Histologic subtypes of non-muscle invasive bladder cancer

The majority of bladder cancers(BCs)are non-muscle invasive BCs(NMIBCs)and show the morphology of a conventional urothelial carcinoma(UC).Aberrant morphology is rare but can be observed.The classification and characterization of histologic subtypes(HS)in UC in BC have mainly been described in muscle in-vasive bladder cancer(MIBC).However,the currently used classification is ap-plied for invasive urothelial neoplasm and therefore,also valid for a subset of NMIBC.The standard transurethral diagnostic work-up misses the presence of HS in NMIBC in a considerable percentage of patients and the real prevalence is not known.HS in NMIBC are associated with an aggressive phenotype.Conse-quently,clinical guidelines categorize HS of NMIBC as“(very)high-risk”tumors and recommend offering radical cystectomy to these patients.Alternative strategies for bladder preservation can only be offered to highly selected patients and ideally within clinical trials.Novel treatment strategies and biomarkers have been established MIBC and NMIBC but have not been comprehensively invest-igated in the context of HS in NMIBC.Further evaluation prior to implementation into clinical practice is needed.

Treatment and surveillance for non-muscle-invasive bladder cancer:a clinical practice guideline(2021 edition)

Non-muscle invasive bladder cancer(NMIBC)is a major type of bladder cancer with a high incidence worldwide,resulting in a great disease burden.Treatment and surveillance are the most important part of NIMBC management.In 2018,we issued“Treatment and surveillance for non-muscle-invasive bladder cancer in China:an evidencebased clinical practice guideline”.Since then,various studies on the treatment and surveillance of NMIBC have been published.There is a need to incorporate these materials and also to take into account the relatively limited medical resources in primary medical institutions in China.Developing a version of guideline which takes these two issues into account to promote the management of NMIBC is therefore indicated.We formed a working group of clinical experts and methodologists.Through questionnaire investigation of clinicians including primary medical institutions,24 clinically concerned issues,involving transurethral resection of bladder tumor(TURBT),intravesical chemotherapy and intravesical immunotherapy of NMIBC,and follow-up and surveillance of the NMIBC patients,were determined for this guideline.Researches and recommendations on the management of NMIBC in databases,guideline development professional societies and monographs were referred to,and the European Association of Urology was used to assess the certainty of generated recommendations.Finally,we issued 29 statements,among which 22 were strong recommendations,and 7 were weak recommendations.These recommendations cover the topics of TURBT,postoperative chemotherapy after TURBT,Bacillus Calmette–Guérin(BCG)immunotherapy after TURBT,combination treatment of BCG and chemotherapy after TURBT,treatment of carcinoma in situ,radical cystectomy,treatment of NMIBC recurrence,and follow-up and surveillance.We hope these recommendations can help promote the treatment and surveillance of NMIBC in China,especially for the primary medical institutions.

Intravesical bacillus Calmette-Guerin(BCG)in treating non-muscle invasive bladder cancer—analysis of adverse effects and effectiveness of two strains of BCG(Danish 1331 and Moscow-I)

Objective:To compare the differences in adverse effects and efficacy profile between bacillus Calmette-Guerin(BCG)Danish 1331 and BCG Moscow-I strain in management of non-muscle invasive bladder cancer.Methods:Clinical data of 188 cases of non-muscle invasive bladder cancer treated with BCG between January 2008 and December 2018 in our institute were collected prospectively and analysed retrospectively,and 114 patients who completed a minimum of 12 months of follow-up were analysed.Patient and tumor characteristics,strain of BCG,adverse effects,and tumor progression were included for analysis.Intravesical BCG was instilled in intermediate-and high-risk patients.Six weeks of induction BCG,followed by three weekly maintenance BCG at 3,6,12,18,and 24 months was advised in high-risk patients.Results:Overall 68 patients received BCG Danish 1331 strain and 46 patients received Moscow-I strain.Patient and tumor characteristics were well balanced between the two groups.The median follow-up period was 42.5 months and 34.5 months in Danish 1331 and Moscow-I groups,respectively.Adverse events like dropout rate,antitubercular treatment requirement,and need of cystectomy were higher in Moscow-I group(n=31,67.4%)when compared to Danish 1331 strain(n=33,48.5%)(p=0.046).On direct comparison between Danish 1331 and Moscow-I strain,there was similar 3-year recurrence-free survival(80.0%vs.72.9%)and 3-year progression-free survival(96.5%vs.97.8%).Conclusion:Study results suggest no significant differences between Danish 1331 and Moscow-I strain in recurrence-free survival and progression-free survival,but a significantly higher incidence of moderate to severe adverse events in BCG Moscow-I strain.

A Th2-score in the tumor microenvironment as a predictive biomarker of response to Bacillus Calmette Guérin in patients with non-muscle invasive bladder carcinoma:A retrospective study

Intravesical Bacillus Calmette Guerin(BCG)is the gold standard therapy for intermediate/high-risk nonmuscle invasive bladder cancer(NMIBC).However,the response rate is~60%,and 50%of non-responders will progress to muscle-invasive disease.BCG induces massive local infiltration of inflammatory cells(Th1)and ultimately cytotoxic tumor elimination.We searched for predictive biomarker of BCG response by analyzing tumor-infiltrating lymphocyte(TIL)polarization in the tumor microenvironment(TME)in pre-treatment biopsies.Pre-treatment biopsies from patients with NMIBC who received adequate intravesical instillation of BCG(n=32)were evaluated retrospectively by immunohistochemistry.TME polarization was assessed by quantifying the T-Bet+(Th1)and GATA-3+(Th2)lymphocyte ratio(G/T),and the density and degranulation of EPX+eosinophils.In addition,PD-1/PD-L1 staining was quantified.The results correlated with BCG response.In most non-responders,Th1/Th2 markers were compared in pre-and post-BCG biopsies.ORR was 65.6%in the study population.BCG responders had a higher G/T ratio and a greater number of degranulated EPX+cells.Variables combined into a Th2-score showed a significant association with higher scores in responders(p=0.027).A Th2-score cut-off value>48.1 allowed discrimination of responders with 91%sensitivity but lower specificity.Relapse-free survival was significantly associated with the Th2-score(p=0.007).In post-BCG biopsies from recurring patients,TILs increased Th2-polarization,probably reflecting BCG failure to induce a pro-inflammatory status and,thus,a lack of response.PD-L1/PD-1 expression was not associated with the response to BCG.Our results support the hypothesis that a preexisting Th2-polarized TME predicts a better response to BCG,assuming a reversion to Th1 polarization and antitumor activity.

Molecular mechanisms and novel therapeutic strategies of BCG-unresponsive non-muscle invasive bladder cancer: Emerging immunotherapy has become a new choice?

Objective:THigh-risk non-invasive bladder cancer(NMIBC)has a high rate of recurrence and disease progression.At present,there are still insufficient effective prevention and treatment methods,especially for patients who have failed BCG treatment.This article reviews the research progress of the molecular mechanism of BCG unresponsive NMIBC,and summarizes the current status and prospects of emerging therapeutic strategies represented by immunotherapy,providing a theoretical basis for the immunotherapy of BCG non-reactive NMIBC.Methods:We searched the PubMed and CNKI journal full-text database search system for keywords"non-muscle invasive bladder cancer,BCG unresponsive,disease recurrence,disease progression,and immunotherapy"with 126 English and 538 Chinese articles.The literature,as well as the relevant clinical research in ClinicalTrials.gov,were integrated together to obtain the results.Results:Immunotherapy was performed in various types of tumors,and the use of immunotherapeutic drugs with different oncotargets administered alone,sequentially or in combination for the treatment of BCG-unresponsive NMIBC have achieved favorable effects,and more Clinical research is still ongoing.Conclusion:Immunotherapy is currently the most promising treatment for cancer,and it is indispensable for patients with NMIBC,both biologically and clinically.We look forward to more laboratory and clinical research in immunotherapy in the future.

Did the Scientific Innovations in the Management of Non-Muscle Invasive Bladder Cancer Patients Improve the Outcome during the Last 2 Decades?

Objectives: Previous reviews reported the outcome of each scientific modality in the management of T1 high-grade bladder cancer. The objective of this review is to assess and evaluate the available scientific modalities used during the last two decades and determine whether they were able to improve the clinical outcome. Literature Search Methodology: A systematic literature review was conducted from 2000-2020 using PubMed, Medline, Embase, and other database sites looking at randomized controlled trials (RCTs), clinical trials, research, review articles, and original articles addressing the different scientific modalities used to diagnose and manage patients with non-muscle invasive Bladder cancer (NMIBC)during the last 2 decades. More than 573 studies were retrieved following the preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines and PICOS criteria (Population, Intervention, Comparators, Outcomes, and Study design). Only 85 articles were selected for review including 19 prospective trials, 44 RCTs, original articles, research articles, one review article, and clinical trials—Retrospective studies were excluded to limit bias as much as possible in the analysis. Results: Randomized controlled trials (RCTs) have become the gold standard for evaluating the efficacy of new treatments. They are considered the highest standard of evidence-based medicine and are the method of choice. Overall, we selected 85 studies for review, among them 63 prospective trials and RCTs, with a total of 21,895 patients, published between 2000 and 2020. Previously conducted studies have shown that identifying rare histological types with poor prognoses can help improve outcomes, mainly the plasmacytoid type. Many articles addressed the role of biomarkers in the early identification of patients with NMIBC for recurrence and progression—P-cadherin expression and others were used to predict recurrence and/or progression with promising results. Despite the need for modifications, risk stratification is an important tool that should be used to improve the outcome of patients with NMIBC. Some found that fluorescence diagnostic cystoscopy (FDC) and Photodynamic diagnosis (PDD) improved recurrence-free survival but not progression and outcome. All authors agree that intravesical BCG is the most effective therapy that changes the course of high-grade T1 mainly progression. Re-TURBT has become one of the recommendations of international societies, but its potential effect on survival improvement is debatable. Most of the articles showed the advantages of early cystectomy in NMIBC but all agree that the selection criteria must be clearly defined. Conclusions: This review analyzed the outcomes provided by the scientific advances in the field of management of NMIBC patients in the last two decades. Patients with T1 bladder cancer have variable outcomes because of tumor heterogeneity and clinical staging. Despite the great development in the field of diagnosis, risk stratification, and management, further large studies are mostly needed to better elucidate this subset of patients and avoid over and under-treatment.

Molecular Assessment of Non-Muscle Invasive and Muscle Invasive Bladder Tumors: Mapping of Putative Urothelial Stem Cells and Toll-Like Receptors (TLR) Signaling

Purpose: The main objectives of this study were to characterize and compare the urothelial stem cells (healthy and cancer cells) and TLRs features in the urinary bladder of men without lesionsand with non-muscle-invasive and muscle invasive urothelial tumors. Materials and Methods: Thirty samples of the urinary bladder of 50 to 80-year-old men, with and without diagnosis of malignant urothelial lesions were used. The 30 samples were divided into 3 groups (n = 10 per group): Normal Group;Non-Muscle Invasive Bladder Cancer Group;Muscle Invasive Bladder Cancer Group. The samples were histopathologically and immunohistochemically analyzed. The study was conducted at teaching Hospital of the University of Campinas (UNICAMP). Results: The CD44 and CD133 immunoreactivities were significantly intense in the muscle-invasive cancer group when compared to the other groups. The ABCG2 biomarker demonstrated intense immunoreactivities in both non-muscle and muscle invasive groups, and absent immunoreactivity in the normal group. All groups showed weak CD117 immunoreactivity. Putative Healthy Stem Cells (CD44/CD133/ CD117+) occurred in all groups. Putative Cancer Stem Cells (CD44/CD133/ABCG2+) only occurred in the non-muscle and muscle invasive cancer groups. TLR2 immunoreactivity was significantly lower in the non-muscle invasive cancer group and absent in the muscle invasive cancer group. TLR4 immunoreactivity was significantly lower in both cancer groups. Conclusions: This study leads us to the conclusion that putative cancer stem cell occurrence was sensitive to the decreased in TLR2 and TLR4 immunoreactivities. Also, TLR2 and TLR4 demonstrated their involvement in the regulation of the different biomarkers for putative healthy and cancer urothelial stem cells, probably acting as negative regulators of urothelial carcinogenesis. Taken together data obtained suggest that use of TLRs agonists could be a promising alternative for the treatment of non-muscle and muscle invasive bladder tumors.

Recirculating chemohyperthermia as a treatment for non-muscle invasive bladder cancer:Current and future perspectives

About 75% of all bladder cancer diagnosed are non-muscle invasive bladder cancer(NMIBC), recurring over 50% of them after transurethral resection of the bladder tumor. In order to prevent recurrences, adjuvant intravesical chemotherapy with mitomycin C and immunotherapy with bacillus Calmette-Gu-érin(BCG) is traditionally used. Unfortunately, many patients relapse after receiving these treatments and a significant proportion of them require surgery. After a one-to-three years BCG maintenance, the risk for progression at 5 years was 19.3% for T1G3 tumors. Many new treatment approaches are being investigated to increase the effectiveness of adjuvant intravesical therapy. One of the developing treatments for intermediate and high-risk NMIBC is the combination of intravesical chemotherapy and hyperthermia, called chemohyperthermia. This article provides a review of the mechanism of action, current status and indications, results and future perspectives.

Reproductive traits associated with invasiveness in Conyza sumatrensis

Conyza sumatrensis （Retz.） E. Walker, a member of Asteraceae, is a highly invasive species. However, its reproduction biology remains poorly known. To understand the role of reproductive traits in successful invasion of the species, we studied several traits of its reproductive system： the miniature capitulum and gynomonoecious sexual system, the biology and phenology of capitula and florets, pollen/ovule ratio, the mating system （selfcompatibility）, flower visitors, physical traits and dispersal potential of achenes, germination potential of achenes from manually pollinated capitula, and the association of these traits with invasiveness. Our study showed that the reproductive traits of autonomous seed production, versatile mating system of self- and cross-pollination, and generalized pollination system might contribute to the species＇ successful invasive capability. The invasiveness was further enhanced by the high and rapid production of achenes, as well as the high percentage, rapid germination rate and high dispersal capability of achenes. It was concluded that in annual or winter-annual weeds, autonomous seed production contributed significantly to the invasiveness of the species.

Lower serum folate is associated with development and invasiveness of gastric cancer

AIM: To evaluate the associations of serum folate levelwith development, invasiveness and patient survival of gastric cancer. METHODS: In this nested case-control study, patients with newly diagnosed gastric cancer undergoing gastrectomy were enrolled, and patients receiving chemotherapy prior to surgery, with other concurrent malignancy, or of the aboriginal and alien populations were excluded. In total, 155 gastric cancer patients and 149 healthy controls were enrolled for determination of serum folate levels and their correlation with gastric cancer. Using the median value of serum folate computed among the overall population as the cutoff value, the associations between serum folate and gastric cancer in all cases and different age and gender subgroups were analyzed by multivariate logistic regression analysis. In the patient cohort of gastric cancer, receiver-operating characteristic analyses were performed to calculate the best cutoff values of serum folate, and the associations between serum folate levels and clinicopathological features were further analyzed by multivariate regression analysis. Survival analyses were conducted using the Cox proportional hazards model.RESULTS: The mean serum folate level was significantly lower in gastric cancer patients than that in controls(3.71 ± 0.30 ng/mL vs 8.00 ± 0.54 ng/mL, P < 0.01), and folate levels were consistently lower in gastric cancer patients regardless of age and gender(all P < 0.01). Using the median serum folate value as the cutoff value, low serum folate was significantly associated with gastric cancer risk in the whole population(OR = 19.77, 95%CI: 10.54-37.06, P < 0.001) and all strata(age < 60 years OR = 17.39, 95%CI: 7.28-41.54, age ≥ 60 years(OR = 21.67, 95%CI: 8.27-56.80), males(OR = 17.95, 95%CI: 7.93-40.62), and females(OR = 20.95, 95%CI: 7.66-57.31); all P < 0.001. In the patient cohort of gastric cancer, the respective cutoff values showed that low serum folate levels were significantly associated with serosal invasion(OR = 2.54, 95%CI: 1.23-5.23), lymphatic invasion(OR = 2.23, 95%CI: 1.17-4.26), and liver metastasis(OR =6.67, 95%CI: 1.28-34.91) of gastric cancer(all P < 0.05). Serum folate level below 1.90 ng/mL was associated with poor patient survival(HR = 1.84, 95%CI: 1.04-3.27, P < 0.05) in univariate analysis.CONCLUSION: Lower serum folate levels were significantly associated with gastric cancer development and invasive phenotypes. The role of folate depletion in gastric cancer invasion warrants further study.

Silencing MTA1 by RNAi Reverses Adhesion, Migration and Invasiveness of Cervical Cancer Cells (SiHa) via Altered Expression of p53, and E-cadherin/β-catenin Complex

It has been reported that metastasis-associated gene 1 （Mta1） is overexpressed in many malignant tumors with high metastatic potential. In addition, some studies indicated that MTA1 participated in invasion, metastasis, and survival of cancer cells by regulating cell migration, adhesion and proliferation. But the role of MTA1 is unclear in vitro in the development of cervical cancer cells. This study investigated whether and how MTA1 mediated cell proliferation, migration, invasion and adhesion in cervical cancer. MTA1 expression level was detected by Western blot in two cervical cancer cell lines of different invasion potentials. The effects of MTA1 expression on SiHa cell apoptosis, cycle, proliferation, migration, invasion and adhesion were tested by flow cytometry, MTT, wound-healing assay, Transwell assay and adhesion assay, respectively. The expression levels of p53, E-cadherin, and β-catenin activity were evaluated in untreated and treated cells. The results showed that MTA1 protein expression was significantly higher in SiHa than in HeLa, which was correlated well with the potential of migration and invasion in both cell lines. Furthermore, the cell invasion, migration and adhesion capabilities were decreased after inhibition of MTA1 expression mediated by Mta1-siRNA transfection in SiHa. However, no significant differences were found in cell apoptosis, cycle, and proliferation. In addition, E-cadherin and p53 protein levels were significantly up-regulated, while β-catenin was significantly down-regulated in SiHa transfected with the siRNA. These results demonstrated that MTA1 played an important role in the migration and invasion of cervical cancer cells. It was speculated that the decreased migration and invasion capability by inhibiting the MTA1 expression in the SiHa cell line may be mediated through the altered expression of p53, and E-cadherin/β-catenin complex. MTA1 could serve as a potential therapeutic target in cervical cancer.

Association of Expression of Leucine-rich Repeats and Immunoglobulin-like Domains 2 Gene with Invasiveness of Pituitary Adenoma

The Leucine-rich repeats and immunoglobulin-like domains-2（LRIG2） gene expression in pituitary adenoma and its correlation with tumor invasiveness were studied.The expression of LRIG2 mRNA and protein in human pituitary adenoma obtained surgically was detected by RT-PCR（39 cases） and immunohistochemical staining（30 cases）.It was found that LRIG2 was mostly localized at the nucleus of the pituitary adenoma cells.Its expression was significantly higher in the invasive cases than in the non-invasive cases.LRIG2 protein was positive in 14 cases out of 21 cases of invasive adenoma,but only 2 cases were positive in 9 cases of non-invasive adenoma.The positive expression rate of LRIG2 mRNA was 91.3% in invasive cases（total 23 cases） and 62.5% in non-invasive cases（total 16 cases）,respectively.LRIG2 gene is overexpressed in invasive pituitary adenoma.It may play an important role in pituitary adenoma invasiveness and further studies are necessary to elucidate the mechanism under this phenomenon.

RhoA and RhoC -siRNA inhibit the proliferation and invasiveness activity of human gastric carcinoma by Rho/PI3K/Akt pathway

AIM: To evaluate the effects of adenovirus-mediated gene transfer of RhoA siRNA and RhoC siRNA on proliferation and invasion of SGC7901 cells by Rho/ PI3K/Akt pathway. METHODS: Plasmid of RhoA siRNA and RhoC siRNA were constructed and transfected into SGC7901 cells. siRNA and LY294002 (PI3K inhibitor) were designed as the control group. The mRNA and protein expressions of RhoA and RhoC were respectively detected with RT-PCR and western blotting. In order to fi nd out the changes of proliferation and invasion power of SGC7901 cell lines, we analyzed the data by MTT, Boyden chamber and evaluated apoptosis of cell with flow cytometry. We treated BALB /C nude mice with RhoA and RhoC-siRNA, and tumor control rate (%) in nude mice was calculated. RESULTS: RhoA and RhoC siRNA transfections specifically down-regulated the corresponding mRNA and protein levels in SGC7901 Cells.The experiment of permeated artificial basal membrane showed that the invasion power of SGC7901 cell lines are on the decline after treatment of Ad-RhoA and RhoC-siRNA (12.64 ± 3.27 vs 87.38 ± 17.38, P < 0.05). The values of 490 nm wavelength light absorption were different in the five groups. The number of alive cells in the group of RhoA and RhoC-siRNA was lower than others in the 6th d (0.71 ± 0.01 vs 3.82 ± 0,11 P < 0.05). The apoptosis rate of transfected RhoA and RhoC-siRNA group with FACS were 19.07% ± 1.78 and there were significant differences between treated and control groups (19.07 ± 1.78% vs 1.23 ± 0.11%, P < 0.01). The tumor transplantation experiment in BALB/C nude mice showed intratumoralinjection of RhoA or RhoC siRNA can inhibit tumor growth. CONCLUSION: RhoA and RhoC siRNA gene therapy mediated by adenovirus may be useful for inhibiting growth and invasion of SGC7901 through a PI3K/Akt pathway. These results provide a novel therapeutic target in preventing gastric cancer cell invasion and metastasis.

Fascin promotes the motility and invasiveness of pancreatic cancer cells

AIM:To explore the role of actin-bundling protein, fascin during the progression of pancreatic cancer. METHODS:The plasmid expressing human fascin-1 was stably transfected into the pancreatic cancer cell line MIA PaCa-2. The proliferation, cell cycle, motility, scattering, invasiveness and organization of the actin filament system in fascin-transfected MIA PaCa-2 cells and control non-transfected cells were determined. RESULTS:Heterogeneous overexpression of fascin markedly enhanced the motility, scattering, and inva-siveness of MIA PaCa-2 cells. However, overexpression of fascin had minimal effect on MIA PaCa-2 cell pro-liferation and cell cycle. In addition, cell morphology and organization of the actin filament system were distinctly altered in fascin overexpressed cells. When transplanted into BALB/c-nu mice, fascin-transfected pancreatic cancer cells developed solid tumors at a slightly slower rate, but these tumors displayed more aggressive behavior in comparison with control tumors. CONCLUSION: Fascin promotes pancreatic cancer cell migration, invasion and scattering, thus contributes to the aggressive behavior of pancreatic cancer cells.

UROKINASE-TYPE PLASMINOGEN ACTIVATOR,ITS RECEPTOR AND INHIBITOR EXPRESSION IN HEPATOCELLULAR CARCINOMA RELATION TO CANCER INVASIVENESS AND PROGNOSIS

Objective: To study the relevance of uPA, uPAR and PAI 1 to hepatocellular carcinoma (HCC) Methods: The expression at protein level of uPA, uPAR and PAI 1 was determined in 48 cases of HCC and 12 cases of benign tumors of liver (as control) by immunohistochemistry Results: When compared to cancer adjacent liver tissue and the control, positive rate of immune staining for uPA, uPAR and PAI 1 on cell membrane were significantly higher in HCC cells ( P <0 05) Positive staining of uPA and uPAR was seen in 16 of 22 and 19 of 22 cases of HCC with invasion, respectively ( P <0 01 and P <0 001) In 8 of 8 cases with cancer embolus, and in 6 of 6 cases with lymph node metastasis was the expression of positive uPAR Compared with 2 of 17 cases without recurrence, uPAR was positive in 15 of 17 recurrent cases ( P <0 01) In 36 cases who survived, 17 was positive uPAR and 15 positive PAI 1, while in 12 cases who died 2 years after surgery, 12 were positive for uPAR and 9 positive PAI 1, respectively ( P <0 01 and P <0 05) In 15 positive cases for all three parameters, 11 had cancer invasion and 7 died within 2 years, while in negative cases, 2 had invasion and none died within 2 years ( P <0 05) Conclusion: Expression of uPA, uPAR and PAI 1 is increased in HCC, uPA and uPAR may contribute significantly to HCC invasion and metastasis uPAR and PAI 1 are associated with poor prognosis of HCC

Identification of CDK6 and RHOU in Serum Exosome as Biomarkers for the Invasiveness of Non-functioning Pituitary Adenoma

Objective To explore circulating biomarkers for screening the invasiveness of non-functioning pituitary adenomas(NF-PAs).Methods The exosomal RNAs were extracted from serum of patients with invasive NF-PA(INF-PA)or noninvasive NF-PA(NNF-PA).Droplet digital PCR was adapted to detect the mRNA expression of candidate genes related to tumor progression or invasion,such as cyclin dependent kinase 6(CDK6),ras homolog family member U(RHOU),and spire type actin nucleation factor 2(SPIRE2).Student’s t-test was used to analyze the statistical difference in the mRNA expression of candidate genes between the two groups.Receiver operating characteristic(ROC)curve was used to establish a model for predicting the invasiveness of NF-PAs.The accuracy,sensitivity,specificity and precision of the model were then obtained to evaluate the diagnostic performance.Results CDK6(0.2600±0.0912 vs.0.1789±0.0628,t=3.431,P=0.0013)and RHOU mRNA expressions(0.2696±0.1118 vs.0.1788±0.0857,t=2.946,P=0.0052)were upregulated in INF-PAs patients’serum exosomes as compared to NNF-PAs.For CDK6,the area under the ROC curve(AUC)was 0.772(95%CI:0.600-0.943,P=0.005),the accuracy,sensitivity,specificity and precision were 77.27%,83.33%,75.00%and 55.56%to predict the invasiveness of NF-PAs.For RHOU,the AUC was 0.757(95%CI:0.599-0.915,P=0.007),the accuracy,sensitivity,specificity and precision were 72.73%,83.33%,68.75%and 50.00%.In addition,the mRNA levels of CDK6 and RHOU in serum exosomes were significantly positively correlated(r=0.935,P<0.001).After combination of the cut-off scores of the two genes,the accuracy,sensitivity,specificity and precision were 81.82%,83.33%,81.25%and 62.50%.Conclusions CDK6 and RHOU mRNA in serum exosomes can be used as markers for predicting invasiveness of NF-PAs.Combination of the two genes performs better in distinguishing INF-PAs from NNF-PAs.These results indicate CDK6 and RHOU play important roles in the invasiveness of NF-PAs,and the established diagnostic method is valuable for directing the clinical screening and postoperative treatment.

Targeted Silencing of Heparanase Gene by Small Interfering RNA Inhibits Invasiveness and Metastasis of Osteosarcoma Cells

The effects of targeted silencing of heparanase gene by small interfering RNA（siRNA） on invasiveness and metastasis of osteosarcoma cells（MG63 cells） were investigated in the present study.Two complementary oligonucleotide strands were synthesized and inserted into pGenesil-1 vector based on the mRNA sequence of heparanase gene.The expression vector containing short hairpin RNA（pGenesil-shRNA） was constructed successfully.MG63 cells were randomly allocated into 3 groups：blank group,empty vector（pGenesil） transfected group and expression vector（pGenesil-shRNA） transfected group.Under the induction of Lipofectamine 2000,the recombinants were transfected into MG63 cells.Heparanase gene expression level was detected by RT-PCR and Western blotting.Cell prolifera-tion was measured by MTT assay.Cell invasiveness and metastasis were examined by cell adhesion and Transwell-ECM assays.HUVECs migration assay was applied for the detection of angiogenesis.As compared with negative controls,the mRNA and protein expression levels of heparanase were down-regulated by 76.1%（P0.01） and 75.3%（P0.01） respectively in the pGenesil-shRNA transfected group.Meanwhile,the proliferation,adhesiveness,invasiveness and angiogenesis properties of MG63 cells were all significantly inhibited.It was suggested that targeted silencing of heparanase gene by siRNA could dramatically inhibit the invasiveness and metastasis of osteosarcoma cells.

Suppression of matrix metalloproteinase-2 via RNA interference inhibits pancreatic carcinoma cell invasiveness and adhesion

AIM:To investigate the inhibitory effects of RNA interference (RNAi) on expression of matrix metalloproteinase-2 (MMP-2) gene and invasiveness and adhesion of human pancreatic cancer cell line,BxPC-3.METHODS:RNAi was performed using the vector (pGPU6)-based small interference RNA (siRNA) plasmid gene silence system to specifically knock down MMP-2 expression in pancreatic cancer cell line,BxPC-3. Four groups of different specific target sequence in coding region of MMP-2 and one non-specific sequence were chosen to construct four experimental siRNA plasmids of pGPU6-1,pGPU6-2,pGPU6-3 and pGPU6-4,and one negative control siRNA plasmid of pGPU6 (-). MMP-2 expression was measured by reverse transcription polymerase chain reaction (RT-PCR) and Western blot. Cell proliferation and apoptosis were examined by methyl thiazolyl tetrazolium (MTT) and flow cytometry,respectively. The abilities of adhesion and invasion were detected by cell adhesion assay and cell invasion assay using Transwell chambers.RESULTS:The expression of MMP-2 was inhibited and the inhibitory effects of different sequence varied. pGPU6-1 group had the most efficient inhibitory effect,followed by pGPU6-2 and pGPU6-3 groups.Invasiveness and adhesion were more significantly reduced in pGPU6-1,pGPU6-2 and pGPU6-3 groups as compared with pGPU6 (-) and blank control groups. However,no difference concerning cell proliferation and apoptosis was observed after transfection between experiment groups and control groups.CONCLUSION:RNAi against MMP-2 successfully inhibited the mRNA and protein expression of MMP-2 in the pancreatic cancer cell line,BxPC-3,leading to a potent suppression of tumor cell adhesion and invasion without affecting cell proliferation and apoptosis. These findings suggest that the RNAi approach towards MMP-2 may be an effective therapeutic strategy for the clinical management of pancreatic tumor.

The Effect of RhoC siRNA on the Invasiveness and Proliferation of Human Cervical Cancer Cell Line SiHa Cells

This study investigated the effect of RhoC GTPase on the proliferation and metastasis of cervical cancer cells, SiHa cells, in vitro. RhoC siRNA was introduced into SiHa cells to silence the RhoC gene. The mRNA and protein expression of RhoC, before and after RhoC siRNA transfection, was examined by RT-PCR and Western blotting, respectively. The proliferation and apoptosis of SiHa cells were examined by MTT assay and flow cytometry （FACS）, respectively. Adhesive rate was evaluated by Matrigel adhesive assay, and the invasive capability and migration capability were assessed by transwell invasive assay and migration assay, respectively. The results showed that after the RhoC siRNA transfection, the mRNA and protein expression of RhoC was down-regulated in SiHa cells. The down-regulation of RhoC GTPase did not affect the cell proliferation and apoptosis （P〉0.05）, but it did suppress SiHa cells＇ adhesion to matrigel （P〈0.01）, the invasive capability （P〈0.01） and the migration capability （P〈0.01）. It was concluded that RhoC obviously promotes the adhesion, invasion and migration of SiHa cells in vitro, but not proliferation and apoptosis, suggesting that RhoC plays an important role in the progression in cervical cancer.

Effects of LY294002 on the invasiveness of human gastric cancer in vivo in nude mice

AIM: To investigate the effects of class phosphatidy-linositol 3-kinase (PI3K) inhibitor LY294002 on the invasiveness and related mechanisms of implanted tumors of SGC7901 human gastric carcinoma cells in nude mice. METHODS: Nude mice were randomly divided into model control groups and LY294002 treatment groups. On days 5, 10 and 15 after treatment, the inhibitory rate of tumor growth, pathological changes in tumor specimens, expression levels of matrix metalloproteinase (MMP)-2, MMP-9, CD34 [representing microvessel density (MVD)] and vascular endothelial growth factor (VEGF), as well as apoptosis indexes in tumor samples were observed.RESULTS: In this study, we showed that treatingthe tumors with LY294002 could significantly inhibit carcinoma growth by 11.3%, 29.4% and 36.7%, after 5, 10 and 15 d, respectively, compared to the control group. Hematoxylin & eosin staining indicated that the rate of inhibition increased progressively (23.51% ± 3.11%, 43.20% ± 3.27% and 63.28% ± 2.10% at 5, 10 and 15 d, respectively) along with apoptosis. The expression of MMP-2 was also downregulated (from 71.4% ± 1.6% to 47.9% ± 0.7%, 31.9% ± 0.9% and 7.9% ± 0.7%). The same effects were observed in MMP-9 protein expression (from 49.4% ± 1.5% to 36.9% ± 0.4%, 23.5% ± 0.9% and 7.7% ± 0.6%), the mean MVD (from 51.2% ± 3.1% to 41.9% ± 1.5%, 30.9% ± 1.7% and 14.9% ± 0.8%), and the expression of VEGF (from 47.2% ± 3.1% to 25.9% ± 0.5%, 18.6% ± 1.2% and 5.1% ± 0.9%) by immunohistochemical staining.CONCLUSION: The classPI3K inhibitor LY294002could inhibit the invasiveness of gastric cancer cells by downregulating the expression of MMP-2, MMP-9, and VEGF, and reducing MVD.