High Effective Detection And Discoveries Of Novel Truncations Of Midkine In Tumors

Midkine (MK),a heparin binding growth/differentiation factor, belongs to Midkine family. Midkine family consists of only two members, namely midkine (MK) and pleiotrophin (PTN). MK is widely expressed during embryonic development and becomes restricted to kidney and small intestine epithelia in the adult. Aberrant MK expression has been reported in a variety of human tumor/cancer tissues. This abnormal expression is not tissue specific, resembling the expression pattern of p53. MK is involved in a variety of biological functions, including promoting embryonic cell growth ,especially in the neural cells,and differentiation. In the adulthood it may play a hormonelike role in activating the differentiation of certain cells and maintaining the normal function of kidney. Besides, it is closely related with tcancer cell growth and differentiation. MK is becoming a novel molecular target for the tumor/cancer therapy.Some studies had suggested that MK expression is correlated with development of tumors/cancers, but few have revealed its spatiotemperal expression and histological information in the progression of diseases. We have first studied MK expression in human adult pancreatic tumor/cancer tissues by immunohistochemistry. (1) MK is significantly up-regulated in the disease tissues; (2) Notably, in the insulinoma it is highly expressed at the leading edge with the disease progression but has only low expression in the tumor body; (3) In the ductal adenocarcinoma, it is highly expressed in the invasive blood vessels, as well as in the abnormal islets and ducts; (4) Note in the advanced disease tissues, it shows aberrant expressions on some fast dividing cells in the expanded ductal epithelia.Interestingly it's frequently expressed in the precancerous lesions. It seems plausible that MK has a role in transforming cells into insulin-expressing cells. Alternatively, it may take part in the migration of insulin-expressing aberrant cells. The probable function of MK as a blood factor ,growth factor and its anti-apoptosis activity can be found obviously in the invasion and migration of ductal gland cancer (ductal carcinoma). That implies that MK may increase the density of vessels and promote the growth of epithelia, which is very important for the progression of cancer.We then used RT-PCR to detect the expression of MK in different human tumor/cancer tissues. We first examined some human tumor/cancer cell lines, including A549 cells, SGC-7901 cells, human 8910 cells and MC-63 cells and then several surgically removed cancer tissues from liver, kidney, galactophore and rectum. To improve the sensitivity and specificity, we have employed nested and touchdown PCR strategy. (1), We havenot only confirmed some previous discoveries about MK's high level expression in cancerous and malignant tumor tissues; (2) but also achieved 100% detection of the expression of truncated MK in the disease specimens examined(the previous are only 60%). There are lots of reports that tMK is an aberrant form of MK lost of the N-terminal and part of the domain encoded by exon III. It is specifically expressed in tumor tissues and takes part in tumorigenesisThus, it is regarded as an effective marker for tumor detection.The simple method used in our experiment improves greatly the possibility of MK to be a marker of tumor detection. (3) In the peripheral cells of liver cancer tissues, we have also detected high _expression of MK and tMK. This coupled with the results from immunohistochemical staining suggests that MK may play an important role in tumorigenesis and metastasis. So MK and tMK could potentially be the early marker for tumor/cancer detection and this is very important for effective detection and therapy; (4) With the improvement in the sensitivity and the specificity, we have found two novel truncations of MK, named tMKB and tMKC in addition totMKA, the previously reported one. The relative length of their mRNA is: tMKA> tMKB>tMKC. Interestedly,we have previously reported three truncated MK in mouse embryo with similar alternative splicing schemes to those found in the above human disease tissues. By comparison, tMK shows some apperent mutation in the diseases, which may provide significant clue to the study about the function of MK in diseases; (5) One of the remarkable characteristics is that the sequences of their splicing sites have some unique sequence specificity:The 3'sequence of the donor has the same sequence as the 5'sequence of the receptor at the splicing site. For example, both of the sequences of the splicing sites in 3'-end and 5'-end are -ACCCCC-. It's interesting that similar situations have been found in the three novel tMKs in mouse embryo. The similarity raises the possibility that there may exist a new mechanism of mRNA splicing;(6) Genetically, tMKA is resulted from the truncation of the whole extron III, tMKB from the whole extron III and a part of IV and tMKC from a part of extron III and a part of IV. Comparison with the three types of truncated MKs found in mouse embryo, may shed light on the the molecule mechanisms of MK in the normal development and tumorigenesis/carcinogenesis; (7) We have applied Real-Time PCR to investigate quantitative relationship of MK & tMKA expression in normal, pre-disease and disease tissues. That has not only confirmed our previous findings but also laid a foundation for future application of MK detection in cancer/disease diagnosis and therapy.In summary, our studies have demonstrated that a methodology for high efficiency of MK detection in tumor/cancer diagnosis has been established.In addition, we have first discovered the three types of truncated MK to be specifically expressed in cancer/tumor tissues. Together, these have provided valuable information for further investigation into the role of MK in embryonic development and especially tumorigenesis/carcinogenesis.