Cell Function And Gene Expression Profiling Of Gastric Cancer Cell Lines In Response To Heparanase

Object:To investigate the impact of heparanase(HPSE) gene on gastric cancer biological behaviors and clarify the underlying molecular mechanisms of HPSE gene in many biological processes of the gastric cancer cells such as signal transduction, tumor angiogenesis, inflammation and tumor metastasis.Methods:1 Effects of HPSE siRNA on HPSE gene expression and biological behaviors of gastric cancer cells①RT-PCR was performed to screen gastric cancer cell lines with high HPSE gene expression.②Both of antisense oligodeoxyribonucleotide (AS-ODN) and control oligodeoxyribonucleotide (NS-ODN) targeting HPSE were designed and chemically synthesized. The screened gastric cancer cells were transfected with optical concentration of AS-ODN and NS-ODN tested by Real-Time PCR, and expression of HPSE at mRNA and protein levels were examined by Real-Time PCR and Western Blot in order to verify the silencing effects.③Effects of silencing HPSE on the morphology of gastric cancer cell SGC-7901 were observed by transmission electron microscope (TEM). The role of silencing HPSE on apoptosis of gastric cancer cell was detected by Annexin V-FITC/PI flow cytometry.④Effects of silencing HPSE on the abilities of invasiveness and migration of gastric cancer cell SGC-7901 were tested by Matrigel invasion assay and cell migration assay.2 Effects of HPSE antisense oligonucleotides on gene expression profiling of gastric cancer cellsAffymetrix Human U133 Plus2 gene chips were used to identify the gene expression changes after HPSE expression knockdown. Total RNA extracted from gastric cancer cells with AS-ODN and NS-ODN was reversely transcripted into cDNA to make chip cross-fertilization after fluorescent labeling. The cross-fertilization results were scanned and analyzed by software and then the Ratio was recorded as Cy3/Cy5 (case group/control group). The screening standards of differential gene were that positive Ratio(Cy3/Cy5)≥2 and negative Ratio (Cy3/Cy5)≤0.5. Some up and down regulated genes were verified by Real-Time PCR after HPSE expression was silenced and gene expression profiling changes of the HPSE-mediated downstream in gastric cancer cell were analyzed. Genes of differential expression that exceeded 2 folds were analyzed by "GO" tool using MAS3.0 software of CapitalBio Corporation, whereas genes that exceeded 1.5 folds were analyzed by "Pathway" tool.Results:1 Effects of HPSE silencing on HPSE gene expression and biological behavior of gastric cancer cells①RT-PCR detection showed that SGC-7901 expressed the highest HPSE levels among three gastric cancer cell lines, that's why SGC-7901 was selected as experiment cell model.②The optimal concentration of siRNA, verified by RT-PCR and Real-Time PCR assay, was lOnM.③Real-Time PCR and Western Blot results showed that expression of HPSE at mRNA and proteins level after siRNA decreased significantly as compared with negative control group.④TEM observation showed that HPSE silenced SGC-7901 exhibited typical apoptosis cell morphology. Annexin V/PI flow cytometry analysis showed that apoptosis was increased remarkably from 1.58% in mock group to 8.45% in HPSE silenced group.⑤Transwell assay indicated that the number of cell migrated through membrane in HPSE silenced group were more less than that of mock group (P<0.01), suggesting that inhibition of HPSE expression could abolish the invasive capabilities of SGC-7901 cells. Scrape migration assay showed that the wounded empty space in HPSE silenced group was obviously wider than that of control group at 24h and 36h after being scraped (P<0.05), indicating that inhibition of HPSE could decrease the migration ability of gastric cancer cells obviously.2 Effects of silencing HPSE on down stream gene expression of gastric cancer cellsAffymetrix analysis showed that expression of PRKCA and PRKCB, central mediator molecule of VEGF signal pathway, was decreased significantly in HPSE silenced group compared with mock group. Meanwhile, the expression of SHB, an adaptor protein of VEGFR-2 pathway, and VE-cadherin which interacted with VEGFR-2, were decreased in HPSE silenced group. It is also founded the inflammatory factor IL-la/IL-lb decreased. However, expressions of innate immunity-mediating molecule DEFB1 and adhesion molecule CD44 increased when HPSE were inhibited.Conclusions:1 Silencing HPSE abolished the abilities of invasion and migration of SGC-7901;2 Expressions of PRKCA and PRKCB, central bridge molecule of VEGF signal cascades, and that of SHB and VE-cadherin, both of which were important member of this pathway, were decreased in HPSE silenced group. Meanwhile, the expression of transmembrane glycoprotein CD44 was increased. These changes might play an important role in the decreased angiogenesis when HPSE expression was attenuated;3 Gene Chip assay showed that decreased expressions of SHB and VE-cadherin, increased expression of CD44, and attenuated FAK pathway when HPSE expression was silenced. All of these might be responsible for increasing cell apoptosis after HPSE was silenced;4 Gene Chip assay also exhibited that decreased expression of inflammatory factor IL-1a/IL-1b, increased expression of anti-inflammation adhesion molecule CD44 and negative inflammation regulatory factor HO-1 in HPSE silenced group relative to mock group. All of these might be involved in the inhibition of inflammatory reaction after HPSE was silenced.