| IntroductionColorectal cancer is the third common cancer worldwide. Lack of distinctive physiological changes and clinical features of the disease underlie a larger proportion of patients being diagnosed at a relatively advanced stage, and hence are deprived of curable treatment. Therefore, the need to discover novel molecular biomarkers to early and effective diagnosis of colorectal cancer is critical to enhance patients’ overall survival. MicroRNA as a revolutionary marker with an astounding potential in the field of cancer diagnostics, prognostics and therapeutic application, is considered to play an important role in discrimination of diseases at various stages with its tissue-specific and spatio-temporal expression characteristics. Our previous studies have revealed a panel of microRNAs:miR-375, miR-424 and miR-92a, which could differentiate between adenoma with high-grade intraepithelial neoplasia and early invasive colorectal cancer, in hope of aiding patients in earlier diagnosis and determining an optimal operative procedure. However, such studies remained at the molecular and cytological level, to further verify the expression level of microRNA from the morphological perspective in order to better understand its alteration and function throughout the development of colorectal cancer, in the present study, the techniques of in situ hybridization and immunohistochemistry were employed to investigate the biological trait of miR-92a, which is one of the candidate microRNAs in differentiating adenoma with high-grade intraepithelial neoplaisa and early invasive colorectal cancer, through its microscopic expression on tissue level.A methodology and optimization of microRNA in situ hybridization in FFPE tissuesIn the establishment of an optimized microRNA in situ hybridization method, there are at least four criteria to be considered:(a) the quantity of the target microRNA, (b) the hybridization temperature, (c) the concentration of the probe, and (d) the temperature of post-hybridization wash, so-called stringency wash. Throughout the experiment, several precautions should be taken against the degradation of target microRNAs, such as the preparation of reagents and tissue sections using 0.1% v/v DEPC-treated (diethylpyrocarbonate, DEPC) water, the sterilization and disinfection of experimental apparatuses according to RNA grade, and the maintenance of an RNase-free working environment. Based upon the original protocol of miRCURY LIMATM microRNAISH Optimization Kit (FFPE) Instruction manual v1.2, various steps in the experiment were further optimized by review and comparison of different in situ hybridization literature. In the present study, the use of LNA-modified,5’-digoxigenin labeled probe of miR-92a at a concentration of 80n,M would efficiently and specifically conjugate itself to the complementary target microRNA sequence at a higher hybridization temperature (about 55℃) and to undergo hybridization for one hour, afterwards, to remove excess unconjugated probes at 4℃ with low concentration of saline sodium citration solution (2×SSC) would produce a high intensity hybridization signal.Analysis of miR-92a expression and its biological behavior in normal colon, adenoma (with high-grade or low-grade intraepithelial neoplasia) and colorectal cancer FFPE surgical tissues by In situ detection and multi-spectral imaging systemThirty-four tissue samples of 24 cases were recruited in the present study, in which 10 of the 34 tissue samples are diagnosed as low-grade intraepithelial neoplasia,11 samples of high-grade intraepithelial neoplasia and 13 samples of colorectal cancer. Third-one normal counterparts from each of the 24 cases (except in three samples where the corresponding normal counterparts are unavailable) are also included in the study. Due to the fact that miR-92a is universally expressed in normal organs and tissues, results of in situ hybridization of miR-92a did not show accurate grading of signal intensity and definite expression area but rather the disclosure of the biological behavior through its expression pattern, which could further be investigated by immunohistochemistry with common CRC-related indicators. As a consequence, the multi-spectral imaging system was introduced in attempt to quantify the signal generated on the tissue sections in terms of optical density at visible wavelength. The application of multiple spectral imaging system in the quantitation of microRNA signal validly distinguished the different expression level of miR-92a in the tested samples and showed statistic significance in some of the interested criteria, except no statistic significance was found between cancer and high-grade intraepithelial neoplastic tissue samples.MiR-92a and the proliferative marker PCNA is correlated by their expression pattern in normal colon FFPE surgical tissuesTwenty-one normal counterparts from the lowest degree of cancerous and non-cancerous lesions from the 24 cases were selected to further study the biological behavior of miR-92a. As the result of in situ detection of miR-92a expression in normal tissue accompanied in each lesioned sample produced a "bottom-thick and top-thin" signal pattern, which is analogous to one of the classic adenoma growth model, the "bottom-up growth" model. It was then postulated that miR-92a may participate in the proliferation and differentiation of colonic stem cell lining the intestinal crypt. Since proliferating cell nuclear antigen, also known as PCNA, is a widely use biomarker in identifying the proliferating state of tissue, so in the study, we examined the proliferation state of the normal colonic tissue by immunohistochemistry and attempted to correlate the two biomarkers in consecutive tissue sections. The morphological expression of miR-92a and PCNA resembled one another, which suggested that miR-92a may take part in proliferation and differentiation of colonic epithelium, thus, in the subsequent study should consider enlarging the sampling size so as to further inspect the relationship between these two biomarkers.MiR-92a and p53 is negatively correlated by expression level in adenoma (with high-grade or low-grade intraepithelial neoplasia) and colorectal cancer FFPE surgical tissuesIn this study, miR-92a was observed to have a correlation with one of the clinical indicators of each case, i.e. p53 expression. To justify this observation, we re-examined the p53 expression of each sample by immunohistochemistry, and compared the result with the intensity of miR-92a expression during in situ detection in consective tissue sections, we found out that in the location where miR-92a was strongly expressed, there would be lower intensity or zero expression of p53 in the corresponding area. Results suggested that these two biomarkers are strongly correlated through statistic analysis using Pearson χ2 test, giving a correlation coefficient of 0.94 with p=0.056. Nevertheless, the interaction between these two biomarkers needs additional investigation for further elaboration of the functional role of miR-92a in colorectal neoplasia. |
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