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Objective:To investigate the effect of the methyltransferase inhibitor azacitidine (5-azaC) on the expression of homeobox A9 (HOXA9) gene in, as well as proliferation, invasion and migration of A375 cells.Methods:In vitro cultured A375 cells were treated with 5-azaC at various concentrations of 1, 5, 10 and 20 μmol/L, while routinely cultured A375 cells receiving no drug intervention served as control group. Methylation-specific PCR was performed to analyze methylation status of the HOXA9 gene promoter region after the treatment with different concentrations of 5-azaC, in order to screen the optimal concentration of 5-azaC for following experiments. Cell counting kit-8 (CCK8) assay was conducted to evaluate the proliferation of A375 cells, Transwell and wound healing assays were performed to estimate the invasion and migration of A375 cells, and real-time fluorescence-based quantitative PCR (qRT-PCR) and Western blot analysis were conducted to determine the mRNA and protein expression of HOXA9 in A375 cells after 5-azaC treatment. Two-independent-sample t test was used for comparisons between two groups. Results:Methylation was observed in the HOXA9 gene promoter region in A375 cells in the control group. After 5-azaC treatment, methylated and unmethylated states coexisted in the HOXA9 gene promoter region in A375 cells, and the higher the concentration of 5-azaC, the higher the degree of demethylation of the HOXA9 gene. Therefore, 20 μmol/L 5-azaC was selected to treat A375 cells for 72 hours, which served as 5-azaC treatment group in subsequent experiments. Compared with the control group, the 5-azaC treatment group showed significantly decreased cellular proliferative ability (72.46% ± 2.19% vs. 100%, t = 28.09, P < 0.001) , significantly decreased number of invasive cells (242.70 ± 29.19 vs. 466.00 ± 22.65, t = 10.47, P < 0.001) , significantly decreased migratory ability (27.56% ± 2.74% vs. 35.69% ± 2.50%, t = 3.79, P = 0.019) , significantly increased HOXA9 mRNA expression (1.73 ± 0.28 vs. 1.01 ± 0.15, t = 3.93, P = 0.017) , and significantly increased HOXA9 protein expression (0.62 ± 0.03 vs. 0.50 ± 0.01, t = 3.82, P = 0.019) . Conclusion:5-azaC can inhibit the proliferative, invasive and migratory ability of A375 melanoma cells, and one of the possible mechanisms underlying this process may be that 5-azaC reverses the methylation in the HOXA9 gene promoter region, activates HOXA9 gene expression, and participates in the regulation of biological behaviors of melanoma cells.
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Objective To investigate the reasons of HOXA5 overexpression in GBM and the molecular mechanism of miR-128-3p regulating the proliferation, invasion and apoptosis of glioblastoma multiforme. Methods After increasing and decreasing miR-128-3p expression in U87 cell lines by lentivirus transfection, the changes of HOXA5 expression were detected by Western blot, to explore the correlation between miR-128-3p and HOXA5 in GBM. The dual-luciferase reporter tests were performed to detect the target interaction of miR-128-3p with HOXA5. Through CCK-8 test, Transwell test, flow cytometric assay and tumor cell xenograft in nude mice, we verified molecular mechanism of miR-128-3p regulating the proliferation, invasion and apoptosis of GBM in vitro and in vivo. Results The expression level of HOXA5 was decreased in U87 cell line after miR-128-3p upregulation. In addition, the expression level of HOXA5 was increased in U87 cell line after miR-128-3p downregulation (P < 0.05). The expression level of HOXA5 was correlated negatively with the expression of miR-128-3p in U87 cell lines. MiR-128-3p targetedly interacted with 3'UTR of HOXA5 and inhibited the expression of HOXA5. The proliferation, invasion and anti-apoptosis of U87 cells were significantly decreased in the miR-128-3p+control group. Conclusion MiR-128-3p regulates negatively the proliferation, invasion and anti-apoptosis of GBM cells by targeting HOXA5. The overexpression of HOXA5 is induced by downregulation of miR-128-3p in GBM.
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OBJECTIVE@#To compare the clinical effect of the combined treatment of acupuncture, moxibustion, Chinese herbal medicine and western medication and simple western medication on polycystic ovary syndrome (PCOS) of kidney deficiency and blood stagnation pattern and explore the effect on endometrial receptivity and the expression of serum homeobox gene A10 (HOXA10).@*METHODS@#A total of 60 patients with PCOS of kidney deficiency and blood stagnation pattern were randomized into a combined treatment group and a western medication group, 30 cases in each one. In the western medication group, on the fifth day of menstruation, clomiphene citrate tablets were taken orally, 50 mg each time, once daily, consecutively for 5 days. On the day when the follicle diameter was ≥ 18 mm, chorionic gonadotrophin for muscular injection, a dose of 10 000 U was given. Before sleep, the aspirin enteric-coated tablets were taken orally, 50 mg (except during menstruation). In the combined treatment group, on the base of the treatment as the western medication group, acupuncture and moxibustion were adopted and the Chinese herbal for tonifying the kidney and activating blood circulation was taken orally. The acupoints were Guanyuan (CV 4), Qihai (CV 6), Zusanli (ST 36), Sanyinjiao (SP 6), Zigong (EX-CA 1), etc. Acupuncture was remained for 30 min each time, once every two days and discontinued during menstruation. Chinese herbal was given from the 3rd day of menstruation till the onset of the next menstruation, one dose each day. After consecutive treatment for 3 menstrual cycles in the two groups, the real-time polymerase chain reaction (RT-PCR) method was adopted to determine the expression of serum HOXA10 before and after treatment in the patients of the two groups. The endometrial thickness at ovulatory phase, uterine arterial flow 7 days after ovulation [including uterine arterial pulsatility index (PI), resistance index (RI), peak systolic velocity (PSV)/end diastolic velocity (EDV), meaning S/D], pregnancy rate and the score of Chinese medicine symptoms before and after treatment were compared in the patients between the two groups.@*RESULTS@#① After treatment, the expression of serum HOXA10 was higher than that before treatment in the patients of the two groups (@*CONCLUSION@#The combined treatment with acupuncture, moxibustion and medication effectively improves endometrial receptivity and uterine arterial flow in the patients with PCOS of kidney deficiency and blood stagnation pattern and increases pregnancy rate. The therapeutic effect is better than the simple western medication and its mechanism is probably related to the regulation of serum HOXA10 expression.
Subject(s)
Female , Humans , Pregnancy , Acupuncture Points , Acupuncture Therapy , Genes, Homeobox , Homeobox A10 Proteins , Kidney , Moxibustion , Polycystic Ovary Syndrome/geneticsABSTRACT
@#[Abstract] Objective: To investigate the expression and clinical significance of long non-coding RNA (lncRNA) HOTTIP in tissues of patients with endometrial carcinoma. Methods: A total of 109 cases of patients with endometrial carcinoma who underwent surgery in Xingxiang Central Hospital from April 2012 to April 2014 were selected. The endometrial carcinoma tissue and its corresponding adjacent tissue (more than 5 cm from the cancer margin) were obtained. The expressions of HOTTIP in endometrial carcinoma and adjacent tissues were detected by qRT-PCR. All patients were followed up from the first postoperative day. The follow-up deadline was April 30, 2019. The end-point event was death and the patient's survival time was recorded. Results: The relative expression level of HOTTIP in endometrial carcinoma tissues was (2.55±0.21), which was higher than that in the adjacent tissue (1.03±0.16) (t=60.631, P<0.01). The differences of the relative expression levels of HOTTIP in endometrial carcinoma tissues between different FIGO stage, histological grade, depth of myometrial invasion, lymphatic vascular infiltration status and lymph node metastasis were statistically significant (all P<0.05). Kaplan-Meier survival analysis showed that the 5-year survival rate and the survival time in the low expression group were higher than those in the high expression group [78.57% vs 37.04%, (70.67±4.94) months vs (42.14±3.65) months], the difference was statistically significant (χ2=12.839, P<0.01). Cox proportional hazards regression model analysis showed that the FIGO stage [HR=2.248 (95%CI: 1.034-4.887)], myometrial invasion depth [HR=3.055 (95%CI: 1.668-5.592)], lymph node metastasis [HR=3.811 (95%CI: 1.786-8.131)] and the expression of HOTTIP [HR=2.649 (95%CI: 1.026-6.842)] were all independent influence factors for the prognosis of patients with endometrial carcinoma. Conclusion: lncRNA HOTTIP is highly expressed in endometrial carcinoma tissues and associated with malignant progression of patients. It is an independent influencing factor for patients’ prognosis.
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Noncoding somatic mutations have been demonstrated to play important role in tumourigenesis. Here we show that there exists an acute myeloid leukaemia associated noncoding somatic mutation at 3′ terminal of conserved HOXA cluster. The mutation was identified in the bone marrow blasts but not peripheral blood mononuclear cells or buccal cells of two M3 (acute promyelocytic leukaemia, APL) type patients from 45 acute myeloid leukaemia patients. The mutation also existed in a pair of twins one of them developed acute myeloid leukaemia M4 (acute myelomonocytic leukaemia) type. The mutation resides in about 2-kb downstream of HOXA1 gene where a functional retinoic acid response element is located and also bound by histone demethylase KDM3B. Reporter assay showed that the mutation results in the upregulation of transcriptional activity and unresponsiveness to retinoic acid receptor. To sum up, we identified a new acute myeloid leukaemia associated noncoding somatic mutation.
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Objective To investigate the expression of HOXA terminal transcript antisense RNA (HOTTIP) in hepatocellular carcinoma (HCC) tissues and to explore its effect on proliferation, invasion and migration in HepG2 cells. Methods A total of 60 cases with HCC tissues undergoing excision surgery and 60 cases of corresponding paracancerous tissues from January 2012 to June 2018 in Dandong First Hospital of Liaoning Province were collected. The expressions of HOTTIP in HCC tissues and paracancerous tissues were detected by using real-time quantitative polymerase chain reaction (RT-qPCR), and the relationship between the expression and clinicopathological features was analyzed. HepG2 cell line with high expression of HOTTIP constructed by cell transfer technique was treated as the experimental group, and the empty plasmid pcDNA3.1-NC was treated as the control group. The effect of HOTTIP on the proliferation of HepG2 cells was detected by using CCK-8 method, and the effect of HOTTIP on invasion and migration of HepG2 cells was detected by using Transwell assay. Results The expression of HOTTIP mRNA in HCC tissues was higher than that in paracancerous tissues, and there was no statistically significant difference (1.9±0.6 vs. 0.9±0.7, t=6.069, P<0.01). The whole HCC cases were divided into the high expression group (30 cases) and the low expression group (30 cases) according to the median value (1.92) of the expression of HOTTIP mRNA. The expression of HOTTIP was related with TNM stage, differentiation degree and lymph node metastasis (χ2 values were 10.800, 8.076, 5.711, all P<0.05), but not with age, gender, tumor diameter, number of tumors, hepatitis and alpha fetoprotein (AFP) levels (all P>0.05). The results of RT-qPCR showed that the expression of HOTTIP mRNA in HepG2 cells was increased after transfection of overexpressed HOTTIP and the differences was statistically significant compared with the control group (63±6 vs. 13±9, t=9.129, P<0.01). The results of CCK-8 method showed that the proliferation activity of cells was enhanced after the overexpression of HOTTIP in HepG2 cells (24 h, 36 h, 72 h at 490 nm absorbance was 1.497 ± 0.017 vs. 0.826 ±0.006, 2.002 ±0.025 vs. 1.211 ±0.020, 3.257 ±0.042 vs. 1.772 ±0.021), and the differences were statistically significant (t values were 5.321, 7.349, 8.793, all P < 0.01). In Transwell invasion assay, the number of cells transferred into the basement membrane in the experimental group was more than that in the control group (101 ±9 vs. 41 ±11), and the difference was statistically significant (t= 6.839, P< 0.01). In Transwell migration assay, the number of cells transferred into the basement membrane in the experimental group was more than that in the control group (112 ±9 vs. 53 ±11), and the difference was statistically significant (t=7.105, P<0.01). Conclusion The expression of HOTTIP in HCC tissues is up-regulated, and the overexpression of HOTTIP can promote the proliferation, invasion and migration of HCC cells.
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Objective To investigate the expressions of HOXA11 and β-catenin proteins in colorectal serrated lesions and their roles in carcinogenesis. Methods A total of 252 cases of colorectal biopsy specimens in Shanxi Dayi Hospital from January 2012 to December 2016 were analyzed retrospectively, including 97 serrated lesions, 46 common adenomas, 109 adenocarcinomas, and 24 normal colorectal mucosa tissues were selected as controls. The expressions of HOXA11 and β-catenin proteins were detected by immunohistochemical EnVision method. Methylation of HOXA11 gene was detected by specific methylation polymerase chain reaction (MSP) in 25 paraffin-embedded adenocarcinoma tissues. Results In 97 serrated lesions, 29 (29.9％) occurred in the left colon;in 46 common adenomas, 27 (58.7％) occurred in the left colon;in 109 adenocarcinomas, 76 (69.7％) occured in the left colon. The difference of the occurrence location among three groups was statistically significant (χ2 = 34.75, P< 0.01). The heterotopic expression rate ofβ-catenin protein in serrated lesions, common adenomas and adenocarcinomas was significantly higher than that in normal mucosae [96.9％ (94/97), 82.6％ (38/46), 86.2％ (94/109) vs. 0 (0/24), P < 0.01]. The heterotopic expression of β-catenin protein was found in serrated lesions and the co-expression of cytoplasm and nucleus was found in common adenomas and adenocarcinomas. The normal expression rate of HOXA11 protein in serrated lesions, common adenomas and adenocarcinomas was lower than that in normal mucosae [33.0％ (32/97), 67.4％ (31/46), 48.6％ (53/109) vs. 100.0％ (24/24), all P< 0.05]. The methylation rate of HOXA11 gene was 84.0％ (21/25). Conclusion The ectopic expression of β-catenin in colorectal serrated lesions suggests that it is associated with serrated lesions, and the low expression of HOXA11 may be an early event in the carcinogenesis of serrated lesions.
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Objective: To detect the methylation levels of homeobox protein Hox A7 (HOXA7) gene promoter in the plasma of non-small cell lung cancer (NSCLC) and to study the value of HOXA7 methylation and serum levels of the tumor markers carcinoembryonic antigen (CEA), carbohydrate antigen 199 (CA199), and cytokeratin 19 fragment (Cyfra21-1) in the auxiliary diagnosis of NSCLC. Methods: Plasma samples were collected from 80 patients with NSCLC and 50 healthy controls, which were enrolled in Henan Cancer Hospital from January 2012 to December 2016. The plasma HOXA7 methylation levels were detected by real-time quantitative methylation-specific PCR, and the plasma levels of CEA, CA199, and Cyfra21-1 were detected by electrochemical luminescence. The ROC curve was constructed to analyze the clinical value of each index in the differential diagnosis of NSCLC, and the relationship between HOXA7 methylation, clinical parameters, and tumor markers was analyzed. Results: The serum levels of HOXA7 methylation in NSCLC patients were significantly higher (χ2=36.972, P<0.000 1) than the healthy control group. The sensitivity and specificity of HOXA7 methylation in the diagnosis of NSCLC were 68.8% (55/80) and 86.0% (43/50), respectively. Plasma HOXA7 methylation was not related to gender, age, smoking history, or pathological type (all P>0.05), but was related to TNM stage (P<0.05). The plasma HOXA7 methylation level of stageⅣpatients (81.8%, 18/22) was the highest. Cyfra21-1 had the highest value in the diagnosis of NSCLC among tumor markers with a sensitivity of 70.00% and a specificity of 90.00%. The combination of HOXA7 methylation with all three tumor markers had the highest efficiency (AUC=0.893, sensitivity 73.75%, specificity 94%) in the diagnosis of NSCLC. The correlation coefficient between HOXA7 methylation and Cyfra21-1 was the highest (r=0.564, P<0.05). Conclusions: HOXA7 gene methylation is related to the degree of metastasis of NSCLC and proves as an efficient diagnostic marker for NSCLC when combined with tumor marker detection.
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@#Objective: To investigate the expression of HOXA13 in non-small cell lung cancer (NSCLC) tissues, and to explore the effect of silencing HOXA13 gene on the growth of A549 cells in vitro and xenograft in nude mice. Methods: A total of 112 pairs of NSCLC tissues and corresponding adjacent normal tissues from patients, who underwent surgical resection at the Department of Thoracic surgery, Central Hospital of Jiaozuo Coal Industry Group from March 2014 toApril 2016, were selected for this study. Real-time fluorescence quantitative PCR was used to detect the expressions of HOXA13 in NSCLC tissues and adjacent tissues. A549 cells were cultured and divided into siRNA-HOXA13 group, negative control group and control group. Real-time quantitative PCR was used to detect the expression of HOXA13 in cells, CCK-8 was used to detect cell proliferation, and Transwell assay was used to detect cell invasion. Xenograft model in nude mice was constructed, and the growth of nude mice was observed.After 5 weeks, the mice was sacrificed and weighed, and the tumor-inhibition rate was calculated. Real-time fluorescence quantitative PCR(qPCR) was used to detect the expression of HOXA13 in xenograft tissues. Results: The relative expression level of HOXA13 mRNAin NSCLC tissues was significantly higher than that in the adjacent tissues (1.83±0.13 vs 1.12±0.10, t=47.008, P=0.000), and its expression was correlated to TNM staging, differentiation and lymph node metastasis (all P<0.05). The relative expression level of HOXA13 mRNA in cells of siRNAHOXA13 group was lower than that in the negative control group and the control group (P<0.05). The cell proliferation level (D values) at 24, 48, 72 and 96 h in the siRNA-HOXA13 group were significantly lower than those in the negative control group and control group (F=30.727, 5.427, 13.816 and 24.454, all P<0.05 or P<0.01); the number of invasive cells in the siRNA-HOXA13 group was lower than that in the negative control group and the control group (all P<0.05). The mass of xenograft in nude mice at week 5 in the siRNA-HOXA13 group was smaller than that in the negative control group and control group, while the tumor-inhibition rate was higher than the negative control group (all P<0.05). The relative mRNA expression level of HOXA13 in xenograft tumor tissue in the siRNA-HOXA13 group was lower than that in the negative control group and the control group (all P<0.01). Conclusion: HOXA13 was highly expressed in the non-small cell lung cancer tissues, and was related to oncogenesis, progression and metastasis of cancer. Specific silencing of HOXA13 gene expression could inhibit the proliferation and invasion of tumor cells and suppress the growth of xenograft in nude mice.
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Objective@#To investigate the expression of HOXA terminal transcript antisense RNA (HOTTIP) in hepatocellular carcinoma (HCC) tissues and to explore its effect on proliferation, invasion and migration in HepG2 cells.@*Methods@#A total of 60 cases with HCC tissues undergoing excision surgery and 60 cases of corresponding paracancerous tissues from January 2012 to June 2018 in Dandong First Hospital of Liaoning Province were collected. The expressions of HOTTIP in HCC tissues and paracancerous tissues were detected by using real-time quantitative polymerase chain reaction (RT-qPCR), and the relationship between the expression and clinicopathological features was analyzed. HepG2 cell line with high expression of HOTTIP constructed by cell transfer technique was treated as the experimental group, and the empty plasmid pcDNA3.1-NC was treated as the control group. The effect of HOTTIP on the proliferation of HepG2 cells was detected by using CCK-8 method, and the effect of HOTTIP on invasion and migration of HepG2 cells was detected by using Transwell assay.@*Results@#The expression of HOTTIP mRNA in HCC tissues was higher than that in paracancerous tissues, and there was no statistically significant difference (1.9±0.6 vs. 0.9±0.7, t = 6.069, P < 0.01). The whole HCC cases were divided into the high expression group (30 cases) and the low expression group (30 cases) according to the median value (1.92) of the expression of HOTTIP mRNA. The expression of HOTTIP was related with TNM stage, differentiation degree and lymph node metastasis (χ2 values were 10.800, 8.076, 5.711, all P < 0.05), but not with age, gender, tumor diameter, number of tumors, hepatitis and alpha fetoprotein (AFP) levels (all P > 0.05). The results of RT-qPCR showed that the expression of HOTTIP mRNA in HepG2 cells was increased after transfection of overexpressed HOTTIP and the differences was statistically significant compared with the control group (63±6 vs. 13±9, t = 9.129, P < 0.01). The results of CCK-8 method showed that the proliferation activity of cells was enhanced after the overexpression of HOTTIP in HepG2 cells (24 h, 36 h, 72 h at 490 nm absorbance was 1.497 ± 0.017 vs. 0.826±0.006, 2.002±0.025 vs. 1.211±0.020, 3.257±0.042 vs. 1.772±0.021), and the differences were statistically significant (t values were 5.321, 7.349, 8.793, all P < 0.01). In Transwell invasion assay, the number of cells transferred into the basement membrane in the experimental group was more than that in the control group (101±9 vs. 41±11), and the difference was statistically significant (t = 6.839, P < 0.01). In Transwell migration assay, the number of cells transferred into the basement membrane in the experimental group was more than that in the control group (112±9 vs. 53±11), and the difference was statistically significant (t = 7.105, P < 0.01).@*Conclusion@#The expression of HOTTIP in HCC tissues is up-regulated, and the overexpression of HOTTIP can promote the proliferation, invasion and migration of HCC cells.
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Objective To study unfractionated heparin (UFH) effect on the expression of HOXA9 in activation of human umbilical vein endothelial cells (HUVECs) induced by lipopolysaccharide (LPS). Methods HUVECs were cultured and they were randomly divided into four groups (n = 5) for the challenge respectively: ① control group (with an equal volume of phosphate buffer saline); ② LPS group (LPS 10 mg/L); ③UFH group (UFH 10 kU/L);④ UFH+LPS group (10 kU/L UFH 30 minutes + LPS 10 mg/L). After treatment for 3 hours, the expressions of HOXA9, E-selectin and nuclear factor-κB (NK-κB) in endothelial cells were detected by Western Blot. Results Compared with the control group, the expression of HOXA9 in LPS group was significantly decreased, the expressions of E-selectin and NF-κB were significantly increased (HOXA9/β-actin: 0.082±0.009 vs. 0.199±0.067, E-selectin/β-actin:0.113±0.055 vs. 0.047±0.030, NF-κB/β-actin: 0.845±0.025 vs. 0.664±0.092, all P < 0.05). Compared with LPS group, the expression of HOXA9 in UFH+LPS group was significantly increased, the expressions of E-selectin and NF-κB were significantly decreased (HOXA9/β-actin: 0.190±0.096 vs. 0.082±0.009, E-selecin/β-actin: 0.057±0.017 vs. 0.113±0.055, NF-κB/β-actin: 0.544±0.060 vs. 0.845±0.025, all P < 0.05). Each protein expression of UFH group were in accordance with the control group. Conclusions In LPS stimulated endothelial cells, HOXA9 expression is down regulated, E-expression is reduced, and endothelial cell activation is inhibited. UFH can inhibit the activation of endothelial cells by decreasing the degree of HOXA9 reduced expression.
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Objective:To investigate the effects of silencing HOXA13 gene on the malignant phenotypes of hepatocellular carcinoma cell lines HepG2and QGY-7703,and to provide a new molecular target for the diagnosis and treatment of hepatocellular carcinoma.Methods:The interference recombinant plasmid of plent-U6-GFP/si-HOXA13 was constructed,then it was stably transfected into the HepG2and QGY-7703 cells.The interfering effects of HOXA13 gene were determined by RT-PCR and Western blotting methods.The HepG2and QGY-7703 cells transfected with HOXA13 knockdown were used as experimental group,and the HepG2and QGY-7703 cells transfected with empty plasmid were used as control group.Then the growth speed was examined by MTT assay, the cell doubling time was examined by double time assay,the colony formation ability was examined by colony formation assay,and the cell cycle was examined by flow cytometry.Results:The MTT assay results showed that compared with control group,the growth speeds of HepG2and GY-7703 cells in experimental group were significantly decreased and the cell cycles were changed;the number of HepG2and QGY-7703 cells in G1phase was increased and the number of cells in S phase was decreased.The doubling time of HepG2and QGY-7703 cells in control group and experimental group were(4.59±0.27),(4.93±0.17),(6.02±0.86),and(6.43±0.66)h, and the differences between control group and experimental group were significant(P<0.05).The colony number of HepG2and QGY-7703 cells in control group and experimental group were 264.00 ± 12.62,269.00 ± 4.55, 165.00± 10.61,and 215.00 ± 4.43,and the differences between control group and experimental group were significant(P<0.01).Conclusion:HOXA13 can increase the proliferation,enhance the clone formation,decrease the number of cells at G1phase and increase the number of cells at S phase in the hepatocellular carcinoma HepG2 and QGY-7703 cells;and it may used as a new molecular target for diagnosis and treatment of hepatocellular carcinoma.
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@# Objective: To study the expression of H O X A 1 0 gene in endometrial carcinoma and its effect on the apoptosis, migration and invasion of Ishikawa cells. Methods: Twenty-one cases of endometrial carcinoma tissue samples and 25 cases of normal endometrial tissue samples from patients treated at the Department of Obstetrics and Gynecology, Nanjing Drum Tower Hospital from 2012 to 2013 were collected for this study. The mRNA and protein expressions of H O X A 1 0 in endometrial carcinoma and normal endometrial tissues were separately tested by Realtime-qPCR (qRT-PCR) and Western blotting. Ishikawa cells were infected with adenovirus-flagHOXA10 at different multiplicity (5, 10, 20 MOI), and infected by adenovirus-flag-lacz (20 MOI) as control; And the cell apoptosis was tested by Flow Cytometry. Ishikawa cells were transfected with 50 nmol/L si-HOXA10 plasmids and 50 nmol/L si-NC plasmids, as down-regulation group and down-regulation control group, respectively. Ishikawa cells were infected with 20 MOI adenovirus-flagHOXA10 and 20 MOI adenovirus-flag-lacz, as up-regulation group and up-regulation control group, respectively. The ability of migration and invasion was detected by transwell assay. Results: The results of qRT-PCR and Western blotting showed that the expressions of H O X A 1 0 mRNA and protein in endometrial carcinoma samples were both significantly lower than normal samples [mRNA: (0.56± 0.14)vs (1.36±0.33), P<0.01; protein: (1.01±0.25) vs (2.10±0.71), P<0.001]. After the up-regulation of H O X A 1 0 gene in Ishikawa cell line, the cell apoptosis rate in ad-flag-HOXA10 groups (5, 10, 20 MOI) was significantly raised, and most of which was in the early apoptosis [(50.92±8.79)%, (55.17±4.07)%, (76.10±3.65)% vs (7.74 ± 0.15)%, all P <0.01]. The number of migrated cells was markedly up-regulated in si-HOXA10 group [(248±25) vs (135±15), P <0.01] but markedly down-regulated in ad-flag-HOXA10 group [(50±6) vs (100±13), P <0.01]. The number of invasive cells was markedly up-regulated in si-HOXA10 group [(131±18) vs (66±9), P <0.01] but markedly down-regulated in ad-flag-HOXA10 group [(34±8) vs (60±4), P <0.01]. Conclusions: Both mRNAand protein expressions of H O X A 1 0 were down-regulated in endometrial carcinoma samples than in normal endometrium. Up-regulation of H O X A 1 0 gene in Ishi kawa cell line can promote cell apoptosis and inhibit cell migration and invasion.
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Objective@#To explore the expression of long noncoding RNA (lncRNA) HOXA11-AS in esophageal squamous cell carcinoma tissues and the relationship of HOXA11-AS level with clinical outcomes.@*Methods@#Quantitative real-time polymerase chain reaction (qRT-PCR) was applied to detect the expression level of HOXA11-AS in cell lines HET-1A, EC9706, EC109, and in tumor tissue and paired adjacent tissue samples from 73 ESCC patients who received surgical resection.The correlations of the expression level of HOXA11-AS with clinicopathological features and prognosis were also analyzed.@*Results@#The relative expression levels of HOXA11-AS in tumor tissue and paired adjacent tissue were 0.832±0.387 and 2.486±1.087, respectively, with significant difference (P<0.001). The expression of HOXA11-AS was upregulated in 63.0%(46/73)ESCC tissues. The relative expression levels of HOXA11-AS in HET-1A, EC-9706 and EC-109 cells were 1.000, 23.553±3.221 and 17.217±1.968, respectively. The expression level of HOXA11-AS was upregulated in ESCC cell lines (P<0.001). High expression level of HOXA11-AS was correlated with histological grade and lymph node metastasis of ESCC patients (P<0.05). However, it was not associated with the age, gender, depth of infiltration and TNM staging (P>0.05). The median overall survival (OS) and median disease-free survival (DFS) of patients with low HOXA11-AS expression were 43 months and 42 months, respectively, significantly longer than 37 months and 28 months of patients with HOXA11-AS high expression (P<0.05). Cox model multivariate analysis showed that the expression of HOXA11-AS and lymph node metastasis were independent factors of poor prognosis of ESCC patients.@*Conclusions@#The expression of HOXA11-AS is upregulated in esophageal cancer cell lines and tissues. High expression of HOXA11-AS is associated with poor prognosis of ESCC patients.Therefore, LncRNA HOXA11-AS may serve as a predictive marker of postoperative ESCC patients.
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Objective@#To identify a pig model with bilateral external ear defects accompanied by aural atresia and investigate its application in plastic surgery.@*Methods@#Erhualian×Shaziling F2 pig inbreeding population was introduced, and examination of external ear morphology was conducted in all individuals. Temporal computed tomography scanning and mutational detection of HOXA1 gene were conducted in one affected and one normal individuals.@*Results@#In Erhualian×Shaziling F2 pig inbreeding population, there were 57 normal and 18 affected individuals among the 75 pigs. Affected subjects presented bilateral external ear defects accompanied by aural atresia; temporal computed tomography scanning showed bilateral aural atresia and dysplasiaof middle ear; and gene detection identified homozygous mutation of HOXA1 gene.@*Conclusions@#Pig model with HOXA1 gene homozygous mutation resembles human microtia at different levels. Our findings provide the theoretical basis for its application to study further pathological mechanism for human microtia.
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Objetive: To investigate the effects of silencing HOXA13 gene on the malignant phenotypes of hepatocellular carcinoma cell lines HepG2 and QGY-7703, and to provide a new molecular target for the diagnosis and treatment of hepatocellular carcinoma. Methods: The interference recombinant plasmid of plent-U6-GFP/si-HOXA13 was constructed, then it was stably transfected into the HepG2 and QGY-7703 cells. The interfering effects of HOXA13 gene were determined by RT-PCR and Western blotting methods. The HepG2 and QGY-7703 cells transfected with HOXA13 knockdown were used as experimental group, and the HepG2 and QGY-7703 cells transfected with empty plasmid were used as control group. Then the growth speed was examined by MTT assay, the cell doubling time was examined by double time assay, the colony formation ability was examined by colony formation assay, and the cell cycle was examined by flow cytometry. Results: The MTT assay results showed that compared with control group, the growth speeds of HepG2 and GY-7703 cells in experimental group were significantly decreased and the cell cycles were changed; the number of HepG2 and QGY-7703 cells in G1 phase was increased and the number of cells in S phase was decreased. The doubling time of HepG2 and QGY-7703 cells in control group and experimental group were (4.59 ± 0.27), (4.93 ± 0.17), (6.02 ± 0.86), and (6.43 ± 0.66) h, and the differences between control group and experimental group were significant (P<0.05). The colony number of HepG2 and QGY-7703 cells in control group and experimental group were 264.00 ± 12.62, 269.00 ± 4.55, 165.00±10.61, and 215.00 ± 4.43, and the differences between control group and experimental group were significant (P<0.01). Conclusion: HOXA13 can increase the proliferation, enhance the clone formation, decrease the number of cells at G1 phase and increase the number of cells at S phase in the hepatocellular carcinoma HepG2and QGY-7703 cells; and it may used as a new molecular target for diagnosis and treatment of hepatocellular carcinoma.
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PURPOSE: Homeobox (HOX) genes are essential developmental regulators that should normally be in the silenced state in an adult brain. The aberrant expression of HOX genes has been associated with the prognosis of many cancer types, including glioblastoma (GBM). This study examined the identity and role of HOX genes affecting GBM prognosis and treatment resistance. MATERIALS AND METHODS: The full series of HOX genes of five pairs of initial and recurrent human GBM samples were screened by microarray analysis to determine the most plausible candidate responsible for GBM prognosis. Another 20 newly diagnosed GBM samples were used for prognostic validation. In vitro experiments were performed to confirm the role of HOX in treatment resistance. Mediators involved in HOX gene regulation were searched using differentially expressed gene analysis, gene set enrichment tests, and network analysis. RESULTS: The underexpression of HOXA11 was identified as a consistent signature for a poor prognosis among the HOX genes. The overall survival of the GBM patients indicated a significantly favorable prognosis in patients with high HOXA11 expression (31±15.3 months) compared to the prognoses in thosewith low HOXA11 expression (18±7.3 months, p=0.03). When HOXA11 was suppressed in the GBM cell lines, the anticancer effect of radiotherapy and/or temozolomide declined. In addition, five candidate mediators (TGFBR2, CRIM1, TXNIP, DPYSL2, and CRMP1) that may confer an oncologic effect after HOXA11 suppression were identified. CONCLUSION: The treatment resistance induced by the underexpression of HOXA11 can contribute to a poor prognosis in GBM. Further investigation will be needed to confirm the value of HOXA11 as a potential target for overcoming the treatment resistance by developing chemo- or radiosensitizers.
Subject(s)
Adult , Humans , Brain , Cell Line , Genes, Homeobox , Glioblastoma , In Vitro Techniques , Microarray Analysis , Prognosis , RadiotherapyABSTRACT
As the crucial member of noncoding RNA family,there is increasing evidence that long noncoding RNA has participated in numerous physiological and pathological processes of organisms.HOXA distal transcript antisense RNA is a long noncoding transcript located at the 5'tip of HOXA.According to research reports,the up-regulation of HOTTIP is closely associated with occurrence and progression of multiple digestive system cancers and promotes the carcinogenesis.The regulatory effect of long noncoding RNA HOXA distal transcript antisense in digestive system neoplasms will be summarized in this article.
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Objective To investigate the regulatory effects of Zishen Yutai Pills on the expression levels of homeboxA10 (HOXA10) and its downstream target gene empty spiracles homebox 2 (EMX2) in the endometria of ovulation-inducing mice at different implantation stages. Methods Seventy-five estrous female Kunming mice were randomly divided into 5 groups, namely normal group, model group 1, model group 2, treatment group 1, treatment group 2, 15 mice in each group. The model group 1 was given short-term protocol for ovulation induction; the model group 2 was given long-term protocol for ovulation induction; the treatment group 1 was given Zishen Yutai pills (at the dose of 0.4 g/mL) on the basis of the protocol for the model group 1; the treatment group 2 was given Zishen Yutai Pills (at the dose of 0.4 g/mL) on the basis of the protocol for the model group 2; the normal group was given intragastric administration or intraperitoneal injection of the same volume of normal saline. The mRNA and protein expression levels of HOXA10 and EMX2 in mouse uterus were detected by real-time fluorescent quantitative polymerase chain reaction (qPCR) and Western blot method, respectively. Results Compared with the normal group, the mRNA and protein expression levels of HOXA10 were decreased, and the mRNA and protein expression levels of EMX2 were increased in model group 1 and model group 2(P< 0.01). Compared with the corresponding model group 1 and 2, the mRNA and protein expression levels of HOXA10 were significantly up-regulated (P < 0.01) , and the mRNA and protein expression levels of EMX2 were decreased in the treatment group 1 and 2 (P < 0.01), respectively. Conclusion Zishen Yutai Pills may improve mouse endometrial receptivity by up-regulating HOXA10 expression and inhibiting EMX2 expression.
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AIM To observe the endometrium receptivity of polycystic ovary syndrome patients and research advance of expression of HOXA10 in endometrium.METHODS Eighty PCOS patients were divided into treatment group and control group,40 persons in each group randomly.The control group was treated with clomiphene (CC) + human menopausal gonadotropin (HMG) + human chorionic gonadotropin (HCG),and the treatment group was treated with CC + HMG + HCG + Yin-Nourishing Yang-Supplementing.After 3 periods,take a record for below:endometrial thickness (Em) in the middle,advanced stage and mid-secretory phase of hyperplasia endometrii,the levels of spiral artery PI/RI in midluteum endometrium,the levels in serum of E2 and P in midluteum endometrium,the expression of HOXA10 mRNA from both groups,clinical pregnancy rate and abortion rate for each group.RESULTS Compared with the control group,endometrial thickness in the treatment group was increased and there is statistical difference in the middle and late follicle phases (P ＜ 0.05),but there was no statistical difference in midluteum endometrium (P ＞ 0.05);the level of E2 and P in the treatment group were raised (P ＜0.05) with statistical significance;PI and RI were obviously contracted (P ＜ 0.01).Expression of HOXA10 mRNA was increased (P ＜ 0.01).Compared with the control group,the treatment group had a significant difference in pregnancy rate (P ＜ 0.05).The abortion rate was lower,but there was no statistical significance (P ＞ 0.05).CONCLUSION Yin-Nourishing Yang-Supplementing Treatment can obtain higher pregnancy rate and lower abortion rate of PCOS patients and the mechanism might be associated with raising the expression of HOXA10 mRNA and reducing spiral artery PI/RI,also improving the function of corpus luteum treatment,then to improve the receptivity of endometrium.Yin-Nourishing Yang-Supplementing Treatment is well worth popularizing in further clinical application.