Abstract
Biomarkers can potentially help in the detection and prognosis of diseases such as cancer, its recurrence, predicting response to therapy, and monitoring of response during and/or after treatment. Endogenous tumor blood biomarkers suffer from low concentrations that are not distinguishable from background noise and, if identified, the localization of the biomarker production site is not known. The use of exogenously introduced or artificial biomarkers can eliminate these issues. In this study, we show that cancer cells can be made to produce an artificial secreted microRNA (Sec-miR) that can be detected in media from cells in culture, and from both blood and urine in living mice. In culture, we show that chaining a number of Sec-miR sequences in a plasmid and transfecting cells with the plasmids could increase Sec-miR secretion as the number of sequences increases. Tumor induction in mice with a stably transfected HeLa cell line shows the presence and significant increase in the Sec-miR with time and tumor growth in plasma (p < 0.001, R2 = 0.5542). The relative half-life of the Sec-miR was seen to be 1.2 h in the plasma of living mice and was seen to appear in urine within 12 h. The transgene for the Sec-miR within a minicircle was introduced via the tail-vein into subcutaneous tumor-bearing mice. As the tumor growth increased with time, further in vivo transfection of the Sec-miR minicircles showed an increase in Sec-miR in both plasma and urine (R2 = 0.4546). This study demonstrated that an exogenous Sec-miR biomarker would allow for early tumor detection using in vitro diagnostics techniques.
Highlights
We demonstrate that the linking of multiple copies of secreted microRNA (Sec-miR) within the plasmid allows for higher detection sensitivity of the miRNA secreted from transfected cells
Both PP-pSurv-Sec-miR 12X-WPRE (PP) and MC-pSurv-Sec-miR 12X-WPRE (MC) were produced according to the protocols proposed by Kay et al Briefly, ZYCY10P3S2T E. coli were transformed with PP, colonies were picked, inoculated into kanamycin-containing
In our previous study [19], we have shown that Sec-miR expression from cells can be amplified by increasing the copy numbers of Sec-miR in a single construct
Summary
Cancer detection has relied on the production of endogenous tumor-specific biomarkers that can be detected in body fluids, mostly in the blood This has proven to be difficult due to the variability of the normal concentration of biomarkers within a patient population and a background level of biomarkers due to normal cells and confounding disease states that prevent the accurate differentiating of an increased biomarker level due to the presence of a tumor [1,2]. This has prevented the introduction of new biomarkers into patient care, even though a relatively high number of them are being discovered [3]. To overcome the above stated limitations of endogenously present biomarkers, we have previously demonstrated the use of an exogenously introduced gene encoding for a protein biomarker that
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
