siRNA dose. The rate of apoptosis also increased with silencing of the Livin gene. In vivo, the tumor size significantly decreased after Livin- siRNA injection at 20 umol/L concentration （P = 0.03）. There were no significant body weight changes of mice after siRNA injection. Histologic examination revealed no significant toxic reaction in kidney, liver and brain of mice. CONCLUSION： siRNA-mediated downregulation of Livin expression can induce apoptosis in colon cancer in vitro and in vivo, which suggests the possibility of new cancer therapeutics using siRNA.

Effect of electroacupuncture on c-IAP1 mRNA and C-IAP2 mRNA in synovial tissues of rats with adjuvant arthritis

Objective: Td observe the therapeutic effect of electroacupuncture (EA) at Zusanli (ST 36), Guanyuan (CV 4) and Ashi points on adjuvant arthritis rats, and explore the mechanism of EA treatment of rheumatoid arthritis (RA). Methods: Sixty male rats were randomly divided into a normal group, a model group, a methotrexate group and an EA group, with 15 rats in each group. Rats in the normal group and the model group were routinely raised and did not receive treatment;rats in the methotrexate group received methotrexate at a dose of 0.35 mg/(kg bw), twice a week for 3 weeks;rats in the EA group received acupuncture at Zusanli (ST 36), Guanyuan (CV 4) and Ashi points, and the bilateral Zusanli (ST 36) and Ashi points were connected to EA apparatus, once a day for 3 weeks. The general status, the swelling degree of the toe, the arthritis index (Al) score, the pathological morphology of the ankle joint, and the mRNA expressions of cellular inhibitor of apoptosis protein (c-lAP) 1 and C-IAP2 in joint synovial tissue cells of the rats in each group were observed. Results: The swelling degree of the toe, Al score and mRNA expressions of c-IAPl and C-IAP2 in the model group were significantly higher than those in the normal group (all P<0.05). Compared with the model group, the swelling degree of the toe, Al score and mRNA expressions of c-IAPl and C-IAP2 in the methotrexate group and the EA group improved (P<0.01 or P<0.05);the expressions of c-IAPl mRNA and C-IAP2 mRNA in rat synovial tissues in the EA group were significantly higher than those in the methotrexate group (P<0.01). Conclusion: EA alleviates joint swelling in rats with adjuvant arthritis. The mechanism may be related to suppress!ng mRNA expressions of c-IAPl and C-IAP2, thus to induce apoptosis of synoviocytes.

Functional analysis of a miRNA‐like small RNA derived from Bombyx mori cytoplasmic polyhedrosis virus

Bombyx mori cytoplasmic polyhedrosis virus(BmCPV)is a major pathogen of the economic insect silkworm,Bombyx mori.Virus‐encoded microRNAs(miRNAs)have been proven to play important roles in host–pathogen interactions.In this study we identified a BmCPV‐derived miRNA‐like 21 nt small RNA,BmCPV‐miR‐1,from the small RNA deep sequencing of BmCPV‐infected silkworm larvae by stem‐loop quantitative real‐time PCR(qPCR)and investigated its functions with qPCR and lentiviral expression systems.Bombyx mori inhibitor of apoptosis protein(BmIAP)gene was predicted by both target prediction software miRanda and Targetscan to be one of its target genes with a binding site for BmCPV‐miR‐1 at the 5′untranslated region.It was found that the expression of BmCPV‐miR‐1 and its target gene BmIAP were both up‐regulated in BmCPV‐infected larvae.At the same time,it was confirmed that BmCPV‐miR‐1 could up‐regulate the expression of BmIAP gene in HEK293T cells with lentiviral expression systems and in BmN cells by transfecting mimics.Furthermore,BmCPV‐miR‐1 mimics could up‐regulate the expression level of BmIAP gene in midgut and fat body in the silkworm.In the midgut of BmCPV‐infected larvae,BmCPV‐miR‐1 mimics could be further up‐regulated and inhibitors could lower the virus‐mediated expression of BmIAP gene.With the viral genomic RNA segments S1 and S10 as indicators,BmCPV‐miR‐1 mimics could up‐regulate and inhibitors down‐regulate their replication in the infected silkworm.These results implied that BmCPV‐miR‐1 could inhibit cell apoptosis in the infected silkworm through up‐regulating BmIAP expression,providing the virus with a better cell circumstance for its replication.

circBIRC6 contributes to the development of non-small cell lung cancer via regulating microRNA-217/amyloid beta precursor protein binding protein 2 axis

Background:Circular RNAs(circRNAs)are considered to be important regulators in cancer biology.In this study,we focused on the effect of circRNA baculoviral inhibitor of apoptosis protein(IAP)repeat containing 6(circBIRC6)on non-small cell lung cancer(NSCLC)progression.Methods:The NSCLC and adjacent non-tumor tissues were collected at Shanghai Ninth People's Hospital.Quantitative real-time polymerase chain reaction was conducted for assessing the levels of circBIRC6,amyloid beta precursor protein binding protein 2(APPBP2)messenger RNA(mRNA),baculoviral IAP repeat containing 6 mRNA(BIRC6),and microRNA-217(miR-217).Western blot assay was adopted for measuring the protein levels of APPBP2,E-cadherin,N-cadherin,and vimentin.Colony formation assay,transwell assay,and flow cytometry analysis were utilized for evaluating cell colony formation,metastasis,and apoptosis.Dualluciferase reporter assay and RNA immunoprecipitation assay were carried out to determine the interaction between miR-217 and circBIRC6 and APPBP2 in NSCLC tissues.The murine xenograft model assay was used to investigate the function of circBIRC6 in tumor formation in vivo.Differences were analyzed via Student's t test or one-way analysis of variance.Pearson's correlation coefficient analysis was used to analyze linear correlation.Results:CircBIRC6 was overexpressed in NSCLC tissues and cells.Knockdown of circBIRC6 repressed the colony formation and metastasis and facilitated apoptosis of NSCLC cells in vitro and restrained tumorigenesis in vivo.Mechanically,circBIRC6 functioned as miR-217 sponge to promote APPBP2 expression in NSCLC cells.MiR-217 inhibition rescued circBIRC6 knockdown-mediated effects on NSCLC cell colony formation,metastasis,and apoptosis.Overexpression of miR-217 inhibited the malignant phenotypes of NSCLC cells,while the effects were abrogated by elevating APPBP2.Conclusion:CircBIRC6 aggravated NSCLC cell progression by elevating APPBP2 via sponging miR-217,which might provide a fresh perspective on NSCLC therapy.

Cloning of human XAF1 gene promoter and assay of its transcription activity in a variety of cell lines

To investigate the regulation of tumor sup-pressor XAF1 gene expression in digestive system cancers,we studied XAF1 gene promoter transcription activity and mRNA level in digestive system cancer cell lines(human hepatoma cell line HepG2,human colon cancer cell line LoVo,and human gastric cancer cell line AGS)and nontumor cell lines(human embryonic liver cell line L02(L02 cells)and human embryonic kidney 293 cells[HEK293 cells])as controls.1395-bp-promoter fragment of XAF1 gene was amplified by polymerase chain reaction(PCR)and cloned into pGL3-basic vector and pEGFP-1 vector to assay its promoter transcription activity.The plasmids were transfected into a variety of cell lines by lipofectamine 2000.The promoter transcription activity was determined by dual-luciferase report assay,and enhanced greenfluorescent protein(EGFP)-positive cells were detected byfluorescence microscope.The expression of XAF1 mRNA in HEK293 and L02 were significantly higher than that in any of the three digestive system cancer cell lines.The dual-luciferase reporter assay showed that the promoter transcription activity in digestive system tumor cell lines transfected with pGL3-XAF1p promoter was apparently lower than that of both HEK293 and L02 cells.Expression of greenfluorescent protein(GFP)under the control of XAF1 promoter in the three digestive system cancer cell lines was lower than that of both HEK293 and L02 cells.The activities of pGL3-XAF1p in the three digestive system cancer cell lines after treatment with heat stress were significantly lower than those in the unstressed cells.The results suggested that remarkably down-regulated XAF1 mRNA expression in digestive system cancer cell lines may be due to loss of transcription activity of XAF1 promoter.