Through rational design of a functional molecular probe with high sequence specificity that takes advantage of sensitive isothermal amplification with simple operation, we developed a one-pot hairpin-mediated quadratic enzymatic amplification strategy for microRNA (miRNA) detection. Our method exhibits ultrahigh sensitivity toward miR-21 with detection limits of 10 fM at 37 °C and 1 aM at 4 °C, which corresponds to nine strands of miR-21 in a 15 μL sample, and it is capable of distinguishing among miRNA family members. More importantly, the proposed approach is also sensitive and selective when applied to crude extractions from MCF-7 and PC3 cell lines and even patient tissues from intraductal carcinoma and invasive ductal carcinoma of the breast.

This protocol describes an isothermal amplification approach for ultrasensitive detection of specific microRNAs (miRNAs). It achieves this level of sensitivity through quadratic amplification of the target oligonucleotide by using a Bst DNA polymerase-induced strand-displacement reaction and a lambda exonuclease-aided recycling reaction. First, the target miRNA binds to a specifically designed molecular beacon, causing it to become a fluorescence emitter. A primer then binds to the activated beacon, and Bst polymerase initiates the synthesis of a double-stranded DNA segment templated on the molecular beacon. This causes the concomitant release of the target miRNA from the beacon--the first round of 'recycling'. Second, the duplex beacon thus produced is a suitable substrate for a nicking enzyme present in solution. After the duplex beacon is nicked, the lambda exonuclease digests the beacon and releases the DNA single strand just synthesized, which is complementary to the molecular beacon, inducing the second round of recycling. The miRNA detection limit of this protocol is 10 fmol at 37 °C and 1 amol at 4 °C. This approach also affords high selectivity when applied to miRNA extracted from MCF-7 and PC3 cell lines and even from breast cancer tissue samples. Upon isolation of miRNA, the detection process can be completed in ∼2 h.

The current study aimed to assess the regulatory mechanism of microRNA-150-5p (miR-150-5p) in the pathogenesis of gastric cancer. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was performed to verify the expression of miR-150-5p in gastric cancer tissues and cell lines, which was revealed to be highly expressed in each. In addition, the expression of miR-150-5p was associated with advanced gastric cancer and lymph node metastasis. The current study then hypothesized that SRC kinase signaling inhibitor 1 (SRCIN1) was the target gene of miR-150-5p, a theory that was confirmed via a dual luciferase reporter gene assay. RT-qPCR and western blotting were then performed to verify the expression of SRCIN1 in gastric cancer tissues and cell lines. The results demonstrated that SRCIN1 was lowly expressed in gastric cancer tissues and cells. To assess the effect of miR-150-5p on gastric cancer cells, experiments were conducted with BGC-823 cells transfected with a miR-150-5p inhibitor or a miR-150-5p inhibitor+SRCIN1-small interfering (si)RNA respectively. A cell counting kit-8 assay and flow cytometry were also used to assess cell viability and apoptosis, respectively. Western blotting and RT-qPCR were further used to measure the expression of specific markers of epithelial mesenchymal transition (EMT), including epithelial cell markers (E-cadherin and zona occluding-1) and interstitial cell markers (vimentin, N-cadherin and β-catenin). The results revealed that the miR-150-5p inhibitor attenuated cell viability, induced apoptosis, decreased the expression of interstitial cell markers and increased epithelial cell marker expression. However, all effects of the miR-150-5p inhibitor were reversed following SRCIN1-siRNA treatment. In summary, the current study indicated that the miR-150-5p inhibitor attenuated cell viability, induced apoptosis and inhibited gastric cancer cell EMT by targeting SRCIN1.

Background: Epithelial ovarian cancer (EOC) is highly lethal gynecological cancer. Forkhead Box Protein C2 (FOXC2) promotes occurrence and development of various malignant tumors. The present study is aimed at exploring the correlation between the polymorphism of FOXC2 and epithelial ovarian cancer susceptibility in Chinese Han population. Methods: A case-control design was used to verify the association between FOXC2 polymorphisms and epithelial ovarian cancer. The genotyping was performed using Taqman® SNP Genotyping kit by qRT-PCR. The genetic variants including rs3751794 C > T, rs1035550 A > G, rs4843163 C > G and rs4843396 C > T in FOXC2 gene were analyzed. The strength of the associations was detected using odds ratios and 95% confidence intervals. Stratification analyses showed the association between the FOXC2 gene polymorphisms rs3751794 C > T, rs4843163 C > G and rs4843396 C > T with epithelial ovarian cancer susceptibility in terms of age, metastasis status, clinical stage, pathological grade, pregnant times, pausimenia, and the expression of ER, PR, wild p53 and mutant p53. Results: Rs3751794 C > T (P = 0.0016), rs4843163 C > G (P < 0.0001) and rs4843396 C > T (P < 0.0001) were significantly associated with increased epithelial ovarian cancer risk. In stratification analyses,rs3751794 C > T, was identified to be dominant in no metastasis patients, clinical stage 4 group, middle grade pathological stage, pregnant time over 3 patients, post-menopause women, strong wild type p53 expression; rs4843163 C > G was dominant in high grade clinical stage, high grade pathological stage, post-menopause women, strong ER expression group and no mutant p53 expression group; rs4843396 C > T was dominant in high grade clinical stage, high grade pathological stage, strong ER expression group. The rs1035550 A > G was not related to epithelial ovarian cancer susceptibility. Conclusions:The results of the current study verified that FOXC2 gene polymorphisms were associated with increased epithelial ovarian cancer risk and suggested that FOXC2 gene polymorphisms might be a potential biomarker for epithelial ovarian cancer susceptibility.