The Expression Of MicroRNA-21?PYEN And P-gp In Colorectal Cancer And The Study Of The Three Relationships

Colorectal cancer(CRC)is one of the most common human malignancy,and it is seriously harmful to human health.Chemotherapy is an important strategy for treating advanced CRC.A major reason that chemotherapy effect is not ideal is multidrug-resistant(MDR).The mechanism of MDR is very complicated,and P-glycoprotein(P-gp)excessive expression by MDR gene MDR1 is an important factor.P-gp overexpression can be used as a predictive factor for drug resistance of tumor chemotherapy.Any factors which could inhibit or reduce P-gp expression are helpful to improve chemotherapy effect.Phosphatase and tensin homolog deleted on chromo some ten(PTEN)gene is a tumor suppressor gene.Promoting the wild-type PTEN gene and protein expression can increase the traditional chemotherapy drugs effect and reverse the multidrug resistance of tumor cells.The interaction network between PTEN protein and P-gp shows that there is correlation between them through search Uni Hi database.It is a fact that PTEN is a target geng of mi R-21 and mi R-21 can regulate PTEN expression.A number of studies have reported that mi R-21 take part in these process such as cell apoptosis,proliferation and migration,poor prognosis,and MDR.However,the mechanism of mi R-21 involved in tumor MDR is unknown yet.We found that there are some base sequences complementary between mi R-21 and MDR1 gene by searching RNA 22 database.Then we speculate that mi R-21 may also regulate the expression of P-gp.In addition,formalin fixed paraffin embedding is the most conventional storage way for clinical pathological tissues.Paraffin-embedded tissue which contains rich clinical information and pathological data is a kind of available resources for molecular biological experiment and retrospective study of human disease,and the molecular structure and mi RNAs chemical properties remain stable.Objective:Analyze the relationship of PTEN protein/P-gp expression level and CRC clinical pathological characteristics respectively and the correlation through detect the expression of PTEN and P-gp in CRC tissue and normal colorectal mucosa(NCM)adjacented to carcinoma.Analyze the relationship between mi R-21 and CRC differentiation degree through extract mi R-21 from paraffin tissues and the correlation between mi R-21 and P-gp.In order to further investigate the fact that mi R-21 take part in CRC MDR is related to P-gp overexpression,which could help to assess the chemotherapy effect and provide new molecular markers and new therapeutic targets for CRC MDR patients.Methods:We collected 65 cases of paraffin-embedded tissues and 20 cases of fresh frozen tissues of CRC and corresponding 5cm outside NCM tissues respectively in Chengde Medical College Affiliated Hospital from January2015 to January 2016 and stored fresh frozen tissues in liquid nitrogen.These patients did not receive preoperative radiation and chemotherapy.Adopt immunohistochemical SP method to detect PTEN protein and P-gp expression of 65 cases paraffin-embedded tissues of CRC and NCM,and analyze experimental results through combined a microscope with Mi Vnt fiber image analysis system.Apply Western Blot technology to detect the expression of PTEN protein and P-gp of 20 cases fresh frozen tissues and analyze the results quantitatively through Quantity One software.Use reverse transcription quantitative real time-polymerase chain reaction(q RT-PCR)to detect and analyze mi R-21 expression of 40 cases paraffin-embedded tissues of CRC and NCM.Carry out statistical analysis through applying SPSS 19.0 software.Results:1 Imunohistochemical results1.1 The expression of PTEN protein and P-gp in CRC tissues and corresponding cancer-adjacent normal tissuesThe positive rates of PTEN in CRC were 52.3% and were significantlylower than those in NCM tissues76.9%(?2 = 8.613,P = 0.003).The positive rates of P-gp in CRC were 89.2% were significantly higher than those in NCM tissues75.4%(? 2= 4.279,P = 0.039).1.2 The relationship between PTEN protein and P-gp expression level respectively and clinicopathological features of CRC tissuesThere is statistical significance between the expression of PTEN protein and these clinicopathological features such as differentiated degree,invasive depth and lymphatic metastasis(P < 0.05).There is statistical significance between the expression of P-gp and these clinicopathological features such as differentiated degree and invasive depth(P < 0.05).1.2.3 The correlation between PTEN protein and P-gp expression in CRC tissuesThere are 27 cases of P-gp positive expression samples(79.4%)among34 cases of CRC tissues of PTEN protein positive expression,and there are 7cases of P-gp negative expression samples(20.6%).There are 31 cases of P-gp positive expression samples among 31 cases of CRC tissues of PTEN protein negative expression(100%).There were significantly negative correlation between the expression of PTEN and P-gp in CRC(r =-0.332,P = 0.007).2 Western Blot resultsPTEN protein expression level in CRC tissues were significantly lower than those in cancer-adjacent normal tissues(t =-1.178,P = 0.000).P-gp expression level in CRC tissues were significantly higher than those in NCM tissues(t =-3.275,P = 0.004),especially in moderate or poor differentiation group.There is statistical significance between moderate or poor differentiation group and high group(t = 7.905,P = 0.000).3 q RT-PCR results3.1 The expression of mi R-21 in CRC tissues and NCM tissuesMi R-21 average relative amount in CRC were higher than those in NCM and there is statistical significance between these two groups(P = 0.000).There is statistical significance comparing mi R-21 average relative amount in moderate/poor differentiation group and their corresponding NCM tissuesrespectively(P = 0.030,P = 0.000).In high,moderate and poor differentiation groups,mi R-21 average expression quantity are 1.25,1.69,2.04 times respectively than corresponding NCM.There is statistical significance between high differentiation group and poor group through compare mi R-21 average relative amount(P < 0.05).3.2 The correlation between mi R-21 and P-gp expression in CRC tissuesThere is positive correlation between the expression of mi R-21 and P-gp in CRC tissues through Spearman correlation analysis.(r = 0.444,P = 0.004)Conclusions:1 PTEN protein low expression is closely related to differentiated degree,invasive depth and lymphatic metastasis.2 P-gp high expression is closely related to differentiated degree and lymphatic metastasis.3 There is significantly negative correlation between the expression of PTEN protein and P-gp in CRC tissues.PTEN gene participate in CRC MDR which may be correlated with P-gp overexpression.4 MiR-21 overexpression is related to CRC differentiated degree.There is positive correlation between the expression of mi R-21 and P-gp in CRC tissues,and mi R-21 involve in CRC MDR which may be also correlated with P-gp overexpression.