Research On The Relationship Between The Expression Of MicroRNA-222 And CDKN1B(P27(kip1))mRNA And The Protein In Oral Squamous Cell Carcinomas

Objective: Oral squamous cell carcinoma(OSCC) is one of the common malignant tumors in Oral and maxillofacial,accounting for above 80% of malignant tumors in the maxillofacial region[1]. And it is the eighth worst in total malignant tumors throughout the human body. Over the recent years its morbidity has increased year by year. At present, the clinical therapies for OSCC are predominated by extended resectionwith adjuvant treatment like radiotherapy, chemotherapy, biological treatment, targeted therapy, immunotherapy and so on. Although the therapeutical methods for OSCC have been diversified and precise, it’s easy to appear local recurrence, lymphatic metastasis and distal metastasis which could affect the prognosis of patients in oral squamous cell carcinomas. Therefore, it is extremely urgent to have a thorough understanding into the pathogenesis, early diagnosis and prognostic analysis of OSCC. Research has confirmed that the abnormal expression of various oncogenes and tumor suppressor genes are possibly related to the occurrence and development of oral squamous cell carcinomas. Micro RNA(miRNAs) is a class of endogenous non-coding small RNA with a length of 18~23nt. Mi RNA can regulate one or more targeted genes mRNA and its expression at the level of translation or transcription[2], due to the feature of non-coding. CDKN1B(p27kip1) is a newly discovered tumor suppressor gene, which is regarded as the newest member of cyclin dependent kinase inhibitor protein(CDKI). CDKN1 B has a close relationshiop with cell cycle and also can block cell progression from G1 period to S period. The objective of this research is to analyze the differential expression of the target gene CDKN1B(p27kip1) mRNA and its protein differences expression in oral squamous cancer cells, and to explore the targeting regulation of miRNA-222 to CDKN1B(p27kip1) in oral squamous cell carcinoma, by the means of studying the synthetic mimics and inhibitor of miR-222 transfected into the human tongue squamous cancer cells(TCA- 83) and setting up a negative control at the same time, which will provid basic knowledge on early detection, precise diagnosis and targeted therapy to OSCC.Method:1The synthetic mimics(up-regulate dgroup) and inhibitor(down-regulated group) of miR-222 are transfected into the human tongue squamous cancer cells(TCA-83) by LipofectamineTM2000, and the negative control group and blank control group are also setted up at the same time. Use Reverse transcription polymerase chain reaction(RT-PCR) to detect respectively the expression of the CDKN1B(p27kip1)mRNA in mi RNA-222 transfected group, negative control group and blank group. Use Flow cytometry(FCM) to detect the protein expression of the taget gene CDKN1B(p27kip1) in each group.2 Statistical methods: SPSS13.0 was used for statistical processing. Measurement data is all normal distribution by the normal test(P>0.05), with the Mean±SD noted. Use the single factor analysis of variance(one-way ANOVA) to make a comparison between two groups, P<0.05, the results have statistical significance.Results:1 Use RT-PCR to detect the expression of mRNA of p27kip1Compared to the miRNA-222 mimics transfected group(0.31±0.04) and the miRNA-222 inhibitor transfected group(0.40±0.01), the results in blank group(0.35±0.02)show P=0.031<0.05, P =0.015<0.05, differences are statistically significant. Compared to the miRNA-222 mimics transfected group and the miRNA-222 inhibitor transfected group, the results in negative control group(0.35±0.03)show P =0.037<0.05, P =0.012<0.05. Compared with the miRNA-222 inhibitor transfected group, the results in the mi RNA-222 mimics transfected group show P =0.037<0.05, differences are statistically significant. Compared to the negative group, the results in The Blank group show that difference was not statistically significant.2 Use Flow cytometry to detected the protein expression of p27kip1The X-mode corresponding value of the blank group is 8.0, FI=lg8.0×340=307.05. The X-mode corresponding value of the miR-222 mimics transfected group is 5.4, FI=lg5.4×340=249.01. The X-mode corresponding value of the miR-222 inhibitor transfected group is 13.4, FI=lg13.4 ×340=383.21. When the miR-222 is over expressed, the protein level of p27kip1 is reduced. Conversely, when the miR-222 is inhibited, the protein level of p27kip1 is increased.3 The regulating function of the miR-222 on p27kip1 in the oral squamous cell carcinoma TCA-83When the mi R-222 is over expressed, the expression of CDKN1B(p27kip1) mRNA is 0.31±0.04, the expression of CDKN1B(p27kip1) protein is 249.01. Compared to the blank group and the negative group, the expression of CDKN1B(p27kip1) mRNA and protein are all decreased. Otherwise, when the mi R-222 is inhibited, compared to the blank group and the negative group, the expression of CDKN1B(p27kip1) mRNA and the expression of CDKN1B(p27kip1) protein are increased.Conclusions:1 After the synthetic mimics and inhibitor of miR-222 are transfected into the human tongue squamous cancer cells(TCA-83) by LipofectamineTM2000, the miR-222 is either overexpressed or inhibited.2 MiR- 222 plays a role in negative regulation of p27kip1 mRNA and its protein.3 CDKN1B(p27kip1) could be the target gene of the miR-222 in the oral squamous cell carcinoma, which demonstrate that mi R-222 is closely related to the oral squamous cell carcinoma and that would be a new tumor marker, thus providing basic knowledge on early detection, precise diagnosis and targeted therapy to OSCC.