Fangchinoline Exerts Anticancer Effects on Colorectal Cancer Cells by Evoking Cell Apoptosis via Endoplasmic Reticulum Stress.

We studied the anti-tumor effect of fangchinoline (FAN) against human colorectal cancer cell lines CCL-244 and SW480 and analyzed the mechanism of FAN action. The cell viability and apoptosis were assessed by MTT test and Annexin V-PI staining; caspase-3 activity was measured by Western blotting. The expression of endoplasmic reticulum stress-related proteins was assessed by real-time PCR, Western blotting, and gene transfection. It was found that FAN inhibited cell growth and induced apoptosis in human colorectal cancer cell lines CCL-244 and SW480 in a dose-dependent manner. The caspase-3 inhibitor Ac-DEVD-CHO could reverse the inhibitory effect of FAN. Moreover, FAN significantly increased the expression of endoplasmic reticulum stress-related proteins p-PERK, p-eIF2α, ATF4, and CHOP in CCL-244 and SW480 cells. In addition, endoplasmic reticulum stress inhibitor 4-phenylbutyric acid or CHOP knockdown could prevent FAN-induced apoptosis. Thus, FAN induced apoptosis of human colorectal cancer through activation of endoplasmic reticulum stress.

CircRNA OXCT1 promotes the malignant progression and glutamine metabolism of non-small cell lung cancer by absorbing miR-516b-5p and upregulating SLC1A5.

Previous study has demonstrated the high expression of circular RNA 3-oxoacid CoA-transferase 1 (circ-OXCT1) in lung adenocarcinoma tumor tissues. However, the role and possible mechanism of circ-OXCT1 in non-small cell lung cancer (NSCLC) progression was unclear.Quantitative real-time PCR (qRT-PCR), western blotting and immunohistochemistry (IHC) staining assay were performed to detect the expression of circ-OXCT1, microRNA-516b-5p (miR-516b-5p), solute carrier family 1 member 5 (SLC1A5) and other indicated protein markers. Cell proliferation was measured by Cell counting kit 8 (CCK8), colony formation and 5-Ethynyl-2'-deoxyuridine (EdU) assays. Flow cytometry was employed to detect the rate of apoptotic cells. Cell migration and invasion were measured using transwell assay. The relative glutamine uptake and α-ketoglutarate (α-KG) production was determined using commercial kits. Interaction between miR-516b-5p and circ-OXCT1 or SLC1A5 was predicted by bioinformatics analysis and confirmed via luciferase reporter and RNA immunoprecipitation (RIP) assays. In vivo assay was implemented to demonstrate the effect of circ-OXCT1 in tumor growth.Circ-OXCT1 and SLC1A5 were upregulated and miR-516b-5p was downregulated in NSCLC tissues and cells. Functional experiments revealed that circ-OXCT1 silencing suppressed cell proliferation, migration and invasion, but promoted cell apoptosis in vitro. Circ-OXCT1 knockdown repressed tumor formation in vivo. Besides, miR-516b-5p was a target of circ-OXCT1, and miR-516b-5p inhibitor could relieve circ-OXCT1 absence-mediated effects in NSCLC cells. SLC1A5 was identified as a target of miR-516b-5p. Circ-OXCT1 promoted SLC1A5 expression by target binding with miR-516b-5p.Circ-OXCT1 facilitated NSCLC progression via miR-516b-5p-dependent regulation of SLC1A5, which provided a possible circRNA-targeted therapy for NSCLC.

PARP-1 modulates the expression of miR-223 through histone acetylation to involve in the hydroquinone-induced carcinogenesis of TK6 cells.

The upstream regulators of microRNAs were rarely reported. Hydroquinone (HQ) is the main metabolite of benzene, one of the important environmental factors contributing to leukemia and lymphoma. In HQ-induced malignant transformed TK6 (TK6-HT) cells, the expression of PARP-1 and miR-223 were upregulated. When in PARP-1 silencing TK6-HT cells, miR-223 was downregulated and the apoptotic cell number correspondingly increased. In TK6 cells treated with HQ for different terms, the expression of miR-223 and PARP-1 were dynamically observed and found to be decreased and increased, respectively. Trichostatin A could increase the expression of miR-223, then the expression of HDAC1-2 and nuclear factor kappa B were found to be increased, but that of mH2A was decreased. PARP-1 silencing inhibited the protein expression of H3Ac, mH2A, and H3K27ac. By co-immunoprecipitation experiment, PARP-1 and HDAC2 were found to form a regulatory complex. In conclusion, we demonstrated that the upregulation of PARP-1 mediated activation of acetylation to promote the transcription of miR-223 possibly via coregulating with HDAC2, an epigenetic regulation mechanism involved in cell malignant transformation resulting from long-term exposure to HQ, in which course, H3K27ac might be a specific marker for the activation of histone H3, which also gives hints for benzene exposure research.

miR-371b-5p promotes cell proliferation, migration and invasion in non-small cell lung cancer via SCAI.

OBJECTIVE: Multiple gene targets have been reported for treatment of non-small cell lung cancer (NSCLC), however, the accompanying genetic tolerance was reported increasingly. Therefore, it is important to find new biomarkers or therapeutic targets in treatment of NSCLC. METHODS: The expression levels of miR-371b-5p were detected by qRT-PCR in NSCLC tissues and cell lines. To evaluate the effect of miR-371b-5p on NSCLC progression, we first transfected the miR-371b-5p inhibitor for construction of the miR-371b-5p down-regulated cell model. Then the cell proliferation, migration, invasion and cell apoptosis were detected. In addition, the expression levels of adhesion factors were detected. The target gene of miR-371b-5p was identified by bioinformatics analysis, and rescue experiment was conducted to validate the effect of miR-371b-5p on proliferation, migration and invasion of NSCLC. RESULTS: Our findings revealed that the miR-371b-5p was overexpressed in NSCLC and could markedly promote the cell proliferation, migration and invasion. Expression levels of both intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) were significantly down-regulated when treated by miR-371b-5p inhibitor. Moreover, dual-luciferase reporter assay showed that the miR-371b-5p targeted SCAI in regulation of cell proliferation, migration and invasion, and the expression of miR-371b-5p was negatively associated with SCAI in NSCLC tissues and cell lines. Rescue experiment revealed that the miR-371b-5p could rescue the effect of SCAI on cell proliferation, migration and invasion. CONCLUSION: Our results suggest that the miR-371b-5p and SCAI may serve as novel prognostic biomarkers and therapeutic targets for NSCLC.

[The value of P16/ki67 double labeling, HPV E6/E7 mRNA detection and their combined application in cytological shunt diagnosis of low-grade squamous intraepithelial lesions].

Objective To explore the value of double labeling of P16/ki67, E6/E7 mRNA of human papillomavirus (HPV) and combined detection in shunt diagnosis of low-grade squamous intraepithelial lesions (LSIL) by thin-layer cervical cytology (TCT). Methods The study enrolled 239 patients who underwent colposcopy and biopsy within 4 weeks after primary TCT diagnosis. The remaining cytological samples were double-labeled with P16/ki67 immunocytochemical staining and the HPV E6/E7 mRNA was detected by Panther automatic HPV E6/E7 mRNA detection system. Using SPSS22.0 software, the positive rates of P16/ki67 double-labeling, HPV E6/E7 mRNA and combined detection were analyzed in different cervical lesions, and the positive rates in the same cervical lesions were compared horizontally to evaluate the efficiency of double labeling of P16/ki67, HPV E6/E7 and combined detection in the diagnosis of high-grade squamous intraepithelial lesions (HSIL) and above lesions. Results The diagnostic results of HE staining for the 239 cases of LSIL were 71 cases of chronic cervicitis (29.71%), 143 cases of LSIL (59.83%), 22 cases of HSIL (9.20%) and 3 cases of cervical cancer (1.26%). There were 46 cases of P16+ki67+ lesions (19.25%), 41 cases of ki67+P16- lesions (17.15%), 33 cases of ki67-P16+ lesions (13.81%) and 119 cases of P16-ki67- lesions (49.79%). The positive rates of P16/ki67 double-labeling, HPV E6/E7 mRNA and combined detection increased with the severity of cervical lesions. The positive rate of combined detection was the highest in the HSIL lesions, which was higher than that of P16/ki67 double-labeling and HPV E6/E7 mRNA detection. The sensitivity of combined detection was higher than that of P16/ki67 double-labeling and HPV E6/E7 mRNA detection. The Youden index of joint detection was 0.7850. Conclusion The combined detection of P16/ki67 double labeling, HPV E6/E7 mRNA and HPV E6/E7 mRNA had a certain clinical value in the management of cell LSIL shunt diagnosis. The combined detection significantly improved the sensitivity and Youden index of HSIL and above lesions, while maintaining a high specificity and coincidence rate.

Regulatory loop between lncRNA FAS-AS1 and DNMT3b controls FAS expression in hydroquinone-treated TK6 cells and benzene-exposed workers.

Hydroquinone (HQ), one of the main metabolites of benzene, is a well-known human leukemogen. However, the specific mechanism of how benzene or HQ contributes to the development of leukemia is unknown. In a previous study, we demonstrated the upregulation of DNA methyltransferase (DNMT) expression in HQ-induced malignant transformed TK6 (HQ-TK6) cells. Here, we investigated whether a regulatory loop between the long noncoding RNA FAS-AS1 and DNMT3b exists in HQ-TK6 cells and benzene-exposed workers. We found that the expression of FAS-AS1 was downregulated in HQ-TK6 cells and workers exposed to benzene longer than 1.5 years via histone acetylation, and FAS-AS1 expression was negatively correlated with the time of benzene exposure. Restoration of FAS-AS1 in HQ-TK6 cells promoted apoptosis and inhibited tumorigenicity in female nude mice. Interestingly, treatment with a DNMT inhibitor (5-aza-2-deoxycytidine), histone deacetylase inhibitor (trichostatin A), or DNMT3b knockout led to increased FAS-AS1 through increased H3K27ac protein expression in HQ-TK6 cells, and DNMT3b knockout decreased H3K27ac and DNMT3b enrichment to the FAS-AS1 promoter region, which suggested that DNMT3b and/or histone acetylation involve FAS-AS1 expression. Importantly, restoration of FAS-AS1 resulted in reduced expression of DNMT3b and SIRT1 and increased expression of FAS in both HQ-TK6 cells and xenograft tissues. Moreover, the average DNMT3b expression in 17 paired workers exposed to benzene within 1.5 years was decreased, but that of the remaining 103 paired workers with longer exposure times was increased. Conversely, DNMT3b was negatively correlated with FAS-AS1 expression. Both FAS-AS1 and DNMT3b influenced the enrichment of H3K27ac in the FAS promoter region by regulating the expression of SIRT1, consequently upregulating FAS expression. Taken together, these observations demonstrate crosstalk between FAS-AS1 and DNMT3b via a mutual inhibition loop and indicate a new mechanism by which FAS-AS1 regulates the expression of FAS in benzene-related carcinogenesis.

Poly(ADP-ribose) polymerase-1 promotes expression of miR-155 by the up-regulation of methyl-CpG binding domain protein 2 in TK6 cells exposed to hydroquinone.

Hydroquinone (HQ), one of the major metabolites of benzene, can induce aberrant gene expression. MiR-155, a tumor activator, participates in various biological processes, including DNA damage response. However, the molecular mechanism of aberrant miR-155 expression is still not completely elucidated. Here, we investigated the mechanism of abnormal expression of miR-155 induced by poly(ADP-ribose)polymerase-1 (PARP-1) expression in HQ-treated TK6 lymphoblastoid cells. We examined the expression of genes related to abnormal expression of miR-155 to explore the reason for this phenomenon. The results of the present study showed that miR-155 was significantly increased and reactive oxygen species (ROS) were decreased in cells treated with HQ for 72 h compared with PBS-treated cells. Meanwhile, E4F1, PARP-1 and PARP-1 related co-regulators (NF-κB, HDAC1, and HDAC2), acetylated histone H3 (H3Ac) were increased in a concentration-dependent manner. Experiments for treatment with 5-AzaC (DNMTs inhibitor), TSA (HDACs inhibitor), DOX (to activate PARP-1) or MG132 (proteasome inhibitor) revealed that the MBDs and PARP-1 was positively associated with miR-155 expression. Moreover, in cells treated with HQ in conjunction with PARP-1 knockdown, expression of miR-155, H3Ac and MBD2 protein were decreased, compared with negative control. In conclusion, PARP-1 activates expression of miR-155 via acetylation by regulating MBD2 in TK6 cells exposed to HQ.

Serum Cystatin C Levels in Twin Pregnancy versus Singleton Pregnancy.

OBJECTIVE: To explore whether there was an increased secretion of cystatin C (Cys C) in twin pregnancy. METHODS: Patients with a total of 281 singleton pregnancies (including 38 patients with preeclampsia) and 72 twin pregnancies, as well as 42 patients who were not pregnant, were included in this study. We tested levels of serum Cys C, creatinine, and uric acid, along with the estimated glomerular filtration rate (eGFR), in different groups. RESULTS: The levels of serum Cys C in all 3 trimesters for women with twin pregnancy were much higher than those in the corresponding trimesters for women with singleton pregnancy. However, we observed little change in eGFR in the corresponding trimesters. Cys C/eGFR in the second and third trimester of twin pregnancy increased, compared with the corresponding trimesters of women with singleton pregnancy. Levels of serum Cys C were higher in the third trimester in women with twin pregnancy than that in patients with preeclampsia. Also, Cys C/eGFR in the third trimester of twin pregnancy was close to the level observed in patients with preeclampsia. CONCLUSIONS: Increased secretion of Cys C could contribute to the elevated serum Cys C levels that we observed in twin pregnancy.

E4F1 silencing inhibits the cell growth through cell-cycle arrest in malignant transformed cells induced by hydroquinone.

Hydroquinone (HQ), one of the most significant metabolic activation products of benzene in an organism, can cause hematological toxicity, such as acute myeloid leukemia. It is a clear carcinogen that can cause changes in the disorder of cell cycle and cell growth. However, its molecular mechanisms remain unclear. E4 transcription factor 1 (E4F1), an important transcription factor, participating in the regulation of cell cycle may be related to the occurrence of tumor. Here, we examined the HQ-induced malignant transformed TK6 cells (TK6-HT) to illustrate the role of E4F1 in carcinogenesis. The present study showed that both the expressions of E4F1 messenger RNA and protein increased obviously in TK6-HT, preliminarily indicating that E4F1 is associated with HQ-induced carcinogenesis. To further explore the role of E4F1, we established E4F1 silencing TK6-HT (pLVX-shE4F1) and its control cells (pLVX-shNC) using lentiviral short hairpin RNA (shRNA) interference expression plasmid vector pLVX-shRNA. Flow cytometry and cell counting kit-8 assay were used to determine the effects of E4F1 silencing on cell cycle and cell growth, respectively. E4F1 silencing inhibited cell growth in TK6-HT. The results from flow cytometry indicated that the inhibitory effect on cell growth may be the results of the E4F1 silencing-induced accumulation in G2/M compared with TK6-HT-shNC. Meanwhile, levels of DNA damage (γ-H2AX), proteins of Rb and phosphorylated Rb, and reactive oxygen species were increased in TK6-HT-shRNA2 cells, which is the critical reason of cell-cycle arrest. In conclusion, E4F1 silencing inhibits the cell growth through cell-cycle arrest in malignant transformed cells induced by HQ.

[Effects of Osthole on Apoptosis of HL-60 Cells Induced by TRAIL and Its Related Mechanism].

OBJECTIVE: To explore the effects of Osthole on apoptosis of HL-60 cells induced by tumor necrosis factor related apoptosis inducing ligand (TRAIL) and its possible mechanism. METHODS: The proliferative inhibition of HL-60 cells treated with different concentrations of Osthole, TRAIL alone and Osthole combined with TRAIL was measured by MTT assay. The HL-60 cells were treated with Osthole, TRAIL alone and Osthole combined with TRAIL at the concentration

miR-7-5p overexpression suppresses cell proliferation and promotes apoptosis through inhibiting the ability of DNA damage repair of PARP-1 and BRCA1 in TK6 cells exposed to hydroquinone.

Hydroquinone (HQ), one of the major metabolic products of benzene, is a carcinogen, which induces apoptosis and inhibit proliferation in lymphoma cells. microRNA-7-5p (miR-7-5p), a tumor suppressor, participates in various biological processes including cell proliferation and apoptosis regulation by repressing expression of specific oncogenic target genes. To explore whether miR-7-5p is involved in HQ-induced cell proliferation and apoptosis, we assessed the effect of miR-7-5p overexpression on induction of apoptosis analyzed by FACSCalibur flow cytometer in transfection of TK6 cells with miR-7-5p mimic (TK6- miR-7-5p). We observed an increased apoptosis by 25.43% and decreased proliferation by 28.30% in TK6-miR-7-5p cells compared to those negative control cells (TK6-shNC) in response to HQ treatment. Furthermore, HQ might active the apoptotic pathway via partly downregulation the expression of BRCA1 and PARP-1, followed by p53 activation, in TK6-miR-7-5p cells. In contrast, attenuated p53 and BRCA1 expression was observed in shPARP-1 cells than in NC cells after HQ treatment. Therefore, we conclude that HQ may activate apoptotic signals via inhibiting the tumor suppressive effects of miR-7-5p, which may be mediated partly by upregulating the expression of PARP-1 and BRCA1 in control cells. The increase of miR-7-5p expression further intensified downregulation of PARP-1 and BRCA1 in TK6-miR-7-5p cells, resulting in an increase of apoptosis and proliferation inhibited.

Bcl-2 protects TK6 cells against hydroquinone-induced apoptosis through PARP-1 cytoplasm translocation and stabilizing mitochondrial membrane potential.

B cell leukemia/lymphoma-2 (Bcl-2) suppresses apoptosis by binding the BH3 domain of proapoptotic factors and thereby regulating mitochondrial membrane potential (MMP). This study aimed to investigate the role of Bcl-2 in controlling the mitochondrial pathway of apoptosis during hydroquinone (HQ)-induced TK6 cytotoxicity. In this study, HQ, one metabolite of benzene, decreased the MMP in a concentration-dependent manner and induced the generation of reactive oxygen species (ROS), the activation of the DNA damage marker γ-H2AX, and production of the DNA damage-responsive enzyme poly(ADP-ribose)polymerase-1 (PARP-1). Exposure of TK6 cells to HQ leads to an increase in Bcl-2 and co-localization with PARP-1 in the cytoplasm. Inhibition of Bcl-2 using the BH3 mimetic, ABT-737, suppressed the PARP-1 nuclear to cytoplasm translocation and sensitized TK6 cells to HQ-induced apoptosis through depolarization of the MMP. Western blot analysis indicated that ABT-737 combined with HQ increased the levels of cleaved PARP and γ-H2AX, but significantly decreased the level of P53. Thus, ABT-737 can influence PARP-1 translocation and induce apoptosis via mitochondria-mediated apoptotic pathway, independently of P53. In addition, we found that knockdown of PARP-1 attenuated the HQ-induced production of cleaved PARP and P53. These results identify Bcl-2 as a protective mediator of HQ-induced apoptosis and show that upregulation of Bcl-2 helps to localize PARP-1 to the cytoplasm and stabilize MMP. Environ. Mol. Mutagen. 59:49-59, 2018. © 2017 Wiley Periodicals, Inc.

Differently expressed long noncoding RNAs and mRNAs in TK6 cells exposed to low dose hydroquinone.

Previous studies have shown that long noncoding RNAs (lncRNAs) were related to human carcinogenesis and might be designated as diagnosis and prognosis biomarkers. Hydroquinone (HQ), as one of the metabolites of benzene, was closely relevant to occupational benzene poisoning and occupational leukemia. Using high-throughput sequencing technology, we investigated differences in lncRNA and mRNA expression profiles between experimental group (HQ 20 µmol/L) and control group (PBS). Compared to control group, a total of 65 lncRNAs and 186 mRNAs were previously identified to be aberrantly expressed more than two fold change in experimental group. To validate the sequencing results, we selected 10 lncRNAs and 10 mRNAs for quantitative real-time PCR (qRT-PCR). Through GO annotation and KEGG pathway analysis, we obtained 3 mainly signaling pathways, including P53 signaling pathway, which plays an important role in tumorigenesis and progression. After that, 25 lncRNAs and 32 mRNAs formed the lncRNA-mRNA co-expression network were implemented to play biological functions of the dysregulated lncRNAs transcripts by regulating gene expression. The lncRNAs target genes prediction provided a new idea for the study of lncRNAs. Finally, we have another important discovery, which is screened out 11 new lncRNAs without annotated. All these results uncovered that lncRNA and mRNA expression profiles in TK6 cells exposed to low dose HQ were different from control group, helping to further study the toxicity mechanisms of HQ and providing a new direction for the therapy of leukemia.

Long non-coding RNAs: The novel diagnostic biomarkers for leukemia.

Long non-coding RNAs (LncRNAs) are a category of non-coding RNAs (ncRNAs) with a length of 200nt-100kb lacking a significant open reading frame. The study of lncRNAs is a newly established field, due in part to their capability to act as the novel biomarkers in disease. A growing body of research shows that lncRNAs may not only useful as biomarkers for the diagnosis and clinical typing and prognosis of cancers, but also as potential targets for novel therapies. Differential expression of lncRNAs has been found in leukemia in the last two years, however, the majority of the lncRNAs described here are transcripts of unknown function and their role in leukemogenesis is still unclear. Here, we summarize the lncRNAs associated with leukemia in order to find a potential classification tool for leukemia, and a new field of research is being explored.

Hydroquinone induces TK6 cell growth arrest and apoptosis through PARP-1/p53 regulatory pathway.

Hydroquinone (HQ), one of the most important metabolites derived from benzene, induces cell cycle arrest and apoptosis. Poly(ADP-ribose) polymerase-1 (PARP-1) participates in various biological processes, including DNA repair and cell cycle regulation. To explore whether PARP-1 regulatory pathway mediated HQ-induced cell cycle arrest and apoptosis, we assessed the effect of PARP-1 suppression on induction of apoptosis analyzed by FACSCalibur flow cytometer in PARP-1 deficientTK6 cells (TK6-shPARP-1). We observed an increase in the fraction of cells in G1 phase by 7.6% and increased apoptosis by 4.5% in PARP-1-deficient TK6 cells (TK6-shPARP-1) compared to those negative control cells (TK6-shNC cells) in response to HQ treatment. Furthermore, HQ might activate the extrinsic pathways of apoptosis via up-regulation of Fas expression, followed by caspase-3 activation, apoptotic body, and sub G1 accumulation. Enhanced p53 expression was observed in TK6-shPARP-1 cells than in TK6-shNC cells after HQ treatment. In contrast, Fas expression was lower in TK6-shPARP-1 cells than in TK6-shNC cells. Therefore, we conclude that HQ may activate apoptotic signals via Fas up-regulation and p53-mediated apoptosis in TK6-shNC cells. The reduction of PARP-1 expression further intensified up-regulation of p53 in TK6-shPARP-1 cells, resulting in an increased G1→S phase cell arrest and apoptosis in TK6-shPARP-1 cells compared to TK6-shNC cells.

LincRNa-p21: function and mechanism in cancer.

In view of the rapid development of gene chips and high-throughput sequencing technology, noncoding RNAs (ncRNas) form a high percentage of the mammalian genome. Two major subgroups of ncRNAs that have been identified are the long ncRNAs (lncRNas) and the microRNAs. A number of studies in the past few years have showed crucial functions for lncRNas in cancer. LincRNa-p21 as a p53-dependent transcriptional target gene and a potential diagnostic marker is involved in proliferation, cell cycle, metabolism and reprogramming. In addition, more researches revealed that lincRNa-p21 is associated with cancer progression and contributed to the treatment and prognosis of cancer. In this review, we briefly summarize the function and molecular mechanisms of lincRNa-p21 in cancer and its regulation for the genes expression .

Fangchinoline inhibits the proliferation of SPC-A-1 lung cancer cells by blocking cell cycle progression.

Fangchinoline (Fan) is a bioactive compound isolated from the Chinese herb Stephania tetrandra S. Moore (Fen Fang Ji). The aim of the present study was to investigate the effect of Fan on the proliferation of SPC-A-1 lung cancer cells, and to define the associated molecular mechanisms. Following treatment with Fan, Cell Counting Kit-8, phase contrast imaging and Giemsa staining assays were used to detect cell viability; flow cytometry was performed to analyze the cell cycle distribution; and reverse transcription-quantitative polymerase chain reaction and western blot assays were used to investigate changes in the expression levels of cell cycle-associated genes and proteins. In the present study, treatment with Fan markedly inhibited the proliferation of SPC-A-1 lung cancer cells and significantly increased the percentage of cells in the G0/G1 phase of the cell cycle in a dose-dependent manner (P<0.05 for 2.5-5 µm; P<0.01 for 10 µm), whereas the percentage of cells in the S and G2/M phases were significantly reduced following treatment (P<0.05 for 5 µm; P<0.01 for 10 µm). Mechanistically, Fan significantly reduced the mRNA expression levels of cyclin D1, cyclin-dependent kinase 4 (CDK4) and CDK6 (P<0.05 for 2.5-5 µm; P<0.01 for 10 µm), which are key genes in the regulation of the G0/G1 phase of the cell cycle. Furthermore, treatment with Fan also decreased the expression of phosphorylated retinoblastoma (Rb) and E2F transcription factor-1 (E2F-1) proteins (P<0.05 for 5 µm; P<0.01 for 10 µm). In summary, the present study demonstrated that Fan inhibited the proliferation of SPC-A-1 lung cancer cells and induced cell cycle arrest at the G0/G1 phase. These effects may be mediated by the downregulation of cellular CDK4, CDK6 and cyclin D1 levels, thus leading to hypophosphorylation of Rb and subsequent suppression of E2F-1 activity. Therefore, the present results suggest that Fan may be a potential drug candidate for the prevention of lung cancer.

A multiplex qPCR gene dosage assay for rapid genotyping and large-scale population screening for deletional α-thalassemia.

The predominant determinants of α-thalassemia are deletions in the human α-globin gene cluster. A rapid DNA-based assay is needed for mass screening in thalassemia-prevention programs. Herein, we established a novel quadruplex TaqMan qPCR gene dosage assay with two separate combination reactions. The assay directly determined the copy number of human α-globin genes based on relative quantitation of three target genes (HBA2, HBA1, and HBZ or HBPA1) versus a control gene (CREBBP). The assay showed good accuracy, with mean intra-assay and interassay variations of 3.31% ± 1.02% and 5.49% ± 0.32%, respectively. The assay was evaluated using 678 pretyped clinical DNA samples containing six α-thalassemia deletions in 13 genotypes and 186 normal samples previously screened by multiplex ligation-dependent probe amplification or gap PCR. As determined by the 2(-ΔΔCq) method, deleted gene dosage ratios were 0.46 to 0.60 in heterozygotes, 0.0 in homozygotes, and 0.97 to 1.07 in nondeleted samples. We found 99.3% concordance between the quantitative PCR and multiplex ligation-dependent probe amplification or gap-PCR results. Furthermore, routine screening for α-thalassemia deletions was performed on 3000 random samples in a blind analysis. Results for all 279 positives, which had different deletions, were fully coincident with results from standard methods. We also identified two novel deletions confirmed by multiplex ligation-dependent probe amplification. Assays using the novel method are simple and suitable for rapid genotyping and mass screening.

[Breeding system of Paris polyphylla var. yunnanensis].

OBJECTIVE: To provide the basic guidance for seed breeding and cross-breeding of Paris polyphylla var. yunnanensis. METHOD: The floral behavior and pollinators were observed; 0.5% TTC solution was used for the pollen viability test and benzidine and -H2O2 was used for estimation of the stigma receptivity. The mating systems were tested by out crossing index (OC1), and pollination experiment was carried out by bagged and emasculated test in the field. RESULT: Commonly, stigma lobes spread slightly, and anthers started presenting the pollen from the outer ring while the flower was just beginning to open. Consequently, the distance between the stigma and its own pollen was relatively far, this "floral behavior" may be conducive to outcrossing. Then the flower entered the later period, while the stigma lobes spread widely, anthers all split, and this "floral behavior" shortened the stigma and its own pollen's distance, which may be conducive to selfing. P. polyphylla was partly protogynous. Stigma life-span was about 10-12 d. After anther dehiscence, the pollen viability maintained about 10% within 2 days, and 20% within 10 days. The value of out crossing index (OC1) was 4. By pollination experiment and pollinators observed, P. polyphylla was self-compatible, but no capacity for autonomous self-fertilization; In natural circumstances, outcrossing fructification rate was low, and mainly anemophilous. Assisted selfing-fertilization fructification rate was higher, spider was the main pollinators. CONCLUSION: P. polyphylla has a mixed mating system with self-pollination and cross-pollination characteristics. Floral behavior has important adaptive significance in avoiding female and male interference, outcrossing, and delayed selfing. P. polyphylla is ambophily (a combination of both wind and insect pollination), pollinators changes due to environment. Pollen limitation is the main cause of low fructification rate under natural conditions.