The Relationship Between The Expression Of Protein IGF-II And HPV Infection In Lung Cancer And The Analysis Of IGF-II Imprinting Status Of Lung Cancer

Objective:1. To investigate the etiological role of human papillomavirus (HPV) in the carcinogenesis of lung cancer, and analysis the relationship of HPV infection and the clinical pathologic characteristics in lung cancer.2. To study the relationship among the high expression of protein IGF-II, the carcinogenesis of lung cancer and their clinical pathologic characteristics.3. To analysis the relationship between HPV infection and the expression of Insulin-like growth factor-II (IGF-II) protein in lung cancer tentatively.4. To determine the imprinting status of Igf2 gene in lung cancer, and analysis the meaning of LOI of Igf2 gene.Methods:1. The immunohistochemicl SP method was used to measure the expression of protein IGF-II and oncoproteins high-risk spectrum HPV E6/E7 in 68 cases of lung cancer and 12 cases of benign lung diseases, respectively;2. The PCR-RFLP and RT-PCR-RFLP methods were used to detect the imprinting status of Igf2 gene in 64 cases of lung cancer and 24 cases of benign lung tissues, respectively.Results:1. The detection of HPV infection and protein IGF-II in 68 cases of lung cancer and 12 cases of benign lung tissuesThe immunohistochemicl SP method combined with PCR were used to detect the high-risk HPV E6/E7 oncoproteins and high risk type of HPV 16/18, in order to select 16/18 type HPV infection with HPV E6/E7 oncoproteins secreted. The results showed that the infection rate of HPV in the lung cancer group was 44.1% (30/68), and that in the lung benign control group was 8.3% (1/12). There was statistical difference between two groups (P<0.05).The overexpression of protein IGF-II was common in the tissue of lung cancer. Its negative, positive, and strong positive expression rate were 10.3% (7/68), 36.8% (25/68) and 52.9% (36/68). In contrast, the negative, positive and strong positive expression rate of IGF-II in benign tissue were 75.0% (9/12), 16.7% (2/12) and 8.3% (1/12), respectively (P <0.05).2. The expression of protein IGF-II between HPV positive group and HPV negative group in 68 cases of lung cancerThe expression level of protein IGF-II in 30 cases of HPV positive lung cancers group was significantly higher than that of HPV negative group (P <0.05). And its negative, positive and strong positive expression rate were 3.3% (1/30), 30.0% (9/30) and 66.7% (20/30); However, the expression rate of protein IGF-II in 38 cases of HPV negative of lung cancers were 15.8% (6/38), 42.1% (16/38) and 42.1% (16/38).3. The relationship between the infection of HPV and the expression of protein IGF-II, and their clinic pathologic characteristics in 68 cases of lung cancerBoth HPV infection and IGF-II protein expression were related to the differentiation grade of lung cancer. The HPV infection rate in poor-differentiation tissues was 64.3% (18/28), and statistically higher than that in moderate-differentiation and well- differentiation (P <0.05), which of that were 33.3% (9/27) and 23.1% (3/13), respectively. The expression of protein IGF-II in poor-differentiation was highest, and its negative, positive and strong positive expression rate were 10.7% (3/28), 17.9% (5/28) and 71.4% (20/28); Its expression rate in moderate-differentiation tissues were 7.4% (2/27), 40.7% (11/27) and 51.9% (14/27), respectively; however, its expression rate in well- differentiation tissues were 15.4% (2/13), 69.2% (9/13) and 15.4% (2/13). There was significant difference among three groups (P <0.05).The high expression of protein IGF-II was related to the lymph node metastasis. Its strong positive expression rate in lymph node metastasis group was 64.3% (27/42), and its strong positive expression rate was just 34.6% (9/26) in lymph node non-metastasis group. There was statistical difference in two groups (P <0.05). The expression level of protein IGF-II was related to the clinical stage, and its negative,positive and strong positive expression rate in stage I lung cancer tissues were 12.5% (2/16), 62.5% (10/16) and 25.0% (4/16); its expression rate in stage II tissues were 13.3% (2/15), 46.7% (7/15) and 40.0% (6/15), respectively; however, its expression rate in III~IV stage were 8.1% (3/37), 21.6% (8/37) and 70.3% (26/37), respectively. There was statistical difference between three groups (P <0.05).4.Identification of the Igf2 gene imprinting status in 64 cases of lung cancer and 24 cases of benign lung tissues by RFLP methodThe PCR-RFLP method was used to screen the informative cases of Igf2 gene. The heterozygotic cases were selected for the further analysis of LOI by RT-PCR-RFLP method. The results showed that the percentage of heterozygotes in lung cancer and benign tissues were 48.4% (31/64) and 37.5% (9 / 24), respectively. The percentage of loss of imprinting (LOI, two-allelic expression) for Igf2 gene in lung cancer heterozygotes was 77.4% (24/31), while 22.6% (7/31) for normal imprinting (single allele expression). However, in the heterozygotes of lung benign lesions tissues, the percentage of LOI of Igf2 gene was 66.7% (6/9), while 33.3% (3/9) exists normal imprinting. The difference between the two groups was not statistically significant (χ~2=0.430, P =0.512).In 31 cases of the matching lung cancer tissues, the percentage of lung tumor tissues for LOI was 77.4% (24/31); the percentage of adjacent (2 cm far) non-tumor tissues for LOI was 61.3% (19/31); however, the percentage of the most distal tissues from the tumor for LOI of Igf2 gene was only 29.0% (9/31). The LOI differences for Igf2 gene among the three tissue groups were statistically significant (P <0.05), and the LOI of Igf2 gene between lung tumor tissues and the adjacent (2 cm far) non-tumor tissues or the most distal tissues from the tumor existed statistical differences, respectively (P <0.05), however, there was no statistical difference between the lung tumor group and the adjacent (2cm far) non-tummor group (χ2=1.897, P =0.168).Conclusion:1. The infection rate of HPV in the lung cancer group was statistically higher than that in the lung benign group, and it was related to the differentiation grade, which indicated that HPV infection may be an important etiological factor in the tumorigenesis of lung cancer.2. The expression level of protein IGF-II in lung cancer was obviously higher than that in benigh control group, and it was related to the lymph node metastasis, differentiation and clinical stage. IGF-II is an important potent mitogen, suggesting that their strong expression may lead to cell malignant transformation and make the lymphatic epithelial cells proned to proliferate, leading to easy adoption of lymph node metastasis of lung cancer. It illustrated that IGF-II played an important role in the development of lung cancer. 3. IGF-II protein expression in lung cancer was associated with HPV infection, the existence of HPV-E6/E7 oncoproteins may induce the loss of imprinting of Igf2 gene, and result in the overexpression of protein IGF-II, which make a role in promoting proliferation and inhibiting apoptosis in the carcinogenisis of lung cancer.4. There was statistical difference among the matching lung cancer for the LOI of Igf2 gene, the LOI of Igf2 gene in lung tumor group and adjacent (2cm far) non-tumor group was statistically higher than that of the most distal tissues from the tumor, suggesting that IGF-II involved in tumor growth mainly with an autocrine and paracrine secretion way.