D-limonene Mediates The Mechanism Of COL8A1 Regulating Tumor Metastasis

Transference is an important factor of malignant tumor that causing death,so it is significance to explore the transfer mechanism of tumor metastasis ,showing a probable way in early diagnosis and treatment of tumer。The main ways for tumer metastasis are from lymph nodes, blood circulation, or transfering plant. More than 50% of tumor metastasis is via lymphatic track,but its mechanisms are still under investigation. Some researches have shown that the transfer of tumor are not only affected by the body micro-environment, but also have a closely relationship with the tumor cells' own characteristics.COL8A1(procollagen, type VIII) is one of extracellular matrix proteins. Collagenα- 1 (VIII) chain precursor (Endothelial collagen) has been found 19 species of human collagen , with the former three-helical peptide molecules is called procollagen collagen which can be hydrolyzed to collagen. COL8A1 is a non-fibrous short chain protein collagen, which can regulate, propagate and adhere to various cells, and tumor cells highly express COL8A1, that may be directly lead to a high degree of tumor cell proliferation. Also COL8A1l high expression is probably suggested as a Specific molecular marker of tumor cells which is hepatic derivation.D-limonene is an effective monomer composition which is extracted from orange peel Chinese medicine, as a sort of single natural terpenes complex。Its chemical name:[1-methyl-4-(1-methyl-ethenyl)-, (4R)-],Molecular formula: C10H16, Molecular Weight: 136.32。D-limonene usually originates from the volatile oil of the Rutaceae plants,such as Citrus, lemon, lime[10]. A large number of experiments have proved that D-limonene has anti-cancer activity,it shows great affevtion on preventative and treatment in chemical-induced tumors in rodent , such as stomach, breast, liver, colon, pancreatic and skin cancer , and its mechanism includs direct cell cytotoxicity, induced tumor cells apoptosis and inhibit farnesyl protein transferase's level and activity inside [1-6]. In vitro experiments,they also discover that D-limonene's cell cytotoxicity on liver cancer, pancreatic cancer, stomach cancer, leukemia and other malignant, through a variety of signaling pathways to induce tumor cell apoptosis [7]. D-limonene may also restrain tumor microvascular and Microlymphatics formation,down rugulate MVD and LMVD, and inhibit the occurrence of tumor metastasis.Hca cells were screened out from 615 mouse ascites type hepatoma cells H22, with the special ability of lymph node metastasis, among which using highly metastatic Hca-F cell species inoculate 615 mice foot pad, result in 80% of the lymphatic transfer, and low metastatic Hca-P cell special in lymph node metastasis rate was 30%, therefore, as a choic model to study the mechanism of lymphatic metastasis, but its specific mechanism is not clear so far。In this study, experimental animals are SPF class C57L, 615 mice,categorized as:high, low, non-lymphatic with metastatic potential of mouse hepatocarcinoma cell Hca-F, Hca-P, Hepa1-6 and normal mice liver cells IAR-20 as model ,to analysis COL8A1 expression in liver cancer mice cells. Then through RNA interference specific technology to Down- regulated expression of COL8A1 in Hca-F cells, observate the effects of lymphatic metastasis in liver cancer cells.Transfect the cDNA into cells Hepa1-6, after COL8A1 is stabilityly expression ,observate the effects of Hepatoma cells in tumorigenicity. D-limonene affects COL8A1 to regulate mechanism of tumor metastasis and tumorigenicity.The aim is to establish the relevance of COL8A1 and tumor metastasis, and to provide a new target for drug design in preventing metastasis .This paper is divided into the following three parts:1 COL8A1 expression in mice liver cancer cellsRT-PCR, Western blot analysis were utilized to analysis the expression of COL8A1 in Hca-F, Hca-P, Hepa1-6, and IAR-20 cells. The results showed that, Hca-F, Hca-P, Hepa1-6, and IAR-20 cells hava COL8A1 expression,and the intensity of COL8A1 expression in Hepa1-6 cells was significantly lower than that of Hca-F and Hca-P cells. Normal mouse liver cell line IAR-20 was detected in a small amount of COL8A1 expression. The conclusion was that, COL8A1 mainly expressed in the cell surface that has a potential of lymph node metastasis; so highly expressing of COL8A1 may be associated with tumor metastasis, and COL8A1 expression is low in the absence of metastatic potential of hepatocellular carcinoma cells in mice.2 The function of COL8A1 expression and regulation in mouse liver cancer cells lymph node metastasis2.1 Down-regulate COL8A1 gene expression through RNAi technology, observed the alteration in the capacity of the Hca-F cells lymphatic metastasis。The results showed that, the mRNA expression of COL8A1 significantly reduced after COL8A1 being inhibited in Hca-F/siRNA cells。But the other two control groups(RNA interference group and ungiven RNA treatment control group ) the expression of COL8A1 mRNA never significantly change。Western blot method further verify the level of protein expression of COL8A1 in the above-mentioned cells.2.2 After RNA interference with the COL8A1 gene in Hca-F cells, using MTT method to test Hca-F cell proliferation activity, designing the two control groups (ungiven RNA interference control group: non-interference Hca-F cells,the RNA interfere control group: control siRNA interference Hca-F cells). The results showed that, Down-regulate COL8A1 gene expression to inhibit Hca-F/siRNA cell proliferation ability in vitro ,the cell growth rate curve was increasingly reduced,while the cell growth rate curve of the control groups is flat trend.2.3 Use the plastic matrix vitro to verify that down-regulate COL8A1 gene expression inhibit Hca-F/siRNA cell's invasive ability in vitro,The results showed that,ungiven RNA interference control group (71±8), the RNA interfere control group (69±9), while the number of Hca-F/siRNA cell significantly reduced that can invade and penetrate the plastic matrix in experimental group (20±5).2.4 Use lymph node metastasis vivo tests to verify that down-regulate COL8A1 gene expression to inhibit Hca-F/siRNA cell's capacity of lymph node metastasis in vivo. The results showed that,the average weight of metastatic lymph nodes in each group,experimental group was significantly reduced, that is COL8A1 RNA interference group(0.26±0.05g), the RNA interfere control group(0.63±0.07g), ungiven RNA interference control group(0.68±0.06g)。2.5 Use the low COL8A1 gene expression in Hepa1-6 cells, have the plasmid Containing the COL8A1-pEGFP-N2 gene to transfect those Hepa1-6 cells,cDNA transfection up-regulate COL8A1 gene expression in Hepa1-6 cells. The results showed that, the Hepa1-6 / COL8A1 cells increase the COL8A1 gene expression after stable transfection, while the cells of the other control groups low-express the COL8A1 gene. this study show that the stable transfection method is feasible,reliable and effective.2.6 Up-regulate the COL8A1 gene expression of the Hepa1-6 cells by the transfection method, using MTT method to test cell proliferation activity,and verify that up-regulate COL8A1 gene expression to increase Hepa1-6/ COL8A1 cell's proliferation capacity in vitro. The results showed that, up-regulate COL8A1 gene expression to icrease Hepa1-6 / COL8A1 cell proliferation ability in vitro,the cell growth rate curve was increasingly ascended,while the cell growth rate curve of the control groups is flat trend。2.7 In vitro invasion test, using the COL8A1 gene over-expression to observe the impact of the invasive ability of the Hepa1-6/ COL8A1 cells。The results showed that the stable COL8A1 expression can promote the invasive ability of the Hepa1-6 / COL8A1 cells in vitro and play an important role in the transfer of tumor.2.8 In vivo tumorigenicity test , using the COL8A1 gene over- expression to observe the tumorigenicity impact of the Hepa1-6 / COL8A1 cells in vivo。The results showed that the stable COL8A1 expression can promote the tumorigenicity ability of Hepa1-6 / COL8A1 cells。3 D-limonene affects COL8A1 to regulate mechanism of tumor metastasis and tumorigenicityTo provide a new target on drug therapy,the drug sensitivity of these cells for the tumor was observed. Inhibiting COL8A1 expression can increase drug sensitivity of Hca-F/siRNA cell to D-limonene. D-limonene affects COL8A1 to regulate tumor Lymphoid cells metastasis in vivo。To inhibit COL8A1 over-expression has increased drug sensitivity of Hepa1-6/COL8A1 cell to D-limonene. D-limonene affects COL8A1 to regulate cell tumorigenicity in vivo。Above all the test results showed that:1. The COL8A1 gene can express in mouse hepatoma cells(Hca-F, Hca-P and Hepa1-6 )the COL8A1 gene expression intensity of the Hca-F cells is significantly higher than that of Hca-P and Hepa1-6 cells. the COL8A1 gene mostly express in malignant cells' surface, and is the main mediator of malignant cells' action。2. The COL8A1 gene expression is closely related with the tumor cell proliferation and invasion.3. Inhibiting COL8A1 expression can increase drug sensitivity of Hca-F/siRNA cell to D-limonene; In contrast, the COL8A1 gene over-expression can inhibit drug sensitivity of Hepa1-6/COL8A1 cell to D-limonene。it is important that D-limonene affects COL8A1 to regulate mechanism of tumor metastasis and tumorigenicity,At the same time,that may provide valuable experimental evidences and clues for the design of new anti-tumor drug.