Objective The objectives of this study were to screen and identify monoclonal antibodies against hepatoma stem cells by screening for hepatoma spheroid cells,and to provide candidate therapeutic monoclonal antibodies for targeting cancer stem cells to treat hepatic cancer.Methods Hepatic cancer stem cells were enriched by serum-free suspension culture.Immunofluorescence,cisplatin resistance assay,Real-time qPCR,subcutaneous tumor formation in nude mice,and other methods were used to screen and identify anti-hepatocarcinoma stem cell monoclonal antibodies.Immunohistochemistry was used to identify the expression of antigen recognized by monoclonal antibody in liver cancer tissues.The antigen was identified by mass spectrometry.Results MHCC97L cells were able to form cell spheres in serum-free suspension culture and were labeled with PKH26 dye.Flow cytometry showed that the expression of CD90⁺ in MHCC97L spheroid cells was 3.4 times higher than that in the parental cells.In the inhibition experiment of serum-free spheroid,6 monoclonal antibodies significantly inhibited MHCC97L cells in serum-free medium,and inhibitory rates were 54.67%,50.33%,45.73%,42.26%,39.11%,and 37.63%,respectively.The results of immunofluorescence showed that monoclonal antibodies 28C10 and CD90 were colocalized in MHCC97L cells.The results of real-time qPCR showed that the expression of Sox-2 and Oct-4 in MHCC97L 28C10⁺ cells was significantly higher than those of MHCC97L 28C10^- cells.Flow cytometry showed that the ratio of 28C10⁺ in MHCC97L cells and its sphere cells were 7.98% and 10.7%,respectively.The ratio of 28C10⁺ cells was increased by 1.34 times.The in vitro globing ability and invasive ability of 28C10⁺ cells obtained by flow cytometry were significantly higher than those of 28C10^- cells.The results of CCK-8 assay showed that 28C10⁺ cells were resistance to cisplatin in 28C10^- cells,which are 1.96 g/ml and 1.16 g/ml,respectively.Tumorigenic assay showed that 28C10⁺ cells were inoculated subcutaneously with 2×10⁴ cells into the nude mice,and tumors were formed in 2 months,with 40% of tumor formation rate.Another nude mouse that did not form a tumor had formed a lung metastasis(1/5).Immunohistochemistry showed that the target antigen positive rate of monoclonal antibody 28C10 in hepatic cancer tissues was about 72.0%(77/107),while it was lowly expressed in adjacent tissues,and the difference was significant.Mass spectrometry showed that the antigen recognized by 28C10 was HSP90α.Conclusion The MHCC97L spheroid cell model is successfully used to identify a monoclonal antibody that specifically recognizes hepatoma stem cells,which provides a foundation for antibody therapy targeting hepatic cancer stem cells.

Objective The Objective of this study was to explore the differences of serum metabolomics between small cell lung cancer(SCLC)patients and healthy volunteers,and to discover serum potential biomarkers for identification and small cell lung cancer staging.Methods Ultra-performance liquid chromatography time of flight mass spectrometry(UPLS-TOF/MS)was used to establish the serum metabolic profile of SCLC.Principal Component Analysis(PCA)and orthogonal hidden variables were analyzed by the EZinfo2.0 software.Orthogonal Partial Least Squares Discriminant Analysis(OPLS-DA)was used to analyze the metabolic differences between the case and normal control groups.Through cluster analysis using HMDB and METLIN database to search for the exact mass-to-charge ratio of the difference,preliminary identification of some substances with significant differences was carried out.Results Ten differential metabolites such as lysophosphatidylcholine between patients and control groups were screened and identified by mass spectrometry and database search.There were 10 different metabolites such as glycocholic acid in the contour analysis of SCLC patients with different stages.Conclusion There is a significant difference in serum metabolism between SCLC patients and healthy controls.The discovery of differential metabolites provides experimental evidence for the identification of small cell lung cancer and potential markers of staging.

Objective The aims of this study were to determine the expression of CXCR family proteins in various subtypes and adjacent tissues of breast cancer and its relationship with prognosis,and to provide a reference for clinical diagnosis,treatment and prognosis of breast cancer.Methods The mRNA expressive profiles of CXCR family proteins in paracancerous tissues and different subtypes of breast cancer tissues were obtained from the TCGA(The Cancer Genome Atlas)database.The prognostic survival analysis of each differentially expressed protein was obtained using the PRECOG website.Results Except for CXCR1,the expression of CXCR family proteins in breast cancer tissues and adjacent tissues was statistically different(P＜0.05).CXCR2P1,CXCR3,CXCR4,CXCR5 and CXCR6 were highly expressed in breast cancer tissues,CXCR2 and CXCR7 were lowly expressed in breast cancer tissues.The expressive levels of CXCR3,CXCR4 and CXCR7 were associated with the prognosis of patients.Conclusion The expressions of CXCR3,CXCR4 and CXCR7 in breast cancer tissues and adjacent tissues is significantly different,and its expression is related to the prognosis of breast cancer patients,which may be a potential target for molecular diagnosis or targeted therapy of breast cancer.

Objective The aim of this study was to investigate the effect of microinjection ibudilast on the appendix pain threshold in nucleus accumbens(NAC)of morphine-addicted rats.Methods Normal rats and morphine-addicted rats were injected with ibudilast(5 μg/2 μL)or saline(2 μL)into NAC through glass microelectrode.The tail-flick period(TFL)was used as an indicator to observe the thermal pain threshold before and after ibuilast injection.Results After injected ibudilast 5 μg into the NAC of normal rats and morphine-added rats,the TFL was prolonged compared with that before injection,and the TFL prolongation of morphine-addicted rats was more significant than that of normal rats.There was no significant change in TFL after 2 μL of saline was injected into the NAC of normal rats and morphine-addicted rats.Conclusion Microinjection of ibudilast into NAC of normal rats and morphine-added rats can cause analgesic effect,and the analgesic effect of morphine-addicted rats is more significant.

Objective The aim of this study was to investigate the expression of CCDC8(coiled-coil domain containing 8,P90)and transforming growth factor β 1(TGF- β1)in none small cell lung cancer(NSCLC),and its relationship with clinicopathological characteristics.Methods The expression of CCDC8 and TGF-β1 in NSCLC was analyzed in the TCGA database.The Kaplan-Meier curve was plotted to analyze its correlation with survival of patient.The expression of CCDC8 and TGF-β1 in 204 cases of conventional paraffin-embedded NSCLC was detected by immunohistochemical staining and their relationship with clinicopathological features of NSCLC was analyzed.The relationship between the expression of CCDC8 and TGF-β1 and the overall survival(OS)of patients was analyzed by Cox regression.Results The expression of CCDC8 in NSCLC patients was positively correlated with TGF-β1 expression in the TCGA data(P＜0.05).OS was low in patients with the low expression of CCDC8(HR=1.32,P=0.0437).In 204 NSCLC tissues,CCDC8 expression was positively correlated with TGF-β1 expression(P=0.023).Cox univariate analysis of CCDC8 expression in NSCLC was associated with OS(P=0.013);whereas the expression of CCDC8 and TGF-β1 was not associated with survival in patients with NSCLC(P=0.967,P=0.816).Cox multivariate analyses of TNM staging and CCDC8 were prognosis factors in patients with NSCLC(P=0.016).Conclusion The expression of CCDC8 is correlated the expression of TGF-β1 in NSCLC.CCDC8 may be a prognostic factor for patients with NSCLC.

Objective The aim of this study was to investigate the relative expression of zinc finger protein ZNF436 in human gastric cancer cells and tissues and its mechanism of ZNF436 on migration and invasion of gastric cancer MKN-28 cells.Methods RT-qPCR and immunohistochemistry were used to determine the expression of ZNF436 at the levels of mRNA and protein in human gastric cancer cells,gastric cancer and matched paracancerous tissues,and normal human gastric tissues.ZNF436 expression and prognostics were analyzed through online data.The ZNF436-siRNA(experimental group)and NC-siRNA(control group)plasmids were transfected into MKN028 cells.RT-qPCR was used to confirm the knockout efficiency of ZNF436 after transfection for 48 hours.The transwell assay was used to analyze the effect of ZNF436 on cell migration and invasion after knockout ZNF436.The effect of ZNF436 on cell metastasis was verified by nude mice metastasis experiments.The effect of ZNF436 on WNT/β-catenin signaling pathway was verified by immunoblotting.Results Compared with normal gastric epithelial cells,ZNF436 was highly expressed in human gastric cancer cells(P＜0.001).ZNF436 was highly expressed in gastric cancer tissues compared with adjacent normal tissues and normal human gastric tissues(P＜0.001).The expression of ZNF436 was associated with the prognosis of gastric cancer from online data(P=0.0028);the high expression of ZNF436 gene,the worse prognosis of patients(P＜0.001);When compared with the control group,the migration and invasion ability in the experimental group was significantly inhibited(P＜0.001);the size and volume of metastasis in nude mice in the experimental group were significantly lower than those in the experimental group(P＜0.001).After the low expression of ZNF436,the WNT/β-catenin signaling pathway and its downstream cytokines were significantly inhibited.Conclusion ZNF436 is up-regulated as a potential oncogene in human gastric cancer and participates in the progression of gastric cancer by promoting WNT/β-catenin signaling pathway.

Objective The Objective of this study was to investigate the role and molecular mechanism of Atractylenolide Ⅰ in the progression of lung cancer.Methods qRT-qPCR and immunohistochemistry were used to detect the expression of TLR4 and MyD88 at levels of mRNA and protein in lung cancer and adjacent tissues.Transwell and MTT assays were used to detect effects of atractylenolide I(100 μM)on the invasion,migration and proliferation of A549 cells.Western blot was also used to detect the effect of atractylenolide I on the expression of TLR4 and MyD88 proteins.Results The expression of TLR4 and MyD88 at levels of mRNA and protein was highly expressed in lung cancer tissues when compared to adjacent tissues(P＜0.05).Compared with the control group,the invasion ability of A549 cells in the Atractylenolide I group was significantly decreased,and the proliferative activity was inhibited(P＜0.05).Atractylenolide I inhibited the expression of TLR4 and MyD88 protein in A549 cells(P＜0.05).Conclusion Atractylenolide I inhibits the invasion and metastasis of lung cancer A549 cells by inhibiting the TLR4/MyD88 pathway.

Objective The Objective of this study was to investigate the relationship between the methylation of phosphodiesterase 4C(PDE4C)gene in peripheral blood leukocytes,and its interaction with environmental factors and the pathogenesis of breast cancer.Methods A case-control study was conducted to select 402 cases of breast cancer and 470 cancer-free controls from October 2010 to December 2014.The specific DNA methylation-modified quantitative PCR method was used to detect the methylation of PDE4C gene.The use of crossover analysis and multivariate logistic regression was used to analyze the relationship between the level of gene methylation and its interaction with environmental factors and the pathogenesis of breast cancer.Results PDE4C methylation was not found to be significantly associated with the onset of breast cancer.High-frequency/high-intake of whole grains,vegetables,onion-based plants,poultry,milk,and regular exercise could reduce the risk of breast cancer;high-frequency/high-intake of pork,irregular menstrual cycle,and a high psychological stress index could increase the risk of breast cancer.PDE4C hypermethylation had a significant combining effect with edible onion-producing plants greater than 3 times/week,with an OR of 0.373(95% CI:0.208~0.668,P=0.001).Conclusion Hypermethylation of PDE4C is not an independent risk factor for breast cancer,and its combination with environmental factors affects the risk of breast cancer.

Objective The aim of this study was to investigate the expression of WEE1 in gastric cancer and its influence on prognosis of patients.Methods Seventy-eight patients with gastric cancer were enrolled.According to the presence or absence of lymph node(LN)metastasis,the patients were divided into LN(-)group and LN(+)group.The expression of WEE1 in gastric cancer tissues was detected by immunohistochemistry and RT-qRCR techniques.The influence of WEE1 expression on LN metastasis and prognosis of gastric cancer were analyzed statistically.Results (1)The positive rate of WEE1 in gastric cancer was 43.6%,of which 13 cases were a low expression and 21 cases with high expression.The positive rates of WEE1 in LN(+)group and LN(-)group were 53.8% and 23.1%,respectively.(2)The results of RT-qPCR showed that the mRNA expression of WEE1 in LN(-)and LN(+)groups was(1.32±0.21)and(3.64±0.41),respectively,and the difference was statistically significant(P＜0.01).(3)The results of ROC curve showed that the area under curve(AUC)of WEE1 mRNA expression level for LN metastasis of gastric cancer was 0.806,the sensitivity was 84.8%,the specificity was 79.6%,and the diagnostic efficacy was good.T stage and LN metastasis were independent risk factors for the expression of WEE1(P＜0.05).(4)Among 78 patients,7 patients were lost to follow-up,the rate of loss of follow-up was 8.9%,and 41 patients died within 5 years.Among them,13,10 and 18 died in the WEE1(-)group,low-expression of WEE1 group and high-expression of WEE1 group,respectively.The 5-year survival rates of the three groups were significantly different(χ²=25.67,P＜0.001).The survival rate in the low expression of WEE1 group was not significantly different from the high expression of WEE1 group(P＞0.05),but both of them were significantly low the WEE1(-)group(P＜0.05).Conclusion WEE1 has a high positive rate in gastric cancer,and is closely related to patient stage and LN metastasis.The positive expression of WEE1 is a strong signal for poor prognosis.

Objective Through the analysis of the clinicopathological features of triple-negative breast cancer(TNBC),the relationship between recurrence,metastasis,the location of metastatic lymph nodes and primary lesions in breast cancer patients with TNBC was investigated.Methods The clinical data of 763 patients with TNBC from December 2008 to December 2012 in our hospital were retrospectively analyzed.Results The onset average age of TNBC breast cancer was 49 years old,and 84.7% of patients with early stage.The recurrence and metastasis rates of lymph node-positive patients were significantly higher than those of lymph node-negative patients,and increased the number of lymph nodes(P＜0.001).In the patient with negative for lymph nodes,the primary tumor recurred in the inner quadrant and the metastasis rates were the highest(9.8% and 13.7%)(P＜0.05).Univariate analysis showed that lymph node status,surgical procedure,and radiation dose were factors influencing recurrence and metastasis in patients with TNBC(P＜0.05).Multivariate analysis showed that clinical stage and lymph node status were independent risk factors for recurrence of TNBC.Clinical stage and surgical approach were independent risk factors for the metastasis of TNBC(P＜0.05).Conclusion (1)The proportion of patients with TNBC at early stage is high.The main pathological type is invasive ductal carcinoma,and the positive expression rate of ki67 is high.(2)The prognosis of patients with positive lymph nodes was related to the number of metastatic lymph nodes,and there was not significant correlated with the location of primary tumor.For patients with negative lymph nodes,the primary tumor was located in the inner quadrant with the highest recurrence and metastasis rates.(3)The recurrence rate of TNBC patients increased with the increase of BMI,but for obese patients,the recurrence rate decreased.(4)Lymph node status,clinical stage,surgical procedure,and radiotherapy dose all affect recurrence and metastasis of patients with TNBC.(5)Clinical stage and lymph node status are independent risk factors for the recurrence of TNBC.Clinical stage and surgical approach are independent risk factors for the metastasis of TNBC.

Objective The SNPs of caspase family have been studied in breast cancer,head and neck cancer,esophageal cancer,lung cancer and other cancer.There are few studies between the SNP of CASP3,7 and the risk of gastric cancer in Chinese population.The aim of this study was to investigate the relationship between the SNP of CASP3,7 genes and the genetic susceptibility of gastric cancer using large samples.Methods Blood samples from 1000 cases of gastric cancer and 1036 cases of normal non-cancer control in Northeast China were collected for genomic DNA extraction.Based on the dbSNP NCBI database and HapMap database,potential SNP sites were selected for CASP3,7,which locate CASP3,CAS′s 3′UTR.The Tasman probe method was used for genotyping of the SNP sites of CASP3,7.The difference between the different variables in the case-control group was analyzed by the bilateral χ² test.After the Hardy-Weinberg genetic balance test for each locus,the χ² test was used to compare the genotype frequency differences between the case and control groups.Univariate and multivariate logistic regression models were used to analyze the association between genotype and disease.The stratification analysis of each locus compared the genotypes between the different sexes,ages,smoking,and drinking status.Results The χ² test compared the differences between the case and control groups.The results showed that there was a significant difference in the smoking,drinking status and smoking in packet number(Pack-years)between the case and control groups(P＜0.05).The genotype frequencies of selected loci were in accordance with Hardy-Weinberg′s law of genetic balance(P＞0.05).Combining and analyzing genotypes with risk alleles,there showed that individuals with two risk genotypes increased by 69.6% in comparison with individuals with 0~1 risk genotype.After adjusted for covariate effects,individuals with three risk genotypes were increased by 27.6% when compared to individuals with 0~1 risk genotypes.Individuals with more than one risk genotype were increased by 35% when compared to individuals with 0-1 risk genotypes.In the stratified analysis,after combination of two genes,the risk genotype in the sub-layer of age ≤ 60 years old,male,never smoking,annual smoking package ≤25,gastric non-cardiac adenocarcinoma was statistically associated with disease,i.e.more risk of gastric cancer.Conclusion Univariate analysis showed that the SNPs at the four sites selected in this study were not associated with the risk of gastric cancer.However,multivariate analysis of CASP3 and CASP7 at the four sites showed that individuals with two risk genotypes increased the risk of gastric cancer in comparison with individuals with 0~1 risk genotypes.In addition,after stratified analysis of the two sites of CASP7,the risk of gastric cancer is more obvious in people aged ≤60 years,never smoking or smoking ≤25 packs per year,and non-gastric cardia adenocarcinoma.

Objective This paper investigated the clinical application of CRT+ARC technique in locally advanced lung cancer.Methods A total of 100 patients with locally advanced lung cancer who underwent radiotherapy in our hospital from March 1,2016 to March 1,2017 were randomly assigned to the experimental and control groups.Each group consisted of 50 patients.The CRT+ARC plans were made for the experimental group,and the CRT + intensity-modulated radiotherapy(IMRT)(CRT+IMRT)plans for the control group.According to the World Health Organization(WHO)criteria,the short-term efficacy of patients was assessed.According to the imaging examination and the Radiation Therapy Organization Group(RTOG)standard,the occurrence of major side effects of radiation pneumonia was identified.Results The effective rate of treatment was 82% in the experimental group and 76% in the control group.There was no difference in the effective rate between the two groups(χ²=0.542,P=0.461).The incidence of pneumonia in the experimental group was 22%,and 18% in the control group.There was also no difference in the incidence of pneumonia between the two groups(χ²=0.250,P=0.617).Conclusion In the clinical application of locally advanced lung cancer,CRT+ARC technique has no difference in the short-term efficacy and the main side effects of radiation pneumonitis compared with CRT+IMRT.

Objective To describe and analyze the incidence and mortality of malignant tumors in Daoli District of Harbin from 2008 to 2012.Methods The tumor registration data of 2008—2012 reported by the Daoli District Cancer Registry was collected.The crude incidence and mortality,China standardization rate,World standardization rate,cumulative rate and truncated incidence rate of major malignant tumors were calculated and trends of the incidence and mortality of malignant tumors in 5 years were analyzed.Results From 2008 to 2012,there were 10 893 cases of malignant tumors in Daoli District of Harbin,including 5879 males and 5 014 females.The incidence rate increased first and then stabilized.Incidence of malignancy,China standardization rate,world standardization rate,cumulative rate and truncated incidence rate in men were higher than those in women.The incidence rates were at a low level in the 0~25 age group,rising rapidly in the 25~80 age group,reaching a peak at the age of 80,and then beginning to decline.From 2008 to 2012,there were 6 801 cases of malignant tumor deaths in Daoli District of Harbin,including 4 069 males and 2 732 females.There was no significant change in mortality over five years.Malignancy mortality,China standardization rate,world standardization rate,cumulative rate and truncated incidence rate in men were higher than those in women.The mortality rates were at a low level in the 0~30 age group,rising rapidly in the 30~75 age group,reaching a peak at the age of 75,and then beginning to decline.The highest incidence of malignant tumors in Daoli District from 2008 to 2012 was lung cancer,followed by liver cancer,colorectal cancer,stomach cancer and breast cancer.The highest mortality rate of malignant tumors was lung cancer,followed by liver cancer,colorectal cancer,stomach cancer and pancreatic cancer.Conclusion The incidence of malignant tumors in Daoli District of Harbin City increased first and then stabilized in 2008-2012,and there was no significant change in mortality.Lung cancer,digestive system malignant tumors,and female breast cancer are the main cancer types of malignant tumors incidence and death in Daoli District of Harbin.

Osteosarcoma(OS)is a malignant tumor that occurs mostly in children and adolescents with a poor prognosis.The 5-year survival rate and the survival rate of patients with lung metastasis or distant metastasis are still unsatisfactory.Currently,the treatment of osteosarcoma is still unsatisfactory under the bottleneck period.Long-chain non-coding RNA(LncRNA)is a non-protein-encoding RNA molecule involved in a variety of processes including gene expression,chromatin remodeling,post-transcriptional processing and transcription.LncRNAs are abnormally expressed in human cancers and they are involved in tumor development,progression and metastasis.This article reviews the research progress of long-chain non-coding RNA in osteosarcoma,which is intended to provide further research for osteosarcoma and propose new diagnosis and treatment strategies.

Wnt7a is a secreted glycoprotein in the Wnt signaling family.It is located on chromosome 3p25 and is easy deleted.It is mainly expressed in placenta,kidney,testis,uterus,fetal lung and brain,and participates in human embryonic development and cell differentiation.More and more studies have found that Wnt7a is also expressed and plays an important role in many tumor cells.As a new tumor target of therapy or an important factor in the tumor growth pathway,Wnt7a has begun to attract the attention of many scientists.

Cancer is one of the major diseases that seriously endanger human health.Recurrence and metastasis may still occur after traditional treatment.The basic reason is the presence of cancer stem cells(CSCs)in tumor tissues.CSCs have a high degree of proliferative ability,self-renewal,multi-directional differentiation,highly tumorigenicity and multi-drug resistance,which control the occurrence and development of tumors,chemotherapy tolerance,recurrence and metastasis.Therefore,clearing CSCs is one of effective ways to improve the curative rate of cancer.Nano-carriers have many advantages such as small size,large specific surface area,good biocompatibility and degradation in vivo.They are often used for targeted delivery and sustained release of drugs,which overcome the toxic side effects of traditional chemotherapeutic drugs,avoid surgical damage and kill CSCs.This paper will review the characteristics of CSCs and nano drug-loading systems as well as their research progress of nano drug-loading systems in CSCs in recent years.

Although surgery,radiotherapy and chemotherapy have made great progress,pancreatic cancer is still the most fatal malignant tumor,with a 5-year overall survival rate of only about 4%.Many studies have demonstrated that the local recurrence of pancreatic cancer is an important cause of high mortality,and the failure rate after local excision is still as high as 50~80%.Therefore,how to improve the local control rate of pancreatic cancer has been a hot spot of concern.Due to the adjacent organs and tissues(such as bone marrow,spinal cord,kidney,liver,intestine,etc.),the dose of external irradiation(EBRT)is limited.Increasing local control rates and reducing treatment side effects are trends in future treatments.Intraoperative radiotherapy has the advantages of dosimetry and direct vision treatment to be increasingly used in the treatment of pancreatic cancer,becoming an important part of comprehensive treatment.This article summarizes the clinical research results of intraoperative radiotherapy for pancreatic cancer in recent years,in order to provide the reference for clinicians.

By now,the clinical methods of cancer treatment are mainly surgery,radiotherapy,chemotherapy,and traditional Chinese medicine methods.However,some tumors are concealed due to onset,initial diagnosis and treatment,which have entered the middle and late stage,lost the operation opportunity and impossible to pass accurate surgical staging by assessment of prognosis.Radical concurrent chemoradiotherapy can be used for tumors that are inoperable and sensitive to radiation therapy.Therefore,it has become a hot spot to explore accurate and economic indicators to evaluate the prognosis of radiotherapy and chemotherapy.Neutrophil to lymphocyte ratio(NLR)refers to the ratio of neutrophils to lymphocytes in peripheral blood before treatment.As a general indicator of systemic inflammatory response,many studies have confirmed that the relationship between the efficacy of radiotherapy and chemotherapy for tumors provides a new way to evaluate the prognosis of tumors.This article briefly reviews the relationship between NLR and the prognosis of tumor-controlled radiotherapy and chemotherapy.