| Objective: Oral Squamous Cell Carcinomas（OSCC） is one of the most common tumors in oral and maxillofacial, which accounts for about 80 percent of the total oral cancer [1], and the incidence of OSCC is rising year by year. Treatment of Oral cancer mainly includes surgical operation, radiation therapy, chemotherapy, biological treatment and so on, but the result shows poor efficacy and bad prognosis. So it is important to explore an efficient treatment method for the prognosis of patients with OSCC. When Ambros[2] and his companions studied worm in 1993, they found the first microRNA（miRNA）, namely, line-4, which took part in the development of the larvae. In 2002, Calin [3] et al first reported the function of miRNA in tumorigenesis. Hereafter, many studies proved that miRNA played an important role in proliferation, origin and apoptosis of tumors. So miRNA provides a new direction for cancer treatment. miRNA-222 is an important member of miRNA family, and some studies find that miRNA-222 is expressed abnormally in various tumors, and the abnormal expression of miRNA-222 affects the biological function of tumor including proliferation, apoptosis and migration. Pang Xin Ya[4] et al found that the expression of miRNA-221 and miRNA-222 were rising in HCC tissue; in the vitro experiments of HepG2 hepatocellular carcinoma cell line, Tao Xuan Chen [5] et al found that when the expression of miRNA-222 was increased, the expression of PTEN was restrained, and the proliferation and invasive ability of HepG2 were enhanced. In the gliomas research, Gillies and LorimerL[6] found that miRNA-221/222 could combine with p27kipl mRNA 3 ’UTR union to reduce the expression of p27kipl. When the expression of miRNA-221/222 was reduced, the amount of p27kipl protein was increased, thus apparently blocking the U251 in the G1 phase, and reducing the proliferation of U251. This experiment aims at investigating the role of miRNA-222 in the occurrence and development of oral squamous cell carcinomas by studying the effect of miRNA-222 on biological functions of OSCC-15 including proliferation, apoptosis and migration, which provides a reference basis for the early diagnosis, treatment and prognosis of oral squamous carcinoma.Methods:1 The recovery, cultivation, passage and frozen storage of OSCC-15 cells.2 Synthetic miRNA-222 inhibitor and miRNA-222 mimics are immediately transfected into OSCC-15, at the same time set up the blank control group.3 MTS kit is used to deal with each group cells at 0 h, 24 h, 48 h and 72 h. Microplate reader tests the absorbance value of miRNA-222 inhibitor group, miRNA-222 mimics group and the blank control group, and then draw the proliferation curve.4 Use Annexin V/PI double dye kits to deal with each group cells. Flow cytometry is used to detect the apoptosis rate of miRNA-222 inhibitor group, miRNA-222 mimics and the blank control group respectively.5 Cell scratch assay tests the migration rate of miRNA-222 inhibitor group, miRNA-222 mimics and the blank control group.6 Result determination and statistical analysis.Apply SPSS 21.0 statistical software to carry out statistical processing, with mean ± standard deviation for the absorbance value of OSCC-15, use T-test to make comparisons on the differences of absorbance value between the groups, and use chi-square test to make comparisons on the differences of apoptosis rate between the groups; the differences of migration rate between the groups are compared by T-test（P<0.05） with statistical significance.Results:1 The influence of miRNA-222 on the absorbancy and proliferation rate of OSCC-15 cells1.1 At 0 hour, the absorbancy of the blank control group is 1.318 ± 0.061, and the absorbancy of miRNA-222 inhibitor group is 1.141±0.040, which is lower than that of the blank control group, with statistical significance（P < 0.05）; the absorbancy of miRNA-222 mimics group optical densities is 1.594 ± 0.138, which is higher than that of the blank control group, with statistical significance（P < 0.05）.1.2 At 24 hour, the absorbancy of the blank control group is 2.128±0.150, and the absorbancy of miRNA-222 inhibitor group is 1.733±0.130, which is lower than that of the blank control group, with statistical significance（P < 0.05）; the absorbancy of miRNA-222 mimics group optical densities is 2.752±0.106, which is higher than that of the blank control group, with statistical significance（P < 0.01）.1.3 At 48 hour, the absorbancy of the blank control group is 2.426±0.124, and the absorbancy of miRNA-222 inhibitor group is 2.155±0.057, which is lower than that of the blank control group, with statistical significance（P < 0.05）; the absorbancy of miRNA-222 mimics group optical densities is 2.791±0.125, which is higher than that of the blank control group, with statistical significance（P < 0.05）.1.4 At 72 hour, the absorbancy of the blank control group is 2.806±0.163, and the absorbancy of miRNA-222 inhibitor group is 2.423±0.053, which is lower than that of the blank control group, with statistical significance（P < 0.05）; the absorbancy of miRNA-222 mimics group optical densities is 3.369±0.409, which is higher than that of the blank control group, with statistical significance（P < 0.05）.1.5 At 0, 24, 48, 72 hours, The proliferation rate of miRNA-222 inhibitor groups is 85.57%, 81.44%, 88.83% and 81.44% respectively, which is lower than that of the blank control group, with statistical significance（P < 0.01）; the proliferation rate of miRNA-222 mimics groups is 120.94%, 129.32%, 115.04% and 129.32% respectively, which is higer than that of the blank control group, but without statistical significance（P > 0.05）.2 The influence of miRNA-222 on apoptosis of OSCC-15 cellsThe apoptosis rate of miRNA-222 inhibitor is 9.58%, which is higher than that of the blank control group（accounts for 6.11%）, with statistical significance（P < 0.01）; The apoptosis rate of miRNA-222 mimics is 2.25%, which is lower than that of the blank control group, with statistical significance（P < 0.01）.3 The influence of miRNA-222 on the migration of OSCC-15 cellsAt 24, 48, 72 hours, the migration rate of miRNA-222 inhibitor groups is 17.9%, 19.4% and 20.6% respectively, the migration rate of the blank control groups is 23.7%, 29.4%, 36.4% respectively, with statistical significance（P < 0.05）.The migration rate of miRNA-222 mimics groups is 47.9%, 67.8% and 78.9% respectively, with statistical significance（P < 0.05）.Conclusions: miRNA-222 can promote the proliferation and migration of OSCC-15 cells, and inhibit apoptosis, which proves that miRNA-222 plays an important role in the proliferation, apoptosis and migration of oral squamous cell carcinomas. |
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