Construction of Recombinant Expression Vector Carrying Human Leukocyte Antigen-G-shRNA and Detection of Its Effect_CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY

Authors：

ZENG Xiancheng ¹ , Chen Wei ² , Zhang Tong ³ , Li Hua ³ , Nie Changfu ⁴ , Zhang Qi ⁴ , Chen Guihua. ³

1. Department of General Surgery， Zengcheng People’s Hospital， Boji Affiliated Hospital of Sun Yat-sen University， Zengcheng 511300， Guangdong Province, China;;
2. Department of General Surgery， the Second Affiliated Hospital， Zhejiang University， Hangzhou 310009， Zhejiang Province， China;;
3. Organ Transplantation Research Institution of Sun Yat-sen University， Liver Transplantation Certer， the Third Affiliated Hospital， Sun Yat-sen University， Guangzhou 510630， Guangdong Province, China;;

Corresponding author：

Keywords：

Human leukocyte antigen-G; RNA interference; Liver cancer

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Objective 　To construct the expression vector of HLA-G-shRNA and investigate the effect of HLA-GshRNA from NK cell lysis.
Methods 　Four HLA-G shRNA plasmids were constructed and transiently transfected to Bel-7402 cell lines， the levels of mRNA and protein of HLA-G were detected by Real-Time PCR and Western blot. The cytotoxicity of NK-92MI cells against the transfected cells was analyzed by LDH releasing assay.
Results 　The gel electrophoresis and sequencing showed that the inserted sequence was identical to the one which we designed, and no aberrations such as mutation，deletion or insertion occurred. The expressions of HLA-G confirmed by Real Time-PCR and Western blot were significantly down-regulated. Bel-7402 cell lines transfected HLA-G shRNA showed higher lytic activity （P＜0.01）. After KIR2DL4 receptor blocked，lytic activity of NK-92 MI cell were decreased （P＜0.01）.
Conclusions 　HLA-G shRNA plasmids are successfully constructed and HLA-G down-regulated can increase NK cytolysis against Bel-7402 cell. After HLA-G combines with KIR2DL4 receptor at the surface of NK cells， the inhibition effect is transferred.

Citation： ZENG Xiancheng,Chen Wei,Zhang Tong,Li Hua,Nie Changfu,Zhang Qi,Chen Guihua.. Construction of Recombinant Expression Vector Carrying Human Leukocyte Antigen-G-shRNA and Detection of Its Effect. CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY, 2012, 19(6): 604-609. doi: Copy

1.	张荣生，王轩，张斌，等. 肝癌肝移植术后复发的危险因素分析 [J]．中国普外基础与临床杂志， 2011， 18(1)：29-32．.
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3.	Yie SM， Hu Z. Human leukocyte antigen-G (HLA-G) as a marker for diagnosis， prognosis and tumor immune escape in human malignancies [J]. Histol Histopathol， 2011， 26(3)： 409-420.
4.	Paul P， Cabestre FA， Ibrahim EC， et al. Identi?cation of HLA-G7 as a new slice variant of the HLA-G mRNA and expression of soluble HLA-G5， -G6， and -G7 transcripts in human transfected cells [J]. Hum Immunol， 2000， 61(11)： 1138-1149.
5.	Carosella ED， Moreau P， Le Maoult J， et al. HLA-G molecules：from maternal-fetal tolerance to tissue acceptance [J]. Adv Immunol，2003， 81： 199-252.
6.	Le Discorde M， Moreau P， Sabatier P， et al. Expression of HLA-G in human cornea， an immune-privileged tissue [J]. Hum Immunol， 2003， 64(11)： 1039-1044.
7.	Lin A， Zhang X， Zhou WJ， et al. Human leukocyte antigen-G expression is associated with a poor prognosis in patients with esophageal squamous cell carcinoma [J]. Int J Cancer， 2011，129(6)： 1382-1390.
8.	Ye SR， Yang H， Li K， et al. Human leukocyte antigen-G expression：as a significant prognostic indicator for patients with colorectal cancer [J]. Mod Pathol， 2007， 20(3)： 375-383.
9.	Du L， Xiao X， Wang C， et al. Human leukocyte antigen-G is closely associated with tumor immune escape in gastric cancer by increasing local regulatory T cells [J]. Cancer Sci， 2011，102(7)： 1272-1280.
10.	Hanak L， Slaby O， Lauerova L， et al. Expression pattern of HLA classⅠantigens in renal cell carcinoma and primary cell line cultures： methodological implications for immunotherapy [J].Med Sci Monit， 2009， 15(12)： CR638-CR643.
11.	Gan LH， Huang LF， Zhang X， et al. Tumor-specific upregulation of human leukocyte antigen-G expression in bladder transitional cell carcinoma [J]. Hum Immunol， 2010， 71(9)： 899-904.
12.	Chen HX， Lin A， Shen CJ， et al. Upregulation of human leukocyte antigen-G expression and its clinical significance in ductal breast cancer [J]. Hum Immunol， 2010， 71(9)： 892-898.
13.	Kren L， Slaby O， Muckova K， et al. Expression of immunemodulatory molecules HLA-G and HLA-E by tumor cells in glioblastomas： an unexpected prognostic significance? [J]. Neuropathology，2011， 31(2)： 129-134.
14.	Schütt P， Schütt B， Switala M， et al. Prognostic relevance of soluble human leukocyte antigen-G and total human leukocyte antigen classⅠmolecules in lung cancer patients [J]. Hum Immunol，2010， 71(5)： 489-495.
15.	Jung YW， Kim YT， Kim SW， et al. Correlation of human leukocyte antigen-G (HLA-G) expression and disease progression in epithelial ovarian cancer [J]. Reprod Sci， 2009， 16(11)： 1103-1111.
16.	Diepstra A， Poppema S， Boot M， et al. HLA-G protein expression as a potential immune escape mechanism in classical Hodgkin’s lymphoma [J]. Tissue Antigens， 2008， 71(3)： 219-226.
17.	Rebmann V， Busemann A， Lindemann M， et al. Detection of HLA-G5 secreting cells [J]. Hum Immunol， 2003， 64(11)：1017-1024.
18.	Mach P， Blecharz P， Basta P， et al. Differences in the soluble HLA-G blood serum concentration levels in patients with ovarian cancer and ovarian and deep endometriosis [J]. Am J Reprod Immunol， 2010， 63(5)： 387-395.
19.	Guimarães MC， Soares CP， Donadi EA， et al. Low expression of human histocompatibility soluble leukocyte antigen-G (HLAG5) in invasive cervical cancer with and without metastasis， associated with papilloma virus (HPV) [J]. J Histochem Cytochem，2010， 58(5)： 405-411.
20.	Lau DT， Norris MD， Marshall GM， et al. HLA-G polymorphisms，genetic susceptibility， and clinical outcome in childhood neuroblastoma [J]. Tissue Antigens， 2011， 78(6)： 421-427.
21.	Paul P， Cabestré FA， Le Gal FA， et al. Heterogeneity of HLA-G gene transcription and protein expression in malignant melanoma biopsies [J]. Cancer Res， 1999， 59(8)： 1954-1960.
22.	Wang Y， Ye Z， Meng XQ， et al. Expression of HLA-G in patients with hepatocellular carcinoma [J]. Hepatobiliary Pancreat Dis Int， 2011， 10(2)： 158-163.
23.	Lin A， Chen HX， Zhu CC， et al. Aberrant human leucocyte antigen-G expression and its clinical relevance in hepatocellular carcinoma [J]. J Cell Mol Med， 2010， 14(8)： 2162-2171.
24.	Naji A， Durrbach A， Carosella ED， et al. Soluble HLA-G and HLA-G1 expressing antigen-presenting cells inhibit T-cell alloproliferation through ILT-2/ILT-4/FasL-mediated pathways [J].Hum Immunol， 2007， 68(4)： 233-239.
25.	Guillard C， Zidi I， Marcou C， et al. Role of HLA-G in innate immunity through direct activation of NF-kappaB in natural killer cells [J]. Mol Immunol， 2008， 45(2)： 419-427.
26.	Zidi I， Guillard C， Carosella ED， et al. Soluble HLA-G induces NF-kappaB activation in natural killer cells [J]. J Physiol Biochem，2010， 66(1)： 39-46.
27.	Ketroussi F， Giuliani M， Bahri R， et al. Lymphocyte cell-cycle inhibition by HLA-G is mediated by phosphatase SHP-2 and acts on the mTOR pathway [J]. PLoS One， 2011， 6(8)： e22776.

1. 张荣生，王轩，张斌，等. 肝癌肝移植术后复发的危险因素分析 [J]．中国普外基础与临床杂志， 2011， 18(1)：29-32．.
2. Le Bouteiller P. HLA-G in the human placenta： expression and potential functions [J]. Biochem Soc Trans， 2000， 28(2)： 208-212.
3. Yie SM， Hu Z. Human leukocyte antigen-G (HLA-G) as a marker for diagnosis， prognosis and tumor immune escape in human malignancies [J]. Histol Histopathol， 2011， 26(3)： 409-420.
4. Paul P， Cabestre FA， Ibrahim EC， et al. Identi?cation of HLA-G7 as a new slice variant of the HLA-G mRNA and expression of soluble HLA-G5， -G6， and -G7 transcripts in human transfected cells [J]. Hum Immunol， 2000， 61(11)： 1138-1149.
5. Carosella ED， Moreau P， Le Maoult J， et al. HLA-G molecules：from maternal-fetal tolerance to tissue acceptance [J]. Adv Immunol，2003， 81： 199-252.
6. Le Discorde M， Moreau P， Sabatier P， et al. Expression of HLA-G in human cornea， an immune-privileged tissue [J]. Hum Immunol， 2003， 64(11)： 1039-1044.
7. Lin A， Zhang X， Zhou WJ， et al. Human leukocyte antigen-G expression is associated with a poor prognosis in patients with esophageal squamous cell carcinoma [J]. Int J Cancer， 2011，129(6)： 1382-1390.
8. Ye SR， Yang H， Li K， et al. Human leukocyte antigen-G expression：as a significant prognostic indicator for patients with colorectal cancer [J]. Mod Pathol， 2007， 20(3)： 375-383.
9. Du L， Xiao X， Wang C， et al. Human leukocyte antigen-G is closely associated with tumor immune escape in gastric cancer by increasing local regulatory T cells [J]. Cancer Sci， 2011，102(7)： 1272-1280.
10. Hanak L， Slaby O， Lauerova L， et al. Expression pattern of HLA classⅠantigens in renal cell carcinoma and primary cell line cultures： methodological implications for immunotherapy [J].Med Sci Monit， 2009， 15(12)： CR638-CR643.
11. Gan LH， Huang LF， Zhang X， et al. Tumor-specific upregulation of human leukocyte antigen-G expression in bladder transitional cell carcinoma [J]. Hum Immunol， 2010， 71(9)： 899-904.
12. Chen HX， Lin A， Shen CJ， et al. Upregulation of human leukocyte antigen-G expression and its clinical significance in ductal breast cancer [J]. Hum Immunol， 2010， 71(9)： 892-898.
13. Kren L， Slaby O， Muckova K， et al. Expression of immunemodulatory molecules HLA-G and HLA-E by tumor cells in glioblastomas： an unexpected prognostic significance? [J]. Neuropathology，2011， 31(2)： 129-134.
14. Schütt P， Schütt B， Switala M， et al. Prognostic relevance of soluble human leukocyte antigen-G and total human leukocyte antigen classⅠmolecules in lung cancer patients [J]. Hum Immunol，2010， 71(5)： 489-495.
15. Jung YW， Kim YT， Kim SW， et al. Correlation of human leukocyte antigen-G (HLA-G) expression and disease progression in epithelial ovarian cancer [J]. Reprod Sci， 2009， 16(11)： 1103-1111.
16. Diepstra A， Poppema S， Boot M， et al. HLA-G protein expression as a potential immune escape mechanism in classical Hodgkin’s lymphoma [J]. Tissue Antigens， 2008， 71(3)： 219-226.
17. Rebmann V， Busemann A， Lindemann M， et al. Detection of HLA-G5 secreting cells [J]. Hum Immunol， 2003， 64(11)：1017-1024.
18. Mach P， Blecharz P， Basta P， et al. Differences in the soluble HLA-G blood serum concentration levels in patients with ovarian cancer and ovarian and deep endometriosis [J]. Am J Reprod Immunol， 2010， 63(5)： 387-395.
19. Guimarães MC， Soares CP， Donadi EA， et al. Low expression of human histocompatibility soluble leukocyte antigen-G (HLAG5) in invasive cervical cancer with and without metastasis， associated with papilloma virus (HPV) [J]. J Histochem Cytochem，2010， 58(5)： 405-411.
20. Lau DT， Norris MD， Marshall GM， et al. HLA-G polymorphisms，genetic susceptibility， and clinical outcome in childhood neuroblastoma [J]. Tissue Antigens， 2011， 78(6)： 421-427.
21. Paul P， Cabestré FA， Le Gal FA， et al. Heterogeneity of HLA-G gene transcription and protein expression in malignant melanoma biopsies [J]. Cancer Res， 1999， 59(8)： 1954-1960.
22. Wang Y， Ye Z， Meng XQ， et al. Expression of HLA-G in patients with hepatocellular carcinoma [J]. Hepatobiliary Pancreat Dis Int， 2011， 10(2)： 158-163.
23. Lin A， Chen HX， Zhu CC， et al. Aberrant human leucocyte antigen-G expression and its clinical relevance in hepatocellular carcinoma [J]. J Cell Mol Med， 2010， 14(8)： 2162-2171.
24. Naji A， Durrbach A， Carosella ED， et al. Soluble HLA-G and HLA-G1 expressing antigen-presenting cells inhibit T-cell alloproliferation through ILT-2/ILT-4/FasL-mediated pathways [J].Hum Immunol， 2007， 68(4)： 233-239.
25. Guillard C， Zidi I， Marcou C， et al. Role of HLA-G in innate immunity through direct activation of NF-kappaB in natural killer cells [J]. Mol Immunol， 2008， 45(2)： 419-427.
26. Zidi I， Guillard C， Carosella ED， et al. Soluble HLA-G induces NF-kappaB activation in natural killer cells [J]. J Physiol Biochem，2010， 66(1)： 39-46.
27. Ketroussi F， Giuliani M， Bahri R， et al. Lymphocyte cell-cycle inhibition by HLA-G is mediated by phosphatase SHP-2 and acts on the mTOR pathway [J]. PLoS One， 2011， 6(8)： e22776.

CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY

Construction of Recombinant Expression Vector Carrying Human Leukocyte Antigen-G-shRNA and Detection of Its Effect

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