Establishment of EGFR-T790M mutation detection method for non-small cell lung cancer based on droplet digital PCR / 中华检验医学杂志

Objective:Use the droplet digital PCR (ddRCR) technology to establish, optimize and evaluate the method of EGFR-T790M mutation detection.Methods:The relevant probes and primers were designed for EGFR-T790M mutations. The ddPCR reaction system was established, the optimal annealing temperature was set and the basic performance of the method was tested. On this basis, from January 2019 to October 2019, 72 cell-free DNA (cfDNA) samples from NSCLC patients were collected from Shandong Cancer Hospital Affiliated to Shandong First Medical University, and clinically verified. The consistency of the gene mutation detections with Bole ddPCR products was analyzed using Kappa test.Results:The ddPCR reaction system was established and optimized. Linear evaluation showed the R2 value was greater than 0.99. Using ddPCR, the blank detection limit was determined to be the numbers of mutant droplets≥2, with excellent specificity. For the sensitivity analysis, the lower limit of mutation detection was determined to be at least 0.05%. In the repeatability and inter-assay precision tests, the results had a coefficient of variation( CV)<20%. The relative deviation of the results was within the range of±10% for the accuracy analysis. Using the established T790M mutation detection method, 72 samples from the NSCLC patients were tested for genetic mutation in cfDNA, and the overall agreement with the Bole ddPCR products was 91.67% (66/72, Kappa=0.749; P<0.001). Conclusion:Using ddPCR, the method of EGFR-T790M mutation detection for NSCLC was successfully established.

Expression and Significance of ACIN1 mRNA in Platelets of Lung Cancer / 中国肺癌杂志

BACKGROUND@#During the occurring and developing of tumor, tumor-educated platelets mRNA profiles were altered. Since platelets are anuclear, the level of mRNAs is probably post-transcriptional regulated by the splicing maturation of pre-mRNA and alternative splicing. Apoptotic chromatin condensation inducer 1 (ACIN1) has been shown to be a component of a splicing-dependent multiprotein exon junction complex (EJC) and was involved in mRNA metabolism associated with splicing. This study analyzed the expression of ACIN1 mRNA in platelets, and explored its potential as a biomarker of lung cancer.@*METHODS@#156 patients with lung cancer and 58 healthy controls in Shandong Cancer Hospital were collected. We isolated platelet pellets by low-speed centrifugation and extracted total RNA. The expression of ACIN1 mRNA in platelets was detected by RT-PCR, the results were analyzed statistically. And the relationship between expression of ACIN1 mRNA and clinical factors were also analyzed.@*RESULTS@#The expression level of ACIN1 mRNA in platelets of patients with lung cancer was significantly higher than that in platelets of healthy controls (P=0.015). The ROC curve showed that the area under the curve of ACIN1 mRNA for detecting lung cancer were 0.608. The expression of ACIN1 mRNA in platelets of lung cancer has no significant relationship with age, gender, pathological type and metastasis or not (P>0.05).@*CONCLUSIONS@#ACIN1 mRNA was highly expressed in platelets of lung cancer patients, and the detection of its expression level might have potential clinical value for the diagnosis of lung cancer.

Predictive Markers for Treating Efficacy of PD-1/PD-L1 Inhibitors in Patients with Lung Cancer: A Review of the 18th World Conference on Lung Cancer / 中国肺癌杂志

Programmed cell death-1 (PD-1)/programmed cell death-ligand 1 (PD-L1) checkpoint blockades have dramatically changed the treatment of non-small cell lung cancer (NSCLC). But we still have no definite biomarkers that may predict the efficacy of treatment by PD-1/PD-L1 inhibitors. In the 18th World Conference on Lung Cancer, the biomarkers that may predict the efficacy of treatment by PD-1/PD-L1 inhibitors in patients with lung cancer has been a popular topic, and it has huge potential in the future. In order to enable more patients to get more benefits from treatment, researchers are looking forward to finding the optimum biomarkers. By organizing and summarizing the information about the biomarkers predicting PD-1/PD-L1 in patients with lung cancer, this review mainly focused on the following six aspects to introduce: expression of PD-L1; tumor mutational burden and the ability of mutation repair, malignant tumor driver mutation, biomarker of immunological effect, blood cell account, comprehensive analysis model. We are hoping to help doctors to find the best biomarker, then much more lung cancer patients could obtain antitumor effects in PD-1/PD-L1 inhibitors treatment.
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Current status of saliva-based biopsy / 中华检验医学杂志

The advantages of saliva-based biomarkers detection in cancer were accurate,simple and noninvasive.Currently,biomarkers validated for saliva detection include protein,mRNA,miRNA and DNA,using PAGE,microarray and sequencing,respectively.Analysis of literatures shows;that saliva biopsy plays an important role in cancer diagnosis,clinical treatment and prognosis.Due to the design of experiments reported werediversification,a large number of validations are necessary to standardize saliva collecting,processing,testing methods and quality assurance.

The preparation and experimental study of a new sentinel lymph node tracer / 中国癌症杂志

Background and purpose:Sentinel lymph node biopsy is regarded as the standard of care in pa-tients without clinical axillary lymph node metastases in early-stage breast cancer. Accurate detection of sentinel lymph node is an important step for staging, prognosis, and treatment. In this study, a new sentinel lymph node tracer was produced by the rituximab to combine with the lfuorescence tracer (indocyanine green, ICG), and to identify the most appropriate combination ratio of the two agents. Its biological property and safety limitation were evaluated.Methods:Rituximab was combined directly with ICG. The new tracer was analyzed for labeled rate by instant thin-layer chroma-tography-silica gel, molecular integrity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and molecular immune activity by ELLAS. The safety limitation was tested according to the Chinese Pharmacopeia. The localization ability of sentinel lymph node was tested in mice.Results:The new tracer was intact and kept the immune activity of rituximab. The ICG labeled rate of rituximab was 100%. The new tracer was bacteria and pyogen free, and was safe to body with location injection. The most appropriate combination ratio of rituximab and ICG was 4∶1 and 6∶1 with the best sentinel lymph node imaging. The location of sentinel lymph node identiifed by the new tracer was accorded with the radiotracer.Conclusion:The preparation method of the new sentinel lymph node tracer is simple and no radioactive injury. The new tracer has no bacteria, no pyogen and no acute toxicity, and can be used in sentinel lymph node visual-ization.

Application of circulating DNA in colorectal cancer / 国际肿瘤学杂志

Circulating DNA is defined as a kind of extracellular DNA that exists in plasma,cerebrospinal fluid and synovial fluid.The concentration of circulating DNA of cancer patients is significantly higher than that in healthy people.The genetic and epigenetic alterations of circulating cell-free nucleic acids are relevant to cancer development and progression,for example,gene mutation,DNA methylation and microsatellite instability and so on.The quantitative and qualitative detection of circulating DNA shows promising potential value in cancer screening,diagnosis,disease monitoring treatment and prognosis.

Influence of LOX downregulation by RNAi on hypoxic metastasis of human lung cancer cell and the underlying molecular mechanism / 中华内分泌外科杂志

Objective To observe the influence of lysyl oxidase(LOX)downregulation via RNAi on hypoxic metastasis of human lung cancer cell 95D and stduy its molecular mechanism.Methods LOX siRNA was used to transfect 95D cell line in normoxia (19％ O2 ).After 24-hour incubation,the cells were cultured in hypoxic incubator (0.5％ O2 ) for 24h.Real-time PCR assay was applied to detect LOX mRNA and Snail mRNA expression.Levels of Src,phosphorylation of Src (P-Src Y418 ) and Snail protein were determined by Western blot assay.Transwell chamber was used to evaluate the cellular invasion potential.Results Compared with 95D cells under normoxic conditions,hypoixa treatment increased LOX mRNA expression by 14 times and invasion ability by 2.12 times respectively.Compared with siRNA control group,LOX siRNA transfection decreased LOX mRNA expression,the invasion ability of hypoxic cells,and the protein expression of P-Src Y418 and Snail by 70％ - 75％,about 30％,and about 40％ respectively (P ＜ 0.05).However,it didn't affect the expression level of Src protein or Snail mRNA ( P ＞ 0.05).Conclusions Impaired metastatic potential of hypoxic human lung cancer cell induced by LOX downregulation is associated with reduced expression level of Src activation and Snail protein.The present data provids experimental evidence for LOX as a potential target for prevention and treatment of lung cancer metastasis under hypoxia.

Effect of RNA interference for OPN on MDA-MB-231 cells and the growth of transplanted tumor in nude mice / 中华普通外科杂志

ObjectiveTo investigate the role of OPN in human breast cancer cell line ( MDA-MB-231) by using small interfering RNA to specifically knockdown OPN expression. MethodsOPN ShRNA expression vector was stably transfected to MDA-MB-231 cell line.The expression of OPN mRNA and protein were analyzed using reverse transcription polymerase chain reaction (RT-PCR)and Western blot,respectively.The growth of MDA-MB-231 cells were observed by MTT.The effect of OPN siRNA on the transplanted tumor growth and tumor hypoxia were assessed in nude mice. ResultsThe expression level of OPN in MDA-MB-231 cells were significantly lower under hypoxia or normoxia(P ＜ 0.05 ).OPN silence with RNAi significantly inhibited the invasion ability and proliferation of MDA-MB-231 cell lines (P ＜ 0.01 ).Inhibition of OPN with RNAi significantly inhibited the growth ability of MDA-MB-231 cells in vivo(P ＜0.05).The tumor hypoxia significantly decreased(P ＜ 0.05). ConclusionsOPN silence with RNAi can effectively inhibit cell proliferation and tumor growth of MDA-MB-231 cells,and decrease the bypoxia level of MDA-MB-231 transplanted tumor in nude mice.

Study on radiosensitivity by targeting HIF-1α in human lung cancer and growth of the transplanted tumors / 中华放射医学与防护杂志

Objective To observe the radiosensitivity by targeting HIF-lα in human lung cancer and the effects on tumor growth in nude mice.Methods Radiosensitivity of A549 and A549/HIF-1α ( － ) cells were tested by clonogenic forming assay.A549/HIF-1 α( － ) cells and A549 cells were injected into the male BALB/C nude mice.Tumor growth was observed.The expression of HIF-1α and microvessel density were detected by immunohistochemistry method.Results SERs of HIF-1α gene silencing were 1.03 in normoxia and 1.65 in hypoxia.The sizes of tumor xenografts derived from A549/HIF-1α( － ) cells were significantly reduced compared to those of the xenografts derived from A549 cells.HIF-1 α protein staining result showed a dramatic decrease in tumors from A549/HIF-1α ( － ) mice.The microvessel densities (MVD) were 19.83 ± 4.09 in A549 group and 11.61 ±3.04 in A549/HIF-lα (-)group(F=15.57,P ＜0.05 ).Conclusions Hypoxia-induced radio-resistance in lung cancer A549 cells could be reversed by silencing the HIF-1α.It also retards the growth of tumor xenografts,decreases HIF-1α expression and reduces the vascularity.

Advances in the research of astrocyte elevated gene-1 / 中国病理生理杂志

Astrocyte elevated gene-1 (AEG-1) was cloned as an human immunodeficiency virus -1-inducible and tumor necrosis factor-α-inducible transcript in primary human fetal astrocytes by a rapid subtraction hybridization approach. AEG-1 down-regulates the expression of the glutamate transporter EAAT2, thus, it is implicated in glutamate-induced excitotoxic damage to neurons as evident in HIV-associated neurodegeneration. Meanwhile, AEG-1 expression is elevated in subsets of breast cancer, prostatic cancer, glioblastoma multiforme and melanoma cells, having a dual specificity phosphatase activity. Overexpression of AEG-1 increases and siRNA inhibition of AEG-1 decreases migration and invasion of human glioma cells, respectively. Recent observations indicate that AEG-1 exerts its effects by activating the nuclear factor kappa B (NF-κB) pathway and AEG-1 is a downstream target of Ha-ras and plays an important role in Ha-ras-mediated tumorigenesis. These findings are intensifying interest in AEG-1 as a crucial regulator of tumor progression and metastasis and as a potential mediator of neurodegeneration.

Construction of the recombinant adenovirus RNA interference vector of human hypoxia-inducible transcription factor-1? gene and its effect on expression of HIF-1? gene in human lung adenocarcinoma cells SPCA-1 / 基础医学与临床

Objective To construct the RNA interference(RNAi)recombinant adenovirus vector targeting at human hypoxia-inducible transcription factor 1?(HIF-1?)and to evaluate its effect on human lung adenocarcinoma cell line SPCA-1.Methods The recombinant adenovirus Ad was constructed.HIF-1? inserted with HIF-1? RNAi fragment via AdEasy system.The virus was purifed by CsCl gradient centrifuge.The functional titer of recombinant adenovirus was measured by transfection test in HEK 293 cells.SPCA-1 cells were transducted with 2 multiplicity of infection(MOI)Ad.HIF1? in vitro,the expression rate of green fluorescence protein(GFP)was recorded by flow cytometry,HIF-1? mRNA and protein level was measured by Real-Time RT-PCR and flow cytometry.ResultsThe recombinant shuttle plasmid PAdTrack.HIF-1? and adenovirus plasmid Ad.HIF-1? were all correct shown by enzyme digestion confirmation.The plasmid pAd.HIF-1? was transducted into HEK293 cells,15%GFP expressionwere seen after 3 days.The final titers of recombinant adenovirus were 5.0?1010 TU/mL.SPCA-1 cells was transducted by Ad.HIF-1? in vitro for 48 h,GFP expression rate was 92%,HIF-1? mRNA and protein level decreased 89% and 87%,respectively.Conclusion RNAi adenovirus vector of human HIF-1? gene has been successfully constructed,which could facilitate the research onHIF-1? gene related gene therapy for lung cancer.

microRNA and human malignant tumors / 基础医学与临床

microRNAs(miRNAs)are a class of 18～26 nt small non-coding single strand RNA molecules,which negatively regulate the expression of a variety of genes at post-transcriptional level by binding to complementary sites on target mRNA.Mutation,deletion or high expression of miRNA was found to correlate with various human cancers.miRNAs involved in tumor cell proliferation,differentiation and apoptosis processes as function as oncogenes or tumor supressors It is predictable that microRNA might be applied in the diagnosis and treatment of malignant tumors.

Recognition of microRNAs and its-targeting genes / 基础医学与临床

microRNAs (miRNAs) are a recently discovered family of small non-coding RNAs, which exist in eukaryotes and regulate gene expression post-transcriptionally by binding to complementary sites on target mRNAs. MiRNAs play important roles in regulation of cell proliferation, development, apoptosis and cell differentiation. It’s predicted that about 1% human known genes encode microRNAs and microRNAs may regulate 10～30%of the genes in the human genome. The paper focus on the knowledge about how to recognize miRNAs and identify their target mRNAs.

Single nucleotide polymorphism of human APM1 detected by using allelic 3′-terminal specific primers real-time PCR / 基础医学与临床

Objective To develop an analyzing technique of single nucleotide polymorphism(SNP) based on non-probe real-time quantitative PCR, and to explore the relationship between SNP in human adiponectin (APM1) gene and type 2 diabetes (T2DM).Methods Design primers with specific 3′-terminal nucleotides and perform Real-time PCR using SYBR Green dye. Genotypes were determined by comparing the amplification efficiency of each pair of primer and verified by sequencing. SNPs 45 and 276 in APM1 of 20 T2DM, 24 obesity and 28 healthy persons were analyzed. Results This technique could discriminate genotype as efficient as sequencing. The genotype of APM1 at +45 site was significantly correlated with T2DM (P

Chemotherapy sensitivity and associated gene expression in human breast cancer cells / 基础医学与临床

Objective To explore the relationship of chemotherapy sensitivity and expression of multidrug resistance genes and apoptosis regulation genes in human breast cancer cell lines.Methods MTT assay was used to detect the sensitivity to adriamycin(ADM),cisplatinum(DDP),mitomycin C(MMC),fluorouracilum(5-Fu),carmustine(BCNU) in five breast cancer cell lines including Bcap37,MCF-7,T47D,MDA-MB-231and MDA-MB-435.Multidrug resistance genes including P-glycoprotein(P-GP),Glutsthione-s-transferases-?(GST-?),Lung resistance protein(LRP),multidrug resistance related protein(MRP),MGMT and apoptosis regulation genes FAS,BCL-2,P53 and P16 were examined by flow cytometry(FCM).Results The chemotherapy sensitivity was obviously divergent in different breast cancer cell lines.The correlation analysis showed that there were positive correlations between the sensitivity to 5-Fu in breast cancer cell lines and the expression of P16.There was no correlation among the sensitivity to other drugs and expression of other genes.Conclusions The sensitivity to 5-Fu is related to the expression of P16 in breast cancer cell lines.

Establisment of BCRP expressing ovary cancer cell line 3AO/BCRP and its biological profile / 基础医学与临床

Objective To explore the function of BCRP in ovary cancer and establisment of BCRP expressing ovary cancer cell line 3AO/BCRP.Methods We construct the BCRP expressing cell line 3AO/BCRP.Extract the total RNA of MCF-7 cells,clone the whole length of BCRP gene by RT-PCR and ligate the gene to pcDNA3.1(-),transfect the positive clones to 3AO cells and select positive cell clones with G418,identify the survival cells by MTT,efflux assay,western blot.Results The multidrug resistance index of 3AO/BCRP to mitoxantrone increased to 11.58 times;the efflux of 3AO/BCRP to mitoxantrone enhanced;the relative level of BCRP mRNA in 3AO/BCRP increased and western blot indicate that the 3AO/BCRP cells can express more protein than 3AO cells.Conclusion 3AO/BCRP is a reliable BCRP expressing cell model,the establishment of this cell line has laid basis for further study of the mechanism of BCRP.

Construction recombinant adenovirus vector expressing murine endostatin and inhibition of blood endothelial cells growth / 生物医学工程学杂志

We constructed recombinant adenovirus vector expressing murine endostatin and evaluated the Inhibition of human umbilical vein endothelial cells (HUVEC). We proved that endostatin significantly suppressed the S phase fraction, inhibited proliferation and increased the apoptotic index of HUVEC.

Anti-cancer effect of cytotoxic T lymphocytes activated by sensitized dendritic cells / 中国病理生理杂志

AIM: To investigate the anti-cancer effect of cytotoxic T lymphocytes (CTL) activated by sensitized dendritic cells (DCs). METHODS: Immature DCs were induced in vitro from peripheral blood monocytic cells (PBMC) and sensitized by adding tumor cells antigen extract. DCs were identified by their morphology and surface markers. MTT assay was used to evaluate the killing activity of CTL activated by sensitized DCs. The effects of specific CTL cells on inhibiting transplanted tumor HT-29 growth and on preventing HT-29 tumor generation were evaluated by injecting CTL into nude mice. RESULTS: After cultured for seven days, a large number of activated DCs were obtained with typical morphology, extensive stimulatory proliferation capacity and high CD80 (63.5%), CD83 (67.6%) and CD3/HLA-DR (83.2%) expressions. The killing activity of CTL at 20∶1 ratio of effective cells to target cells was more than 75% to tumor cells, 35%-45% to homologous cell line and weaker to other germ cell line (P

Inhibitory effect of arsenic trioxide on human glioma cell growth / 中国病理生理杂志

AIM: To investigate the inhibitory effect of arsenic trioxide on human glioma cell line U251 growth, change of gene expression and intracellular calcium content. METHODS: MTT method was used to observe the growth inhibition effect. Cell cycle, positive rate of proliferation cell nuclear antigen (PCNA), apoptosis associated protein Fas and Bcl-2, and intracellular calcium ion (IECa~ 2+ ) levels were measured by flow cytometry in U251 cells treated with different doses of As_2O_3. Apoptosis was detected with annexin V-FITC+PI dual parameter. RESULTS: As_2O_3 inhibited the growth of U251 cells dramatically. There were obvious dosage-effect and time-effect correlations (P0.05). The cell cycle was arrested in G_2M phase. Apoptosis occurred in U251 cells treated with As_2O_3 by annexin V-FITC+PI dual parameter detection. CONCLUSION: As_2O_3 inhibits the growth of U251 cells in vitro dramatically and induces apoptosis. The mechanism is probably associated with the improvement of Fas expression and IECa~ 2+ levels, decrease in PCNA protein expression and cell cycle arrest.