The Synergy Of Id1 And NF-κB Subunits P65 In The Pathogenesis Of Oral Squamous Cell Carcinoma

Objectives:Oral Squamous Cell Carcinoma(OSCC)is one of the most common malignanttumor in head and neck worldwide, with a high incidence, and with a serious threat tohuman health. The pathogenesis of OSCC is not clear at present. Studies show thatdifferentiation inhibitory factor-1(Id1) and nuclear factor kappa B(NF-κB) in oralsquamous cell carcinoma(OSCC) were higher expression,The study is designed toexplore the role of Id1 and NF-κB in the pathogenesis of OSCC.Materials and methods:1.The surgical specimens were collected from clinical patients, Control specimenswere paracancerous tissue specimens.2.Isolated cells from the OSCC specimens, and cultured.3.RT-PCR detected the expression of the both CD133 and BMI-1 m RNA transcriptsin OSCC clinical specimens, Western blot confirmed CD133 and BMI-1 proteinexpression in OSCC tissue specimens.4.With the use of FACS and MACS for cell sorting, CD133 +BMI-1+ cells was sortedout and then cultivated.5.Constructed Id1 and P65 gene vector.6.Transfection with efficient electricity, Id1 and P65(individually or simultaneously)transfected CA9-22 and HOK16 B cells respectively, and detect the expression ofCD133 and BMI- 1 after the transfection.7.According to the different number of cells, CD133+BMI-1+ cells(from eachCA9-22, HOK16 B and OSCC cells) were injected into the SCID mice, to observe thegrowth of xenograft tumor in SCID mice.Results:1.RT-PCR demonstrated the expression of the both CD133 and BMI-1 m RNAtranscripts in OSCC clinical specimens, and then demonstrated the co-expression ofthe both CD133 and BMI-1 proteins in cultured cells from clinical OSCC tissues.2.Successful sorting out the original generation of CD133+BMI-1+cells.3.Successful constructed Id1 and P65 gene vector.4. In the cell cultures of CA9-22, the expression of BMI-1 protein was not detected inId1 or p65 transfected cells alone, but in cells with the transfection of both Id1 andp65 in combination,the expression of CD133、Bmi-1、Cdc2 and CD1 was significantlyincreased;this synergy regulate the expression could be blocked by inhibitors of CD1 arginine vasopressin, Cdc2 catharidic aci inhibitors and protease inhibitorsCycloheximide respectively; the expression of CD133 protein was detected incells with the transfection of Id1、p65or Id1+p65.5.After transfected with Id1 and p65, CA9-22 cells affect the cell cycle process andpromote cell proliferation significantly.6.At a number of 10,000 CD133+ BMI-1+ keratinocytes of CA9-22 and OSCC cells,there were one and two xenograft tumors, respectively, from CA9-22 and OSCCinjected animals, At a number of 100,000 CD133+ BMI-1+ keratinoyctes, there weretwo and five xenograft tumors, respectively, from CA9-22 and OSCC injectedanimals.Conclusions:1.The subgroup of CD133+BMI+ cells were observed in the OSCC fresh specimens2.Id1 and NF-κB strengthened the expression of both CD133 and BMI-1 via thecyclin D1 and Cdc2 signaling pathways in OSCC cell cultures, and promote the celldifferentiation and proliferation3.CD133+BMI-1+ keratinocytes from OSCC tissues and CA9-22 cell cultures initiatedthe growth of xenograft tumors in SCID/Beige mice.4.Id1 and NF-κB subunits p65 may play a collaborative role in the pathogenesis ofOSCC...