Retrovirus Mediated-stable Expression Of Human GW112 Gene In Human Gastric Cancer SGC-7901 Cells

Gastric cancer is the fourth most common cancer and the second leading cause of cancer-related death in the world. Despite the significant advances in surgical techniques and oncology treatment regimens,the prognosis of patients with gastric cancer remains dismal. It is urgent to develop a new treatment for such malignancy.GW112 ,an olfactomedin- related glycoprotein, also called OLFM4, hOlfD, hGC-1,was originally cloned from human hematopoietic precursor cells and identified as human G-CSF- stimulated clone-1 (hGC-1).Recently, the role of GW112 has been extensively examined in many sorts of human cancers. GW112 was over- expressed in gastric cancer and associated with tumour stage and metastasis. As a secreted N- linked glycoprotein, GW112 facilitates cell adhesion by binding cadherin and lectins. GW112 was also reported to inhibit various apoptotic pathways and to promote proliferation of cancer cells. Overexpression of the GW112 can attenuate retinoic acid/ interferonβ-mediated cellular apoptosis and repress expression of apoptosis-related genes via binding to the potent apoptosis inducer GRIM-19.However, a limited number of investigations on roles of OLFM4 in gastric cancer have been reported so far. It remains to be determined the function of OLFM4 gene in gastric cancer cells.In this study, First,we used Real-time PCR to analyze the expression of GW112 in gastric tissues, adjacent tissues and normal stomach tissues . Second, a recombinant Retrovirus carrying GW112 gene pLEGFP- N1-GW112 was constructed, packaged and purified. The stable overexpression of GW112 in SGC-7901 cells was established.This study may lay a foundation for the further study on the role and its antiapoptotic mechanism of GW112 in gastric cancer.The contents and results include the following four aspects:1. To compare the expression of GW112 gene in gastric tissues, gastric adjacent tissues and normal stomach tissues .Firstly, 23 gastric tissues, gastric adjacent tissues and normal stomach tissues were selected and preserved. The specific Real-time PCR primer were designed according to the sequences of GW112 gene andβ-actin in Genebank. And then, Total RNA was extracted from the gastric tissues, gastric adjacent tissues and normal stomach tissues . Real-time PCR was used to analyze GW112 mRNA levels.The results show that GW112 mRNA levels in gastric tissues were significantly increased, compared with that in adjacent tissues and normal stomach tissues(3-6-fold).There is no significant difference about GW112 expression in adjacent tissues and normal stomach tissues(1.3-fold).These data indicate that GW112 is overexpressed in gastric tissues campared with gastric adjacent tissues and normal stomach tissues, consisting to the other reports.2. To establish the cells model that stably over-expressed GW112.To establish the model of SGC-7901 cells that stably over-expressed GW112, a retrovirus-mediated system, pLEGFP-N1 was used to deliver and express human GW112 gene to upregulate its expression in SGC-7901 cells. To this end, a recombinant retrovirus vector carrying GW112 gene pLEGFP- N1-GW112 was constructed, packaged. The stable PT67-GFP and PT67-GW112 clones were established by the screen of G418. After purified, recombinant retrovirus PT67-GFP control and PT67-GW112 were used to transfect SGC-7901 cells to obtain 7901-GFP control and 7901-GW112 cells that GFP and GW112 gene were stably over- expressed respectively. Last, GW112 mRNA levels was assessed by Real-Time PCR.1). GW112 gene was amplicated The PCR product was digested and then cloned into a retroviral vector-pLEGFP-N1 at SalI and BamHI sites. The results of restriction enzymes digestion and sequencing for recombinant plasmid pLEGFP-N1-GW112 demonstrated that the sequence of GW112 was identical to that in GeneBank. No. 006418 and were constructed correctly.2). The pLEGFP- N1-GW112 and pLEGFP- N1 plasmids were transfected into the PT67 packing cell using Lipofectamine 2000. After screened by G418-selective medium, stable PT67-GFP and PT67-GW112 clones were established.3). SGC-7901 cells were tranfected with recombinant retrovirus from PT67-GFP and PT67-GW112 clones , respectively. Stable SGC-7901-GFP control and SGC-7901- GW112 clones were abtained. GW112 levels were then detected by Real-time PCR. The results show that Retrovirus-mediated expressing GW112 could markedly and durably upregulate its expression in SGC -7901cells.