Gene Expression Profile Of Salivary Adenoid Cystic Carcinoma Associated With Perineural Invasion And Related Studies

Salivary adenoid cystic carcinoma (ACC) has a characteristic of perineural invasion (PNI), it often invades nerves and distantly metastasizes by growing along with nerves. A few of studies have reported the potential effects of some certain genes in the process of PNI in ACC, but most of the studies are limited, so they cannot elucidate the molecule mechanism of PNI in ACC thoroughly. The process of PNI in ACC was complicated which related to multiple genes, component elements, and pathways. So, there is important significance to clarify gene expression profile associated with PNI which can enrich basic theory and guide clinical treatment in ACC.Objectives:1. To investigate the range of gene expression differences between PNI and non-PNI ACC cell groups which procured by laser capture microdissection (LCM) and to correlate gene expression profile associated with PNI to identify potentially new biomarkers of ACC with PNI.2. CD146(MCAM), one of the up-regulated genes from gene expression profile, was chose to be the target for RNAi. To analyze its biological effects in reducing in vitro perineural invasion on ACC-M cell by specifically knock-down the transcription of CD146.Methods:1. Combined use of H&E staining and immunohistochemistry (IHC) method in consecutive frozen sections from primary ACC specimens, obvious PNI was assessed in the cancerous tissues. Pure cancer cells adjacent to the nerve tracts from cancerous tissues judged as PNI were procured by LCM. Then we constructed gene expression profile associated with PNI by combining use of RNA linear amplification and high-throughput cDNA microarray technique.2. Patterns of gene expression were verified by quantitative real-time PCR (QRT-PCR) and IHC. Gene expression profile associated with PNI was analyzed by bioinformatics methods. Target gene CD146 was chose for the following RNAi research.3. According to the CD146 cDNA in the Genebank, we designed two different shRNA targeting the coding sequence of the CD146. The pGe-CD146-shRNA1, 2 were constructed by inserting the designed shRNA in the eukaryotic expression vector pGenesil-1. The vector was identified by restriction endonuclease digestion and partial nucleotide sequencing. ACC-M cells were transfected with different vectors respectively. Monoclones of stable transfectants, were procured by limiting dilution assay, then selected by G418. The expression results of CD146 mRNA and protein were detected after RNAi.4. Effects of CD146 gene silencing on ACC-M cells were detected, including cell proliferation activity analyzed by MTT assay, and cell cycle change detected by flow cytometry (FCM) analysis. The inhibitory effect of RNAi on ACC-M cell's PNI in vitro was demonstrated in modified Boyden chambers.Results: 1. Obvious PNI was found in 6 out of the 15 primary cancerous tissues. Pure cancer cells adjacent to the nerve tracts from 6 cancerous tissues judged as PNI were laser captured, and pure cancer cells from the same 6 tumors distant from the nerve tracts were also procured. Differential expression genes were detected by Agilent human 1A cDNA microarray. Of 18,716 interrogated genes and 1,457 ESTs, analyzed data showed 53 genes were differentially expressed (fold difference≥2.0 or≤1/2) among all 6 arrays that could distinguish PNI and non-PNI cancer cell groups. Of the 53 genes found differentially expressed, 38 were up-regulated and 15 down-regulated. Thus we constructed gene expression profile associated with PNI.2. Of the 53 differentially expressed genes, we performed QRT-PCR and IHC analysis for a group of selected genes to validate the findings of the microarray analysis. Hierarchical clustering showed the 53 genes could be divided into five clusters discriminating the PNI. Functional gene classes highly represented in PNI cell group included those involved in development, signal transducer activity, neurogenesis, oncogene, inflammatory response, immune response, binding, cell adhesion, and extracellular matrix functions. Several genes previously identified as overexpressed in ACC were confirmed by our study in addition to identifying several novel genes with potential roles in the pathobiology of ACC associated with PNI.3. We successfully constructed CD146 shRNA eukaryotic expression vector system based on target gene CD146, including pGe-CD146-shRNA1, 2; a negative control vector pGe-negative-shRNA and a positive control vector pGe-GAPDH-shRNA. Combined use of limiting dilution assay and G418 pressure selection, we successfully constructed monoclones of CD146 shRNA stable transfectants. It has been proved that the mRNA and protein expression levels of CD146 were significantly inhibited in the stable transfectants.4. Findings based on MTT and FCM analysis indicated that the knockdown of CD146 expression could inhibit proliferation of ACC-M cells by modulating G0/G1 and S cell cycle regulators. Modified Boyden chambers experiment demonstrated that the knockdown of CD146 expression could inhibit PNI activity of ACC-M cells, and that different shRNA transfectants showed striking unequal efficiency in inhibiting PNI activity.Conclusions:1. Combined use of LCM and high-throughput cDNA microarray techniques, we successfully constructed in vivo gene expression profile of salivary ACC and to correlate the profile with PNI. Hierarchical clustering and functional gene classes can analyze the gene expression profile all around. Several novel genes with potential roles in the pathobiology of ACC associated with PNI deserved further study.2. CD146 shRNA eukaryotic expression vectors and their stable transfectants can specifically and effectively inhibit the expression of CD146, suggesting that it could be a good platform applied for functional research.3. The knockdown of CD146 expression by RNAi can successfully inhibit the malignant behaviors of ACC-M cells, especially PNI ability in vitro, implicating that CD146 may be a new candidate target gene for treatment of ACC.