The Studies On MUC4Expression In Precancerous Lesions Of The Pancreas And The Relationship With Pancreatic Cancer Cells’ Activity

Background&ObjectiveCurrently, pancreatic cancer (PC) is one of the highest mortality rates of solid tumors.Due to the lack of effective early detection indicators, and when the disease is limited to thepancreas, most of the patients did not show clinical symptoms. Therefore, most of thepatients can be diagnosed until the advanced disease, but this time the tumor has beentransferred to other organs. Though undergo with surgical resection immediately, they alsocouldn’t get satisfactory results. Recently, more and more studies have confirmed thatpancreatic cancer is primarily comes from one of several precancerous lesions. Chief amongthese are the pancreatic intraepithelial neoplasia (PanINs). Therefore, further study of themolecular changes in precancerous lesions of pancreas, which is help for early diagnosis ofpancreatic cancer, and implement treatment of pancreatic cancer during the early stage.MUC4is a transmembrane mucin, which not expressed in normal pancreas, whereasoverexpressed in pancreatic cancer. More and more studies show that MUC4may be used asa new diagnostic, prognostic and therapeutic target in pancreatic cancer. Due to currentresearches on its expression in the precancerous lesions of pancreas are few in our country,therefore, its mechanism of action in the precancerous lesions of pancreas is not clear. In thisexperiment, we use immunohistochemistry to detect MUC4protein expression in a variety ofpancreatic tissues (Normal, PanINs and PC), revealing the changes of MUC4protein in theevolution of pancreatic cancer, providing support for further research of the mechanism ofaction of MUC4protein in the precancerous lesions of pancreas.There has been a lot of studies have shown that, MUC4overexpressed in pancreaticcancer cells, but not expressed in normal pancreatic cells, indicating that MUC4involved inthe process of malignant transformation of normal cells of the pancreas. But the studies on itsrelationship with pancreatic cancer cells’ activity are very few in our country, moreover, theresearches on the association of MUC4overexpressed in pancreatic cancer with cellproliferation and apoptosis, invasion and metastasis activity are fewer. CD18/HPAF is ahighly invasive pancreatic cancer cell lines. This experiment through compares MUC4overexpressed CD18/HPAF (CD18/HPAF-Scr) with MUC4silent CD18/HPAF(CD18/HPAF-siMUC4), which reveals the relationship between MUC4overexpressed inpancreatic cancer and cell proliferation and apoptosis, invasion and metastasis activity, providing support for further research of the mechanism of MUC4protein as a therapeutictarget in pancreatic cancer.Methods1. Detection of the MUC4protein expression in a variety of pancreatic tissues (Normal,PanINs and PC) by immunohistochemistry.2. By the stable expression of MUC4-specific short hairpin RNA expression to make theMUC4silence in CD18/HPAF cells.3. By Western blotting analysis and immunofluorescence technique to detect the expressionof MUC4protein.4. By double thymidine block method，the cells were synchronized in G1-S phase; Cellswere stained by propidium iodide (PI) and then followed by flow cytometry to detect thecell cycle changes and the cell proliferation of CD18/HPAF-siMUC4cells andCD18/HPAF-Scr cells.5. Cells were stained by annexin V and propidium iodide (Annexin V-FITC/PI) and thenfollowed by flow cytometry to detect the cell apoptosis of CD18/HPAF-siMUC4cells,CD18/HPAF-Scr cells and not stained cells.6. By cell transmembrane experiment in vitro to detect the invasion and metastasis activity ofCD18/HPAF-siMUC4cells and CD18/HPAF-Scr cells.Results1. The results of MUC4protein expression by immunohistochemistry in various pancreatictissue show that the increasing of MUC4protein expression accompany with an increasingin the degree of organization profiled, from normal pancreas to PanIN-1to PanIN-2toPanIN-3and then to PC, even during all of the evolution progress of the PC. The differencehas statistical significance.2. The results of Western blotting and immunofluorescence technique analysis show that,compared with control cells, expression of MUC4in CD18/HPAF-siMUC4down by about80%. Confocal immunofluorescence microscopy results are also consistent with the Westernblotting analysis, further confirmed that, compared with the control cells, the MUC4expression has decreased significantly in CD18/HPAF-siMUC4cells.3. Double thymidine block method+flow cytometry (propidium iodide staining) test’sresults show that CD18/HPAF-Scr cells go into the S phase of the total proportion of42.51%,compared to CD18/HPAF-siMUC4cells was17.71%. This explains that, MUC4-overexpressing (CD18/HPAF-Scr) cells have a higher cell proliferation rates.On theother hands, it indicates that MUC4overexpression makes the number of proliferatingCD18/HPAF cells increase.4. Flow cytometry (annexin V-propidium iodide staining) test’s results show thatCD18/HPAF-siMUC4cells have nearly twice multiplier of cell apoptosis index thanCD18/HPAF-Scr, but the necrosis was almost the same between the two indices. Thissuggests that over-expression of MUC4beneficial CD18/HPAF pancreatic cell survival.5. The results of transmembrane experiment in vitro indicate that the number ofMUC4-silence (CD18/HPAF-siMUC4)cells that invade the basement membrane are onlynearly a third of the number of control (CD18/HPAF-Scr) cells. This suggests that followedby the silence of MUC4expression, the invasion of CD18/HPAF cells declined significantly.ConclusionMUC4protein expression along with the entire process of malignant transformation inthe pancreas; MUC4protein expression levels increased with increasing degree of abnormaltissue, which suggesting that it has a role of promoting the evolution of pancreatic cancer;Compared with the MUC4-silencing cells, MUC4-overexpressing cells have a higherproliferation rate, this suggests that MUC4protein is associated with the unlimited growth ofpancreatic cancer cells(CD18/HPAF); Compared with MUC4-overexpressing cells,MUC4-silent cells have a higher apoptotic index, This suggests that, MUC4protein canpromote the survival of pancreatic cancer cells (CD18/HPAF); In terms the ability of cellinvasion, when silence MUC4expression, the invasive activity of pancreatic cancer cells(CD18/HPAF) has decreased significantly. After the silence of MUC4protein expression inpancreatic cancer, the changes of proliferation and apoptosis, invasion and metastasis activityin pancreatic cancer cells(CD18/HPAF) suggest that MUC4can be used as a potentialtherapeutic target for pancreatic cancer.