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Objective:To construct human immortalized keratinocytes stably expressing human papillomavirus type 16 (HPV16) E6/E7 gene, and provide a cell model for studying mechanisms underlying HPV16 E6/E7-induced cell immortalization and malignant transformation.Methods:Primary human foreskin keratinocytes (HFKs) were isolated by sequential two-step enzymatic digestion. Cultured HFKs were stably transfected with a HPV16 E6/E7 gene-overexpressing lentiviral vector LV5-HPV16 E6/E7, and consecutively cultured for more than 30 passages. Then, immortalized keratinocytes were screened out and divided into 3 groups: (1) blank control group: second-passage primary HFKs; (2) experimental group: HFKs transfected with LV5-HPV16 E6/E7 at different passages, and the second-passage primary HFKs transfected with LV5-HPV16 E6/E7 were referred to as A0 cells, thereafter, the transfected HFKs were named according to their passage number, such as A1, A2, ... A30; (3) positive control group: the HPV16-positive cervical cancer cell line SiHa. Real-time fluorescence-based quantitative PCR (qRT-PCR) and Western blot analysis were performed to determine the mRNA expression of HPV16 E6/E7 and protein expression of HPV16 E6/E7 and CK14, respectively, in the blank control group, experimental group and positive control group. Cell counting kit-8 (CCK8) assay and Transwell insert invasion assay were conducted to assess the cellular proliferative and invasive activity. In vivo tumor formation experiment in nude mice was conducted to investigate the tumorigenicity of A30 cells in the experimental group and SiHa cells in the positive control group. Results:Primary HFKs were successfully isolated. After the primary HFKs were transfected with the recombinant plasmid LV5-HPV16 E6/E7, the blank control group showed no fluorescence in the cells, but showed senescent phenotypes after serial passages, while in the experimental group, the volume and morphology of A30 cells were similar to those of the primary HFKs with the proportion of fluorescence-positive cells being 100%. Compared with the blank control group, the experimental group showed significantly increased mRNA expression levels of HPV16 E6 and E7 in A1, A10, A20 and A30 cells (HPV16 E6: t = 7.12, 8.07, 6.53, 5.66, P < 0.001, < 0.001, = 0.001, = 0.005, respectively; HPV16 E7: t = 3.20, 4.29, 3.75, 4.22; P = 0.024, 0.008, 0.013, 0.014, respectively) . The protein expression of HPV16 E6/E7 was absent in the blank control group, but was observed in A30 and SiHa cells. CCK8 assay showed that the proliferative activity of A10, A20 and A30 cells was significantly higher than that of the blank control group ( t = 6.49, 7.55, 9.43, P = 0.003, 0.002, 0.001, respectively) , while there was no significant difference in the proliferative activity between A1 cells and the blank control group ( t = 2.40, P = 0.074) . Transwell insert invasion assay showed that A30 cells could not cross the basement membrane, but SiHa cells could pass through the basement membrane and were stained blue. Two months after the inoculation with A30 cells in the nude mice, no visible tumors were found, which was confirmed by a histological study. Subcutaneous tumors were formed in the nude mice after the inoculation with SiHa cells. Conclusion:Human immortalized keratinocytes were successfully established by lentivirus-mediated transfection with HPV16 E6/E7 gene, and can serve as an ideal cell model for HPV-related research.
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Objective To investigate the relationship between the composition of vaginal microbiota and the course of cervical precancerous lesion.Methods A total of 64 vaginal swabs were collected from 22 healthy women, 18 CINⅠ patients and 24 CINⅡ/Ⅲ patients who visited Obstetrics and Gynecology of the Second Xiangya Hospital of Central South University during July 2014 and July 2015.The Bacterial genomic DNA was extracted and the V3 and V4 hypervariable regions of 16S rRNA were amplified and high-throughput sequenced.The abundance and composition of vaginal microbiota were analyzed by Uparse, Mothur and LefSe statistical software.Results There was no significant difference in Alpha diversity index between CINⅡ/Ⅲ group(Chao:63±32;ACE:72±38;Simpson:0.70±0.27;Shannon:0.70±0.63) and control group ( Chao:48±24;ACE:54±25;Simpson:0.71±0.27;Shannon:0.65±0.58)(W=192,P=0.11;W=189,P=0.10;W=281,P=0.72;W=241,P=0.62).The ACE(85±37) and Chao(66±25) values of CINⅠgroup were significantly different from those of the control group (ACE:54±25;Chao:48±24)(W=99,P=0.006;W=113,P=0.02).At the phylum level, 78.69%(309 020/392 722) of the vaginal microbiota in the control group was Firmicutes, 16%(62 846/392 722) was Actinobacteria.Firmicutes was reduced to 64.86%(208 422/321 318) and Actinobacteria increased to 27.71%(89 040/321 318) in CINⅠgroup.The composition of vaginal microbiotain in CINⅡ/Ⅲ group was similar to those of control group.At the genus level, the composition of vaginal microbiota were similar between CINⅡ/Ⅲ group and control group, with Lactobacillus as predominant genus[71.81%(307 658/418 424)], Gardnerella[12.91%(55 299/428 424)], others such as Prevotella, atopobium were less.In the CINⅠ group, the abundance of Lactobacillus was decreased to 56.26%(180 787/321 318), Gardnerella was increased to 19.62%(63 057/321 318), and Listeria was increased to 7.7%(24 746/321 318).The composition of vaginal microbiota in the most samples was classified as CSTⅢ and CSTⅠ, with Lactobacillus inersand and Lactobacillus crispatus were dominant respectively.There was no significant difference in the composition of vaginal microbiota between the three groups(χ2=2.72, P=0.949).LEfSe analysis showed that the abundance of bacteria in CIN group and control group were varied.At the genus level, there were significant differences in the abundance of Geobacter, Atopobium and Ureaplasma (P<0.05, P<0.05, P<0.01, respectively).At the species level, there was significant difference in the abundance of Ureaplasma urealyticum serotype 9 (P<0.01).Conclusion The diversity and the composition of vaginal microbiota were similar between CIN patients and healthy women, but the abundances of some bacteria were varied, with Ureaplasma increased in patients with CIN.
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Objective To compare the safety and efficacy of direct and remedial rotational atherectomy in the treatment of heavily calcified coronary artery lesions.Methods We retrospectively reviewed 58 patients admitted in the Shanghai Chest Hospital and Liaocheng People Hospital from May 2012 to July 2015 who had received stent implantation and rotational atherectomy.The 58 patients were divided into two groups which were the direct atherectomy group (n =27) and the remedial atherectomy group (n =31).General clinical date,lesion and procedural characteristics,intraoperative complications,in-hospital and follow-up MACCE were compared between the two groups.Results There were no differences between the two groups in general clinical date intraoperative complications,amount of contrast agent used,proceduraltime,rates of in-hospital and follow-up MACCE.Nevertheless,compared with the direct artherectomy group,the remedial group had more number of balloon dilations during procedure [3 (1,5) vs.2 (1,2),P ＜ 0.001] and higher peak cardiac troponin levels [1.1 (0.3,3.0) μg/L vs.0.5 (0.1,2.3) μg/L,P =0.032].Conclusions Remedial rotational atherectomy with drug-eluting stent had the same safety and efficacy as direct atheretomy with drug-eluting stent in treating patients with heavily calcified coronary lesions.It is reasonable and safe to transform routine PCI to remedial rotational atherectomy when the 2.0 mm semi compliant balloon or/and 2.5 mm non-compliant balloon cannot pass through or dilate the lesions.
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DNA from 32 cases of tongue squamous cell carcinoma(TSCC)specimens and the neck lymph node metastasis specimens were processed by bisulfite treatment.The methylation of the specimens was examined by Q-MSP amplification.The consistency of p1 6 methyla-tion of primary TSCC with that of lymph node metastasis tumor was 90.62%(P <0.05).We assumed that p1 6 gene promoter CpG island methylation rates in primary TSCC and metastatic lymph nodes are consistent,p1 6 gene methylation status can be a methylation predictor be-tween premary and lymph node metstasized TSCC.
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OBJECTIVE@#To explore the differentially expressed gene profile in cervical squamous cell carcinoma tissues with and without pelvic lymph node metastasis by microarray technique and to screen tumor metastasis-related genes.@*METHODS@#Cancer tissue samples were collected from 4 cervical squamous cell carcinoma patients with pelvic lymph metastasis and 6 patients without metastasis before the treatment. Differentially expressed gene profile was identified between cervical squamous cell carcinoma tissues with and without pelvic lymph node metastasis by NimbleGen microarray. Fluorescent real-time quantitative PCR was employed to validate the accuracy of the microarray by detecting the expression of genes picked from the differentially expressed gene profile. We screened tumor metastasis-related genes through the gene bank, PUBMED and gene ontology analysis.@*RESULTS@#With the application of microarray, 329 genes were identified as markedly differentially expressed genes between cervical squamous cell carcinoma tissues with and without pelvic lymph node metastasis. Real-time PCR was in accordance with microarray. Forty-four genes associated with tumor metastasis were identified from the differentially expressed gene profile.@*CONCLUSION@#Gene profile in cervical squamous cell carcinoma tissues associated with pelvic lymph node metastasis is preliminarily identified, indicating that lymph node metastasis of cervical squamous cell carcinoma is a process involving numerous genes.
Subject(s)
Adult , Aged , Female , Humans , Middle Aged , Carcinoma, Squamous Cell , Genetics , Pathology , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Lymphatic Metastasis , Oligonucleotide Array Sequence Analysis , Uterine Cervical Neoplasms , Genetics , PathologyABSTRACT
Objective To examine the frequency of coexisting endometrial carcinoma with preoperative diagnosis of endometrial atypical hyperplasia (EAH).Methods From Apr 2002 to Apr 2007,61 consecutive patients diagnosed as EAH who underwent total hysterectomy in our department were retrospectively analyzed.Patients were stratified as 44 premenopausal cases and 17 menopausal cases.The patients were divided into 2 groups depending on whether or not cancer cells were found by histology in removed uterus (cancer group vs EAH group) to explore valuable predictive risk factors for concomitant endometrial carcinoma through reviewing the clinical data.Logistic regression analysis and logistic regression equation are used to evaluate the risk of coexisting endometrial carcinoma with preoperative diagnosis of endometrial atypical hyperplasia.Results Endometrial thickness and the type of EAH were selected into the equation in premenopausal cases,showing statistically significant differences(P ＜ 0.001) and the overall accuracy was 86.4％.Menopausal years and the type of EAH were selected into the equation in menopausal women,also showing statistically significant differences (P ＜ 0.04)and the overall accuracy was 82.4％.Conclusions Endometrial thickness and the type of EAH are valuable in predicting the risk of endometrial carcinoma in premenopausal women.Menopausal years and the type of EIN are valuable in prediciting the risk of coexistance with endometrial carcinoma in menopausal women.
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Objective To investigate the effect of CD147 monoclonal antibody (mAb) on the natural resistance to paclitaxel (TAX) in the human cervical cancer line (HCE1) multicellular spheroid (HCE1/MCS) model and if CD147 mAb can reverse the HCE1/MCS resistance to TAX. Methods HCE1/MCS was obtained by liquid overlay and rotating technique. HCE1/MCS morphological changes were observed before or after the interference of CD147 mAb. The effects of TAX on HCE1/MCS (including inhibition ratio, IC50 and index of multicellular resistance) before or after CD147 mAb treatment were determined by the method of WST-1 and the inhibition ratio curve was mapped. Cell cycle and apoptosis were detected by flow cytometer (FCM). The expression of CD147 and P-gp of both HCE1/MC and HCE1/MCS was detected by immunocytochemistry. Results HCE1/MCS was established successfully. CD147 mAb could inhibit HCE1/MCS from forming spheroids. CD147 mAb could enhance the sensitivity of HCE1/MCS to TAX. IC50 in different concentrations of CD147 mAb (5,10,20 μg/mL) HCE1/MCS group were (40.31±3.73), (32.43±1.56), and (30.69±1.01) μg/mL. CD147 mAb resulted in G1/G0 arrest in HCE1/MCS. CD147 mAb of low concentrations (0-10 μg/mL) caused a dose-dependent inhibition of HCE1/MCS (P<0.05). Combined with TAX, CD147 mAb could also induce HCE1/MCS cell cycle arrest in both G1/S and G2/M stage. The expression of CD147 and P-gp was consistent in HCE1/MCS groups. Conclusion CD147 plays an important role in muliticellular resistance of cervical cancer and inhibition of CD147 can synergistically reverse the multicellular drug resistance (MCR) in cervical cancer. The MCR of HCE1/MCS mediated by CD147 is related to P-gp.
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OBJECTIVE@#To investigate the relationship between matrix matrix metalloproteinases (MMP)-9 expression in HCE1/multicellular spheroids (MCS) and invasion of cervical carcinoma, and to explore the effect of MMPs inhibitor GM6001 on the model that HCE1/MCS invade live human umbilical vein endothelium cell (HUVEC).@*METHODS@#We established the invasion model by coculturing HCE1/MCS and HUVEC, and detected the MMP-9 expression in HCE1 monolayer cells (HCE1/MC), HCE1/MCS and HUVEC using immunohistochemistry and treated the HCE1/MCS invasion model with GM6001.@*RESULTS@#A cervical squamous carcinoma cell HCE1 infiltrating model was established. Compared with that in HCE1/MC, MMP-9 expression in the HCE1/MCS was significantly higher (P<0.05). Compared with the control group, the invasion of HCE1/MCS was inhibited by 26.09% and 92.95% (P<0.05) in the GM6001 2.5 μmol/L and 12.5 μmol/L group. MMP-9 expression in HCE1/MCS in the GM6001 12.5 μmol/L group was obviously lower than that in the control group (P<0.05). With the increase of the concentration of GM6001, the inhibition increased.@*CONCLUSION@#The enhancement of HCE1/MCS invasion may be related to the up-regulation of MMP-9 expression in HCE1/MCS. GM6001 can down regulate the MMP-9 expression in HCE1/MCS and partially block the HCE1/MCS invasion to HUVEC.
Subject(s)
Female , Humans , Carcinoma, Squamous Cell , Pathology , Cell Line , Cell Line, Tumor , Dipeptides , Pharmacology , Endothelial Cells , Pathology , Matrix Metalloproteinase 9 , Metabolism , Neoplasm Invasiveness , Spheroids, Cellular , Metabolism , Pathology , Umbilical Veins , Cell Biology , Uterine Cervical Neoplasms , PathologyABSTRACT
Objective To investigate the effect of the ubiquitin-proteasome pathway inhibitor MG132 on the natural resistance to cisplatin in the human cervical cancer line (HCE1) muhicellular spheroid (HCE1/MCS) model and to probe it if MG132 could reverse the HCE1/MCS resistance to cisplatin, as well as the possible mechanism of drug resistance.Methods (1) HCE1/MCS was obtained using liquid overlay and rotating technique.(2)Four groups were established (MG132 group, cisplatin group, MG132 + cisplatin group, the control group).Cell viability were measured by trypan blue exclusion assay.Cell cycle and apoptosis were detected by flow cytometry.(3) The expression of nuclear factor (NF) kB of both HCE1 monolayer cells (HCEI/MC) and HCE1/MCS was detected by western blot, and the expression of B cell lymphoma/leukemia-2 (bcl-2) was detected by immunohistochemistry.Results (1) HCE1/MCS was established successfully.(2) Cell inhibited rate of HCE1/MC and HCE1/MCS was: in MG132 group, (11.67 ± 2.34) % vs (10.78 ± 1.17) % (P > 0.05) ; in MG132 + cisplatin group, (92.67 ± 2.52)% vs (91.33 ±2.18)% (P>0.05); in cisplatin group, (45.01±7.44)% vs (9.45±5.98)% (P<0.05).(3)The rate of apoptosis of HCE1/MC and HCE1/MCS were: in MG132 group, 8.14% and 5.97% ; in MG132 + cisplatin group, 99.01% and 95.22% ; in cisplatin group, 33.61% and 0.88%.(4)The expression level of NF-kB and the high expression rate of bcl-2 were: in HCE1/MCS of control group, 0.67 and 60% ; in HCE1/MCS of cisplatin group, 0.85 and 83% ; in HCE1/MCS of MG132 group, 0.39 and 20% ; in HCE1/MCS of MG132 + cisplatin group, 0.47 and 33%.Conclusions (1) HCE1/ MCS present natural resistance to cisplatin and may become a good model for the study of cervical cancer drug resistance in vitro.(2) MG132 could induce the inhibition and apoptosis of HCE1/MCS cells and partially reverse the natural resistance of HCE1/MCS to cisplatin, of which partially reverse the natural resistance may be in relation to the down-regulation of NF-kB and bcl-2 expression.
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Objective: To probe into the relationship between integrin β1 expression in human cervical carcinoma HCE1 multicellular spheroids (HCE1/MCS) and their invasion into human umbilical vein endothelium cells (HUVEC), so as to assess the inhibitory effect of anti-integrin β1 monoclonal antibody P5D2 on the invasion of HCE1/MCS into HUVEC. Methods: A model of HUVEC monolayer invaded by HCE1/MCS was established (in brief, the HCE1 invading model). Morphology changes of the HCE1 invading model were observed under inverted microscope and analyzed by Motic Med System after P5D2 treatment. The expressions of β1 integrin on HCE1 monolayer cells, HCE1/MCS, HVUEC monolayer cells, and P5D2-treated HCE1 invading models were measured by immunocytochemistry SABC assay. Results: A HCE1 invading model was successfully established. The HCE1/MCS proliferated rapidly after culture, and on the 7th day the invading area of HCE1/MCS was 40.42 folds larger than the original HCE1/MCS. Invading areas of P5D2-treated HCE1/MCS were significantly smaller than that of the control group after 1, 4, 7 day (P<0.05, or P<0.01), with the invading area after 7 days reduced by (84.68±0.08) % compared with the control group. Integrin β1 expression in HCE1/MCS was significantly higher than that in HCE1 monolayer cells (P<0.01), and the expression was negative in HUVEC. Integrin β1 expression in P5D2-treated HCE1/MCS was significantly lower than that the in untreated HCE1/MCS (P<0.01). Conclusion: Upregulated expression of integrin β1 in HCE1/MCS and HCE1 invading model may be associated with their enhanced adhesion and invasion abilities. Anti-integrin β1 monoclonal antibody P5D2 can partially block the invasion of HCE1/MCS into HUVEC.