Expression And Clinicopathological Significance Of MiR-501 And CYLD Protein In Cervical Squamous Cell Carcinoma

Research background: Cervical cancer is a gynecological malignant tumor that seriously threatens women’s health worldwide,and its incidence has been increasing year by year recently.About 87% of cervical cancer deaths occur in developing countries,and it is the most common cause of cancer death among women in developing countries.Cervical cancer mainly includes squamous cell carcinoma(squamous cell carcinoma)and adenocarcinoma.The cause and mechanism of cervical cancer are not fully understood.Currently,surgery and radiotherapy,combined with chemotherapy,are mainly used in clinical practice.However,a large number of cases have poor prognosis due to metastasis and recurrence.Therefore,it is necessary to understand the mechanism of the development and progression of cervical cancer and provide new targets for the diagnosis and treatment of cervical cancer.Micro RNAs(miRNAs)are a class of single-stranded non-coding small RNA molecules encoded by endogenous genes.The length is usually 19-25 nucleotides.It can degrade or inhibit the translation of target m RNA by completely or partially binding to target m RNA 3’UTR.It plays a regulatory role in cell proliferation,differentiation,metabolism and apoptosis.Studies have found that miRNAs can also participate in the development of tumors as oncogenes or tumor suppressor genes.Micro RNA-501-5p(miR-501)is a newly discovered non-conserved miRNA.Currently,there are few studies on miR-501.It has been reported that miR-501 is significantly increased in hepatocellualr carcinoma(HCC),lung adenocarcinoma and gastric cancer.The high expression of miR-501 is positively correlated with the TNM stage of lung adenocarcinoma and is a predictor of poor prognosis of gastric cancer.CYLD gene(cylindromatosis)is found in headscarf tumor syndrome in recent years,and the protein it encodes is thought to be a tumor suppressor.Mutations in the CYLD gene are associated with multiple familial trichoepithelioma and multiple myeloma.The results of comparative genomic hybridization showed that the CYLD copy number decreased in HCC,renal spiradenocylindroma and two new hpv18-positive cervical clear cell carcinoma strains.Decreased CYLD expression was also found in human colon cancer,HCC and breast cancer cell lines and tissues.It has been found that CYLD is a direct target of miR-501 in HCC.Mi R-501 can down-regulate the expression of CYLD,inhibit apoptosis and promote the proliferation of HCC cells.Other scholars found that the AG/GG genotypes of miR-501 in Chinese han population induce higher risk of liver cancer compared with the AA genotypes the growth of tumor is accelerated.A allele reduces the binding ability of miR-501 to CYLD.AA genotype can inhibit the proliferation of HCC cells and promote apoptosis.However,the expression and role of mir-501 and CYLD proteins in cervical squamous cell carcinoma have not been reported.Objectives: To compare the expression of miR-501 and CYLD proteins in cervical squamous cell carcinoma and its adjacent tissues,To investigate the relationship between miR-501 and CYLD protein and the development of cervical squamous cell carcinoma and its clinicopathological significance.Methods:(1)From October 2014 to October 2017,49 cervical squamous cell carcinoma formalin-fixed,paraffin-embedded(FFPE)specimens were selected from Dalian Municipal Central Hospital,Dalian,China.There were 22 cases above 50 years old and 27 cases below 50 years old.According to the 2014 FIGO staging standard of the international federation of obstetricians and gynecologists,there were 30 cases in stage I and 19 cases in stage II;there were 31 cases with tumors smaller than 4 cm and18 cases larger than 4 cm;HPV positive 21 cases,negative 28 cases;there were 17 cases with lymph node metastasis and 32 cases without lymph node metastasis;there were 15 cases of highly differentiated squamous cell carcinoma,10 cases of moderately differentiated squamous cell carcinoma,and 24 cases of poorly differentiated squamous cell carcinoma.Adjacent normal cervical tissue from the tumor edge of each patient was collected as a negative control.(2)TRIzol reagent was used to extract RNA from cervical squamous cell carcinoma wax mass tissues.Real-time quantitative RT-PCR(real-time qRT-PCR)was used to detect the expression of miR-501 in cervical squamous cell carcinoma and its paracancerous cervical tissue,and U6 was used as internal reference.(3)The expression of CYLD protein in cervical squamous cell carcinoma and its para-carcinoma cervical tissue was detected by immunohistochemical SP method.Results:(1)Real-time qRT-PCR method was used to detect the expression of miR-501.The relative expression of miR-501 was obtained by 2-(35)(35)Ctmethods.Our results showed that the expression level of miR-501 in cervical squamous cell carcinoma tissues was significantly increased,which was 1.54 times higher than that in the adjacent cervical tissues(P = 0.021).(2)The relative expression level of miR-501 in cervical squamous cell carcinoma tissues of each example was compared with that in the corresponding paracancerous tissues.The mean value of the obtained ratio was 2.31,which was used as the cut-off point value to divide all the cases into two groups: < 2.31 group(n = 21)and 3 2.31group(n = 28).Among 18 cases with 3 4 cm of tumor size,15 cases(15/18)had a miR-501 relative expression of 3 2.31,which was significantly different from those with < 4 cm of tumor size(13/31)(P = 0.026).In 19 cases of FIGO staging II,there were 15 cases(15/19)with miR-501 relative expression of 3 2.31,compared with stage I(13/30),the difference was statistically significant(P = 0.019).In 17 cases with lymph node metastasis,there were 15 cases(15/17)with miR-501 relative expression of 3 2.31,compared with that no lymph node metastasis(13/32),the difference was statistically significant(P = 0.010).In high,medium and low differentiation of cervical squamous cell carcinomas,miR-501 relative to expression of 3 2.31 were 40.00%(6/15),40.00%(4/10)and 75.00%(18/24)respectively,and the difference was statistically significant(P = 0.043).There was no significant difference between miR-501 expression and patient age of cervical squamous cell carcinoma(P = 0.517)and HPV infection(P = 0.072).(3)The expression of CYLD protein was detected by immunohistochemical SP assay.CYLD positive staining was mainly located in the cytoplasm and CYLD protein expression was also observed in some cell nuclei,with yellow or brown-yellow granules.In accordance with Fromowitz’s semi-quantitative grading method,the score of immunohistochemical results was obtained by staining percentage and staining intensity:(1)dyeing strength: negative(0 points),no staining;weak positive(1 point),namely light yellow;medium positive(2 points),namely yellow;strong positive(3 points),namely brown and yellow color;(2)positive range: < 25% = 1,25%-50% = 2,51%-75% = 3,>75% = 4.The immunohistochemical score is multiplied by the above two.The mean CYLD protein score was calculated and used as the cut-off point value.The score > 2was assessed positive,and ￡ 2 negative.The results showed that the immunohistochemical score of CYLD protein in normal cervical tissue was significantly increased,which was 2.15 times higher than that in cervical squamous cell carcinoma tissues(P < 0.01).The positive rate of CYLD protein in normal cervical tissue was 83.67%(41/49),significantly higher than that in cervical squamous cell carcinoma tissues(34.69%,17/49)(P < 0.01).(4)When comparing the relationship between CYLD protein and clinicopathological factors in cervical squamous cell carcinomas,the CYLD protein score of each cervical tissue adjacent to cancer was compared with that of cervical squamous cell carcinomas.All cases were divided into two groups: positive(n = 20)with > 1 and negative(n = 29)with ￡ 1 using the average of the ratio as cut-off point.According to the results of the 31 cases of tumor < 4 cm,CYLD protein positive expression was in 16 cases(16/31),and tumor 3 4 cm of 18 cases,CYLD protein positive expression was in 4 cases(4/18),and the difference had statistical significance(P = 0.041);in 30 cases of cervical squamous cell carcinomas with FIGO staging I,CYLD protein positive expression was in 16 cases(16/30),and in 19 cases of stage II,CYLD protein positive expression was in 4 cases(4/19),and the difference had statistical significance(P = 0.037);among the 17 patients with lymph node metastasis,there were 2 patients with positive CYLD protein expression,and among the 32 patients without lymph node metastasis,there were 18 patients with positive CYLD protein expression,and the difference was statistically significant(P = 0.016).Among the 15 patients with high differentiation,there were 9patients with positive CYLD protein expression;among the 10 patients with medium differentiation,there were 6 patients with positive CYLD protein expression;among the24 patients with low differentiation,there were 5 patients with positive CYLD protein expression,and the difference was statistically significant(P = 0.020).There was no significant difference between CYLD protein expression and age of cervical squamous cell carcinoma(P = 0.380)and HPV infection(P = 0.292).Conclusions:(1)Mi R-501 expression is significantly increased in cervical squamous cell carcinoma.Mir-501 expression was positively correlated with the size of primary cervical squamous cell carcinoma,FIGO stage and lymphatic metastasis,and negatively correlated with the degree of differentiation.(2)CYLD protein expression was significantly decreased in cervical squamous cell carcinoma.CYLD protein expression was negatively correlated with the size of primary cervical squamous cell carcinoma,FIGO stage and lymphatic metastasis,and positively correlated with the degree of differentiation.(3)Mi R-501 can promote the development and progression of cervical squamous cell carcinoma,while CYLD protein can inhibit the development and progression of cervical squamous cell carcinoma.Mi R-501 and CYLD may be potential candidate targets for the treatment of cervical squamous cell carcinoma.