Histologial Features Of Tumor-bearing Tissues Foemed By Human Fibroblasts After Reprograming By Piwil2 And Prognosis-related Research Of Piwil2/Piwil4 Protein In Hepatocellular Carcinoma

Objective To observe the histological features of tumor-bearing tissues formed by human fibroblasts after reprograming by spermatogonial stem cell self-renewal key regulating gene Piwil2(Piwil2-i CSC),and explore the relation between Piwil2/Piwil4 protein with hepatocellular carcinoma(HCC) patients’ prognosis.Methods Piwil2-i CSC tumors spheroids-like colonies were selected for tumor formation assay in four nude mices,Pathological features of Piwil2-i CSC tumors were observed by histology. Stem cell markers and common triploblastic markers were detected by reverse transcriptase-polymerase chain reaction(RT-PCR) assay and immunohistochemistry. Germ cell tumor markers were detected by immunohistochemical examination.Piwi family member Piwil2,Piwil4 protein expression in HCC tissues microarray were detected by double immunofluorescence histochemistry.Explore the effect of markers differential expression rang Piwil2,Piwil4 to Piwil2/Piwil4 in neoplastic cells to HCC prognosis.Results1 Two weeks after inoculation,subcutaneous tumors were formed in all the four nude mice with a tumor foamation rate of 100%. In the Piwil2-i CSC tumor tissues,Piwil2-GFP+ cells showed high-density nuclear expression and were widely observed in DAPI-stained sections.Numerous mitotic figure of the neoplastic cells were seen(>10cells/field of vision under high magnification) in HE-stained sections.Enlarged abnormal cell nuclei were observed.2 RT-PCR assay showed that Piwil2-i CSC tumors still expressed Piwil2 and some self-renewal and pluripotent markers of stem cell and some markers of triploblastic differentiation. Immunohistochemical staining showed that the tumors expressed stem cell markers,triploblastic markers and germ cell tumor markers AFP and HCG.3 Double immunofluorescence histochemistry in HCC tissues microarray showed that Piwil2 protein mainly expressed in nuclear, and the expression intensity is higher than cytoplasm. However,Piwil4 protein mainly expressed in cytoplasm, and the expression intensity is higher than nuclear.4 To observe the co-expressed orientation rule of Piwil2/Piwil4 molecular chaperone in HCC tissues,four co-expression patterns were found,included Nuclear co-expression(Piwil2+/-Piwil4+/+ and Piwil2+/-Piwil4+/-), and Nuclear-cytoplasm co-expression(Piwil2+/+Piwil4+/+),and cytoplasm co-expression(Piwil2+/+Piwil4-/+), and no co-expression in Nuclear or cytoplasm(Piwil2+/-Piwil4-/-,Piwil2+/-Piwil4-/+and Piwil2+/+Piwil4-/-).Conclusions1 Piwil2-i CSC tumors are probably undifferentiated embryonic small cell carcinoma,most likely to be immature teratoma, mixed with yolk sac tumor and choriocarcinoma components. It can be used as a useful model for the research of origin or genesis mechanism of cancer stem cells and the treatment of relevant tomors.2 The positive expression of Piwil2 or Piwil4 in nucleus of HCC neoplastic cells suggested worse prognosis, Piwil2/Piwil4 molecular chaperone co-expressed in nuclear were related to the prognosis of patients with HCC.3 Piwil2/Piwil4 molecular partner has the potential to be an important molecular marker which predict the prognosis of HCC,singleness Piwil2 or Piwil4 marker cannot serve as evaluation index.