Expression Of PinX1 Gene In Renal Cell Carcinoma And The Biological Significance

Objective: Renal cell carcinoma (RCC) is one of the frequently malignant tumors in the urinary carcinoma. Surgical treatment early is the most efficient. RCC has more malignancy and insensitivity to radiotherapy and chemotherapy. Today the gene therapy to RCC has been the hotspot in tumor therapy. Telomerase activation contributes greatly to human cells carcinogenesis. Consequently, the suppression to the telomerase activation will induce the tumor cell to apoptosis. The human telomerase reverse transcriptase (hTERT) is one of the most important elements in telomerase. PinX1 is one of telomere binding proteins, which can interact with hTERT and TRF1. PinX1 has been proposed as a putative tumor suppressor due to a negative effect on cellular telomerase activity. Our previous study showed that hPinX1 possesses a novel effect of mediating telomerase nucleolar localization.To address the roles of PinX1 in human cancer, we elucidated the expression of PinX1 in RCC tissues and their adjacent normal renal tissues, and further investigate its relationship to the clinical feature. Then the inhibition to RCC cell line A498 and the sensitivity to chemotherapeutical drugs after the over- and low- expression of PinX1 was explored.Methods: 1. The PinX1-C gene was inserted into plasmid to construct the expression vector of pET28a(+)-PinX1-C and expressed protein in E. Coli BL-21, then PinX1-C-His fusion protein purification was performed through affinity chromatograph. KunMing mouse were immunized to produce polyclonal antibody (mouse-anti PinX1), and antibody specificity is analyzed by Western blotting.2. 56 samples of resected RCC tissue and adjacent renal normal tissue were examined by RT-PCR, then the relationship of PinX1 expression to the clinical stage and pathological grade were analyzed.3. The RCC cell lines A498 were treated with anti-tumor reagents eptoside, cisplatin and 5-FU at each indicted dosages, respectively. PinX1 expression in A498cell lines were examined by RT-PCR. Then the sensitivity and inhibition of A498 to chemotherapeutical drug were further examined by MTT and Trypan blue exclusion assay after over-expression PinX1 gene and siRNA against PinX1 in A498 induced by 5-Fu. The telomerase activity were analyzed to elucidate the relationship the expression of PinX1 and the therapeutic reaction in A498 induced by 5-Fu.Result: 1. We constructed pET28a(+)-PinX1-C expression vector successfully. High expression of Fusion protein PinX1 -C-His was induced in E.Coli BL21 and purificated through affinity chromatograph. A mouse polyclonal anti-PinX1-C protein serum was successfully obtained.2. The expression of PinX1 in RCC carcinoma tissues was lower than the carcinoma besides tissues (P<0.05). PinX1 expression is significantly correlated with the pathologic grade, clinical stage and lymph node metastasis of RCC (P<0.05). The higher clinical and pathologic stage in RCC, the lower expression of PinX1.3. Inducible down regulation of PinX1 is implicated in the anticancer effect of 5-FU in A498 cell lines. The down regulation of PinX1 is coincidence to cell apoptosis . The effect of 5-FU on the induction of PinX1 mRNA down-regulation displayed a dose- and time-dependent manner . But we did not observed a significant alteration in the cellular telomerase activity in A498 cells before and after the 5-FU treatments. Ectopic overexpression of PinX1 in A498 cells conferred these cells a resistance to the 5-FU-induced cellular damages. Contrarily, low-expression of PinX1 with siRNA in A498 upgraded the sensitivity to 5-Fu.Conclusion: Polyclonal anti-PinX1 protein is the basis for investigation of PinX1 gene. The differential and specific polyclonal antibody provides credible precondition for futher experimentation. The down-expression of PinX1 was founded in RCC. The down grade is depended for the clinical stage and pathological grade in RCC. PinX1 gene may be used as one of the molecular markcrs to determine the malignant degree, metastasis potency and predicting progression of RCC. Suppressing PinX1 expression by siRNA significantly enhances the sensitivity of the A498 cells to the 5-Fu treatment , decrease telomerase activation and shorten telomere. Therefore PinX1 gene may become the target to genetherapy in RCC. PinX1 gene may have adouble function. One is negative for telomere /telomerase for the PinX1-C frangment suppresses telomerase activation and induces apoptosis. The other is positive for telomere/telomerase .That is to say PinX1 possesses a cellular protective activity and protect cell against aggression.