Expression Of Tissue Factor And Its Alternatively Splice In The Gastric Cancer Tissue And The Regulating Mechanism

Tissue factor(TF), comprising of a 47-k Da transmembrane glycoprotein, mediates a variety of physiologic- and pathophysiologic-relevant functions, such as tumor cell proliferation and metastasis, besides its primary function as the initiator of extrinsic coagulation cascade. Laboratory and clinical evidence showed the deviant expression of TF in several cancer systems and its tumor-promoting effects. It is expressed in two naturally occurring isoforms: membrane-bound “full-length”(fl)TF and soluble “alternatively spliced”(as)TF. The expression and function of both isoforms are influenced by several factors. Tumor-associated stimuli can regulate the transcription of TF. And as posttranscriptional modulator, micro RNAs are ofimmense importance for modulating their expression and function directly or indirectly. Gastric cancer is one of the most common malignant tumors. Evidences suggested aberrant expression of TF in gastric cancer, which was related to the biological behavior of gastric cancer. But the role of as TF in gastric cancer and its regulation are not clear yet. So in this study, we designed the primer and probe of as TF and detected the expression of fl TF and as TF m RNA by real time fluorescence quantitative polymerase chain reaction(PCR)in human gastric cancer tissues. The correlations among clinical data of patients with gastric cancer, fl TF and as TF were analyzed. Multi-factor Cox model was used to analyze their correlation with prognosis, including survival time. fl TF and as TF m RNA were also detected in MKN28 gastric cancer cells in vitro after stimulation with TNF-α. For long non-coding RNAs(lnc RNAs)could regulate the expression of target genes as competing endogenous RNAs(ce RNAs)with micro RNAs and played an important role in tumorigenesis, we investigated the regulation of lnc RNA H19 on the expression of fl TF and as TF in gastric cancer cells via its interaction with mi RNA19a(mi R-19a), thus providing clues for target therapy towards posttranscriptional modulation of TF.Our study showed the following results:(1) Meta-analysis suggested a significant relationship between positive TF expression and regional lymph node metastasis(P=0.001). Univariate survival analysis and multivariate survival analysis indicated a significant relationship between positive TF expression and prognosis, as well as between positive TF expression and survival(both P <0.0001). However, we didn’t observe its correlation with the size and proliferating time of tumor. As to the role of as TF in tumor, we failed to do meta-analysis for lack of original data.(2) We designed the primer and Taq Man probe of af TF, the forward primer located at the conjugate of the fourth and sixth exons, in order to avoid the amplification of fl TF and facilitate the detection of fl TF and as TF more efficiently and accurately.(3) The relative expression of fl TF m RNA [7.45(0.34~33.68)] in human gastric cancer tissues was significantly higher than those in healthy gastric tissues [3.00(1.36~5.02)](P=0.035), as well as as TF m RNA [0.88(0.07~26.00)] in gastric cancer tissues than those in healthy gastric tissues [0.33(0.03~0.97)](P=0.006). There was no correlation between the expression of fl TF, as TF and relevant elements of prognosis such as gender, age, TNM stage, pathological grade, tumor size, histological type or chemotherapy sensitivity(P>0.05). Single factor analysis(Log-rank survival analysis) demonstrated that the average survival time of gastric cancer patients with fl TF low expression was 15.66 month-longer than those with high expression([33.83(25.24~42.43)] vs [18.18(13.32~23.04)], χ2=6.185,P=0.013), and the average survival time of gastric cancer patients with as TF low expression was 4.82 month-longer than those with high expression([25.32(21.19~29.45)] vs [20.50(12.51~28.48)], χ2=4.604,P =0.032). The average survival time was 5.56 month-longer in the group with both fl TF and as TF low expression than in the group with either fl TF or as TF high expression([28.96(24.90~33.00) vs [23.40(16.27~30.51)], χ2=4.548,P=0.033]). And the difference was 19.94 months between the group with both fl TF and as TF low expression and the group with both fl TF and as TF high expression([31.07(23.21~38.92)] vs [11.13(6.90~15.36)], χ2=13.005,P=0.000), and 16.92 months between the group with either fl TF or as TF high expression and the group with both fl TF and as TF high expression(χ2=6.346,P=0.012). Multi-factor Cox model analysis demonstrated that both fl TF(HR=6.03,95%CI=1.02~35.71, P<0.05) and as TF(HR=10.74, 95%CI=2.32~49.59, P<0.01) expression could be considered predictors of survival time in gastric cancer patients.(4) In vitro experiments demonstrated that with the stimulation of TNF-α, MKN28 cancer cells had higher expression of lnc RNA H19, fl TF and as TF, but lower expression of mi R-19 a. With construction of lentivirus p LVX-H19 lnc RNA-sh RNA downregulated vector and TF overexpressed vector, we found mi R-19 a could bind to 3’UTR of TF and inhibit TF expression. mi R-19 a decreased TF protein level in gastric cancer cells and suppressed the function of fl TF/as TF to promote gastric cancer cell proliferation and invasion. Downregulation of lnc RNA H19 increased mi R-19 a expression, decreased as TF/fl TF expression level and suppressed the proliferation and invasion of gastric cancer cells.In summary, the designed as TF primer and Taq Man probe could detect the expression of as TF well. And it was first reported that as TF m RNA was overexpressed in gastric cancer tissues as fl TF m RNA, which was closely related with prognosis, though not with pathology. as TF m RNA level was negatively related with the survival time of patients with gastric cancer post surgery, and could be a better predictor of survival time if combined with fl TF m RNA level. Additionally, TNF-α increased TF expression in MKN28 cancer cells in vitro, which was posttranscriptionally regulated by lnc RNA H19 and mi R-19 a, indicating its role in promoting the genesis and development of gastric cancer. So as TF, fl TF and lnc RNA H19 are important biomarkers for predicting the prognosis of gastric cancer. Our study not only demonstrated the posttranscriptional regulation of TF by lnc RNA H19 and mi R-19 a, but also provided clues for target therapy towards as TF, fl TF and lnc RNA H19 in gastric cancer, which was of clinical meaning.