| Hepatocellular carcinoma (HCC) is the fifth deadly tumor in the world, and the third tumorreduce the death in China. Development of HCC is associated closed with hepatic B virus.Because of the non-specifity of symptom in HCC patients, many patients lost the best opportunityof operation. Non-surgery therapies such as transaterial chemotherapy embolization (TACE),radiofrequency ablation (RFA), percutaneous ethanol injection (TEI) only has limited effects.Many patients succumb to the recurrence and metastasis at last. Biotherapy and gene therapysuppose the new direction for therapy of metastasis of HCC. However, invasion and metastasis ofHCC is a process of multi-step and multi-gene, which is concerned to biological characteristics oftumor cells, the local microenviroments in tumor such as matrix metalloprotease (MMP), integrin,stroma-cell and blood vessel, and local and systematic immunology.Chemokines and chemokine receptors are the critical molecular in homing and directionalchemotaxis of immunocells. To date, about 45 chemokines and 18 chemokine receptors have thedefinite functions. Another chemokine and chemokine receptors have not clear in function. In2001, Muller first reported the expressions of chemokine receptors in breast cancer were related tothe metastasis of tumor cells, particularly CXCR4 in lung metastasis, and CCR7 in lymph nodemetastasis. By now, more and more researches have shown that chemokine receptors can befunctionally expressed in tumor cells, particularly in tumor cells with high metastasis potentials.Therefore, hypothesis has been raised that metastatic behaviors to specific organs ortissues of tumor cells own the similar mechanisms with directional chemotaxis ofimmune cells. Try to find the relatively specific chemokine receptors associatedclosely with lung metastasis may provide the new potential therapy approach formetastasis of HCC.HCC often metastasis to lung, bone, brain and adrenal bland in clinic, particularlyto lung which occurred about thirty-five percent of metastasis. Our previous researchhas shown that lung crude extraction can chemotaxis to HCC cell with strong lungmetastasis potential.Here, we compared systematically chernokines and chemokine receptors expressedin HCC cell lines with differential lung metastasis potential, screened out the interesting chemokine and chemokine receptors related closely with lung metastasisof HCC tumor cells, and further demonstrated the screened chemokine and receptorsPart OneAnalysis and screen of expression profiles of ehemokine receptors inHCC cell lines with different lung metastasis potentialThe main aim of this part of study was to compare the chemokine and chemokinereceptors profiles of HCC cell lines with differential lung metastasis potentials, and toscreen the chemokines and chemokine receptors.Firstly, we designed eighteen pairs of primers of chemokine receptors by Premiersoftware, then analyzed chemokine receptors profiles of five human HCC cell lineswith elevated lung metastasis potentials (SMMC-7721, MHCC97-L, MHCC97-H,HCCLM3, and HCCLM6). Chemokine& receptors oligo gene chip were used tocompare and analyze the human HCC cell lines with increased lung metastasispotentials. Results of RT-PCR showed that Expression profiles of chemokinereceptors on four group HCC cell lines with different metastatic potentials havesignificant difference (p＜0.01), in which CCR10, CXCR4 and CXCR6 expresseddecreased gradually when metastatic potential of cell line increased. The expressionsof CCR3, CCR4, CCR10, CCR12 and XCR1 on HCCLM6 were significantly reducedcompared with SMMC-7721 (P＜0.01), whereas the expressions of CXCR1 (p=0.006)and CXCR5 (p=0.003) exceeded SMMC-7721. Except for CXCR2, CXCR6 andXCR1, most of chemokine receptors on MHCC97-H expressed differently comparedwith MHCC97-L(p＜0.05), in which expressions of CCR1 (p=0.002), CCR2(p=0.004) and CCR5 (p=0.046) exceeded MHCC97-L. Results of gene chipshowed there was prominent statistical difference in chemokines and chemokinereceptor profiles of four groups detected cell lines with different lung metastasispotentials (p＜0.01). Gene chips showed that there were 8 genes up-regulated exceed 2folds in HCCLM3 compared with MHCC97L. There were 9 genes up-regulated and42 genes down-regulated exceed 2 folds in HCCLM3 compared with SMMC-7721respectively. There were 17 genes up-regulated exceed 2 folds in MHCC97Lcompared with SMMC-7721. For 18 definite chemokine receptors, gene chips showedthe similar results to RT-PCR. CXCR1, orphan receptor RDC 1, IL-8 expressed highlyin HCCLM3 and were associated with the lung metastasis potentials of liver cancer.In contrast, CXCR4 showed the opposite trend. In addition, MMP2, MMP7 expressed also highly in HCCLM3.The above experiments showed that chemokine and chemokine receptor profileshad differential expression at mRNA level and eDNA level in HCC cell lines withdifferent lung metastasis potentials. Chemokine IL-8, chemokine receptorRDC1(CMKOR1),CXCRlexpressed highly and were correlated positively to lungmetastasis of HCC, whereas the expression of CXCR4 was correlated negatively tothe lung metastasis of HCC. Further functional test and clinical test should be done.Part TwoExpression of RDC1 gene in human liver cancer and human HCCcell lines with different lung metastatic potentialOne: Expression validation and functional analysis of CXCR4 by ligand SDF-1To date, CXCR4 is one of the chemokine receptor reported to relate to tumormetastasis most frequently by literatures. Many researches had showed that CXCR4up-regulated in tumor tissue and were correlated closely with metastasis potential.CXCR4 and RDC1 locate the near zone in the same chromosome, and have the greathomology in sequence. And they both are the co-receptor of HIV virus infected CD4+T lymphocyte. The results of first part showed CXCR4 was down-regulated whenlung metastasis potential increased, whereas expression of RDC1 was correlatedpositively to the increased lung metastasis potentials. So in order to study deeply thepotential function of RDC1 in lung metastasis of liver cancer, we made the expressionand function analysis of CXCR4 in this part.The work of this part continued use MHCC97-H and MHCC97-L with high andlow lung metastasis as objects, studied the function and significance of CXCR4 inlung metastasis of HCC. We used RT-PCR and Western Blotting to compare andanalyze the mRNA and protein expression level of CXCR4 in the two cell lines.SDF-1, the ligand of CXCR4, and lung extracts were used in chemotaxis and antibodyinhibition assays to observe the function of SDF-1 and lung extracts of nude mouse toehemotaxis of MHCC97-H. Results showed that CXCR4 in MHCC97-H wasexpressed lower than expression in MHCC97-L, and protein level was in eonsistantwith the mRNA level. MHCC97-H can respond to SDF-1 a between 1 ng/ml to100ng/ml, particularly at 50ng/L. Lung extraction can ehemotaxis to MHCC97-H ,compared with other groups (p＜0.05), however, the chemotaxis effect decreased little when added CXCR4 antibody. The above experiments showed that CXCR4 canexpress in liver cancer cells and may take part in the lung metastasis of liver cancer,but not the main receptor molecular in invasion and metastasis of liver cancer.Two: Validation RDC1 at mRNA level and protein levelBinding the results of first part, in this part we further validated the mRNA leveland protein level of RDC1.We use the real-time PCR to analyze the differential mRNA expression level ofRDC1 in SMMC-7721, MHCC97-L, MHCC97-H, and HCCLM3 with gradually increased lungmetastasis potentials. Western Blotting was used to validation the difference in protein level. Theresults showed that Ct value of HCCLM3 was 22.58±0.29, the relative value to GAPDH was(1.96±0.15)×10-3, which were both higher than MHCC97-L(Ct value: 23.57±0.40, relativevalue:(1.12±0.22)×10-3) and SMMC-7721(Ct value: 28.08±0.48, relative value: (0.13±0.10)×10-3) (p＜0.01). The protein level of RDC1 showed the uniformity to mRNA level.The above results suggested that RDC1 was highly expressed in HCC cell line withhigh lung metastasis potential, and expression of RDC1 was correlated to invasioncharacteristic of liver cancer cells.Three: To use clinic tissue microarray (TMA) to verificate the screenedchemokine and chemokine receptorsThe work of this part used the clinical specimen to verificated the screenedchemokine IL-8 and chemokine receptor RDC1, CXCR1 and CXCR4. According tofollowed-up data of the lung metastasis post-operation, we constructed tissuemicroarray from selected 116 cancer tissue and 46 cancer-side tissues from patientswith radical excision from January 2000 to 2004. SABC immunohistochemistry wasused to detect the expression of IL-8, RDC1, CXCR1 and CXCR4. CrossTab wasused to analyze the 3-year-survival, Kaplan-Meier method was used to analyze therelationship of RDC1, CXCR4, IL-8, CXCR1 with 3-year accumulative survival ofHCC patients.The results showed that the four detected protein mainly stained intra-cytoplasm oftumor cells. The orphan receptor RDC1 expressed more in primarily focus of lungmetastasis group than in non-lung metastasis group (p＜0.05), and expressed higherprominently in tumor tissue than cancer-side (p=0.006). in addition, expression levelof RDC1 is related to tumor size (p＜0.05), but not related to sex, tumor number, embolus, integrity of tumor membrane and TNM stage. The IL-8 expressed more inprimarily focus of lung metastasis group than in non-lung metastasis group (p=0.018),but no statistical significance between cancer-side and tumor tissue (p=0.10). similarto RDC1, expression level of IL-8 is related to tumor size (p＜0.05), but not related tosex, tumor number, embolus, integrity of tumor membrane and TNM stage. CXCR4can be detected in all patients in used experimental conditions, but expression levelhad not statistical significance between lung metastasis group post-operation andnon-lung metastasis group, and not at pathology characteristics and clinical stage.Expression of CXCR4 in tumor-side exceeded the primary focus (p=0.034).Expression of CXCR4 in tumor-side tissue had not statistical difference between lungmetastasis group and non-lung metastasis (p=0.917). Similar to CXCR4, expressionlevel of CXCR1 had not statistical significance between lung metastasis grouppost-operation and non-lung metastasis group, and not at pathology characteristicsand clinical stage under our experimental conditions. The expression of all detectedproteins in cancer-side had not statistical significance between lung metastasis groupand non-lung metastasis group. Patients with lower expression of RDC1 (64%) hadhigher 3-year-survival post-operation than patients with higher expression of RDC1(51%). Patients with elevated expression of IL-8 decreased 3-year-survival. Patientswith low expression of IL-8 had higher accumulative survival than with highexpression of IL-8 (p=0.034), whereas patients with low expression of CXCR1 hadlower accumulative survival than high expression of CXCR1. However, RDC1 andCXCR4 had no statistical significance in groups.The above experiments suggested that increased expression of RDC1 and IL-8 inpatients with HCC was associated with lung metastasis and tumor size. And higherexpression of IL-8 was correlated with 3-year-survial.Part ThreeStudy on effects of RDC1 on lung metastasis characteristics ofHCCLM3 in vitro and in vivo after RNA interferenceThe results of ahead two part study had suggested that the orphan receptor RDC1was the chemokine receptor related closely to lung metastasis of HCC. Combined therecent literature suggested that RDC1 maybe one of the main receptor molecular participated in the lung metastasis of primary liver cancer. So we selected RDC1 asthe object of this part had the important significance in theory and clinic. In this part,we utilized the RNA interference technique to down-regulated the RDC1 gene inHCClm3 with high lung metastasis potential, further observed the effect of RDC1 totumor cell growth, blood vessel formation, matrix degradation, invasion and lungmetastasis behavior in vivo to study the mechanism of RDC1 participated in lungmetastasis of liver cancer and to explore the therapy effects of RDC1 siRNA in lungmetastasis of HCC.Experimental in vitro first designed and screened the effective RDC1 smallinterference RNA, and used the RNA interference to down-regulated RDC1 geneexpression in HCCLM3. RT-PCR and Western Blotting were used to verify the RNAinterference. Immunofluorescence was used to detect the RDC1, CXCR4, IL-8 andsurviving after RDC1 RNAi. Immunoenzymology experiments were used to detectchanges of OPN and VEGF. RT-PCR was used to detect OPN, survivin. Gelatinasezymogram was used to observe the effects of down-regulated of RDC1 to matrixdegradation. Transwell was used to detect the effects of RDC1 to invasion potential ofHCCLM3. MTT was used detect the effects of RDC1 to proliferation of tumor cells.Nude mice were used to observe the effects of RDC1 RNAi to growth and lungmetastasis of liver cancer in vivo.Results showed that designed siRNA of RDC1 gene all had some degreeinterference. RT-PCR and Western Blotting verified siRNA-286 have strongestinterference effects (65%). Immunoinfluencense showed RNAi of RDC1 candown-regulated the expression of RDC1, IL-8, surviving, but had no effect onexpression of CXCR4. Immnoenzyme showed decreased level of OPN, VEGF afterRNAi. Gelatinase spectrum analysis showed decreased expression of MMP2 andMMP9 after RNAi at 24 hours and 48 hours. Transwell showed group siRNA had lessnumber invasion cells compared with control groups (p＜0.01). MTT showed growthof group RNAi slowed after 72 hours (p＜0.01). Nude mice experiments showedformation of cancer reached 100% in all groups. The weight and volume group RNAi(1.20±0.75g) (372.83±265.02mm3) was less than the group non-interference(3.02±1.68g) (1265.42±900.01mm3). Similarly, group RNAi and i.v. (1.18±0.77g) (355.42±383.10mm3) was also less than group non-interference. However,group RNAi and intra-tumor (2.27±1.03g) (940.58±593.89mm3)had no statisticalsignificance at weight and volume compared with group non-interference. Non-interference had 100% formation pulmonary metastasis (8.0±2.8 numbers),group RNAi had 4 mice observed pulmonary metastasis (2.5±2.0 numbers), whichhad statistical significance compared with group control (p＜0.01). Group RNAi andi.v. only had 3 mice observed metastasis (2.3±2.2 numbers), which also hadstatistical significance (p＜0.01). Group RNAi and intra-tumor had 5 mice was foundpulmonary metastasis (6.3±3.6numbers), which had no statistical significancecompared negative group.The above experiments suggested that RDC1 may take part in the growth andmetastasis of primary liver cancer by promoting the proliferation of cancer cells,inhibiting the survivin, increase the extra-cellular matrix degradation, and promote theblood vessel formation. In addition, down-regulate the RDC1 gene expression caninduce the growth inhibition of tumor and decreased lung metastasis potential in vivo,and RDC1 maybe a potential molecular target for lung metastasis of HCC therapy.Conclusions1. Chemokine and chemokine receptors expressed differently at mRNA level andcDNA level in human liver cancer cell lines with different lung metastasispotential. Chemokine IL-8, chemokine receptor RDC1, CXCR1 were correlatedpositively with lung metastasis of liver cancer, whereas CXCR4 was vise visa.2. CXCR4 can express in liver cancer tissue and cell lines and may take part in thelung metastasis of liver cancer, but not the main receptor molecular in invasionand metastasis of liver cancer.3. RDC1 was highly expressed in HCC cell line with high lung metastasis potential,and was correlated to invasion characteristic of liver cancer cells.4. The elevation of RDC1 and IL-8 expression were related with elevated lungmetastasis of HCC patients in clinic.5. RDC1 may take part in the growth and metastasis of primary liver cancer bypromoting the proliferation of cancer cells, inhibiting the survivin, increase theextra-cellular matrix degradation, and promote the blood vessel formation. Novelty1. Using hepatocellular carcinoma models with lung metastasis potential from highto low, we found and identified that many chemokine and chemokine receptorsmay take part in the lung metastasis of primary liver cancer, which supposed thetheory and experiment basis for understand the selected metastasis of HCC.2. Up-regulation of chemokine receptor RDC1 expression was found to beassociated closely with lung metastasis of primary liver cancer first time, andrecognized as a tumor-related gene participated in lung metastasis of liver cancer.3. Using RNA interference to explore the mechanism of RDC1 participated in lungmetastasis of HCC, and RDC1 may stimulate the growth and lung metastasis ofHCC by promoting the proliferation of HCC tumor cells, inhibiting the apoptosis,promoting the matrix degradation, and microvessel generation.4. To explore the merit of siRNA as potential drugs for therapy of lung metastasis ofHCC.Potential merits for clinical application1. RDC1, one of the G protein coupled receptors, maybe the potential therapeutictarget of metastasis of primary liver cancer in clinic because of the intimaterelationship with HCC.2. To supply the theory basis for deep research of invasion and metastasis of HCC. |
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