Effects Of Osteopontin On Growth, Invasiveness And Chemosensitivity For Gastric Carcinoma

Gastric carcinoma is a common malignant carcinoma in our country, ranking the second cancer in male, and the third in female. Almost one third of the gastric carcinoma are at the late stage on diagnosis, lost the chance of surgical radication. Even those with surgical therapy, 60%-70% of the patients would have local recurrence and metastasis. However, chemotherapy, although as the main method, only have the response rate around 40%-50% in all the regimens. Thus, it is essential to seek any new method to increase the chemosensitivity. Osteopontin(OPN) has been reported in some cancers to be related with cancer cell growth and progression. Our previous clinical study also showed OPN was higher expressed in the gastric carcinoma tissue than the para-carcinoma normal tissue. Thus, this study was to investigate the OPN effect on the gastric carcinoma growth and invasiveness, and to evaluate whether OPN silencing could enhance the chemosensitivity of the cancer cells.Part one: Osteopontin expressed in the different differentiated gastric cancer cell lines Objective: To explore osteopontin(OPN) expression in the varied differentiated gastric carcinoma cell lines (MKN28, SGC7901 and BGC823). Methods: Real-time PCR and Western-blot methods were used to detect the OPN expression at gene level and protein expression level, separately. Results: Morphological pictures showed the MKN28 cell line with highly differentiation, SGC7901 cell line with mediated differentiation, and BGC823 with poorly differentiation; Real-time PCR results showed MKN28 and SGC7901 had OPN expression, while no expression detected in the cell line of BGC823; Western-blot also showed the OPN protein expression existed in the cell line of MKN28 and SGC7901, but nothing detected in the cell line of BGC823. Conclusion: Osteopontin(OPN) expressed in the gastric carcinoma cell lines of MKN28 and SGC7901, but not expressed in the cell line ofBGC823.Part two: Down-regulation of osteopontin by siRNA on the biological behavior of gastric carcinoma cell linesObjective: To explore the effects of down regulation of OPN on the biological behavior of MKN28 and SGC7901 cell lines. Methods: OPN siRNA was designed according to the relevant literature and was transfected into the two cell lines. Fluorescent labeling was used to test the transfected efficiency. Western blot was used to detect the down-regulation of OPN protein. Real-time PCR was used to test the ratio and time difference of down-regulation of OPN mRNA after siRNA transfected. Different methods were used to test the biological changes before and after OPNsiRNA transfected into these two cell lines, like flow cytometry to test cell cycle and apoptosis, MTT method to test the proliferation for the consecutive seven days and compare the difference between the two groups (OPNsiRNA transfected or non-transfected) with mixed-model, Transwell method to test the capability of moving and invasion of cancer cells and the difference tested by t-test. Results: FITC labeling showed under fluorescent microscope the transfected efficiency of OPNsiRNA were more than 90% in the two cell lines; Real-time PCR showed OPNmRNA was at most down-regulated 47% down-regulated at the 72^nd hour in SGC7901, while 40% at the 48^th hour in MKN28; Western blot showed the expression of OPN protein were down-regulated after siRNA transfected both in the two cell lines while compared with GAPDH andβ—actin; MTT results showed the proliferation decreased after transfected with OPNsiRNA both in MKN28 and SGC7901 (P<0.01) , however, significant difference were existed from day 5 to day 7 in SGC7901 cell line and day 1 to day 5 in MKN28 cell line; flow cytometry tested the cell cycle were interfered by transfection with OPNsiRNA, the proportion of M phase cells decreased from 4.96% to 0.39% in MKN28, while from 18.78% to 17.02% in SGC7901, no apoptosis induced after transfection in the two cell lines. Transwell experiment showed less cells (SGC7901: t=5.172, P<0.01; MKN28: t=11.365, P<0.01) moving through the artificial man-made basement membrane after transfected with OPNsiRNA, suggested the capability of moving and invasion decreased after OPN down-regulated in the cell lines. Conclusion: The siRNA designed and synthesized in the study showed the capability of down-regulation of OPN in the cell lines of MKN28 and SGC7901. OPN suggested be the factor related with proliferation, movement and invasion in MKN28 and SGC7901 cell lines.Part three: Effect of down-regulation of OPN on the chemosensitivity of gastric carcinoma cell linesObjective: To explore whether OPN could be the target of gastric cancer therapy. Methods: Gastric cancer cell line SGC7901 and MKN28 were separated into two groups whether OPN had been down-regulated by the small interfering RNA described in part 2, and adding cytotoxic drugs of 5FU, DDP, ADM and MMC in the four dosage levels, separately. MTT method was to detect the extinction value of each sample at the 48th hour, 72nd hour, 96th hour and time without adding drugs. At each time point, the curves of each drug concentrations and extinction values in the two groups were made. Mixed-model was used to test the difference between the two groups. Results: In SGC7901 cell line, while adding 5FU,ADM,MMC, the OPN siRNA transfected group expressed the lower proliferation capability than the non-transfected group(P<0.05),which suggested the OPN interference could enhance the chmosensitivity of SGC7901 to these drugs. However, such effect was not existed while adding cisplatin(DDP). In MKN28 cell line, the enhancing effects were observed while adding the drug 5FU, DDP and ADM (P<0.05), but not existed while adding MMC. Conclusions: OPN interfering seems to increase some cytotoxic drug sensitivity to SGC7901 and MKN28 cell lines. OPN might be a potential target of gastric cancer therapy, but further researches are needed, especially oriented to certain drugs.Part four: SummaryOur study demonstrated osteopontin expressed in most of the gastric carcinoma cell lines. Then, the biological behavior of gastric carcinoma cell lines were compared before and after OPN protein down-regulation by RNA interfering. The results revealed that OPN was an important factor participating in gastric carcinoma cell proliferation and invasiveness. Moreover, down-regulation of OPN expression by siRNA could enhance the chemosensitivity of gastric carcinoma cells to certain cytotoxic drugs, which suggested OPN might be a new therapeutic target in gastric cancer.