Cell Journal (Yakhteh)

215-221

Authors: Samieh Asadian; Abbas Piryaei; Zahra Farzaneh; Bagher Aziz Kalantari; Mehdi Azad; Sahar Moghbeli Nejad; Mohamad Reza Davarpanah; Morteza Mohamadi; Anastasia Shpichka; Nematolah Gheibi; Peter Timashev; Massoud Vosough

Objectives: Epigenetic alterations, including any change in DNA methylation pattern, could be the missing link of understanding radiation-induced genomic instability. Dapper, Dishevelled-associated antagonist of β-catenin homolog 2 (DACT2) is a tumor suppressor gene regulating Wnt/β-catenin. In hepatocellular carcinoma (HCC), DACT2 is hypermethylated, while methylation status of its promoter regulates the corresponding expression. Radionuclides have been used to reduce proliferation and induce apoptosis in cancerous cells. Epigenetic impact of radionuclides as therapeutic agents for treatment of HCC is still unknown. The aim of this study was to evaluate epigenetic impact of 188Rhenium perrhenate (188ReO4) on HCC cells. Material and Methods: In this in vitro experimental study, HepG2 and Huh7 cells were treated with 188ReO4, receiving 55 and 73 Mega Becquerel (MBq) exposures, respectively. For cell viability measurement, live/dead staining was carried out 18, 24, and 48 hours post-exposure. mRNA expression level of β-Catenin, Wnt1, DNMT1, DACT2 and WIF- 1 genes were quantified by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Then, possible regulatory impact of DACT2 upregulation was investigated through evaluating methylation-specific PCR (MS-PCR). Results: Results showed that viability of both cells was reduced after treatment with 188ReO4 at three time points postexposure compared to the control groups. The qRT-PCR results showed that DACT2 mRNA level was significantly increased at 24, and 48 hours post-exposure in HepG2 cells compared to the control group, while, no significant change was observed in Huh7 cells. Methylation pattern of DACT2 promoter remained unchanged in HepG2 and Huh7 cells. Conclusion: Treatment with 188ReO4 reduced viability of HepG2 and Huh7 cells. Although DACT2 expression was increased after 188ReO4 exposure in HepG2 cells, methylation pattern of its promoter remained unchanged. This study assessed impacts of the 188 ReO4 β-irradiation on expression and induction of DACT2 epigenetic aberrations as well as the correlation of this agent with viability of cells.

230-238

Authors: Guangbin Wu; Peilong Sun; Chunzhi Qin

Objective: Growing evidences have exposed the important roles of long noncoding RNAs (lncRNAs) in the triple negative breast cancer (TNBC) inhibition. The function of glucuronidase beta pseudogene 11 (GUSBP11) in the TNBC occurrence remains obscure. To detect the function of GUSBP11 in TNBC progression and explore its downstream molecular mechanism. Materials and Methods: In this experimental study, using quantitative reverse transcription real-time polymerase chain reaction (RT-qPCR), we measured the GUSBP11 expression in the TNBC cell lines. Gain-of-function assays, including colony formation, flow cytometry, and western blot were used to identify the probable effects of GUSBP11 overexpression on the malignant behaviors of TNBC cell lines. Moreover, mechanism assays, including RNA immunoprecipitation (RIP), RNA pull down and luciferase reporter assays were taken to measure the possible mechanism of GUSBP11 in the TNBC cell lines. Results: GUSBP11 expressed at a low RNA level in the TNBC cell lines. Overexpression of GUSBP11 RNA expression inhibited the proliferation, migration, epithelial-to-mesenchymal transition (EMT) and stemness while elevated the apoptosis of the TNBC cell lines. GUSBP11 positively regulated the expression of sphingolipid transporter 2 (SPNS2) via acting as a competing endogenous RNA (ceRNA) of miR-579-3p, thereby suppressing the development of TNBC cell lines. Conclusion: GUSBP11 impedes TNBC progression via modulating the miR-579-3p/SPNS2 axis.

245-254

Authors: Jiacheng Sun; Lei Wang; Xinhai Zhu; Molei Shen

Objective: Circular RNAs (circRNAs) are identified as key modulators in cancer biology. Nonetheless, the role of circ_0006427 in non-small cell lung cancer (NSCLC) and its modulatory mechanism are undefined. This study aimed to investigate the potential function and mechanism of circ_0006427 in NSCLC. Materials and Methods: In this experimental study, circ_0006427, miR-346 and vestigial like family member 4 (VGLL4) mRNA expressions were analyzed in NSCLC tissues and cells, using quantitative reverse transcription polymerase chain reaction (qRT-PCR). Multiplication, migration and invasion of NSCLC cells were examined using the CCK-8 method and Transwell experiment, respectively. Dual-luciferase reporter gene experiments were conducted to identify the paring relationship between circ_0006427 and miR-346. Western blot was employed to determine expressions of VGLL4 and epithelial-mesenchymal transition (EMT) markers on protein levels. Immuno-histochemistry (IHC) method was adopted to assess VGLL4 protein expression in NSCLC tissues. Results: Circ_0006427 expression was down-regulated in NSCLC tissues and cells, and circ_0006427 suppressed multiplication, migration, invasion and EMT of NSCLC cells. miR-346 expression was upregulated in NSCLC tissues and cells, and miR-346 worked as a target of circ_0006427. VGLL4 was down-regulated in NSCLC tissues and cells, and knockdown of VGLL4 accelerated multiplication, migration, invasion and EMT of NSCLC cells. Circ_0006427 enhanced VGLL4 expression by competitively binding with miR-346. Conclusion: Circ_0006427/miR-346/VGLL4 axis regulated NSCLC progression.

261-266

Authors: Amirhossein Izadpanah; Nowruz Delirezh; Rahim Mahmodlou

Objective: The induction of immunity against cancer stem cells (CSCs) can boost the efficiency of cancer vaccines. Heat shock proteins (HSPs) are required for the successful activation of anti-tumor immune responses. Glycoprotein 96 (gp96) is a well-known HSP that promotes the cross-presentation of tumor antigens. The aim of the present study was to optimize the temperature for induction of gp96 in grade 3 breast cancer spheres. Materials and Methods: In the experimental study, CSCs were enriched from breast tumor tissue samples and cultured in DMEM-F12 with epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), B27, and bovine serum albumin (BSA) for 22 days. The expression level of CD24 and CD44 as CSC markers was measured by flow cytometry in secondary mammospheres, and the expression of NANOG, SOX2, and OCT4 genes in CSCs was also analyzed using the real-time polymerase chain reaction (PCR). To find the optimal temperature regulation of gp96, the mammosphere was incubated at different temperatures for 1 hour, and gp96 expression was measured using the western blotting assay. Results: Primary mammospheres were obtained after seven days of culture, and secondary spheres formed 22 days after passage. Flow cytometry analysis showed that cells with CD24- CD44+ phenotype were enriched in the culture period (from 2.6% on day 1 to 32.6% on day 22). Real-time PCR indicated that OCT4, NANOG, and SOX2 expression in mammospheres were increased by 3.8 ± 0.6, 17.8 ± 0.6, and 7.7 ± 0.8 fold respectively in comparison to the MCF-7 cell line. Western blot analysis showed that gp96 production was significantly upregulated when mammospheres were incubated at both 42°C and 43°C in comparison to the control group. Conclusion: Altogether, we found that heat-induced upregulated expression of gp96 in CSCs enriched mammospheres from breast tumor tissue might be used as a complementary procedure to generate more immunogenic antigens in immunotherapy settings.

277-284

Authors: Zahra Amirkhani; Mansoureh Movahedin; Nafiseh Baheiraei; Ali Ghiaseddin

Objective: It was in the early 20th century when the quest for in vitro spermatogenesis started. In vitro spermatogenesis is critical for male cancer patients undergoing gonadotoxic treatment. Dynamic culture system creates in vivo-like conditions. In this study, it was intended to evaluate the progression of spermatogenesis after testicular tissue culture in mini-perfusion bioreactor. Materials and Methods: In this experimental study, 12 six-day postpartum neonatal mouse testes were removed and fragmented, placed on an agarose gel in parallel to bioreactor culture, and incubated for 8 weeks. Histological, molecular and immunohistochemical evaluations were carried out after 8 weeks. Results: Histological analysis suggested successful maintenance of spermatogenesis in tissues grown in the bioreactor but not on agarose gel, possibly because the central region did not receive sufficient oxygen and nutrients, which led to necrotic or degenerative changes. Molecular analysis indicated that Plzf, Tekt1 and Tnp1 were expressed and that their expression did not differ significantly between the bioreactor and agarose gel. Immunohistochemical evaluation of testis fragments showed that PLZF, SCP3 and ACRBP proteins were expressed in spermatogonial cells, spermatocytes and spermatozoa. PLZF expression after 8 weeks was significantly lower (P<0.05) in tissues incubated on agarose gel than in the bioreactor, but there was no significant difference between SCP3 and ACRBP expression among the bioreactor and agarose gel culture systems. Conclusion: This three-dimensional (3D) dynamic culture system can provide somewhat similar conditions to the physiological environment of the testis. Our findings suggest that the perfusion bioreactor supports induction of spermatogenesis for generation of haploid cells. Further studies will be needed to address the fertility of the sperm generated in the bioreactor system.