LncRNA HOTAIR participates in the development and progression of adrenocortical carcinoma via regulating cell cycle.

OBJECTIVE: To explore the role of HOTAIR in the pathogenesis of adrenocortical carcinoma (ACC) and its underlying mechanism. PATIENTS AND METHODS: Differentially expressed lncRNA (HOTAIR) in ACC was screened out from the GEO database. The survival analysis and ROC curve were performed according to HOTAIR expressions in ACC patients. The correlation between HOTAIR expression and clinical information of ACC patients was analyzed by chi-square test. The univariate and multivariate COX regression analysis was carried out to analyze the relationship between HOTAIR expression, disease-free survival (DFS) and overall survival (OS) of ACC patients. We then detected HOTAIR expression in 77 ACC tissues and 30 normal tissues by qRT-PCR (quantitative Real-time polymerase chain reaction). ACC cell lines were further screened out for the following in vitro experiments. After altering HOTAIR expression in ACC cells by plasmid transfection, proliferation and cell cycle were detected by Cell Counting Kit-8 (CCK-8) and colony formation assay, respectively. Finally, Western blot was utilized to detect expressions of cell cycle-related genes in ACC cells. RESULTS: HOTAIR was overexpressed in ACC tissues than that of normal tissues. HOTAIR expression was remarkably increased in ACC with T3 and T4 stage than that of T1 and T2 stage. Moreover, HOTAIR expression was remarkably increased in ACC with stage III and IV than that of stage I and II. HOTAIR was an independent prognostic factor for DFS and OS of ACC patients. For in vitro experiments, inhibited proliferation and arrested cell cycle were observed in H295R cells transfected with si-HOTAIR. Opposite results were obtained after SW-13 cells were transfected with HOTAIR overexpression plasmid. Furthermore, expressions of cell cycle-related genes, including Cyclin D1, p-Rb and p-GSK3ß were remarkably decreased after HOTAIR knockdown. CONCLUSIONS: We demonstrated for the first time that HOTAIR is overexpressed in ACC and is a prognostic risk factor in ACC patients. HOTAIR participates in the development and progression of ACC via shortening cell cycle and promoting proliferation of ACC cells.

Construction of a Hep-2 cell line stably transfected with Livin shRNA.

OBJECTIVE: The aim of this study was to construct a eukaryotic expression plasmid with a short hairpin RNA (shRNA) targeting Livin in order to obtain a stably transfected Hep-2 cell line with a reduced expression of Livin. METHODS: The shRNA targeting Livin mRNA was designed, and a shRNA plasmid and a negative control plasmid were constructed. After amplification in E. coli, restriction endonuclease digestion and sequence confirmation, the plasmids were transfected into Hep-2 cells using Lipofectamine 2000. The stably transfected cell line was screened using G418, and inhibition of Livin mRNA and protein levels were detected using real-time PCR and western blotting, respectively. RESULTS: pGenesil-Livin-shRNA eukaryotic expression plasmid was successfully constructed and identified by sequencing. Green fluorescent protein (GFP) expression was observed in Hep-2 cells transfected with shRNA plasmids by fluorescence microscopy. The expression levels of Livin mRNA and protein decreased significantly in Hep-2 cells transfected with the shRNA recombinant plasmid. The mRNA level was reduced by 47.17 %, and the protein level was reduced by 34.25 %. CONCLUSION: The shRNA eukaryotic expression plasmid targeting Livin was successfully constructed, which could significantly inhibit the expression of Livin in laryngeal cancer Hep-2 cells. This provides a basis for future research on the function of Livin in Hep-2 cells, and gene therapy for laryngeal cancer.

[Comparison between poly hydroxy acrylic acid and Van-clear replacing the traditional reagents to detect the cervical hTERC genes by adopting FISH technique].

OBJECTIVE: To observe the difference of the human telomeres RNA component (hTERC) genes' amplification in the cervical tissue by applying the environment-friendly fixative poly hydroxy acrylic acid and the transparent dewaxing solution Van-clear separately or jointly to replace the traditional fixative 4% (volume fraction) neutral buffered formalin and the conventional transparent dewaxing solution xylene in the use of fluorescence in situ hybridization (FISH) for detection. METHODS: In the study, 255 cases of cervical tissue specimens submitted by the Department of Gynecology in Zhongshan Boai Hosipital were collected from Mar. 2013 to Apr. 2015. Four samples were taken from the same lesion site. All the cases were divided into 4 groups and named group A, B, C, and D. Group A used 4% neutral buffered formalin fixed and xylene dewaxing to make slices. Group B used poly hydroxy acrylic fixed and xylene dewaxing to make slices. Group C used 4% neutral buffered formalin fixed and Van-clear transparent to make slices. Group D used poly hydroxy acrylic fixed and Van-clear transparent dewaxing to make slices. The amplification of hTERC genes in the four groups of cervical specimens was also detected by FISH technique. RESULTS: When the hTERC genes were detected by FISH method under the fluorescence microscope, it was obvious that the tissue profile and the background of group A, B, C and D were all clear. The probe was fixed in the accurate position so that the bright red or green fluorescence signals were easily found in these four groups. Compared with the positive rate of group A, there was no statistical significance in that of group B, C and D (P>0.05). At the same time, the coincidence rate of the FISH results was high, which showed that the new environment-friendly reagent had no significant difference in the detection of cervical hTERC genes by FISH technique. CONCLUSION: It is possible for the environment-friendly reagent poly hydroxy acrylic acid and Van-clear to replace 4% neutral buffered formalin and xylene separately or jointly to detect the cervical hTERC genes by adopting FISH technique.