MUC4Gene Expression In The Bladder Cancer Cell Lines And Bladder Cancer Tissues And Targeting Killing Effect Of Recombinant Adenovirus Carrying HSV-tk Driven By Its Promoter On Bladder Cancer Cells

Bladder cancer is one of the most common malignant tumor of urinary system inChina, and the transitional cell carcinoma takes90%of bladder cancer. The occurrenceand growth of bladder cancer are pathological changes of complexity, multi-factor andmulti-step, involving both internal genetic factors and external environmental factors. Thecharacteristic of the bladder cancer is heterology, multi-center growth, high recurrence rateand the increase of malignant degree after recurrence. At present, most of the researches onthe etiology of bladder cancer focused on gene changes. The occurrence of the normalbladder cells is triggered by the change of DNA. The mutation of cancer gene fromproto-oncogene results in immoderate division of the cells, which leads to recurrence anddeterioration of bladder cancer. Another important molecular mechanism of bladder canceris the code regulation of cells growth, DNA repair or apoptosis protein suppress tumorgene inactivation. As a result, DNA damaged cells do not undergo apoptosis, which leadsto uncontrollable growth of cells. In addition, amplification or overexpression of thenormal gene encoding the some growth factors or their receptors are also related to theoccurrence and growth of bladder cancer.At present, the bladder cancer is mainly treated with surgery, combined withpostoperative intravesical chemotherapy. However, the high recurrence rate afteroperation has seriously affected the patient’s life quality. Therefore, themolecular mechanism research about the occurrence, development, invasion andmetastasis of bladder cancer will cast great significance to explore the ideal treatmentof bladder cancer. The present study shows that the recurrence, invasion and metastasis ofbladder cancer are decided by the tumor cells biological behavior, among which theadhesion of tumor cells plays an important role. The study also finds that mucin (mucin, MUC) is related to several kinds of tumors’ recurrence, invasion andmetastasis. MUC4, an important member of transmembrane adhesion protein, possesses acharacteristic of potential oncogene and can be used as a marker of some tumorsdevelopment process. According to the present research, there exists abnormal expressionof MUC4in malignant tumors like pancreatic cancer, ovarian cancer, prostate cancer,esophageal squamous cell carcinoma and squamous cell carcinoma of head and neck.Meanwhile, MUC4is involved in tumorigenesis, development through a variety ofmechanisms, including intercellular adhesion, cell signal transduction, cell apoptosis andso on.In recent years, the study finds that MUC1is closely related to genitourinary tumors.It is found that the expression rate of MUC1is significantly higher in bladder cancertissues than non cancer tissues by testing the bladder cancer tissues through monoclonalantibody, which proves that MUC1abnormal high expression in bladder cancer tissuesmay be a symbol of bladder malignant progression and lymph node metastasis. There arealso reports about MUC1gene abnormal expression in prostate cancer and renal cellcarcinoma and its expression level in renal cell carcinoma being related to metastasis andprognosis. MUC1gene is in close relation with the genitourinary tumors, especially thebladder cancer. We have found the MUC4gene’s abnormal high expression in bladdercancer tissues by applying the second generation sequencing technology to capture its exonin the early research stage. There are few studies about the MUC4gene in bladder cancer,whether domestic or abroad. The question is whether MUC4, as one kind of sticky protein,associate with bladder cancer. Therefore, to investigate the question, we carried out thefollowing experiments, which is divided into two parts:Part one: MUC4gene expression in the bladder cancer cell lines andbladder cancer tissuesObjective: To investigate the MUC4gene expression in HT1376, T24bladder cancercell lines, bladder cancer tissues, para-carcinoma tissues and normal bladder mucousmembrane tissues.Methods: Adopting the methods of QT-PCR and Western blot to detect MUC4genemRNA and protein expression in HT1376, T24bladder cance cell lines and normalbladder epithelial cell lines. Detecting the MUC4mRNA expression, totals to27cases of bladder cancer tissue specimens, in9matched bladder cancer tissues, para-carcinomatissues and normal bladder mucous membrane tissues through QT-PCR. Among the cases,all of the bladder tissue are transitional cell carcinoma,6superficial,3invasive.Immunohistochemical method is adopted to test the MUC4protein expression in64bladder tissues (including34cancer tissues,20para-carcinoma tissues and10normalbladder mucous membrane).Results: The relative expression in HT1376, T24bladder cance cell lines and normalbladder epithelial cell lines detected of MUC4mRNA by QT-PCR, is respectively13.746±0.309,3.501±0.286,0.914±0.100. In9matched bladder cancer tissues, the relativeexpression in the cancer tissues, para-carcinoma tissues and normal bladder mucousmembrane tissues is10.673±2.797,2.679±0.488,0.610±0.294respectively. Comparedwith the normal bladder mucous membrane, the expression of MUC4mRNA is higher inbladder cancer cell lines and bladder cancer tissues. The difference is of statisticalsignificance(P <0.05). Compared with para-carcinoma tissues, MUC4mRNA has higherexpression in bladder cancer tissues, which is also of statistical significance (P <0.05).Compared with the normal bladder mucous membrane, MUC4mRNA has higherexpression in the para-carcinoma tissues and with same statistical significance(P<0.05). ByWestern blotting, compared with the normal bladder mucous membrane, MUC4proteinhas higher expression in HT1376and T24bladder cancer cell lines, and expression inHT1376cell line is higher than that in T24cell line. The difference is also statisticallysignificant (P<0.05). Among the34bladder cancer tissues,27show MUC4protein positiveexpression through immunohistochemical detection, which means the positive rate is79.4%;10shows positive expression of MUC4in20para-carcinoma tissues, and thepositive rate is50%;3shows positive expression of MUC4in10normal tissues, bringingthe positive rate to30%. The difference between the groups is of statistical significance(P<0.05). The MUC4protein expression in para-carcinoma tissues of bladder cancer andnormal bladder tissues is similar to that of mRNA. The MUC4protein expression hasnothing to do with bladder cancer clinical stage, but is related to bladder cancerpathological grading (P <0.05).Conclusions: The expression of MUC4gene is high in HT1376and T24bladdercancer cell lines and bladder cancer tissues. The MUC4protein expression level isassociated with bladder cancer pathological grading, which means MUC4, serves as a candidate target gene of advanced grading bladder cancer, can play an important role in theprocess of development of bladder cancer.Part two: The targeting killing effect of recombinant adenovirus carryingHSV-tk driven by MUC4promoter on bladder cancer cellsObjective: The study is designed to discuss MUC4promoter driven HSV-tkadenovirus targeting killing bladder cancer cells and inducing apoptosis.Methods: MUC4promoter active sequence is cloned, and the transcription activity ofit in T24and HT-1376bladder cancer cells is detected through the recombinant luciferaseassay system. Taking adenovirus system as the carrier to build the HSV-tk recombinantadenovirus promoter driven by human MUC4promote,which activated thetranscription activity of MUC4promoter signal regulation,and driven its downstreamHSV-tk gene,which is combined with GCV to detect the cytotoxicity upon the above twokinds of bladder cancer cells.Results:Constructed the the recombinant adenovirus plasmid, which can stablyexpress promoter regions which lie in upstream of ATG of MUC4gene, ithas strong transcriptional activity in T24and HT-1376cells, but has almost notranscriptional activity in HFL1cells(the control group),which indicate that MUC4promoter transcriptional regulation its downstream gene of HSV-tk has the characeristicof tumor cells specificity. The results indicate that HSV-tk driven by MUC4promoterrecombinant adenovirus combined with GCV could induce HT-1376and T24bladdercancer cells apoptosis, and specific targeting cytotoxic effect.Conclusions:,HSV-tk driven by MUC4promoter recombinant adenovirus combinedwith GCV could produce considerable targeting killing effect on HT-1376and T24bladdercancer cells, by activating the signal regulation transcription activity of MUC4promoter.