miR-375 Regulates the Proliferation, Apoptosis and Colony Formation of Thyroid Cancer Cells via Targeting YAP1

Objective: Yes-associated protein 1 (YAP1) regulates cell proliferation and apoptosis. Abnormal miR-375 level was related to thyroid cancer. Software predicted a relationship between miR-375 and YAP1. Our study investigated whether miR-375 regulates YAP1 expression and affects thyroid cancer cells. Methods: The tumor tissues and adjacent tissues of thyroid cancer patients were collected to measure miR-375 and YAP1 expression. The dual luciferase reporter experiment verified the regulation between miR-375 and YAP1. Thyroid cancer cell line B-CPAP and TPC-1 cells were divided into miR-NC group and miR-375 mimic group followed by analysis of cell proliferation by flow cytometry, caspase-3 activity, and cell clone formation ability by plate cloning assay. Results: Compared with adjacent cancer tissues, miR-375 in thyroid cancer tissues was decreased and YAP1 was increased. miR-375 targets YAP1. Compared with Nthy-ori 3-1 cells, miR-375 in B-CPAP and TPC-1 cells was significantly reduced and YAP1 was increased. Transfection with miR-375 mimic significantly inhibited cell proliferation, increase caspase-3 activity, and reduced the ability of cells to form clones. Conclusion: miR-375 can inhibit YAP1 expression, decrease the proliferation of thyroid cancer cells, induce cell apoptosis, and reduce clone formation.

HPV16 E6-178G/E7-647G Promotes Proliferation and Inhibits Apoptosis in Cervical Cancer C33A Cells

Background：HPV16 is the main cause of cervical cancer. In our study, we aimed to investigate the role of HPV mutants HPV16 E6-178G/E7-647G in the proliferation and apoptosis of cervical cancer C33A cells. Methods：Plasmids encoding the HPV16 E7 prototype (E7-647A)-GV144, E7 mutant (E7-647G)-GV144, HPV16 E6/E7 prototype (E6-178T/E7-647A)-GV144, and E6/E7 mutant (E6-178G/E7-647G)-GV144 were stably transfected into cervical cancer C33A cells. Western blot analysis, CCK8 proliferation assay, cell cloning assay and flow cytometry were used to detect the effects of the different polymorphism sites in HPV16 on cell proliferation and apoptosis. Results：HPV16 mutations promoted the proliferation and inhibited the apoptosis of cervical cancer C33A cells, and the effect of the E6-178G/E7-647G co-mutation was significantly greater than that of the single E7-647G mutant (P<0.05). Conclusions：HPV16 E6-178G/E7-647G can thus promote the proliferation and inhibit the apoptosis of cervical cancer cells.

Emergence of a KPC Variant Conferring Resistance to Ceftazidime-Avibactam in a Widespread ST11 Carbapenem-Resistant Klebsiella pneumoniae Clone in China

Carbapenem-resistant Klebsiella pneumoniae (CRKP) infection poses a great threat to public health worldwide, and KPC-2-producing strains are the main factors responsible for resistance to carbapenems in China. Ceftazidime/avibactam (CZA) is a novel β-lactam/β-lactamase inhibitor combination with good activity against KPC-2 carbapenemase and is becoming the most important option for treating KPC-producing CRKP infection. Here, we report the emergence of a novel KPC-2 variant, designated KPC-74, produced by K. pneumoniae strain KP55, that conferred CZA resistance in a patient after CZA exposure. The novel blaKPC–74 variant showed a deletion of 6 nucleotides at positions 712–717 compared with blaKPC–2, and this deletion resulted in the consequent deletion of glycine and valine at positions 239 and 240. Antimicrobial susceptibility testing showed that KP55 presents multidrug resistance, including resistance to CZA and ertapenem, but is susceptible to imipenem, meropenem, and colistin. The blaKPC–74 gene was located on a plasmid, as determined by S1-nuclease pulsed-field gel electrophoresis followed by southern blotting, and confirmed to be 133,766 bp in length by whole-genome sequencing on both the Illumina and MinION platforms. The CZA resistance phenotype of the novel KPC variant was confirmed by both transformation of the blaKPC–74-harboring plasmid and a blaKPC–74 gene cloning assay, showing a 64-fold higher CZA minimum inhibitory concentration (MIC) than the recipient strains. The G239_V240del observed in KPC-74 was outside the omega-loop region but was still close to the active site Ser70 and omega-loop in the protein tertiary structure. The enzyme kinetic parameters and IC50 values further indicated that the hydrolytic activity of the KPC-74 enzyme against ceftazidime was potentiated twofold and that the affinity between KPC-74 and avibactam was alleviated 17-fold compared with that of the KPC-2 allele. This CZA resistance mediated by KPC-74 could be selected after CZA therapy and evolved to be more diverse and heterogeneous. Surveillance of CZA resistance is urgently needed in clinical settings.

Wnt7a Promotes the Occurrence and Development of Colorectal Adenocarcinoma

ObjectiveThe expression of Wnt7a in colorectal cancer tissues and cell lines was analyzed, and the effect of Wnt7a on the proliferation of colorectal cancer cells was studied, so as to confirm the relationship between Wnt7a and the occurrence and development of colorectal cancer.Methods(1) Immunohistochemical method was used to compare the expression of Wnt7a in different tissues and its relationship with the clinicopathology of colorectal adenocarcinoma. (2) The expression levels of Wnt7a in colorectal cancer cell lines HT-29 and HCT 116 were detected by qRT-PCR. (3) The down-regulated Wnt7A expression vector was constructed, and the down-regulated Wnt7A expression cell line was established. The regeneration ability of cancer cells was detected by stem cell ball formation assay, and the influence of plate cloning assay on the proliferation ability of colorectal cancer cells was detected.Results(1) The positive rates of Wnt7a in normal colorectal mucosa, colorectal adenoma and colorectal adenocarcinoma tissues gradually increased,Wnt7a are closely related to the degree of colorectal adenocarcinoma differentiation, lymph node metastasis and Duke stage. (2) The expression level of Wnt7a in colorectal cancer cells was higher than that in normal colorectal epithelial cells. (3) The down-regulation of Wnt7A reduced the proliferation ability of colorectal cancer cells.ConclusionsWnt7a promotes the occurrence and development of colorectal adenocarcinoma.

HPV16 E6-178G/E7-647G Promotes Proliferation and Inhibits Apoptosis in Cervical Cancer C33A Cells

BackgroundHPV16 is the main cause of cervical cancer. In our study, we aimed to investigate the role of HPV mutants HPV16 E6-178G/E7-647G in the proliferation and apoptosis of cervical cancer C33A cells. MethodsPlasmids encoding the HPV16 E7 prototype (E7-647A)-GV144, E7 mutant (E7-647G)-GV144, HPV16 E6/E7 prototype (E6-178T/E7-647A)-GV144, and E6/E7 mutant (E6-178G/E7-647G)-GV144 were stably transfected into cervical cancer C33A cells. Western blot analysis, CCK8 proliferation assay, cell cloning assay and flow cytometry were used to detect the effects of the different polymorphism sites in HPV16 on cell proliferation and apoptosis. ResultsHPV16 mutations promoted the proliferation and inhibited the apoptosis of cervical cancer C33A cells, and the effect of the E6-178G/E7-647G co-mutation was significantly greater than that of the single E7-647G mutant (P<0.05). ConclusionsHPV16 E6-178G/E7-647G can thus promote the proliferation and inhibit the apoptosis of cervical cancer cells. Importance: HPV16 is the most common type and the main cause of cervical cancer. It is a fatal threat to the health of millions of women. The genetic differences among HPV16 sub-types may be related to their carcinogenic potential. HPV mutations can differ in biology and etiology, leading to differences in tumor development and behavior. However, little is known about the carcinogenic potential of the HPV16 variant in Asian women compared to many studies in European and American populations.

Irradiation-Induced Polyploidy Giant Cancer Cells Mediate Tumor Cell Repopulation via Neosis

Background Tumor repopulation generally describes the phenomenon that residual tumor cells surviving therapies tenaciously proliferate and reestablish the tumor, presenting an embarrassing plight for cancer treatment. However, the cellular and molecular mechanisms underlying this process remains poorly understood. In this study, we proposed polyploidy giant cancer cells (PGCCs)-mediated and neosis-based tumor repopulation after radiotherapy.Methods The formation of PGCCs after irradiation was examined in vitro and in vivo. The demise of X-ray irradiated cells was detected by flow cytometry, clonogenic cell survival assay and transmission electron microscopy. Western blot was used to test cell proliferation and death related protein expression level of these irradiated cells. Time lapse microscopy was adopted to observe the destiny of PGCCs. The property of these PGCCs was identified by TUNEL assay, Brdu chasing assay, western blot, immunocytochemical and immunofluorescence staining. The relationship of HMGB1 with PGCCs-derived tumor repopulation was conducted via HMGB1 chemical inhibitors. Finally, animal model was used to verify the formation of PGCCs, and the relevance of HMGB1 in this process was investigated by immunohistochemical staining.Results The majority of PGCCs induced by irradiation move towards cell demise, whereas some of them intriguingly possessed proliferative property. Utilizing time-lapse microscopy and single-cell cloning assay, we observed that neosis derived from those PGCCs with proliferative capacity contributed to tumor cell repopulation after irradiation. Using the conditioned media collected from dying tumor cells to perform single-cell cloning assay, we unexpectedly demonstrated that HMGB1 released from dying tumor cells participated the process of neosis-based tumor repopulation. In irradiation treated animal tumor bearing model, the expression level of HMGB1 increased after irradiation compare with non-irradiated group. Moreover, some PGCCs presented high HMGB1 expression. Interestingly, we also observed that the proliferation potential of PGCCs varied. Some PGCCs proliferated at early stage, while some PGCCs proliferated at late stage.Conclusion X-ray irradiation could induce the formation of PGCCs, which could move towards both cell death and survival; irradiation-generating PGCCs mediated tumor cell repopulation after irradiation via neosis; HMGB1 released from dying cells stimulated the process of neosis and participated in tumor repopulation after irradiation.

Thymidylate synthase gene expression in solid tumors predicts for response to pemetrexed in vitro

13058 Background: Pemetrexed (P) is a novel antifolate which targets thymidilate synthase (TS), dihydrofolate reductase (DHFR), and glycinamide ribonucleotide formyltransferase (GARFT). The aim of the present study was to identify gene expression thresholds for these enzymes in human tumor specimens in order to separate P-sensitive from P-resistant patients. Methods: Soft-agar cloning assays were performed on freshly biopsied tumor cells exposed one hour to clinically achievable concentrations of P. In parallel, RNA was isolated, transcribed to cDNA and subsequently used for multiplex real-time PCR. Gene expression data were normalized against beta-actin transcripts followed by correlation against cloning assay results. Iterative calculations (fourfold analysis) were done for each enzyme separately to find the best cutoff for prediction of sensitivity to P. Results: Sensitive and resistant tumor samples were statistically significant different in gene expression of TS, DHFR, and GARFT (p < 0.003). 81% of all tumors with a TS copy number < 144 (related to 104 copies β-actin) were sensitive to P in vitro. (specificity = 0.69; chi2 = 14.14). Statistical tests demonstrated that gene expression of TS, DHFR, and GARFT are dependent variables and that TS transcription is the leading variable. The combination of TS, DHFR, and GARFT expression data was not superior to TS alone. Conclusions: TS expression is the most meaningful predictor for sensitivity (≤ 144 copies) or resistance (> 144 copies) to Pemetrexed in fresh tumor tissue. This observation forms a rationale for clinical trials using TS expression as predictor for clinical response. No significant financial relationships to disclose.