Investigation of mechanistic role of HOX transcript antisense intergenic RNA and Protocadherin10 in gastric carcinogenesis

Abstract

HOX transcript antisense intergenic RNA (HOTAIR), as a long non-coding RNA (lncRNA) has been reported to regulate the carcinogenesis by epigenetic mechanism in many cancers. Protocadherin 10 (PCDH10) is one of well-known tumor suppressor genes and it is frequently methylated in gastric cancers (GC). Although both HOTAIR and PCDH10 play a major role in gastric carcinogenesis, there are no reports on the interactions between the two in the carcinogenic mechanism. Therefore, we tried to find out the function of HOTAIR related to the gastric carcinogenesis of PCDH10 in this thesis. For this purpose, we investigated mechanism of HOTAIR on apoptosis, cell proliferation, and invasiveness as indicators of carcinogenesis and metastasis of GC. Quantitative real-time reverse transcription polymerase chain reaction (PCR) and western blotting were used to examine the expression of HOTAIR and its related genes. The effect of HOTAIR on the promoter methylation of PCDH10 was analyzed by Methylation-specific PCR(MS-PCR) method. In addition, we investigated the interaction between miR-148b and HOTAIR by dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay. The expression of HOTAIR was significantly up-regulated in GC tissues (P<0.05) and GC cell lines (P<0.01), while PCDH10 was down-regulated in GC tissues (P<0.05). Knockdown of HOTAIR by siRNA of HOTAIR (si-HOTAIR1 and 2) significantly upregulated the mRNA/protein expression of PCDH10 and reduced the methylation of PCDH10 compared to scramble in MKN 28 and MKN 74 cells. Si-HOTAIR1 and 2 significantly reduced DNA methyltransferase 1 (DNMT1) expression, and over-expression of HOTAIR enhanced DNMT1 expression. In RIP, we found that miR-148b interacted with HOTAIR. Si-HOTAIRs increased miR-148b expression, and miR 148b mimic inversely reduced HOTAIR expression. Si-HOTAIRs and miR-148b mimic reduced DNMT1 expression and increased PCDH10 expression compared to control. Finally, we found that DNMT1 was downregulated after si-HOTAIRs and it was restored after treatment with miR-148b inhibitor. We demonstrate that HOTAIR interacts with miR-148b and DNMT1, eventually leading to PCDH10 methylation, which contributes to the progression of GC. The current data provide a better understanding for the key roles of lncRNA HOTAIR in epigenetic mechanism of GC.