**6. Future direction**

We have been demonstrated the usefulness and accuracy of sequencing in genetic research of the liver. One of the main problems with applying sequencing to the miRNA transcription research is that sequencing is a time-consuming procedure. And an important consideration for the discovery of miRNA by sequencing is the difficultly in identifying miRNAs that are expressed at low levels, at highly specific stages or in rare cell types. Moreover, a serious problem is that some miRNAs are difficult to profile precisely due to their physical properties or post-transcriptional modifications, such as RNA editing. In principle, these limitations can be overcome by extensive sequencing of small RNA libraries from a broad range of samples. For differential display, the sequencing-based method has the theoretical advantage in that it has the capability to discover and detect novel miRNAs. Based on our sequence variability results, especially with regard to RNA modifications, the accuracy of the sequence-based method is expected to be superior to that of the hybridization-based method. For the prediction of novel miRNAs, methods that rely on phylogenetic 1 genes. To overcome this problem, we made use of a computational approach for structural conservation criteria using the thermodynamic stability and intrinsic structural features of miRNAs. In clinics, pathologists often meet difficult situations in which they cannot clearly tell whether the tissue specimens they are observing are malignant or benign. Thus, in our opinion, using some miRNAs as a tumor marker would help clinicians to clearly determine whether that tissue is cancerous. miRNA sequences followed by bioinformatics have greater power than individual miRNAs or other clinic-pathological variables for the detection of high risk patients' groups with poor prognoses. There is currently little data available as to how we can use each miRNA to predict high risk groups; however, additional future miRNA work and data accumulation will elucidate such criteria. And further investigation is warranted to clarify the mechanism of aberrant expression of miRNAs in cancer and its participation in carcinogenesis. Nevertheless, these findings show that sequence-based miRNA profiling has potential for the confirmation of precise miRNA dynamics in a specific disease. In addition, it will increase our understanding of the mechanisms and factors involved in human liver cancer.
