The diagnostic value of serum lncRNA CATG00000112921.1 as a marker of multiple myeloma.

BACKGROUND: Multiple myeloma (MM) is a malignant plasma cell disease. At present, numerous studies have shown that lncRNA plays a very important role in the occurrence, development and even drug resistance of multiple myeloma. It may become a potential diagnostic and prognostic marker of multiple myeloma and provide new ideas for targeted therapy. Based on the above research background, this study used gene chip technology to screen out the differentially expressed lncRNA in the serum of MM patients and healthy people, and verified more clinical serum samples to screen out the lncRNA with the largest difference as a biomarker for further research. METHOD: In this research, the data of hospitalized patients diagnosed with MM and healthy people in the Affiliated Hospital of Guangdong Medical University were retrospectively collected. The lncRNA expression profile of serum samples from patients with multiple myeloma and healthy controls was analyzed by lncRNA chip technology. The serum samples were verified by real-time fluorescence quantitative PCR, and the candidate diagnostic markers were screened out. The ROC working curve was drawn to evaluate the diagnostic efficacy of the candidate markers and to determine their stability at different temperatures and time. RESULT: A total of 44 MM patients and 37 healthy people were involved in this research. Among them, 4 patients with MM and 4 patients with HD were sent for microarray analysis. According to Fold Change ≥ 2 and P < 0.05, a total of 17 differentially expressed lncRNA molecules were screened, of which 9 were up-regulated RNA molecules and 8 were down-regulated RNA molecules. Through real-time fluorescence quantitative PCR verification, it was found that lncRNA CATG00000112921.1 was highly expressed in the healthy control group and diminished in patients with multiple myeloma, P < 0.001. The ROC curve demonstrated that the area under the curve (AUC) was 0.749, the sensitivity was 0.636, the specificity was 0.789, and the 95 % CI was 0.636-0.862 (P < 0.001). In addition, in order to verify the effects of temperature, time and repeated freezing and thawing on lncRNA, it was placed at 25°C, 4°C, -20°C, -80°C for 0 h, 24 h, 48 h, 72 h, and placed at-80°C repeated freezing and thawing 0 times, 2 times, 4 times, 8 times, and the expression level was not significantly changed. CONCLUSION: Serum lncRNA CATG00000112921.1 may be a potential candidate diagnostic marker for multiple myeloma. The ROC curve shows that it has good diagnostic value, and its high stability at different temperatures and different times is a required condition for becoming a diagnostic marker. As far as we know, this is the first study in the world to find differential expression of lncRNA CATG00000112921.1 in peripheral serum of healthy people and newly diagnosed multiple myeloma patients. This study also highlights the application of gene chip technology in screening differentially expressed genes.

New biopsy after antibiotic treatment: effect on outcomes of assisted reproduction in patients with infertility and chronic endometritis.

RESEARCH QUESTION: What is the effect of chronic endometritis on patients with infertility, the necessity of endometrial re-examination and the effect of improving chronic endometritis after one cycle of antibiotic treatment on pregnancy outcomes? DESIGN: Infertile patients (n = 4003) who underwent IVF and intracytoplasmic sperm injection treatment were included. Pregnancy outcomes of groups positive for chronic endometritis were compared with groups that were negative (group 1). Patients that were positive were divided into the chronic endometritis new biopsy group (group 2) and chronic endometritis non-re-examination group (group 3). After doxycycline treatment and re-examination, the chronic endometritis new biopsy group was divided into improved chronic endometritis group (ICE) and not-improved chronic endometritis group (NICE), and their general indicators and reproductive outcomes were compared. RESULTS: No significant difference was observed in embryo implantation, early or late pregnancy loss, ectopic pregnancy, clinical pregnancy and live birth rates between groups 2 and 3. The clinical pregnancy and live birth rates in the NICE group were significantly lower than those in the ICE group (P = 0.008 and P = 0.001, respectively). After controlling for potential confounding factors, age, average number of high-quality embryos, endometrial thickness on the day of embryo transfer and number and type of embryo transfer were factors associated with live birth rates. CONCLUSIONS: Endometrial re-examination of women with chronic endometritis treated with doxycycline had no effect on pregnancy outcomes. The first cycle of doxycycline treatment could effectively improve reproductive outcomes of women with five or more CD138+ cells/high-power field.

Deficiency of kin17 Facilitates Apoptosis of Cervical Cancer Cells by Modulating Caspase 3, PARP, and Bcl-2 Family Proteins.

Background: The treatment of cervical cancer in the late stage is still quite challenging, because of nonspecificity in conventional therapies and the lack of molecular targeted drugs. It is necessary to find novel biomarkers for cervical cancer treatment. Methods: In the present study, cervical cell lines HeLa and SiHa with kin17 knockdown were constructed by transfection of the recombinant lentiviral vector carrying KIN17 siRNA and screened by puromycin. The established cells with kin17 knockdown were determined by fluorescence observation and western blotting. Cell apoptosis and the mitochondrial membrane potential (MMP) were detected by flow cytometry. The activity of caspase 3 enzyme was tested by spectrophotometry. The expression profile of apoptosis-associated proteins was analyzed by western blotting. Finally, we used bioinformatics and proteomic data to analyze KIN-related genes in cervical cancer. Results: The results showed high fluorescent positive rates (>90%) and high gene silencing efficiency (>65%) in HeLa and SiHa cells transfected with gene silencing vectors. Moreover, kin17 deficiency decreased the MMP and increased the apoptosis rates in HeLa and SiHa cells, respectively. Furthermore, knockdown of kin17 enhanced the activity of caspase 3 enzyme, increased the expression of cleaved PARP and Bim, while decreasing the expression of Bcl-xL and phosphorylated BAD in HeLa and SiHa cells. Identification of KIN-related prognostic genes in cervical cancer revealed that a total of 5 genes (FZR1, IMPDH1, GPKOW, XPA, and DDX39A) were constructed for this risk score, and the results showed that CTLA4 expressions were negatively correlated with the risk score. Conclusion: Our findings demonstrated that kin17 knockdown facilitates apoptosis of cervical cancer cells by targeting caspase 3, PARP, and Bcl-2 family proteins. Besides, kin17 could regulate cancer cell apoptosis through the mitochondrial pathway and could be used as a novel therapeutic target for the regulation of cell apoptosis in cervical cancer.

ECHS1, an interacting protein of LASP1, induces sphingolipid-metabolism imbalance to promote colorectal cancer progression by regulating ceramide glycosylation.

Sphingolipid metabolic dysregulation has increasingly been considered to be a drug-resistance mechanism for a variety of tumors. In this study, through an LC-MS assay, LIM and SH3 protein 1 (LASP1) was identified as a sphingolipid-metabolism-involved protein, and short-chain enoyl-CoA hydratase (ECHS1) was identified as a new LASP1-interacting protein through a protein assay in colorectal cancer (CRC). Gain- and loss-of-function analyses demonstrated the stimulatory role played by ECHS1 in CRC cell proliferation, migration, and invasion in vitro and in vivo. Mechanistic studies of the underlying tumor-supportive oncometabolism indicate that ECHS1 enables altering ceramide (Cer) metabolism that increases glycosphingolipid synthesis (HexCer) by promoting UDP-glucose ceramide glycosyltransferase (UGCG). Further analysis showed that ECHS1 promotes CRC progression and drug resistance by releasing reactive oxygen species (ROS) and interfering mitochondrial membrane potential via the PI3K/Akt/mTOR-dependent signaling pathway. Meanwhile, the phenomenon of promoting the survival and drug resistance of CRC cells caused by ECHS1 could be reversed by Eliglustat, a specific inhibitor of UCCG, in vitro and in vivo. IHC assay showed that ECHS1 was overexpressed in CRC tissues, which was related to the differentiation and poor prognosis of CRC patients. This study provides new insight into the mechanism by which phospholipids promote drug resistance in CRC and identifies potential targets for future therapies.

LASP1 interacts with N-WASP to activate the Arp2/3 complex and facilitate colorectal cancer metastasis by increasing tumour budding and worsening the pattern of invasion.

LIM and SH3 protein 1 (LASP1) is a metastasis-related protein reported to enhance tumour progression in colorectal cancer (CRC). However, the underlying mechanism is still elusive. As the major biological and pathological functions of LASP1 are accomplished by its LIM and SH3 domains via protein-protein interactions, a yeast two-hybrid system was employed to screen novel LASP1-interacting proteins. N-WASP, a member of the Wiskott-Aldrich syndrome protein (WASP) family, was screened and identified as a LASP1-interacting protein overexpressed in CRC tissues. N-WASP could stimulate the migration and invasion of CRC cells in vitro and increase the formation of subcutaneous tumours, mesenteric implanted tumours and hepatic metastatic tumours. N-WASP could interact with and activate the Arp2/3 complex to stimulate actin polymerization, thus changing the migratory and invasive capabilities of CRC cells. The interaction of LASP1 with N-WASP did not influence the expression of N-WASP but recovered the reduced actin polymerization induced by N-WASP silencing. High N-WASP expression was detected in most clinical colorectal samples, and it was positively correlated with the expression of LASP1 and ARP3, as well as the tumour budding and pattern of invasion, but negatively correlated with host lymphocytic response. Our study suggests a new mechanism for LASP1-mediated CRC metastasis determined by exploring LASP1-interacting proteins and identifies N-WASP as a potential therapeutic target for CRC.

Kin17 knockdown suppresses the migration and invasion of cervical cancer cells through NF-κB-Snail pathway.

Cervical cancer is one of the most common cancers in women worldwide. Metastasis in cancer has been a Gordian knot due to unsatisfactory clinical treatments. KIN17, a highly conserved gene from yeast to human, up-regulation is associated with the pathogenesis and development of several common cancers. Our previous works revealed that elevated expression of kin17 observed in cervical cancer tissues showed a close association with lymph node metastasis. This study aimed to explore roles and mechanisms of kin17 in the migration and invasion of cervical cancer cells. Cervical cancer cell lines HeLa and SiHa with kin17 knockdown were constructed by using recombinant lentiviral vector that carry specific siRNA targeting KIN17 gene. The mRNA and protein levels of kin17 in cells were determined by RT-qPCR and western blotting, respectively. Wound healing assay and transwell assays were performed to assess the migration and invasion abilities of the cancer cells, respectively. The expression of signaling proteins involved in the NF-κB-Snail pathway was analyzed by western blotting. As our results showed, the mRNA and protein levels of kin17 in HeLa cells and SiHa cells showed a significant decrease by transfection with recombinant lentiviral vector carrying specific siRNA. Compared with control group, the migration rates were decreased in the kin17 knockdown group in both HeLa and SiHa cell lines in wound healing assay as well as transwell assay without matrigel. Kin17 knockdown also reduced the cell invasion number of both HeLa and SiHa cells. In addition, the phosphorylation of nuclear factor Kαppa B (NF-κB) p65, IKαppa B kinase α (IKKα), and IKαppa B α (IκBα) in NF-κB pathway and the expression of Snail were decreased in HeLa cells and SiHa cells by kin17 knockdown. Our results demonstrated that knockdown of kin17 in cervical cancer cells suppressed cell migration and invasion, and inhibited the activity of NF-κB signaling pathway and the expression of Snail. These findings suggested kin17 as an essential regulator of the cell migration and invasion and the underlying molecular mechanism involved NF-κB-Snail pathway in cervical cancer. This might serve as a novel molecular therapeutic target for treating cervical cancer metastasis.

Knockdown of DNA/RNA-binding protein KIN17 promotes apoptosis of triple-negative breast cancer cells.

Effective therapy for breast cancer has been extensively studied worldwide, particularly for triple-negative breast cancer and drug-resistant subtypes. DNA/RNA-binding protein KIN17 (kin17) has been reported to be significantly upregulated in breast cancer cells, and is proposed to serve a role in the regulation of cell proliferation. The present study further investigated the association of kin17-knockdown with breast cancer cell apoptosis. Cell Counting kit-8, flow cytometry, TUNEL assay and caspase 3/7 analysis were performed on MDA-MB-231 cells to determine the association between kin17 and breast cancer cell apoptosis. In addition, western blot analysis was performed to investigate the mechanism of kin17 in the apoptosis of MDA-MB-231 cells. The results revealed that knockdown of kin17 inhibited proliferation and promoted apoptosis of MDA-MB-231 cells, and suggested a poly (adenosine diphosphate-ribose) polymerase-related mechanism behind the apoptosis of the cells. These findings suggested that kin17 could become a novel target for breast cancer therapy.

Upregulation of KIN17 is associated with non-small cell lung cancer invasiveness.

Kin17 DNA and RNA binding protein (Kin17) is a highly conserved protein that participates in DNA replication, DNA repair and cell cycle progression. Recently, the tumor-promoting function of Kin17 has been demonstrated and increasingly studied. In the present study, the role of Kin17 in the invasion and metastasis of non-small cells lung cancer (NSCLC) was investigated. Elevated Kin17 mRNA and protein expression was identified in a total of 97 NSCLC and benign lung lesion tissue specimens. Kin17 overexpression was significantly correlated with high tumor grade and lymph node metastasis, indicating poor patient prognosis. Scratch and Transwell assays demonstrated that the knockdown of KIN17 inhibited the ability of NSCLC cells to migrate and invade. Furthermore, reverse transcription-quantitative polymerase chain reaction and western blot analyses confirmed that knockdown of KIN17 decreased the expression of matrix metalloproteinase 7, epidermal growth factor receptor and v-myc avian myelocytomatosis viral oncogene homolog. The results of the present study indicate that Kin17 is markedly overexpressed in NSCLC tissues compared with benign lung lesion and peritumoral tissue. The upregulation of KIN17 may serve an important role in the metastasis of NSCLC cells. These results indicate that Kin17 is a novel diagnostic and prognostic biomarker of NSCLC, in addition to being a potential therapeutic target for the treatment of patients with NSCLC.

Elevated Expression of Kin17 in Cervical Cancer and Its Association With Cancer Cell Proliferation and Invasion.

BACKGROUND: Cervical cancer is one of the most common cancers in women worldwide. Emerging evidence suggests that kin17 is a tumor-promoting protein in some types of solid tumors. However, whether kin17 contributes to cervical cancer carcinogenesis remains unknown. METHODS: Kin17 expression in clinical samples from Guangdong Women and Children's Hospital and Health Institute was detected by immunohistochemical staining. A series of functional experiments including 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide assay, 5-bromo-2'-deoxyuridine assay, colony formation, transwell assay, flow cytometry of apoptosis, and cell cycle were performed to explore the roles of kin17 in cervical cancer cells HeLa. RESULTS: In this study, we showed for the first time that the expression of kin17 was significantly increased in clinical cervical cancer samples, and associated with tumor differentiation, lymph node metastasis, and ki-67 expression in a clinicopathologic characteristics review. Furthermore, silence of kin17 in HeLa cells inhibited cell proliferation, clone formation, cell cycle progression, migration, and invasion, and also promoted cell apoptosis. CONCLUSION: Our findings demonstrate that kin17 is closely related to the cell proliferation and invasion of cervical cancer and could be a novel diagnostic and therapeutic target for cervical cancer management. The underlying mechanisms should be elucidated in future research.

The research on screening differentially expressed genes in Hirschsprung's disease by using Microarray.

OBJECTIVE: To study the differential expression of genes between Hirschsprung's disease (HSCR) and normal tissue by using microarray for exploring the mechanism of HSCR development and establishing the gene expression profiles of HSCR. METHODS: Colon tissues (aganglionic and normal segments) of 4 patients with HSCR were detected by the Agilent SurePrint G3 Human GE 8x60K Microarrays. RT-PCR was used to verify the results of Microarray test. Then, immunohistochemistry was used to demonstrate the expression of HAND2 in the myenteric plexus of the colon from 46 patients with HSCR to further explore the relationship between HAND2 and development of HSCR. RESULTS: A total of 12,125 meaningful expressed genes were screened out. 4 pairs of specimens had 622 differentially expressed genes, 584 (93.89%) of which were up-regulated while 38(6.11%) were down-regulated. 6 of the 622 genes were tested by RT-PCR, which were consistent with the results detected by Microarray. The average optical density of positive expression of HAND2 in myenteric plexus was compared between the aganglionic, transitional, dilated, normal segments and control group. The average optical density in the aganglionic segments was obviously reduced. Statistical analyzed data showed that it has significant deviation (P<0.01). CONCLUSION: 1. A set of differentially expressed genes between aganglionic and normal segments of HSCR was obtained. Our data may provide significant information to research the pathogenesis of HSCR. 2. Reduced protein expression of HAND2 in the myenteric plexus of the aganglionic would suggest that HAND2 was involved in the pathogenesis of HSCR.

[Infection of cytomegalovirus and herpes simplex virus and morphology of the infected spermatogenic cells in infertile men].

OBJECTIVE: To study the infection of human cytomegalovirus (HCMV) and herpes simplex virus type II (HSV-I) and the morphological characteristics of the infected spermatogenic cells in the semen of infertile men. METHODS: We washed and concentrated the spermatogenic cells obtained from 83 semen samples of infertile men, extracted DNA and then screened HCMV and HSV-II by polymerase chain reaction (PCR). Immunocytochemistry (ICC) was used to detect the expression of correlative virus antigens of the positive semen cells, and the cytology smear was employed to observe the morphological changes of the spermatogenic cells under the microscope after cytology staining. RESULTS: Of all the semen samples, 8 were HCMV positive, 4 HSV-II positive, but none were both HCMV and HSV-II positive. HCMV late antigens were positively and HCMV early antigens negatively expressed in the spermatogenic cells of the 8 HCMV positive cases. In the 4 HSV-II positive cases, 3 were positively and 1 weakly positively expressed. In the semen of the 12 positive cases were found large numbers of immature spermatogenic cells, with different manifestations of apoptosis, such as chromatin pycnosis, vacuoles, damaged nuclear membrane, and apoptotic bodies, but without virus infection-induced specific morphological alteration. Sperm concentration of the positive group was significantly lower than that of the negative (P < 0. 05). CONCLUSION: Spermatogenic cells infected by HCMV and HSV-II may cause pathologic lesions and affect spermatogenesis. Morphologically, the infected spermatogenic cells may undergo some pathologic alteration, such as apoptosis. The rate of HCMV infection is higher among infertile males with pathologic cells in the semen.

[Expression and action of vascular endothelial growth factor and its receptor in a rat model of retinopathy of prematurity].

OBJECTIVE: To explore the expression of vascular endothelial growth factor (VEGF) and its receptors (flt-1 and flk-1) in the retina of retinopathy of prematurity (ROP), and its relation to the alteration of retinal blood vessels. METHODS: Eighty-six newborn Sprague-Dawley rats were randomly divided into hyperoxia and air groups, then each group was further divided into 1, 3, 7 and 14 days subgroups. The rats in hyperoxia group inhaled 75% oxygen and ROP model was thus set up. These animals were sacrificed respectively after 1, 3, 7 and 14 days, then the retinal endothelial cells were marked by CD34 to observe the change of retinal blood vessels. The expression of VEGF, flt-1 and flk-1 in the retina was measured by immunohistochemistry. RESULTS: The retinal capillary density index (RCDI) in control group increased as days went on (F = 21.589, P < 0.01, but it was the least on the 7th day in hyperoxia group, after the rats had been returned to air for 7 days, RCDI increased significantly (F = 67.885, P < 0.01); In the control group, the expression of VEGF and flk-1 was the strongest in the retina on the 7th day, the result had significant difference as compared with the 1st and 14th day (P < 0.05). The expression of VEGF and flk-1 on the 7th day in hyperoxia group was weaker than that of control group (P < 0.05). But on the 14th day in hyperoxia group, they were stronger than that of control (P < 0.05). The localization of the expression of flt-1 was changed when blood vessels altered, but there was no significant difference in expression intensity as a whole (P > 0.05). CONCLUSION: When the premature retina was exposed to hyperoxia, the expression of VEGF and flk-1 was reduced, and retinal blood vessels were also decreased; but the expression of VEGF and flk-1 was stronger in retina when premature rats were exposed to relative hypoxia, and the retinal blood vessels also increased significantly. It is concluded that VEGF and flk-1 may play important roles in the development of retinal blood vessels and its change in ROP. However, flt-1 has less effect compared with flk-1.