Expression of Annexin A3 in Primary Cultured Parenchymal Rat Hepatocytes and Inhibition of DNA Synthesis by Suppression of Annexin A3 Expression Using RNA Interference

Abstract

Annexin A3 is a member of the lipocortin/annexin family, which binds to phospholipids and membranes in a Ca 2؉ -dependent manner. Although annexin A3 has various functions in vitro, its cellular significance is completely unknown. Annexin A3 is not found in rat liver in vivo. In the present study, we investigated the expression of annexin A3 in primary cultured parenchymal rat hepatocytes. Annexin A3 protein was detected in 48-h, but not 2.5-h, cultured hepatocytes using Western blot analysis. The annexin A3 level further increased after an additional 24 h of culture. Annexin A3 mRNA was not detected in 2.5-h cultured hepatocytes but was detected 22 h after the start of culture by RT-PCR analysis, reaching a maximum value after 48 h of culture. To define the role of Annexin A3 in DNA synthesis, RNA interference was used to reduce annexin III gene expression in hepatocytes. The transfection of small interfering RNAs targeting annexin A3 in the hepatocytes reduced the corresponding mRNA and protein expression by approximately 80% and more than 90%, respectively, at 24 h after transfection. In the annexin A3 small interfering RNAs-transfected cells, DNA synthesis, as assessed by [ 3 H]thymidine incorporation, decreased by approximately 70% not only in the control cultures, but also in the hepatocyte growth factor-or epidermal growth factor-treated cells. These findings show that annexin A3 is expressed in primary cultured parenchymal rat hepatocytes and that the suppression of annexin A3 expression using RNA interference inhibits DNA synthesis.

Change in Annexin A3 Expression by Regulatory Factors of Hepatocyte Growth in Primary Cultured Rat Hepatocytes

“…The close correlation between known actions of various stimulators and inhibitors on hepatocyte growth and change in AnxA3 expression is consistent with our recent finding that AnxA3 acts as a positive regulator of hepatocyte growth in cultured hepatocytes. 12) In addition, we discovered that enhanced expression of AnxA3 was observed in the proliferative hepatocytes after carbon tetrachloride-induced rat liver damage and 70% partial hepatectomy (unpublished observation). This may lead to speculation that regulation of AnxA3 expression by these factors may be involved in their regulation of hepatocyte growth.…”

“…[7][8][9][10][11] We have recently reported that AnxA3 is expressed in cultured rat hepatocytes and that inhibition of AnxA3 expression by RNA interference results in a significant inhibition of hepatocyte growth. 12) This evidence indicates that AnxA3 acts as a positive regulator on hepatocyte growth in cultured hepatocytes. In relation to our report, it is noteworthy that hepatocytes placed under culture conditions acquire a growth potential characterized by enhancement of hepatocyte growth dependent on several growth factors, [13][14][15] whereas adult hepatocytes are normally quiescent in vivo.…”

“…For 18S rRNA, the PCR program consisted of 40 cycles of 10 s at 94°C, 10 s at 60°C, and 20 s at 72°C. The sequences of AnxA3-specific primers were 5Ј-CAA-ATTCACCGAGATCCTGT-3Ј and 5Ј-TGCTGGAGTGCTG-TACGAAA-3Ј, 12) those of the albumin-specific primers were 5Ј-AAGGCACCCCGATTACTCCG-3Ј and 5Ј-TGCGAAGT-CACCCATCACCG-3Ј, 26) and those of 18S rRNA-specific primers were 5Ј-CCAGAGCGAAAGCATTTGCCA-3Ј and 5Ј-GGCATCACAGACCTGTTATTGCTC-3Ј. The 18S rRNA PCR product specificity was confirmed by DNA sequencing using an ABI Prism 377 Sequencer (Applied Biosystems, Foster City, CA, U.S.A.).…”

Impact of Annexin A3 expression in gastric cancer cells

“…It has been shown that disorders of Annexin expression are indirectly linked to different human diseases, such as rheumatoid arthritis, lung cancer, and diabetes [24][25]. The expression level of Annexin is closely related to the proliferation, differentiation, and invasion/migration of tumors as well as the clinical stages of tumors [12,20], and therefore has recently become a hot spot in the tumor research field. Human Annexin A3 genes are dispersed throughout the genome on chromosomes 4 q13-q22, including the two main isoforms of 33 kD and 36 kD [20].…”

“…Quantitative real-time PCR was performed using the SYBR Green RT-PCR Kit (Applied Biosystems, Foster City, CA, USA) in a total volume of 20 μl containing 2 μl of reverse transcription product, 5 pmol of each primer, 10 µl of 2x SYBR Green mix (Applied Biosystems), and 0.4 µl of a 1,000x diluted reference dye (Applied Biosystems). The primers [20] used to detect Annexin A3 expression were 5'-CAAATTCACCGAGATCCTGT-3' (forward) and 5'-TGCTGGAGTGCTGTACGAAA-3' (reverse), to detect the β-actin expression were 5'-ACCACAGTCCATGCCATCAC-3' (forward) and 5'-TCCACCACCCTGTTGCTGTA-3' (reverse). The reactions were performed in an Applied Biosystems Prism model 7900HT Sequence Detection System with the following settings: Prior to amplification, an initial denaturation step was performed (95°C for 5 min) ensuring complete denaturation of the DNA and activation of the Taq polymerase.…”