P16^INK4a Overexpression And Its Relationship With HPV Infection In Cervical Carcinomas

Background and Objective Tumor suppressor gene INK4a encodes p16^INK4a protein and aberrant expression of p16^INK4a protein has been reported in various malignancies, whereas p16^INK4a overexpression has been detected in almost all precancerous and cancerous lesions of the cervix and it's reason has been indefinite. Human papilloma virus(HPV) infection was essential for genesis of cervical carcinomas. About 50% of cervical cancers was related with HPV type 16(HPV16).After infection, E6 and E7 gene of HPV were integrated to genome of cervical epithelium. Continued expression of transforming oncoprotein E6 and E7 not only drives the neoplastic progression in cervical epithelium but also plays important role in maintaining malignant phenotype of cervical cancer cells. Correlations between p16^INK4a overexpression and HPV infection has no accepted argument. This study was carried out to assess the correlations between p16^INK4a expression and the HPV infection by HPV16E7 gene silencing using small interfering RNA(siRNA) in human cervical carcinomas cell lines SiHa and CaSki and by comparing p16^INK4a expression in CaSki(HPV16 infection), HeLa(HPV18 infection), C-33A(no HPV infection); to ascertain the role of p16^INK4a immunostaining as an early biomarker of uterine cervix carcinomas.Methods1. The expression of p16^INK4a protein was detected in 126 cervical lesions samples by immunohistochemical staining, including cervical cancer(CC),cervical intraepithelial neoplasia(CIN) and cervicitis with high-risk Human papillomavirus(hr-HPV) infected or no, and to investigate the relationships of p16^INK4a protein expression with hr-HPV state.2. RNA interference(RNAi) originated by chemically synthetic small interference RNA (siRNA) could induce HPV16E7 gene silencing in cervical carcinomas cell lines SiHa and CaSki.3. Expressions of HPV16E7 and INK4a mRNA were examined by semiquantitative reverse transcription polymerase chain(RT-PCR) in SiHa-Nontransfection, SiHa -siHPV16E7, SiHa-siNControl and CaSki-Nontransfection, CaSki-siHPV16E7,CaSki-siNControl cells respectively.4. Expressions of HPV16E7 and INK4a protein were examined by Western blot in SiHa-Nontransfection, SiHa-siHPV16E7, SiHa-siNControl and CaSki-Nontransfection, CaSki-siHPV16E7, CaSki-siNControl cells respectively.5. Cell cycle analysis were assessed by flowcytometry(FCM)in SiHa-Nontransfection, SiHa-siHPV16E7 and CaSki-Nontransfection, CaSki-siHPV16E7, cells respectively.6. Expressions of INK4a mRNA and protein were examined by RT-PCR and Western blot in CaSki, HeLa and C-33A cells with or without HPV16E7 infection,respectively.Results1. Immunostaining for p16^INK4a and relationship between p16^INK4a immunoreactivity and HPV in 126 cervical lesions samples: (1) The positive expression rates of p16^INK4a in CC,CIN and cervicitis samples were 100.00(50/50), 71.93%(41/57) and 42.11%(8/19) respectively, and there was obvious difference between the three groups(p<0.05). (2)The positive expression rates of p16^INK4a in CC,CIN and cervicitis samples(n=89)with HPV infection were 100.00%(42/42), 76.32%(29/38) and 44.44%(4/9) respectively, and there was obvious difference between the three groups(p<0.05). (3) The positive expression rates of p16^INK4a in CC,CIN and cervicitis samples(n=37) without HPV infection were 100.00% (8/8), 63.16%(12/19) and 40.00%(4/10) respectively, and there was obvious difference between the three groups(p<0.05). (4) The positive expression rates of p16^INK4a in CIN1,CIN2 and CIN3 samples(n=57) were 36.84%(7/19), 81.82%(18/22) and 100.00%(16/16), respectively, and there was obvious difference between the three groups(p<0.05). (5) The positive expression rates of p16^INK4a in CIN1,CIN2 and CIN3 samples(n=57) with HPV infection were 37.50%(3/8), 73.33%(11/15) and 100.00%(15/15) respectively, and there was obvious difference between CIN3 and CIN2 or CIN1, but there was no obvious difference between CIN2 and CIN1(p>0.05). (6) In 107 CC and CIN, 71(66.36%) cases showed simultaneous positive expression of p16^INK4a proteins and hr-HPV DNA, 21(19.63%) cases showed positive expression of p16^INK4a and negative expression of hr-HPV DNA, 8(7.57%) cases showed negative expression of p16^INK4a and positive expression of hr-HPV DNA, the positive rates of p16^INK4a and/or hr-HPV DNA expressions was 93.46%, whereas there was no significant relationship between p16^INK4a expression and hr-HPV DNA infection(p>0.05). (7) p16^INK4a protein was positive in all of CC(n=47) and CIN3(n=16) cases with or without HPV infection. The positive expression rates of p16^INK4a in hr-HPV(+) and hr-HPV(-) CIN samples were 76.32%(29/38) and 63.16%(12/19) respectively, and there was no obvious difference between the two groups(p<0.05), as well as in hr-HPV(+) and hr-HPV(-) cervicitis tissues, the positive rates of p16^INK4a expression were 44.44%(4/9) and 40.00%(4/10) respectively. This expression increases from cervicitis, CIN to CC, and the significant difference was observed between the groups of CC, CIN and cervicitis with hr-HPV infected or no(p<0.05).2. When CaSki cells were interfered by siHPV16E7 001,siHPV16E7 002 and siHPV16E7 003, the levels of mRNA encoding HPV16E7 in cells was reduced, especially siHPV16E7 001 can make HPV16E7 mRNA reduce by 80%. Moreover, the most suitable multiplicity of infection of siHPV16E7 001 for CaSki cells was 60nM.3. Compared with SiHa-Nontransfection(0.6033±0.0186) and SiHa-siNControl (0.5500±0.0173), HPV16E7 mRNA expression(0.3067±0.0120) surveyed by RT-PCR decreased markedly in SiHa-siHPV16E7 group(p<0.01), and similar alterations were found in HPV16E7 protein expression among these three groups. The above mentioned results suggested that HPV16E7 expression in SiHa cells could be inhibited efficiently by RNAi.4. Compared with CaSki-Nontransfection(1.1733±0.0667) and CaSki-siNControl (1.3933±0.1667), HPV16E7 mRNA expression(0.4033±0.0328) surveyed by RT-PCR decreased markedly in CaSki-siHPV16E7 group(p<0.01), and similar alterations were found in HPV16E7 protein expression among these three groups. The above mentioned results suggested that HPV16E7 expression in CaSki cells could be inhibited efficiently by RNAi.5. Compared with SiHa-Nontransfection(1.0033±0.0233) and SiHa-siNControl (0.9267±0.0371), INK4a mRNA expression(0.5833±0.0133) surveyed by RT-PCR decreased markedly in SiHa-siHPV16E7 group(p<0.01), and similar alterations were found in INK4a protein expression among these three groups. The above mentioned results suggested that INK4a expression in SiHa cells could be inhibited efficiently by siHPV16E7 interference.6. Compared with CaSki-Nontransfection(0.7833±0.0088) and CaSki-siNControl (0.8533±0.0353), INK4a mRNA expression(0.4767±0.0145) surveyed by RT-PCR decreased markedly in CaSki-siHPV16E7 group(p<0.01), and similar alterations were found in INK4a protein expression among these three groups. The above mentioned results suggested that INK4a expression in CaSki cells could be inhibited efficiently by siHPV16E7 interference.7. Cell cycle analysis showed that siHPV16E7 induced accumulation of SiHa and CaSki cells in G0-G1 phase by 31.7% and 15.6% with a decrease in the percentage of cells in S-phase by 6.4% and 14.9% relative to control. These sequence specific siRNAs showed a blockbuster effect in arrestment of the cell cycle.8. Compared with CaSki(0.3967±0.0491) and HeLa(0.5567±0.1037), INK4a mRNA expression(0.5633±0.0722) surveyed by RT-PCR had no obvious difference in C-33A cells (p>0.05), and similar alterations were found in INK4a protein expression among these three groups. The above mentioned results suggested that INK4a expression was at equal pace in CC cells with or without HPV infection.Conclusion1. Overexpression of INK4A gene in precancerous and cancerous lesions of the cervix with or without HPV infection.2. These sequence specific siRNAs showed a blockbuster effect in downregulation of HPV16E7 gene expression in CC cells SiHa and CaSki.3. INK4a expression in SiHa and CaSki cells could be inhibited efficiently by siHPV16E7 interference,moreover, these sequence specific siRNAs showed a blockbuster effect in arrestment of the cell cycle.4. INK4a expression was with one accord in CC cells with or without HPV infection. Therefore/ In a word, the above mentioned conclusion suggested that INK4a expression was at equal pace in CC cells with or without HPV infection. Overexpression of p16 INK4a protein is associated with HPV in cervical cell carcinoma and dysplasia with HPV infection,whereas there are else mechanisms on overexpression of p16 INK4a in CC and dysplasia without HPV infection. Overexpression of INK4a gene help to define the screening and early diagnosis of cervical cancer cases.