1.Role Of SALL4 In Gestational Trophoblastic Neoplasia And Its Expression In Clinical Patients Based On CTC Platform 2.Application Of Melting Curve Analysis In IGH Clonality Detection Of B Cell Lymphoma

This paper contains two parts,in the first part we aim to investigate how SALL4 affect the functions of malignant trophoblast and detect SALL4 expression in clinical gestational trophoblastic neoplasia(GTN)based on circulating tumor cell technology.Choriocacinoma cell line JAR was cultured,and its SALL4 expression was stably knockdown or overexpressed by transfection of lentivirus particles;CCK-8 and transwell invasion and migration assays were used to assess the proliferative,invasive and migrating functions.Results show that SALL4 knockdown decreased these functions,whereas SALL4 overexpression improved these functions.Cell cycle distribution was detected by flow cytometric analysis showing that SALL4 knockdown induced cell cycle arrest at G0/G1 phase.Also,Western blot and RT-qPCR analysis show that the expression of c-Myc,MMP2,CCND1 and β-catenin in SALL4 knockdown cells were down regulated at both protein and RNA level.Circulating tumor cells were detected using micro-filtration combined with RNA FISH;SALL4 expression of single CTC was further detected.CTC detection results demonstrate that GTN group had a higher count of total CTC,biphenotypic CTC and mesenchymal CTC than the normal intrauterine pregnancy(NIUP)group,ROC analysis for these three indexes showed area under curve(AUC)value as 0.776(p=0.001),0.719(p=0.012)and 0.695(p=0.025),respectively.CTC based SALL4 detection reveals that SALL4 high CTC was detected in 6 of 19 GTN patients but none was observed in benign or NIUP group.So we conclude that SALL4 might be an oncogene in GTN and has potential in regulating c-Myc,CCND1,MMP2 via Wnt/β-catenin pathway;the higher CTC count in GTN suggests that CTC is a promising adjunct biomarker for diagnosis of GTN;the specific detection of SALL4 high CTC in GTN patient,again provides new evidence,supporting that SALL4 plays a role in trophoblast carcinogenesis.In the second part,we aim to establish an optimized protocol by applying FR2 primers of BIOMED-2 into melting curve analysis to detect IGH rearrangement and evaluate its clinical application value in B cell lymphomas.40 formalin-fixed paraffin-embedded(FFPE)tissues from B cell non-Hodgkin lymphomas,31 FFPE tissues from reactive lymphoid hyperplasia were collected.Two sets of primers referring to BIOMED-2 design were synthesized including IgH variable framework region 2(FR2)and framework region 3(FR3)plus joining region(JH).Amplification and melting curve analysis were successively performed in LightCylcer system with SYBR green dye containing reaction mixture.Traditional PCR followed by polyacrylamide gel electrophoresis(PAGE)and capillary gel electrophoresis(CE)was used to validate the results from MCA.As result,MCA of combined FR2 and FR3 primer sets yields sensitivity and specificity equal to 70%and 100%,respectively.The addition of FR2 primers increases the sensitivity by 12.5%.PAGE was performed to detect IGH rearrangement in 40 FFPE lymphoma samples to validate MCA.Results from MCA and PAGE reach a consistency as high as 95%.Two cases were positive in MCA,but indefinite in PAGE.These two samples were further tested by capillary electrophoresis,which turned out consistent with MCA.Detection of serial dilutions of lymphoma cell line reveals that the analytical sensitivity of MCA was 3.125%.So we conclude that optimized MCA is reliable,high-throughput,faster and cross-contamination free.We believe that optimized MCA has potential for evaluating monoclonal IgH rearrangement in a clinical environment.