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Developing sensitive point-of-care testing (POCT) of oncogenic nucleic acids from HPV infection is essential in preventing cervical cancer, especially in resource-limited settings. Rolling circle amplification (RCA) is attractive in achieving POCT; however, still exists a bottleneck requiring multiple amplification steps with multiple detection probes due to the limited sensitivity. Here we developed a minimum secondary structured RCA technique (MSS-RCA) by designing a unique circular template, demonstrating significantly enhanced detection sensitivity with only one amplification step and one primer. We demonstrated that RCA amplification efficiency could be kinetically manipulated by controlling the secondary structure of the circular template. Introducing the invertase probe to MSS-RCA, Human Papillomavirus 16 (HPV16) E6/E7 nucleic acid target was detected with a personal glucose meter (PGM) with a sensitivity of 5 fM (50 zmol in 10 µL). This integrated MSS-RCA-PGM detection system was successfully applied to detect HPV16 E6/E7 mRNA extracted from 54 cervical swab samples reaching a positive predictive value of 100.00% and negative predictive values of 96.00% (77.77% to 99.40%, 95% CI). MSS-RCA-PGM provides a sensitive POCT platform for the detection of biomarkers for screening of cervical cancer or other diseases.

Nicotine addiction and the occurrence of lymph node spread are two major significant factors associated with esophageal cancer's poor prognosis; however, nicotine's role in inducing lymphatic metastasis of esophageal cancer remains unclear. Here we showed that OTU domain-containing protein 3 (OTUD3) was downregulated by nicotine and correlated with poor prognosis in heavy-smoking esophageal cancer patients. Nicotine-mediated OTUD3 downregulation promoted lymphatic metastasis by inducing substantial tumor-induced lymphangiogenesis. At the mechanistic level, OTUD3 directly interacted with ZFP36 ring finger protein (ZFP36) and stabilized it by inhibiting FBXW7-mediated K48-linked polyubiquitination. ZFP36 bound with the VEGF-C 3-'UTR and recruited the RNA degrading complex to induce its rapid mRNA decay. Thus, downregulation of OTUD3 and ZFP36 was essential for nicotine-induced VEGF-C production and lymphatic metastasis in esophageal cancer. This study establishes that the OTUD3/ZFP36/VEGF-C axis plays a vital role in nicotine addiction-induced lymphatic metastasis. It also suggests that OTUD3 may serve as a prognostic marker, and induction of the VEGF-C mRNA decay might be a potential therapeutic strategy against human esophageal cancer.