Inorganic phosphate (Pi) is shown to be involved in excretion of methylglyoxal (MG) in the promastigote form of Leishmania donovani parasite. Absence of Pi leads to its accumulation inside the parasite. Accumulation of MG is toxic to the parasite and utilizes glyoxylase as well as excretory pathways for its detoxification. In addition, Pi is also reported to regulate activities of ectoenzymes and energy metabolism (glucose to pyruvate) etc. Thus, it is known to cumulatively affect the growth of Leishmania parasite. Hence the transporters, which allow the movement of Pi across the membrane, can prove to be a crucial drug target. Therefore, we characterized two phosphate transporters in Leishmania (i) H+ dependent myo-inositol transporter (LdPHO84), and (ii) Na+ dependent transporter (LdPHO89), based on similar studies done previously on other lower organisms and trypanosomatids. We tried to understand the secondary structure of these two proteins and confirm modulation in their expression with the change in Pi concentration outside. Moreover, their modes of action were also measured in the presence of specific inhibitors (LiF, CCCP). Further analysis on the physiological role of these transporters in various stages of the parasite life cycle needs to be entrenched.

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The emergence of large-scale transcriptomic data provides the opportunity for identifying novel putative targets of microRNAs (miRNAs). In this study, we followed a computational pipeline to predict the candidate gene targets of the miR-34 family. This approach integrates the expressions of miR-34 with genes of heterogeneous primary cervical epithelial squamous cell carcinomas (CESC). Integration of miR-34b and epithelial-mesenchymal transition (EMT) regulated genes has also been focussed, EMT being a reversible process that fuels cancer metastasis. An in-silico approach involving three processes was carried out with CESC datasets of the cancer atlas genome (TCGA), which includes correlation analysis, target prediction database lookup, functional enrichment, network analysis, survival analysis, and EMT score derivation. The results indicate that the miR-34 family may regulate the candidate genes of the mTOR pathway, cell cycle (CCND2) and cell adhesion functions (FZD4). Further, the study reveals the possible regulation of EMT signature genes, namely BMP7, CAV1 and ID2by miR-34b. Further, these transcriptomic signatures were validated in a subset of CESC from the South Asian Indian population (n = 10) and in non-cancerous cervical tissues (n = 5). Upon stably expressing miR-34b in cervical cancer cells (C33A and HeLa), we found repression of these candidate genes and a low negative correlation (r2 = 0.07) between miR-34b and EMT score indicating FN1 as its putative target. Together, these studies revealed the potential targets of the miR-34 family, especially miR-34b, with the hope that they would emerge as potential biomarkers and/or promising therapeutic targets in CESC.

Background: Cervical cancer is one of the most common cancers in women worldwide, particularly in low- and middle-income countries. Due to low surveillance and late detection, it is one of the major health issues leading to high mortality rates. MicroRNA-34 family (miR-34a, b, c) is thought to be tumor suppressive in several cancers but the mechanistic basis is not clear. Methods: Stable cell lines were generated with inducible hsa-miR-34b lentiviral vector from Dharmacon and maximal time and dose for induction of miR-34b with doxycycline were determined. Cell viability studies on HeLa, SiHa and C33A were investigated by MTT assay. Acridine orange/Ethidium bromide (AO/EB), senescence associated β-galactosidase (SA- β gal) assays for apoptosis and senescence and assessment of DNA damage (γH2AX) were performed. Total reactive oxygen species (ROS), superoxide, hydroxyl and peroxynitrite and H2O2 radicals were quantitated by DCFDA, DHE, HPF dyes and pHyper-dMito plasmid, respectively. Results: Overexpression of miR-34b reduced cell viability in cervical cancer cell lines except SiHa. Further investigation by AO/EB staining revealed no significant increase in the proportion of apoptotic cells on overexpression of miR-34b. Analysis of SA- β gal on overexpression of miR-34b revealed an increase in the proportion of senescent population in all 3 cell lines. Increased DNA damage is also observed in agreement with an increase in cellular senescence. In addition, an increase in oxidative stress was observed in miR-34b overexpressing cell lines as total ROS was elevated in all 3 cell lines with increased superoxide, hydroxyl and peroxynitrite and H2O2 radicals in HeLa and C33A while SiHa showed increased levels of H2O2 radicals with no significant change in other radicals. Conclusions: Overexpression of miR-34b can promote an increase in cellular senescence and oxidative stress in cervical cancer. The differential action in different cell lines may be due to the difference in expression of miR-34b in these cell lines. Our results highlight the potential of miR-34b to be used for therapeutic intervention in cervical cancer. Citation Format: K J Sindhu, Devarajan Karunagaran. MiR-34b promotes cellular senescence and ROS generation in human cervical cancer cells. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3774.