Background: Tumor cells proliferation and apoptosis inhibition are the mechanisms through which the Colorectal Cancer (CRC) progression, metastasis and chemoresistance are promoted pathologically, offering clinical advantages for characterizing their molecular regulators.Objectives: In this study, to unravel the role of PIWIL2 as a potential CRC oncogenic regulator, we examined the effect of its overexpression on proliferation, apoptosis and colony formation of SW480 colon cancer cell line.Material and Methods: Established SW480-P (overexpression of PIWIL2) and SW480-control (SW480-empty vector) cell lines were cultured in DMEM containing 10% FBS with 1% penicillin-streptomycin. The total DNA and RNA was extracted for further experiments. Real-Time PCR and western blotting assay were performed to measure the differential expression of proliferation associated genes including the expression of cell cycle and anti-apoptotic genes as well as Ki-67 and PIWIL2 in both cell lines. Cell proliferation was determined using MTT assay, doubling time assay and the colony formation rate of transfected cells was measured with the 2D colony formation assay.Results: At the molecular level, PIWIL2 overexpression was associated with significant up-regulation of cyclin D1, STAT3, BCL2-L1, BCL2-L2 and Ki-67 genes. MTT and doubling time assay showed that PIWIL2 expression induced time-related effects on proliferation rate of SW480 cells. Moreover, SW480-P cells had markedly greater capacity to form colonies.Conclusions: PIWIL2 plays important roles to promote cancer cell proliferation and colonization via the cell cycle acceleration and inhibition of apoptosis, the mechanisms through which this gene seems to contribute to CRC development, metastasis and chemoresistance, hence potentially highlighting PIWIL2 targeted therapy as a valuable tool for CRC treatment.

Background: In order to improve culture conditions for optimal spermatogenesis, quantitative assessment of the male germ cell gene expression profile upon spontaneous ES cell differentiation is necessary. In this study, the quantitative expression profile of PIWIL2, germ-line specific marker, during the early stage of embryoid body (EB) formation and differentiation (0-3-day-old EB) was studied.Methods: CCE mouse embryonic stem cells (ESCs) were cultured in DMED containing 20% fetal bovine serum (FBS) for 1, 2 and 3 days. The total RNA was isolated from ESCs, 1-3-day-old EBs, and adult testis as positive control. cDNA was prepared and quantitative real-time PCR was done for Oct-4 to study the pluripotency of this cell line. Also, the molecular pattern of PIWIL2 expression in developing EB was investigated.Results: Our quantitative results confirmed the pluripotency of CCE mouse ESC line and showed that PIWIL2 was expressed in undifferentiated CCE mouse ESC line. Our results also showed that expression of PIWIL2 increased significantly during the process of EB formation and differentiation up to 2-day-old EB and decreased non- significantly in 3-day-old EB.Conclusion: Our results suggest that EB provide a cellular environment similar to the early embryonic microenvironment and cause the efficient and progressive germ cell lineage differentiation in this system.

PURPOSE: Human PIWIL2, a member of Piwi subfamily of Argonaute proteins, is primarily expressed in testis, where it regulates self-renewal of germ cells. However, its ectopic expression has been reported with several tumors, including breast cancer. The upregulation of PIWIL2 in various stages of breast cancer suggested its suitability as a novel tumor biomarker. Considering the vital role of microRNAs (miRNAs) in regulating the expression of most human genes, we hypothesized a concomitant downregulation of the bioinformatically-predicted PIWIL2-targetting microRNAs in breast cancer.METHOD: We employed different bioinformatic tools to predict PIWIL2-targeting miRNAs. Then, from the list of predicted miRNAs, we chose two less studied miRNAs (miR-1267 and miR-2276) for experimental validation. Using a real-time RT-PCR approach, we quantified the relative expression of the miRNAs in 31 pairs of formalin-fixed paraffin-embedded tumor/non-tumor tissue samples.RESULTS: Our data revealed a noticeable but not statistically significant (P=0.133) down regulation of miR-1267 in tumor samples, compared to non-tumor samples obtained from the same patients. Down regulation of miR-1267 was more significant in higher grades of malignancies (fold change = 2.39, P=0.033) and also in lymph nodes containing high-grade tumor cells (fold change = 6.66, P=0.02). Interestingly, a significant upregulation of miR-1267 was observed in tumors at high stages (stage 3a, 3b), compared to low stages (stage 2a, 2b) (fold change = 8.05, P=0.048). Similar patterns of expression alteration were also observed for miR-2276.CONCLUSION: Altogether, our findings suggest a probable tumor suppressor role for miR-1267 and miR-2276 in breast tumor initiation and progression, but a probable promoting role for them in invasion and metastasis.

Objective: PIWIL2, a member of Ago/Piwi gene family containing Piwi and PAZ domains, has been shown to be ectopically expressed in different cancer cells, especially its remarkable expression in cancer stem cells (CSCs), and is also known to be essential for germ line stem cell self-renewal in various organisms. The hypothesis that CSC may hold the key to the central problem of clinical oncology and tumor relapse leads to more anticancer treatment studies. Due to emerging controversies and extreme difficulties in studying of CSC, like the cells using in vivo models, more attempts have expended to establish different in vitro models. However, the progress was slow owing to the problems associated with establishing proper CSC cultures in vitro. To overcome these difficulties, we prompted to establish a novel stable cell line over-expressing PIWIL2 to develop a potential proper in vitro CSC model.Materials and Methods: In this experimental study, mouse embryonic fibroblasts (MEFs) were isolated and electroporated with a construct containing PIWIL2 cDNA under the control of the cytomegalovirus promoter (CMV). Stable transfectants were selected, and the established MEF-PIWIL2 cell line was characterized and designated as CSC-like cells using molecular markers. Functional assays, including proliferation, migration, and invasion assays were performed using characterized CSC like cells in serum-free medium. Additionally, MEF-PIWIL2 cell density and viability were measured by direct and indirect methods in normoxic and hypoxic conditions.Results: The results of reverse transcriptase-polymerase chain reaction (RT-PCR), western blot, and immunocytochemistry revealed an overexpression for PIWIL2 in the transfected PIWIL2 cells both in the RNA and protein levels. Furthermore, analysis of the kinetic and stoichiometric parameters demonstrated that the specific growth rate and the yield of lactate per glucose were significantly higher in the MEF-PIWIL2 group compared to the MEF cells (ANOVA, p<0.05). Also, analysis of functional assays including migration and invasion assays demonstrated a significantly higher number of migrated and invaded cells in the MEF-PIWIL2 compared to that of the MEF cells (ANOVA, p<0.05). The MEF-PIWIL2 cells tolerated hypoxia mimetic conditions (CoCl2) with more than 95% viability.Conclusion: According to the molecular and functional studies, it has been realized that Piwil 2 plays a key role (s) in tumor initiation, progression and metastasis. Therefore, PIWIL2 can be used not only as a common biomarker for tumor, but also as a target for the development of new anticancer drug. Finally, the main outcome of our study was the establishment of a novel CSC-like in vitro model which is expected to be utilized in understanding the complex roles played by CSC in tumor maintenance, metastasis, therapy resistance or cancer relapse.

Background: Piwi-like RNA-mediated gene silencing 2 (PIWIL2) is a member of AGO/PIWI gene family, which is enriched in cancer stem cells (CSCs). The purpose of this research was to investigate the overexpression of PIWIL2 and its role in the induction of EMT and CSC properties in MCF7 breast cancer cell line. Materials and Methods: MCF7 cells were transfected with the human gene PIWIL2 (Hili) under the control of CMV promoter utilizing the neon electroporation method. Subsequently, the selection was conducted using G418, and doubling time was calculated in the transformed and control cells. RT and real-time PCR were also performed to analyze the expression of epithelial and mesenchymal genes and those related to CSCs. Results: According to the observations from this study, transfecting MCF7 cells with PIWIL2 triggered the conversion of epithelial cells to mesenchymal cells and induced the genes specific for breast CSCs, which was coincident with 9-h reduction in the doubling time of the transfected cells. Furthermore, the molecular analyses revealed a significant reduction in the expression of epithelial markers, while a significant increase was detected in the expression of mesenchymal genes and many CSC biomarkers. Conclusion: PIWIL2 protein acts as a master regulatory protein that is able to manipulate the transcription through specific signaling pathways, which allow the cells to gain stem cell-like properties.

Background: Piwi-like RNA-mediated gene silencing 2 (PIWIL2) is a member of AGO/PIWI gene family, which is enriched in cancer stem cells (CSCs). The purpose of this research was to investigate the overexpression of PIWIL2 and its role in the induction of EMT and CSC properties in MCF7 breast cancer cell line. Materials and Methods: MCF7 cells were transfected with the human gene PIWIL2 (Hili) under the control of CMV promoter utilizing the neon electroporation method. Subsequently, the selection was conducted using G418, and doubling time was calculated in the transformed and control cells. RT and real-time PCR were also performed to analyze the expression of epithelial and mesenchymal genes and those related to CSCs. Results: According to the observations from this study, transfecting MCF7 cells with PIWIL2 triggered the conversion of epithelial cells to mesenchymal cells and induced the genes specific for breast CSCs, which was coincident with 9-h reduction in the doubling time of the transfected cells. Furthermore, the molecular analyses revealed a significant reduction in the expression of epithelial markers, while a significant increase was detected in the expression of mesenchymal genes and many CSC biomarkers. Conclusion: PIWIL2 protein acts as a master regulatory protein that is able to manipulate the transcription through specific signaling pathways, which allow the cells to gain stem cell-like properties.

Aim: This study aims to confirm previous fundamental in vitro findings about the PIWIL2 gene by investigating the effects of its overexpression on cell cycle, proliferation, apoptosis, and stem cell expression markers in colorectal cancer cells (CRC cells) at in vivo level. Background: PIWIL2 has a critical role in maintaining cellular stemness and proliferation. PIWIL2 is an oncogene whose expression in CRC is associated with the occurrence, metastasis, and poor prognosis. Methods: SW480 cells harboring expression vectors with/without PIWIL2 were cultured and inoculated in BALB/c nude mice. Tumor formation and growth were monitored every 3 days. On the 28th day after inoculation, the tumors were harvested for their total RNA extraction, and the expression profiling of the candidate genes was performed by Real-time PCR. Results: Our results for the expression profiling of the xenografted tumors showed a significant increase in the expression of cancer stem cell markers, including CD24, CD133, and pluripotency marker SOX2 in the PIWIL2 over-expressing xenografts, compared to the control cell line. Moreover, PIWIL2 dramatically promoted the anti-apoptotic pathway by inducing STAT3 and BCL2-L1 genes in the PIWIL2 over-expressing xenografts, along with the up-regulation of Cyclin D1 and Ki-67 genes. Conclusion: This research supports our prior in vitro findings, highlighting the critical role that PIWIL2 plays in the development of CRC and its substantial promise as a leading candidate for CRC-targeted therapy.