Although HPV infection is the major cause of tumorigenesis in cervical cancer; interestingly it has been noted that HPV infection does not always lead to cervical cancer. This suggests that there could be some unknown factor that directly or indirectly interacts with these HPV oncoproteins leading to tumorigenesis. Our study aims to understand whether any correlation exists between the Piwi homologs with HPV oncoproteins in cervical cancer. To begin with, the expression pattern of Piwi proteins were evaluated in cultured cervical cancer (C33A, SiHa, and CaSki) cell lines. We observed that all the Piwi variants were differentially expressed in all the selected cervical cancer cell lines with PiwiL1 showing the highest expression in CaSki cells, suggesting a positive correlation with HPV oncoproteins. On investigating this correlation further, we found that PiwiL1 was co‐expressed and physically interacted with HPV oncoproteins E6 and E7. Not only E6 and E7 physically interact with PiwiL1, but also accentuated its expression when over‐expressed in HaCaT cells. Since PiwiL1 protein is known for its overexpression in many cancers, we wanted to know how this molecule is regulated in cancer. For this, we have carried out an in silico analysis of the PiwiL1 promoter to find the possible binding TFs using multiple online tools such as Alibhabha, AllegenPromo, and TFsitescan. We further shortlisted the TFs based on their function in cancer, interestingly we found both p53 and E2F in the list. Further analysis of these selected factors suggested that PiwiL1 promoter activity was significantly upregulated in the presence of E2F, whereas it was downregulated with p53. The binding of these transcription factors and their differential regulation of PiwiL1 promoter could be one of the reasons for its over‐expressed status in cancer. In addition to this, it is also known that the Piwi‐piRNA complex act as guiding signals for factors that play a significant role in tumorigenesis. Therefore, we have constructed a pipeline to predict the piRNAs from a small RNA Sequencing Data (SRP119662) obtained from the NCBI database, we identified 2274 piRNAs from both control and tumor samples, out of this, only 9 piRNAs were found to be significantly differentially expressed compared to control. Even though in silico analysis predicted 6 piRNAs to be significantly upregulated and 3 piRNAs to be significantly downregulated, the qPCR analysis was not entirely in accordance to the prediction. Out of the 3 down‐regulated piRNAs, hsa_piR_007863 showed elevated levels in CaSki in comparison to HaCaT while hsa_piR_002320 which was predicted to be upregulated in tumor showed lower expression. Our preliminary data suggest that PiwiL1 protein and associated piRNAs may have an important role in HPV‐associated tumorigenesis, whose exact molecular mechanism needs to be further evaluated.
Regulatory factor X1(RFX1) is a unique transcription factor that can act both as an activator and repressor in a context‐dependent manner. In most of the cases, it has been shown to exert an inhibitory action on its targets. It belongs to the evolutionarily conserved RFX (Regulatory Factor binding to the X‐box) family of transcription factors consisting of eight homologs (RFX1‐8) in humans. RFX1 is seen down‐regulated in many cancers and is speculated to have a critical role in regulating stem cell properties. Cancer stem cells are major hurdles in cancer therapy as they are capable of repopulating a tumor after treatment. We aim to understand the regulation of cancer stemness by RFX1 in human teratocarcinomal (NTERA‐2) cancer stem like cells. We have chosen NTERA2 cl.D1 (NT2) embryonic carcinoma cells since they are the malignant counterparts of embryonic stem cells and are pluripotent in nature, making them an ideal candidate to be used as a cancer stem cell model. The cells were differentiated using DISC (Days in suspension culture) method to obtain differentiated populations (NT2D). Using real time analysis, the expression of RFX1 was evaluated in proliferating (NT2P) and differentiating (NT2D) cell populations. A significant increase in RFX1 expression was observed in NT2D cells compared to NT2P cells. For further validation, the same was done in stem cell population enriched using Abcg2 promoter where the stem (GFP+ve) and non‐stem (GFP‐ve) cell populations were sorted out by FACS. Further RFX1 expression was perturbed in NTERA cells, and the assessment of cardinal properties of CSCs, such as clonogenicity/self‐renewal properties, proliferation potential, and migration/invasive properties in vitro,revealed that RFX1 plays a significant role in cancer stemness. Therefore, understanding the regulation of cancer stemness by RFX1 will allow us to formulate better diagnostic and therapeutic approaches to address chemoresistant cancers.
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