Promoting effects on the proliferation and metastasis of ACC tumor cell with XAGE-1 b overexpression

������� ������ ��������� �� �������� ����� ���������� �h��������z�� b� �������� ��� ���������� ���w�h �h�� �xh�b��� ��h����� ���������� �� �u�����, �h����h���p� ��� ������h���p� ��� �� ���� �xp������ ����� ���������� w��h well-defined carcinogenic and metastasis processes. There �� �� ����� ���� ����b���h�� f�� X�GE-1b, wh��h �� � ���b�� �f �h� ������ ������ ������� f����� �� �u����������� ��� the metastasis process of ACC. We studied and elucidated �h� ����������� b��w��� �h� p����f������� ��� ���������� �f ACC and XAGE-1b. The eukaryotic vector was constructed f�� X�GE-1b �����xp������� �� �CC-2 �����, wh��h w��� u��� f�� ��u����� �h� p����f������� ��� ��������� ph�����p� in vivo ��� in vitro. RNAi technology was used to suppress �h� �xp������� �f X�GE-1b �� �h� �CC-M ���� ����, ��� �hRN� �xp������� ������ w�� ���� ������u���� ��� �������� f�� �����f����� X�GE-1b �xp������� �pp���� �� �CC-M ���� lines. The effects on cell migration activity with XAGE-1b overexpression were determined by QCMTM 24-Well Cell I������� ����� in vitro, and a lung metastatic model in mice. We found decreased effects on the proliferation phenotype �f �CC-M ���� in vivo ��� in vitro w��h X�GE-1b ��w����u������, ��� X�GE-1b �����xp������� p������� �h� p����f������� �f �CC-2 ����� in vivo ��� in vitro, wh��� ��� �����xp������� p������� �h� ��������b���� �������� �f �CC-2 ����� in vitro ��� ���������� in vivo of the nude mice. Th� p����f������� in vitro �f �CC-M ����� ��� �ub�u�����u� �u��� ���w�h �f �u�� ���� w�� ��h�b���� b� X�GE-1b �����ference. The ACC-2 cell line with XAGE-1b overexpression ���p����� ���� ��p�� p����f������� ��� h��h�� ��������b���� ��� ���������� �b����� in vivo ��� in vitro, w��h ���� angiogenesis in the tumor tissues. XAGE-1b gene was able �� ��f�u���� ������������ �������� �� ����������, ������� �� tumorigenesis and metastasis of ACC.


Figure 4 .
Figure 4. IHC staining of XAGE-1 protein in tumorigenesis originated from ACC-pE-M cells.Primary antibody diluted 100 times and 200 times, respectively.M, tumorigenesis of ACC-pE-dM cells; N, the control group of tumorigenesis of ACC-pE-N cells.

Figure 5 .
Figure 5.The effect on the transmembrane ability of ACC-2 cells and the lung migration of the nude mice with XAGE-1b overexpression.(A) The standard curve representing the correlation between the transmembrane cell number and relative fluorescence units (RFU).(B) The comparison of the transmembrane ����� ����u����� f��� �h� �������� �u��� ( ** P<0.01).(C) The evident lung tumor nodules of the nude mice in ACC2-Xb-19 and ACC2-Xb-M group are shown in the first and second line, no tumor nodules were observed in the control group.H&E staining of tumor tissues are shown in the third line.The tumor tissues show the typical characteristics of ACC in ACC2-Xb-19 and ACC2-Xb-M group, but no tumor nodules in the negative group.IHC staining of XAGE-1b protein with lung metastases is shown in the fourth line.IHC staining of the negative control is shown in the last line.

Figure 7 .
Figure 7.The endogenous interference efficacy of the shRNA expression vector against XAGE-1b.(A) The interfering effects were identified by semi-quantitative RT-PCR.Lipo group, the blank cell control group with Lipofectamine 2000 and no plasmid; Blank, empty vector control group; N group, negative interfering control; sh-a, sh-b, sh-c: the interfering vector on the different position of XAGE-1b.(B) The interference effects were identified by quantitative PCR and performed in triplicate.

Figure 8 .
Figure 8.The effects on the proliferation of ACC-M cells in vitro and in the nude mice with XAGE-1b interference.(A) C1 and C2 representing the two resistant cell clones with 100% expression of red fluorescent protein, respectively, and as the final stable cells, named the ACC-M-pG-Sh-a and ACC-M-pG-Negative.(B) The results of MTT and performed in triplicate with three repeat wells per time ( ** P<0.01).(C) The results of cloning.(D) The tumor growth curves and representing the significant difference between ACCM-pG-Sh-a and empty vector group ( * P<0.05).(E) The comparison of tumor weight (mean ± SD, �=3, ** P<0.01).(F) The comparison of tumor volume (mean ± SD, n=3, ** P<0.01).

Figure 9 .
Figure 9.The subcutaneous tumor of ACC in the nude mice with XAGE-1b interference.(A) The subcutaneous tumor in nude mice (n=4) and the scale bar characterizing the tumor size as 1 cm is in the right corner.(B) The imaging of live animals.(C) H&E staining of the tumor tissues (x200).(D) IHC of XAGE-1b in tumor tissues.Left, normal staining control; right, negative control without primary antibody.(E) The comparison of the blood vessel number in tumor between ACCM-pG-Sh-a and ACCM-pG-Negative (P>0.05).(F) The typical tumor vascular staining of ACCM-pG-Sh-a and ACCM-pG-Negative group.

Table III .
The primer sequences for semi-quantative PCR detection.

Table IV .
The groups of subcutaneous tumorigenesis of the nude mice in vivo.