AIM: To study the basic mechanism of transcriptional regulation, NKX3.1 gene promoter was cloned and its promoter activities in prostate cancer cell lines and other cancer cell lines were tested. METHODS: 1.04 kb - promoter fragment of NKX3. 1 gene was obtained by PCR and cloned into pGL3 - basic and pEGFP - 1 that are promoter - less reporter vectors to examine its promoter activity driving the reporter gene transcription. The promoter activity was determined by dual -luciferase reporter assay and the expression of GFP reporter observed under fluorescence micro scope. RESULTS: The sequence of the cloned 1.04 kb promoter proved to be correct by DNA sequencing. The dual - lu ciferase reporter assay (M1/M2) showed that the promoter activity in LNCaP cell transfected with pGL3 - 1.04 kb promoter was about 1.5 - fold higher than that of pGL3 - control transfection and 50 - fold higher than that of pGL3 - basic transfec tion. To investigate the 1.04 kb - promoter activity in different tumor cell lines, the constructed pGL3 - 1.04 kb promoter and pEGFP - 1.04 kb promoter were transfected into several cell lines, respectively. The results showed that the activity of 1.04 kb promoterin LNCaP was highest among the tested cell lines. Multiple consensus sequence elements have been iden tified within the 1.04 kb fragment using TRANSFAC database. Further experiments will be done to determine their founc tions. CONCLUSION: Cloned 1.04 kb fragment upstream of NKX3.1 gene presented a strong promoter activity and its activity was highest in LNCaP cell among the tested tumor cell lines.

AIM: To observe the effect of exogenous androgen responsive element decoy on the promoter of prostate specific antigen (PSA) and the growth of LNCaP cells for searching the possibility of gene therapy for prostate cancer. METHODS: Firstly, pGL3 - PSA luciferase expression vector containing 640bp - promoter fragment of PSA gene was constructed. Then, a 23 -mer phosphorothioated ARE decoy based on the deduced ARE sequence at the promoter region of PSA gene was synthesized. pGL3 - PSA and ARE decoy DNA were cotransfected into PC3 - M cell by lipofectamineTM 2000. Through detecting the activity of luciferase, the effect of ARE decoy on the promoter of PSA was studied. Then the ARE decoy DNA was transfected into LNCaP cells. The effect of decoy DNA on the proliferation of LNCaP cells was examined by using MTT assay. The effects of apoptosis were detected by phase contrast microscopy, DNA agrose gel electrophoresis and flow cytometry. Meanwhile, the nuclear extract was prepared from LNCaP cells and DNA - protein interactions were examined by electrophoretic mobility shift assay (EMSA). RESULTS: The reporter assay showed that the aetivity of luciferase was significantly reduced in the ARE decoy - transfected cells, bnt not in the cells transfected with the control decoy. EMSA demonstrated specific binding of the ARE decoy to androgen receptor. The growth of LNCaP was remarkably inhibited and apoptotic morphological changes as well as DNA fragmentation were observed in the ARE decoy- transfected cells. The rate of apoptosis was 22.4% detected by FCM. CONCLUSION: The ARE decoy is capable of inhibiting the promoter of PSA gene and inducing the apoptosis in prostate cancer cells. It may become a potential therapeutic tool for prostate cancers.

AIM: To observe the effect of E2F decoy DNA on proliferation and apoptosis of androgen-independent prostate cancer cell line PC-3M.METHODS: E2F decoy DNA,ARE decoy DNA and control decoy DNA were transfected into PC-3M cells with lipofectamine,respectively.Their effects on cell proliferation were detected by MTT assay.The changes of cell morphology were observed by inverted phase contrast microscope.The cell apoptotic rate was determined by flow cytometry(FCM) analysis and chromosome DNA ladder was detected by DNA gel electrophoresis.The expression of c-Myc mRNA and cyclin D1 mRNA was detected by RT-PCR.The protein levels of c-Myc and cyclin D1 were detected by Western blotting.RESULTS: The growth of PC-3M cells was inhibited after transfection.The transfected PC-3M cells displayed typical apoptotic morphological changes.The apoptotic rate was 26.35% and DNA ladder was observed after transfection.The expression of c-Myc and cyclin D1 were inhibited.CONCLUSION: These results indicate that E2F decoy DNA induces apoptosis of androgen-independent prostate cancer cell lines PC-3M and inhibits cell proliferation via inhibiting expression of c-Myc and cyclin D1.

AIM: To study the basic mechanism of transcriptional regulation,NKX3.1 gene promoter was cloned and its promoter activities in prostate cancer cell lines and other cancer cell lines were tested.METHODS:(1.04 kb)-promoter fragment of NKX3.1 gene was obtained by PCR and cloned into pGL_3-basic and pEGFP-1 that are promoter-less reporter vectors to examine its promoter activity driving the reporter gene transcription.The promoter activity was determined by dual-luciferase reporter assay and the expression of GFP reporter observed under fluorescence microscope.RESULTS: The sequence of the cloned(1.04 kb) promoter proved to be correct by DNA sequencing.The dual-luciferase reporter assay(M1/M2) showed that the promoter activity in LNCaP cell transfected with pGL_3-(1.04 kb) promoter was about 1.5-fold higher than that of pGL_3-control transfection and 50-fold higher than that of pGL_3-basic transfection.To investigate the 1.04 kb-promoter activity in different tumor cell lines,the constructed pGL_3-(1.04 kb) promoter and pEGFP-1.04 kb promoter were transfected into several cell lines,respectively.The results showed that the activity of(1.04 kb) promoter in LNCaP was highest among the tested cell lines.Multiple consensus sequence elements have been identified within the(1.04 kb) fragment using TRANSFAC database.Further experiments will be done to determine their founctions.CONCLUSION: Cloned 1.04 kb fragment upstream of NKX3.1 gene presented a strong promoter activity and its activity was highest in LNCaP cell among the tested tumor cell lines.

Objective To investigate the chondrogenic feasibility of the human umbilical cord derived mesenchymal stem cells (hUCMSCs)as cartilage tissue engineering seed cells ,type Ⅱ collagen composite glycosaminoglycan scaffold as the cellular carrier and cell‐scaffold complex .Methods The type Ⅱ collagen composite glycosaminoglycan scaffolds was prepared .The pore diameter , porosity and hydrophilia of scaffold materials were observed and measured by electronic microscope .The corresponding histological analysis on the scaffold materials was performed .hUCMSCs of P3 generation were cultured and identified .The hUCMSCs suspen‐sion was inoculated in the type Ⅱ collagen composite glycosaminoglycan scaffold for conducting culture without adding inducer .The samples were taken out after 3 weeks and performed the toluidine blue and safranin O staining ,type Ⅱ collagen immunohistochemi‐cal staining and SEM scanning .Results hUCMSCs of P3 generation highly expressed the mesenchymal cell marker CD29 and CD105 ,while hardly expressed endothelial cells of CD34 and hematopoietic cell markers .The type Ⅱ collagen composite glycosami‐noglycan scaffold presented white porous foam like ,the porosity was (91 .8 ± 2 .17)% ,the average pore diameter was 110‐230 μm , which was homogeneously distributed and had interpenetration .The scaffold showed good hydrophilicity with the water absorption expansion rate of (213 .71 ± 1 .31)% .The scaffold staining of toluidine blue ,safranin O and type Ⅱ collagen was positive .The car‐tilage‐like tissues were observed ,and gradually increased in the surface of cell‐scaffold complex along with culture ,which were posi‐tive in Toluidine blue ,safranin O and type Ⅱ collagen staining ,the electronic microscopic observation displayed that the cells were actively proliferated in the scaffold ,closely adhered with the materials ,the cartilage‐like cells and a large number of peripheral colla‐gen fibers with zigzag connection could be seen .Conclusion Compositing hUCMSCs and type Ⅱ collagen composite glycosamin‐oglycan scaffold could construct tissue‐engineering cartilage in vitro without induction ,which lays a certain experimental foundation for the repair of cartilage damage .

Objective To analyze the infiltration abundance of macrophage M2 in breast cancer tissues and explore the correlation between VSIG4 and macrophage M2 and the potential mechanism of regulating the invasion and migration of breast cancer patients. Methods We downloaded the RNA-seq data of TCGA-BRCA and assessed the infiltration abundance of immune cells in the samples by CIBERSORT, and established a prognostic risk prediction model. Then, we analyzed the effect of macrophage M2 and VSIG4 on the prognosis of breast cancer patients. In addition, we analyzed the signaling pathway associated with VSIG4 by gene set enrichment analysis and predicted its upstream regulation of miRNA. Results The infiltration abundance of macrophage M2, age, PR status and pathological stage were involved in the establishment of risk prediction model, and the model had a good prediction performance (AUC=0.816). High infiltration of macrophage M2 (HR=1.35, P < 0.05) and high expression of VSIG4 (HR=1.4, P=0.039) suggested poor prognosis of breast cancer patients. VSIG4 could be regulated by upstream miR-29a-3p and significantly correlated with Toll-like receptor, cell adhesion, production and release of cytokine. Conclusion VSIG4 is significantly associated with breast cancer patients' prognosis and infiltration of macrophage M2, regulated by the upstream miR-29a-3p and promotes the invasion and migration of breast cancer cells. It can be used as a potential prognostic marker for breast cancer.

【Objective】 To evaluate the clinical characteristics and risk factors of adverse transfusion reactions (ATR) in Chinese adults, and to provide evidence-based medical evidence for early prevention. 【Methods】 The controlled trial (CT) of risk factors for ATR in Chinese adults were collected through PubMed, Embase, Cochrane Library, CNKI, CMB, VIP and Wanfang database, and the retrieval time was from the establishment of those databases to January 31, 2021 Literature was selected and extracted by 2 researchers according to inclusion and exclusion criteria. Meta-analysis was performed by RevMan5.3 software. 【Results】 A total of 28 049 patients in 12 literature were included, 1 190 patients were included into the ART group and 26 859 into the non-ART group. Meta-analysis results showed that the incidence of ART was 1.63% (410/24 361), mainly allergic reaction (43.90%, 188/410) and non-hemolytic fever (40%, 164/410). Primary hematologic disease (OR=27.11, 95%CI=21.64~33.96, P<0.01), allergy history(OR=15.52, 95% CI=2.20~109.38, P<0.01), transfusion history(OR=9.36, 95% CI=7.77 ~11.28, P<0.01), numbers of blood transfusion > 2 (OR=7.06, 95% CI=5.64~8.84, P<0.01), >30 min interval between blood issuing and transfusion (OR=3.40, 95% CI=2.88~4.00, P<0.01), transfusion of plasma (OR=2.67, 95%CI=2.20~3.25, P<0.01) and cryoprecipitate (OR=1.43, 95%CI=1.21~1.68, P<0.01) were risk factors for ART, while the transfusion of red blood cells/white blood cells/platelets (OR=0.29, 95% CI=0.24~0.35, P<0.01) was the protective factor. Sensitivity analysis showed that the results were stable. 【Conclusion】 According to the correlation intensity, the risk factors for ART in Chinese adults from high to low are primary blood disease, history of allergy, transfusion history, numbers of blood transfusion >2, >30 min interval between blood issuing and transfusion, transfusion of plasma and cryoprecipitates, while transfusion of red blood cells/white blood cells/platelets was the protective factor.

Objective: To explore the short-term efficacy and prognosis of palliative surgical treatment for malignant bowel obstruction (MBO) caused by peritoneal metastasis of colorectal cancer (mCRC). Methods: A retrospective cohort study was conducted. The inclusion criteria for patients were as follows: (1) primary colorectal cancer; (2) massive peritoneal metastasis; (3)obstructive site located below Treitz ligament by imaging; (4) obstruction refractory to conservative treatment; (5) estimated rese survival time more than 2 months; (6) patients and their families had strong willingness for operation; (7) surgical treatment included stoma/bypass and debulking surgery. In accordance with the above criteria, clinicopathological data of 46 patients undergoing palliative surgery at Peking University Gastrointestinal Cancer Center, Unit III from January 2016 to October 2018 were retrospectively collected. Postoperative symptomatic relief rate, morbidity of complication within 30 days, complication classification (Clavien-Dindo classification), mortality and survival after operation were analyzed. Kaplan-Meier method was used to evaluate survival and Cox regression analysis was used to identify prognostic factors. Results: Among 46 patients, 30 were male and 16 were female with median age of 63 (19-87) years; 23 patients received stoma/bypass surgery (stoma/bypass group), and 23 cases received tumor debulking surgery (debulking group). The overall symptom relief rate was 76.1% (35/46), while symptom relief rate in the debulking group was 91.3% (21/23), which was significantly higher than 60.9% (14/23) in the stoma/bypass group (χ²=4.301, P=0.038). Postoperative complications occurred in 25 patients. The complication rate was 52.2% (12/23) in the debulking group and 56.5% (13/23) in the stoma/bypass group, without statistically significant difference (χ²=0.088, P=0.767). Morbidity of complication beyond grade III was 8.7% (2/23) and 13.0% (3/23) in the debulking group and stoma/bypass group respectively, without statistically significant difference (χ²=0.224, P=0.636). Four patients died within 30 days after operation, 2 (8.7%) in each group. Twenty-four patients underwent 1-8 cycles of chemotherapy ± targeting therapy (regimens: CapeOX ± Bevacizumab, FOLFOX/FOLFIRI ± Bevacizumab/Cetuximab), including 10 cases in the stoma/bypass group and 14 cases in the debulking group. Two patients of debulking group received postoperative radiotherapy and chemotherapy (50.6 Gy/22 f, with concurrent oral capecitabine). Till the last follow up of April 2019, 34 patients died (34/46, 73.9%) with a median overall survival time of 6.4 months, and the 6-month and 1-year survival rate was 54.5% and 29.2% respectively. The median survival time in the debulking group was significantly longer than that in the stoma/bypass group (11.5 months vs. 5.2 months, χ²=5.117, P=0.024). The median survival time of the 35 patients with symptomatic relief after operation was significant longer than that of 11 patients without relief (7.1 months vs 5.1 months, χ²=3.844, P=0.050). Multivariate analysis showed stoma/bypass surgery (HR=2.917, 95%CI:1.357-6.269, P=0.006) and greater omental metastasis (HR=4.060, 95%CI:1.419-11.617, P=0.009) were independent risk factors associated with prognosis of patients with MBO caused by peritoneal mCRC. Conclusions For patients of MBO caused by peritoneal mCRC, tumor debulking surgery may achieve higher symptom relief rate and prolong survival. Greater omental metastasis indicates poor prognosis.